Note: Descriptions are shown in the official language in which they were submitted.
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NOVEL BRASSICA VARIETY
FIELD OF THE INVENTION
The present invention is related to the production and cultivation of novel
Brassica cultivars.
BACKGROUND OF THE INVENTION
Brassica
The Brassicaceae (or Cruciferae) is a family of approximately 350 genera and
3,200 species of pungent or acrid plants commonly referred to as the "mustard
family."
Included within this family are many edible species, including cabbage,
broccoli,
cauliflower, kale, kohlrabi, turnips, radish, and rutabaga. The edible genera
include
Brassica (rape, cabbage, and mustard), Lepidium (cress), Nasturtium
(watercress),
Raphanus (radish), Romarmoracia (horseradish), and Wasabia (Japanese
horseradish or
wasabe). The ornamental genera include Aethionema (stonecress), Alyssoides
(bladderpod), Alyssum (madwort), Arabis (rock cress), Aubrieta, Aurinia,
Brassica
(ornamental kale), Cardamine, Cheiranthes (wallflower), Crambe (colewort),
Diplotaxis (rocket), Draba, .~rysimum (wallflower or blister cress), Hesperis
(rocket),
Iberis (candytuft), Lobularia, Lunaria (honesty), and Matthiola (stock).
Brassica sp. include plants commonly referred to as "rape" "rapeseed," or
"canola." These plants are annual or biennial, and resemble mustard in
appearance.
Rape is primarily grown as an oil source, as well as for stock feed and green
manure.
Brassica napus and Brassica rapa, as well as other species, are important
worldwide
as oilseed crops, with the oil found in mature seeds known as canola oil.
Canoia must
meet standards based on seed, meal and oil characteristics. For example, the
seed
must be of the geniis Brassica and contain <1.0% of all fatty acids as erucic
acid
(22:1) and <18 ,moles of glucosinolates per gram of whole seed at a moisture
content
of 8.5%; and the meal must contain no more than 30 moles of glucosinolates per
gram of oil free meal, at a moisture content of 8.5% (Canadian Canola
Council.1997;
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and Canola Growers Manual, 1991 ). Thus, there is great interest in producing
Brassica plants that produce large quantities of high quality canola oil.
The rape Brassica can be grouped into one of two different growth habits,
referred to as annual spring (or summer) cultivars and biennial winter
cultivars. The
winter types are planted in the fall, overwinter as seedlings, and then
complete their
development the following spring. The winter type cultivars require
vernalization at
low temperatures to induce flowering and seed development. Concurrent with
vernalization in winter cultivars is the acquisition of a degree of freezing
tolerance.
The spring types do not have a vernalization requirement.
The vernalization requirement reduces the chance of premature flower induction
that may occur following brief periods of warm weather during the winter.
While this
is advantageous to producers, it is detrimental to breeders, who must add six
weeks to
every generation time for winter cultivars. However, compared to spring types,
winter
types have numerous attributes such as superior lodging resistance, increased
yield,
better disease resistance, and higher inherent and acclimation-specific
freezing
tolerance.
Low Temperature Effects and Freezing Tolerance
As biological stress includes any change in environmental conditions that
might
reduce or adversely affect growth or development, exposure of plants to low
temperatures places stress on plants. This stress triggers a series of
responses apparent
in freezing tolerant species, including low temperature stress, low
temperature
adjustment, the acquisition of acclimation-specific freezing tolerance
(acclimation), and
vernalization. Adjustment to low temperature stress and acclimation occur
simultaneously, making them difficult to differentiate.
A number of mechanisms have evolved that enable plants to cope with a
variety of adverse environmental conditions. In order to survive exposure to
freezing
temperatures, plants must possess high levels of both inherent and acclimation-
specific
tolerance. Inherent tolerance is genetically determined and allows plants to
cope with
short periods of high, sub-zero temperatures. Acclimation, developed in
response to
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prolonged exposure to low temperatures, increases the degree of freezing
tolerance in
plants capable of acclimating. This process involves complex physiological,
molecular, and biochemical processes (See e.g., Singh and Laroche, Biochem.
Cell.
Biol., 66:650-657 j1988J).
Low temperature acclimation has been shown to cause alterations at all gene
expression levels (See, Thomashow, Adv. Genet., 28:99-130 [1990]). The genes
encoding a number of low temperature-induced proteins have been isolated from
several plants including B. napes (Orr et al., Plant Physiol., 98:1532-1534
[1992]; and
Weretilynk et al., Plant Physiol., 101:171-177 [1993]), Arabidopsis thaliana
(Kurkela
and Frank, Plant Mol. Biol., 15:137-144 [1990]; Gilmour et aL, Plant Mol.
Biol.,
18:13-21 [1992]; Lin and Thomashow Plant Physiol., 99:519-525 [1992]; and
Palva,
Gene expression under low temperature stress, in Basra (ed.), Stress-Induced
Gene
Expression in Plants, Harwood Academic, New York, pp. 103-130 [1994]),
Spinacia
oleracea {Guy, Ann. Rev. Plant Physiol., Plant Mol. Biol., 47:187-223 [1990]),
Hordeum vulgare {Cativelli and Bartels, Plant Physiol., 93:1504-1510 [1990]),
and
Medicago sativa (Monroy et al., Plant Physiol., 102:873-879 [1993]). Gene
subsets or
families appear to be regulated at the level of RNA transcription or protein
translation
during exposure to low temperature (Guy, supra; Johnson-Flanagan et aL, Plant
Physiol., 81:301-308 [1991]; and Hawkins et al, Genome 39:704-710 [1996]).
However, no function has been assigned to any of the low temperature-induced
genes
described.
Selection for freezing tolerant lines or cultivars has been difficult because
of
the numerous responses associated with exposure to low temperature, including
low
temperature adjustment and vernalization. Further, many breeders working to
improve
freezing tolerance have relied upon field survival as the major selection
criterion
(Marshall, in Christiansen and Lewis (eds)., Breeding For Tolerance to Heat
and
Cold. Breeding Plant for Favorable Environments, John Wiley and Sons, New
York,
pp. 47-69 [1982]). This is difficult to assess, as there are many other
factors that
control field survival, including ice encasement and the consequent anoxia
(Andrews
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and Morrison, Agronomy J., 84:960-962 [1992]). Selection is further
complicated by
the fact that the acclimation process is cumulative and can be stopped,
reversed or
restarted (Gusts et al., Can. J. Bot., 60:301-305 [1982]; and Roberts, Can. J.
Bot.,
57:413-419 [1979]). The fact that plant material must be acclimated for up to
six
weeks for maximum attainment of freezing tolerance has also complicated the
breeding
and selection process for hardier canola cultivars.
Furthermore, data collected to date indicate that freezing tolerance is
complex
and follows a polygenic pattern (See, Stushnoff et al., Breeding and selection
of
resistance to low temperature, in Voss (ed.), Plant Breeding--A Contemporary
Basis,
pp. 115-13b [1984]). For example, Sutka (Sutka, Theor. Appl. Genet., 59:145-
152
[ 1981 ]) showed that 1 S of 2 i chromosomes can be implicated in freezing
tolerance in
Triticum aestivum (winter wheat). In B. napus, there are a minimum of four
linkage
groups that appear to segregate with freezing tolerance (See, Teutonico et
al., Crop
Sci., 33:103-107 [1995]).
It is difficult to ascertain the highest level of inherent and acclimation-
specific
freezing tolerance achieved to date in winter canola, as many breeding
programs do
not assess freezing tolerance. Nonetheless, it is possible that the highest
tolerances are
in the range of -5°C and -15°C, based on the fact that most
tolerant canola quality
rape was produced in the Pacific Northwest, a region that can be very cold. In
the
. early years of the development of tolerant canola, Cascade was used as a
foundation
stock, as it was the most freezing tolerant material available at the time.
Since then,
freezing tolerance in canola has not been significantly improved. This appears
to be
the result of a lack of suitable germplasm, an inability to rapidly and
accurately assess
tolerance, and the complexity of plant responses to low temperature. Thus, due
in part
to the increased demand for quality canola oil, there remains a need in the
art to
increase the freezing tolerance of winter canola cultivars, including an
increased need
for an understanding of the genetic control of agronomic traits (Ferreira et
al., Theor.
Appl. Genet., 89:615-621 [ 1994). Development of cultivars better suited to
the often
harsh conditions in the temperate growing regions (e.g., drought, heat,
disease and in
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particular, low temperature), has become an important consideration for canola
breeders.
SUMMARY OF THE INVENTION
The present invention is related to novel Brassica cultivars, and methods for
developing novel Brassica plants.
The present invention provides methods for producing improved cultivars. In
preferred embodiments, the improved cultivars are members of the genus
Brassica. In
particularly preferred embodiments, the cultivars are canola. In one
embodiment, the
present invention provides a method for producing an improved Brassica
cultivar,
comprising the steps of: providing a first parent Brassica line and a second
parent
Brassica line; crossing the first and second parent Brassica lines to produce
a
reciprocal cross; culturing the reciprocal cross to produce a microspore
culture; and
treating the microspore culture to produce a doubled haploid cultivar.
In another embodiment, the method further comprises the step of vernalizing
the reciprocal cross. In yet another embodiment, the method further comprises
the
step of vernalizing the doubled haploid. Thus, it is contemplated that either
or both
the reciprocal cross and doubled haploid undergo vemalization.
In an alternative embodiment, the first and second parent Brassica Iines are
winter type. In one preferred embodiment, the first parent Brassica line is
selected
from the group consisting of Cascade and Rebel. In yet another alternate
embodiment,
the present invention further comprises the step of growing the doubled
haploid
cultivar to produce seeds. In another embodiment, the present invention
provides the
step of harvesting the seeds.
The present invention also provides the doubled haploid cultivars. In
preferred
embodiments, the doubled haploid cultivar is vernalization minus and/or
freezing
tolerant.
The present invention further provides a method for producing Brassica
cultivars, comprising the steps of crossing first and second Brassica plants,
where at
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least one of the Brassica plants is characterized by at least one genetic
factor which
confers freezing tolerance, the genetic factor being capable of transmission
to the
Brassica cultivar substantially as a recessive gene.
In addition, the present invention provides the cultivars produced according
to
the method. In one preferred embodiment, the genetic factor confers
vernalization
independence (i. e., the plant does not require vernalization). In an
alternative preferred
embodiment of the method, the Brassica plant is selected from the group
consisting of
Cascade and Rebel. In a most preferred embodiment, the Brassica plant is
selected
from the group consisting of Rebel, VERN-, and any line that does not require
vernalization (e.g., FZ progeny containing the approximately 2.8 kb band, but
not the
approximately 3.0 kb band described in Example 9, and shown in Figures 6A-C).
The present invention also provides a winter Brassica cultivar expressing
characteristics of spring Brassica cultivars. In preferred embodiments, the
cultivar is
vernalization independent, and/or freezing tolerant. In a particularly
preferred
embodiment, the cultivar is VERN- or Rebel.
The present invention also provides methods and models to elucidate the
genetic components of Brassica associated with various traits, including, but
not
limited to freezing tolerance and vernalization. It is contemplated that the
present
invention will find use in establishing the genetic elements associated with
these and
other economically important traits and characteristics. In particular, it is
contemplated
that the vernalization independent cultivars (i. e., spring-like
characteristics) that retain
the desirable qualities of winter types will be used to develop additional
cultivars. The
present invention will also find use in the development of improved cultivars
of other
members of the Brassicaceae.
It is further contemplated that the cultivars of the present invention will
find
widespread use in agricultural areas in which freezing tolerance and/or
vernalization
are considerations in making choices as to the type of canola to cultivate.
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DESCRIPTION OF THE FIGURES
Figures lA-C are graphs of freezing tolerance for three DH, lines, including
VERN-. Figures 1D and lE are graphs showing the inherent and acclimation-
specific
freezing tolerance of Cascade, Rebel, F,, and the DH, line, VERN-.
Figure 2 shows a graph of an LOD plot of flowering time.
Figure 3 shows the segregation of acclimation-specific freezing tolerance.
Figures 4A and 4B provide representative blots probed with the cDNA clone
BN28 from B. napes.
Figure 5 is a representative blot probed with genomic clones A) EC2E5, and B)
WG1F6.
Figure 6 is a table showing the recombination analysis of DH, lines.
Figure 7A is a blot showing RFLP complementation analysis of parents and F,.
Figures 7B and 7C are a drawing and blot (respectively), showing the
complementation
analysis of the parent lines, VERN-, an F, plant, and multiple FZS.
Figure 8 is a blot showing RFLP analysis of BC,FZ segregants.
Figure 9A shows the RFLP results obtained with the EcoRI-digested samples, and
Figure 9B shows the results obtained with the PvuII-digested samples.
Figure 10 shows a Northern blot demonstrating expression of BN28.
Figure 11 shows an immunoblot analysis of BN28 protein accumulation.
Figure 12 provides a graph showing the marker loci of Cascade, Rebel, and
VERN-.
GENERAL DESCRIPTION OF THE INVENTION
The present invention provides methods of producing new Brassica cultivars
that
retain a high degree of both inherent and acclimation-specific freezing
tolerance, but lack
2S a vernalization requirement.
The present invention provides several distinct advantages over currently used
winter cultivars, as it provides plant lines with acclimation-specific
freezing tolerance in
excess of -18 °C, representing an improvement of at least 3 °C
over previously reported
tolerance levels. However, the phenotypic differences associated with
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inherent and acclimation-specific freezing tolerance in Brassica napus
represent but
one of the improvements provided by the lines of the present invention.
As the improved lines are derived from canola quality material, the present
invention also provides methods to increase freezing tolerance in winter
canola in
demand for high quality oil production. The methods of the present invention
are also
useful for improving the freezing tolerance of spring canola. Spring-type
canola
cultivars often suffer from late spring frosts, while the plants are at the
seedling stage,
and then in the fall during maturation. For example, the inherent tolerance of
-8.5°C
observed during the development of the present invention could be transferred
to
spring canola, representing significant progress in developing tolerance to
these frosts.
Definitions
Prior to providing a further description of the invention, and to facilitate
understanding the invention, a number of terms are defined below.
As used herein, the term "Brassica" refers to any member of the genus
Brassica. The term "Brassicaceae" (or "Cruciferae") refers to the family of
plants
commonly referred to as the "mustard family."
As used herein, the term "winter type" refers to plants which typically have a
vernalization requirement. These plants are "vernalization dependent."
As used herein, the term "spring type" (or "summer type") refers to plants
that
do not have a vernalization requirement. These plants are "vernalization
independent."
As used herein, the term "cultivar" refers to any cultivated variety produced
by
horticultural techniques. The term encompasses any horticultural variety,
strain, or
race that has originated and persists under cultivation.
As used herein, the term "strain" refers to an intraspecific group of
organisms
that possess only one or a few distinctive traits, are usually genetically
homozygous
(i. e., pure breeding) for those traits, and are often maintained as an
artificial breeding
group for domestication (e.g., agricultural applications), or for genetic
experimentation.
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The term also encompasses "variety," although this term is applied when the
differences between the intraspecific groups is substantial.
As used herein, the term "inflorescence" refers to the flowering part of a
plant,
the arrangement of flowers on a plant, as well as the process of coming into
bloom
(i. e., blossoming).
As used herein, the term "anthesis" refers to the period during which a flower
opens (or the act of a flower opening), or coming into full bloom.
As used herein, the term "stigma" refers to the receptive surface usually
located
at the apex of the style of a flower, on which compatible pollen grains
germinate. The
term "pollen grain" refers to microspores in flowering plants that germinate
to form
male gametophytes (pollen grain and pollen tube).
As used herein, the term "graft" refers to the union of a piece of one plant
to
another, established plant.
As used herein, the term "meristem" refers to regions where cells are actively
dividing.
As used herein, the term "bolt" refers to the rapid growth of a stem prior to
flowering.
As used herein, the term "annual" refers to plants that complete their life
cycle
within a single growing season. The term encompasses plants that grow from
seed,
bloom, fruit, and die in the course of the same year. However, it is also
intended to
encompass plants that may be carried over two or more years by preventing them
from
setting seed.
As used herein, the term "biennial" refers to plants that require two seasons
of
growth.
As used herein, the term "vernalization" refers to plants that require
exposure to
low temperature in order to complete their life cycle.
As used herein, the term "freezing tolerance" refers to the ability of a plant
to
withstand the effects of subzero temperatures (i.e., temperatures below
freezing [0°C]).
As used herein, the term "double haploid" refers to plants produced by
manipulation of the developing pollen grains to induce development of a
sporophyte
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plant (i. e., a haploid plant), that is further treated so that its single
haploid set of
chromosomes is doubled. These plants are 100% homozygous. In the case where
the
"parent" plant used as a source of developing pollen is heterozygous, each
doubled
haploid plant produced is genetically different, since these plants represent
the haploid
products of different meioses where recombination of alleles of different
genes may
occur. Inheritance analysis of the alleles of genes using doubled haploid
plants is
equivalent to the direct analysis of the gametes produced by a heterozygote.
As used herein, the term "haploid" refers to a plant having one set of
unpaired
chromosomes.
As used herein, term "diploid" refers to plants having two sets of
chromosomes.
As used herein, the term "allopolyploidy" refers to hybrids arising from the
combination of chromosomes from two different species or strains.
As used herein, the term "aneuploidy" refers to the condition in which the
chromosome numbers are not exact multiples of the haploid set (i. e., there
are missing
or extra chromosomes present within the nucleus).
As used herein, the term "hybrid" refers to the offspring of two plants of the
same, different, or closely related species that differ in one or more genes.
The term
"heterosis" refers to hybrid vigor" (i.e., increased vigor, size, and/or
fertility of a
hybrid, as compared with its parents). "Hybridogenesis" refers to a form of
clonal
reproduction in species hybrids, whose gametes carry only the nuclear genome
derived
from one of the parental species. A "hybrid swarm" is a continuous series of
morphologically distinct hybrids resulting from hybridization of two species
followed
by crossing and backcrossing of subsequent generations. "Hybrid sterility"
refers to
the failure of hybrids between different species to produce viable offspring.
"Hybrid
breakdown" refers to the reduction in fitness of FZ and/or backcross
populations from
fertile hybrids produced by intercrossing genetically disparate populations or
species.
The term is also used in reference to a postzygotic reproductive isolating
mechanism.
When used in relationship to the creation of hybrids, the term "hybridization"
refers to
the mating of individuals belonging to genetically disparate populations or to
different
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species, or in Mendelian terms, hybridization is the mating of any two unlike
genotypes or phenotypes.
As used herein, the term "reciprocal cross" refers to the crossing of two
plants
of different varieties or cultivars. The term encompasses crosses of the forms
A
S (female) x B (male) and B (female) x A male), where the "A" and "B"
individuals
differ in genotype and/or phenotype. For example, the term encompasses the
crossing
of Cascade and Rebel, as described in Example 2. "Reciprocal genes" refer to
complementary genes, while "reciprocal hybrids," are hybrid offspring derived
from
reciprocal crosses of parents from different species. "Reciprocal
recombination" refers
to the production of new linkage arrangements that are different from those of
the
maternal and paternal homologues, that occur in the gametes of dihybrids.
As used herein, the term "self' refers to an individual plant produced by self
fertilization, as opposed to cross-breeding.
As used herein, the term "cross-fertilization" or "cross-breeding" refers to
the
I S fertilization of the ovules of one flower by the pollen of another,
between individuals
of the same or different species, resulting in the production of hybrid
plants.
As used herein, "F," refers to offspring resulting from the first experimental
crossing of plants (i. e., the first filial generation). The parental
generation with which
the genetic experiment starts is referred to as "P," or "P."
As used herein, the term "backcross" refers to a cross between an offspring
and
one of its parents or an individual genetically identical to one of its
parents. A
"backcross parent" is that parent of a hybrid with which it is again crossed,
or with
which is is repeatedly crossed. A backcross may involve individuals identical
to the
anent rather than the anent itself. "B " "B " "B " etc. refer to the first
second
P ~ P a 2~ a> > > >
third, etc. backcross generations. The first backcross is created by mating an
individual with one of its parents or with an individual of that identical
genotype. The
offspring resulting from this cross are the "B, generation." The second
backcross is
created by crossing B, individuals again with individuals of a genotype
identical to the
parent referred to in the first backcross, etc.
As used herein, the term "back mutation" refers to reverse mutations.
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As used herein, the term "polymorphism" refers to the occurrence within and
among populations of several phenotypic forms associated with the alleles of
one gene
or homologues of one chromosome. The term is also used in reference to the
large
number of variants seen in different individuals.
As used herein, the terms "pleotropism" and "pleotropy" refer to the
production
of multiple phenotypic effects by a single gene. The term encompasses multiple
phenotypic effects that are distinct and seemingly unrelated. "Pleomorphism"
refers to
the occurrence of variable phenotypes in a genetically uniform group of
organisms.
As used herein, the term "homologous chromosomes" refers to plants that have
IO matching chromosome pairs. The term encompasses paired chromosomes, one of
paternal and one of maternal origin. Homologous chromosomes are
morphologically
alike and contain loci for the same genes. It also refers to chromosomes that
pair
during the process of meiosis. Each homologue is a duplicate of one of the
maternally
or paternally contributed chromosomes. Homologous chromosomes contain the same
I S sequence of genes. Therefore, each gene is present in duplicate.
As used herein, the term "homozygous" refers to plants that have identical
genes on homologous chromosomes. The term is also used in reference to the
situation in which, at a particular locus, the alleles on a chromosome pair
are identical.
As used herein, the term "heterozygous" refers to plants that have both
20 dominant and recessive genes for a particular characteristic on homologous
chromosomes. The term is also used in reference to the situation in which, at
a
particular locus, the alleles on a chromosome pair are different.
As used herein, the term "heredity," refers to the transmission of traits to
successive generations.
25 As used herein, the term "alternation of generations" refers to the
reproductive
cycles in which a haploid phase and a diploid phase alternate, during the
course of a
plant's life cycle. In mosses and vascular plants, the haploid phase is the
gametophyte,
while the diploid phase is the sporophyte.
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As used herein, the term "independent segregation," refers to the independent,
random inheritance of genes on different chromosomes. It is also sometimes
referred
to as "independent assortment."
As used herein, the term "karyotype" refers to the chromosomal constituents of
a cell or individual. The term is often used in reference to the arrangement
of
chromosomes during a particular stage in meiosis, in which the chromosomes may
be
observed, counted, and potential heredity problems identified.
As used herein, the term "meiosis" refers to the process during which one
round of chromosomal replication (duplication) is followed by two rounds of
division,
to produce four haploid cells. Nuclear division of sex cells results in the
formation of
cells with half the normal amount of genetic information. Thus, each of these
"haploid" cells contain one of each pair of the individual's chromosomes. The
term
"mitosis" refers to the process of cell division in which two cells ("daughter
cells") are
produced from one cell ("parent" or "mother" cell) in which each of the
daughter cells
contains the same genetic complement as the parent cell.
As used herein, the term "phenotype" refers to the observable characteristics
of
an individual, which results from the expression of the individual's genotype.
The
term "genotype" refers to the genetic makeup of the individual. Often, it is
used in
reference to the alleles of particular gene or limited number of genes under
investigation.
As used herein, the term "clones" refers to genetically identical organisms
produced vegetatively from a single parent.
As used herein, the term "spore" refers to a reproductive cell that grows
directly into a new plant. The term "microspore" refers to a spore that
develops into a
male gametophyte. The term "gametophyte" refers to the haploid phase, in which
mitosis results in the production of gametes in plants undergoing an
alternation of
generation. The term "sporophyte" refers to a diploid, spore-producing plant
in the
alternation of generations.
As used herein, the term "germination" refers to the beginning of growth of a
seed, spore, or pollen grain.
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As used herein, the term "stably maintained" refers to organisms that maintain
-
at least one of their unique properties or elements (i. e., the element that
is desired)
through multiple generations. For example, it is intended that the term
encompass
recombinant winter Brassica plants that have lost the vernalization
requirement typical
S of winter canola. It is not intended that the term be limited to any
particular species,
cultivar or strain.
The term "gene" refers to a DNA sequence that comprises control and coding
sequences necessary for the production of a polypeptide or precursor. The
polypeptide
can be encoded by a full length coding sequence or by any portion of the
coding
sequence so long as the desired enzymatic activity is retained.
The term "allele" refers to alternate forms of a gene that can exist at a
particular locus.
The term "gene frequency" refers to the percentage of a given type of allele
in
a population. The term "gene pool" refers to the total complement of all genes
present
in members of the population. The term "genetic equilibrium" refers to the
situation in
which allele frequencies are maintained at the same rate in successive
generations (i. e.,
the allele frequencies are neither increasing nor decreasing in successive
generations
within the population).
As used herein, the term "genome" refers to the array of genes carried by an
individual organism. In plants, the genome is comprised of multiple
chromosomes.
As used herein, the term "intron," refers to non-coding segments of DNA
located between coding regions in a gene which is transcribed, but does not
appear in
the mRNA, nor final gene product. The term "exon" refers to coding regions of
DNA.
As used herein, the term "inverted repeat refers to two copies of the same DNA
sequence which are in oriented in opposite directions on the same molecule.
As used herein, the term "linkage" refers to the situation in which two or
more
non-allelic genes tend to be inherited together. Linked genes are are located
on the
same chromosome, although they can be separated by crossing over. The term
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"linkage disequilibrium" refers to the finding that some gene pairs are found
together
more frequently than would be expected by chance. Thus, they are present more
often
that the product of their individual gene frequencies. The term "linkage
group" refers
to a group of genes with loci on the same chromosome. The term "locus" refers
to the
S site on a chromosome at which a certain gene is located (plural: loci).
The term "wild-type" refers to a gene or gene product which has the
characteristics of that gene or gene product when isolated from a naturally
occurring
source. A wild-type gene is that which is most frequently observed in a
population
and is thus arbitrarily designed the "normal" or "wild-type" form of the gene.
In
contrast, the term "modified" or "mutant" refers to a gene or gene product
which
displays modifications in sequence and or functional properties (i. e.,
altered
characteristics) when compared to the wild-type gene or gene product. It is
noted that
naturally-occurring mutants can be isolated; these are identified by the fact
that they
have altered characteristics when compared to the wild-type gene or gene
product.
1 S The term "oligonucleotide" as used herein is defined as a molecule
comprised
of two or more deoxyribonucleotides or ribonucleotides, preferably more than
three,
and usually more than ten. The exact size will depend on many factors, which
in turn
depends on the ultimate function or use of the oligonucleotide. The
oligonucleotide
may be generated in any manner, including chemical synthesis, DNA replication,
reverse transcription, or a combination thereof.
Because mononucleotides are reacted to make oligonucleotides in a manner
such that the 5' phosphate of one mononucleotide pentose ring is attached to
the 3'
oxygen of its neighbor in one direction via a phosphodiester linkage, an end
of an
oligonucleotide is referred to as the "S' end" if its 5' phosphate is not
linked to the 3'
oxygen of a mononucleotide pentose ring and as the "3' end" if its 3' oxygen
is not
linked to a 5' phosphate of a subsequent mononucleotide pentose ring. As used
herein, a nucleic acid sequence, even if internal to a larger oligonucleotide,
also may
be said to have 5' and 3' ends.
When two different, non-overlapping oligonucleotides anneal to different
regions of the same linear complementary nucleic acid sequence, and the 3' end
of one
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oligonucleotide points towards the 5' end of the other, the former may be
called the
"upstream" oligonucleotide and the latter the "downstream" oligonucleotide.
The term "primer" refers to an oligonucleotide which is capable of acting as a
point of initiation of synthesis when placed under conditions in which primer
extension
is initiated. An oligonucleotide "primer" may occur naturally, as in a
purified
restriction digest or may be produced synthetically.
A primer is selected to be "substantially" complementary to a strand of
specific
sequence of the template. A primer must be sufficiently complementary to
hybridize
with a template strand for primer elongation to occur. A primer sequence need
not
reflect the exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer, with the
remainder of
the primer sequence being substantially complementary to the strand.
Non-complementary bases or longer sequences can be interspersed into the
primer,
provided that the primer sequence has sufficient complementarity with the
sequence of
the template to hybridize and thereby form a template primer complex for
synthesis of
the extension product of the primer.
"Hybridization" methods involve the annealing of a complementary sequence to
the target nucleic acid (the sequence to be detected). The ability of two
polymers of
nucleic acid containing complementary sequences to find each other and anneal
through base pairing interaction is a well-recognized phenomenon. The initial
observations of the "hybridization" process by Marmur and Lane, Proc. Natl.
Acad.
Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461
(I960)
have been followed by the refinement of this process into an essential tool of
modern
biology. Nonetheless, a number of problems have prevented the wide scale use
of
hybridization as a tool in diagnostics. Among the more formidable problems
are: 1 )
the inefficiency of hybridization; 2) the low concentration of specific target
sequences
in a mixture of genomic DNA; and 3) the hybridization of only partially
complementary probes and targets.
With regard to efficiency, it is experimentally observed that only a fraction
of
the possible number of probe-target complexes are formed in a hybridization
reaction.
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This is particularly true with short oligonucleotide probes (e.g., less than
100 bases in
length). There are three fundamental causes: a) hybridization cannot occur
because of
secondary and tertiary structure interactions; b) strands of DNA containing
the target
sequence have rehybridized (reanneaIed) to their complementary strand; and c)
some
target molecules are prevented from hybridization when they are used in
hybridization
formats that immobilize the target nucleic acids to a solid surface.
Even where the sequence of a probe is completely complementary to the
sequence of the target (i. e., the target's primary structure), the target
sequence must be
made accessible to the probe via rearrangements of higher-order structure.
These
higher-order structural rearrangements may concern either the secondary
structure or
tertiary structure of the molecule. Secondary structure is determined by
intramolecular
bonding. In the case of DNA or RNA targets this consists of hybridization
within a
single, continuous strand of bases (as opposed to hybridization between two
different
strands). Depending on the extent and position of intramolecular bonding, the
probe
can be displaced from the target sequence preventing hybridization.
Solution hybridization of oligonucleotide probes to denatured double-stranded
DNA is further complicated by the fact that the longer complementary target
strands
can renature or reanneal. Again, hybridized probe is displaced by this
process. This
results in a low yield of hybridization (low "coverage") relative to the
starting
concentrations of probe and target.
With regard to low target sequence concentration, the DNA fragment
containing the target sequence is usually in relatively low abundance in
genomic DNA.
This presents great technical difficulties; most conventional methods that use
oligonucleotide probes lack the sensitivity necessary to detect hybridization
at such low
levels.
One attempt at a solution to the target sequence concentration problem is the
amplification of the detection signal. Most often this entails placing one or
more
labels on an oligonucleotide probe. In the case of non-radioactive labels,
even the
highest affinity reagents have been found to be unsuitable for the detection
of single
copy genes in genomic DNA with oligonucleotide probes. (See Wallace et al.,
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Biochimie 67:7ss [1985]). In the case of radioactive oligonucleotide probes,
only
extremely high specific activities are found to show satisfactory results.
(See
Studencki and Wallace, DNA 3:1 [1984] and Studencki et al., Human Genetics
37:42
[198s]).
s With regard to complementarity, it is important for some diagnostic
applications to determine whether the hybridization represents complete or
partial
complementarity. For example, where it is desired to detect simply the
presence or
absence of DNA encoding a particular protein, it is only important that the
hybridization method ensures hybridization when the relevant sequence is
present;
conditions can be selected where both partially complementary probes and
completely
complementary probes will hybridize. Other diagnostic applications, however,
may
require that the hybridization method distinguish between partial and complete
complementarity. It may be of interest to detect genetic polymorphisms.
Unless combined with other techniques (such as restriction enzyme analysis),
1 s methods that allow for the same level of hybridization in the case of both
partial as
well as complete complementarity are typically unsuited for such applications;
the
probe will hybridize to both the normal and variant target sequence.
Hybridization,
regardless of the method used, requires some degree of complementarity between
the
sequence being assayed (the target sequence) and the fragment of DNA used to
perform the test (the probe). (Of course, one can obtain binding without any
complementarity but this binding is nonspecific and to be avoided.)
The "complement" of a nucleic acid sequence as used herein refers to an
oligonucleotide which, when aligned with the nucleic acid sequence such that
the 5'
end of one sequence is paired with the 3' end of the other, is in
"antiparallel
2s association." Certain bases not commonly found in natural nucleic acids may
be
included in the nucleic acids of the present invention and include, for
example, inosine
and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may
contain mismatched base pairs or unmatched bases. Those skilled in the art of
nucleic
acid technology can determine duplex stability empirically considering a
number of
variables including, for example, the length of the oligonucleotide, base
composition
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and sequence of the oligonucleotide, ionic strength and incidence of
mismatched base -
pairs.
As used herein the tenors "protein" and "polypeptide" refer to compounds
comprising amino acids joined via peptide bonds and are used interchangeably.
S The terms "native gene" or "native gene sequences" are used to indicate DNA
sequences encoding a particular gene which contain the same DNA sequences as
found
in the gene as isolated from nature. In contrast, "synthetic gene sequences"
are DNA
sequences which are used to replace the naturally occurnng DNA sequences when
the
naturally occurring sequences cause expression problems in a given host cell.
For
example, naturally-occurring DNA sequences encoding codons which are rarely
used in
a host cell may be replaced (e.g., by site-directed mutagenesis) such that the
synthetic
DNA sequence represents a more frequently used codon. The native DNA sequence
and the synthetic DNA sequence will preferably encode the same amino acid
sequence.
"Nucleic acid sequence" as used herein refers to an oligonucleotide,
nucleotide
or polynucleotide, and fragments or portions thereof, and to DNA or RNA of
genomic
or synthetic origin which may be single- or double-stranded, and represent the
sense or
antisense strand. Similarly, "amino acid sequence" as used herein refers to
peptide or
protein sequence. "Peptide nucleic acid" as used herein refers to an
oligomeric
molecule in which nucleosides are joined by peptide, rather than
phosphodiester,
linkages.
A "deletion" is defined as a change in either nucleotide or amino acid
sequence
in which one or more nucleotides or amino acid residues, respectively, are
absent.
An "insertion" or "addition" is that change in a nucleotide or amino acid
sequence which has resulted in the addition of one or more nucleotides or
amino acid
residues, respectively, as compared to, naturally occurring sequences.
As used herein, the term "non-polar" ("nonpolar") insertion refers to an
insertion of a DNA fragment that does not negatively affect the expression of
genes
located downstream of the insertion.
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As used herein, the term "insertional inactivation" refers to the abolition of
the
functional properties of a gene product by insertion of a foreign DNA sequence
into
the coding or regulatory portion of the gene.
A "substitution" results from the replacement of one or more nucleotides or
amino acids by different nucleotides or amino acids, respectively.
As used herein, the term "substantially purified" refers to molecules, either
nucleic or amino acid sequences, that are removed from their natural
environment,
isolated or separated, and are at least 60% free, preferably 75% free, and
most
preferably 90% free from other components with which they are naturally
associated.
An "isolated polynucleotide" is therefore a substantially purified
polynucleotide.
As used herein, the term "probe" refers to an oligonucleotide (i.e., a
sequence
of nucleotides), whether occurring naturally as in a purified restriction
digest or
produced synthetically, which is capable of hybridizing to another
oligonucleotide or
polynucleotide of interest. Probes are useful in the detection, identification
and
isolation of particular gene sequences. It is contemplated that any probe used
in the
present invention will be labelled with any "reporter molecule," so that is
detectable in
any detection system, including, but not limited to enzyme (e.g., ELISA, as
well as
enzyme-based histochemical assays), fluorescent, radioactive, and luminescent
systems.
It is further contemplated that the oligonucleotide of interest (i. e., to be
detected) will
be labelled with a reporter molecule. It is also contemplated that both the
probe and
oligonucleotide of interest will be labelled. It is not intended that the
present invention
be limited to any particular detection system or label.
As used herein, the term "target" refers to the region of nucleic acid bounded
by the primers used for polymerase chain reaction. Thus, the "target" is
sought to be
sorted out from other nucleic acid sequences. A "segment" is defined as a
region of
nucleic acid within the target sequence.
"Amplification" is defined as the production of additional copies of a nucleic
acid sequence and is generally carried out using polymerase chain reaction
(PCR) or
other technologies well known in the art (e.g., Dieffenbach and Dveksler, PCR
Primer,
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a Laboratory Manual, Cold Spring Harbor Press, Plainview NY (1995]). As used
herein, the term "polymerise chain reaction" ("PCR") refers to the method of
K.B.
Mullis (U.S. Patent Nos. 4,683,195, 4,683,202, and 4,965,188, hereby
incorporated by
reference), which provides methods for increasing the concentration of a
segment of a
target sequence in a mixture of genomic DNA without cloning or purif cation.
This
process for amplifying the target sequence consists of introducing a large
excess of
two oligonucleotide primers to the DNA mixture containing the desired target
sequence, followed by a precise sequence of thermal cycling in the presence of
a DNA
polymerise. The two primers are complementary to their respective strands of
the
double stranded target sequence. To effect amplification, the mixture is
denatured and
the primers then annealed to their complementary sequences within the target
molecule. Following annealing, the primers are extended with a polymerise so
as to
form a new pair of complementary strands. The steps of denaturation, primer
annealing and polymerise extension can be repeated many times (i.e.,
denaturation,
annealing and extension constitute one "cycle"; there can be numerous
"cycles") to
obtain a high concentration of an amplified segment of the desired target
sequence.
The length of the amplified segment of the desired target sequence is
determined by
the relative positions of the primers with respect to each other, and
therefore, this
length is a controllable parameter. By virtue of the repeating aspect of the
process, the
method is referred to as the "polymerise chain reaction" (hereinafter "PCR").
Because
the desired amplified segments of the target sequence become the predominant
sequences (in terms of concentration) in the mixture, they are said to be "PCR
amplified".
As used herein, the term "polymerise" refers to any polymerise suitable for
use
in the amplification of nucleic acids of interest. It is intended that the
term encompass
such DNA polymerises as Taq DNA polymerise obtained from Thermus aquaticus,
although other polymerises, both thermostable and thermolabile are also
encompassed
by this def nition.
With PCR, it is possible to amplify a single copy of a specific target
sequence
in genomic DNA to a level detectable by several different methodologies (e.g.,
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hybridization with a labeled probe; incorporation of biotinylated primers
followed by
avidin-enzyme conjugate detection; incorporation of'ZP-labeled deoxynucleotide
triphosphates, such as dCTP or dATP, into the amplified segment). In addition
to
genomic DNA, any oligonucleotide sequence can be amplified with the
appropriate set
of primer molecules. In particular, the amplified segments created by the PCR
process
itself are, themselves, efficient templates for subsequent PCR amplifications.
Amplified target sequences may be used to obtain segments of DNA (e.g., genes)
for
insertion into recombinant vectors.
As used herein, the terms "PCR product" and "amplification product" refer to
the resultant mixture of compounds after two or more cycles of the PCR steps
of
denaturation, annealing and extension are complete. These terms encompass the
case
where there has been amplification of one or more segments of one or more
target
sequences.
As used herein, the term "nested primers" refers to primers that anneal to the
target sequence in an area that is inside the annealing boundaries used to
start PCR.
(See, K.B. Mullis, et al., Cold Spring Harbor Symposia, Vol. LI, pp. 263-273
[1986]).
Because the nested primers anneal to the target inside the annealing
boundaries of the
starting primers, the predominant PCR-amplified product of the starting
primers is
necessarily a longer sequence, than that defined by the annealing boundaries
of the
nested primers. The PCR-amplified product of the nested primers is an
amplified
segment of the target sequence that cannot, therefore, anneal with the
starting primers.
As used herein, the term "amplification reagents" refers to those reagents
(deoxyribonucleoside triphosphates, buffer, etc.), needed for amplification
except for
primers, nucleic acid template and the amplification enzyme.
As used herein, the terms "restriction endonucleases" and "restriction
enzymes"
refer to bacterial enzymes, each of which cut double-stranded DNA at or near a
specific nucleotide sequence.
As used herein, the terms "vector" and "vehicle" are used interchangeably in
reference to nucleic acid molecules that transfer DNA segments) from one cell
to
another. The terms "shuttle vector" or "bifunctional vector" refer to a
cloning vector
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(i. e., vector) that is capable of replication in two different organisms.
These vectors -
can "shuttle" between the two hosts. For example, the present invention
encompasses
shuttle vectors that are capable of replicating in various cultivars of
Brassica.
The terms "expression vector" or "expression cassette" as used herein, refers
to
a recombinant DNA molecule containing a desired coding sequence and
appropriate
nucleic acid sequences necessary for the expression of the operably linked
coding
sequence in a particular host organism. Nucleic acid sequences necessary for
expression in prokaryotes usually include a promoter, an operator (optional),
a
ribosome binding site, and an initiation codon, often along with other
sequences. The
term "expression" may refer to "gene expression" and/or "protein expression."
As used herein, the term "multiple cloning site module" or refers to nucleic
acid that contains multiple cloning sites (i. e., "restriction sites," "MCS,"
or
"polylinker"). It is intended that the term encompass DNA that contain unique,
as well
as non-unique restriction sites. It also is intended to encompass multiple
cloning site
modules that contain foreign (i. e., exogenous) DNA inserted within the DNA
containing the MCS. This foreign DNA may be inserted within the MCS by
recombinant techniques. The DNA may also contain foreign DNA that is inserted
in
locations other than the MCS.
The terms "in operable combination," "in operable order," and "operably
linked" as used herein, refer to the linkage of nucleic acid sequences in such
a manner
that a nucleic acid molecule capable of directing the transcription of a given
gene
and/or the synthesis of a desired protein molecule is produced. The term also
refers to
the linkage of amino acid sequences in such a manner so that a functional
protein is
produced.
As used herein, the term "replicon" refers to a genetic element that behaves
as
an autonomous unit during DNA replication. The term also encompasses nucleic
acid
regions or units that have a single site for origin of replication.
As used herein the term "portion" when in reference to a gene refers to
fragments of that gene. The fragments may range in size from a few nucleotides
to
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the entire gene sequence minus one nucleotide. Thus, "a nucleotide comprising
at least
a portion of a gene" may comprise fragments of the gene or the entire gene.
As used herein, the terms "complementary" or "complementarity" are used in
reference to polynucleotides (i. e., a sequence of nucleotides) related by the
base-
pairing rules. For example, for the sequence "A-G-T," is complementary to the
sequence "T-C-A." .Complementarity may be "partial," in which only some of the
nucleic acids' bases are matched according to the base pairing rules. Or,
there may be
"complete" or "total" complementarity between the nucleic acids. The degree of
complementarity between nucleic acid strands has significant effects on the
efficiency
and strength of hybridization between nucleic acid strands. This is of
particular
importance in amplification reactions, as well as detection methods which
depend upon
binding between nucleic acids.
The term "homology" refers to a degree of complementarity. There may be
partial homology or complete homology (i. e., identity). A partially
complementary
sequence is one that at least partially inhibits a completely complementary
sequence
from hybridizing to a target nucleic acid is referred to using the functional
term
"substantially homologous." The inhibition of hybridization of the completely
complementary sequence to the target sequence may be examined using a
hybridization
assay (Southern or Northern blot, solution hybridization and the like) under
conditions
of low stringency. A substantially homologous sequence or probe will compete
for
and inhibit the binding (i. e., the hybridization) of a completely homologous
to a target
under conditions of low stringency. This is not to say that conditions of low
stringency are such that non-specific binding is permitted; low stringency
conditions
require that the binding of two sequences to one another be a specific (i.e.,
selective)
interaction. The absence of non-specific binding may be tested by the use of a
second
target which lacks even a partial degree of complementarity (e.g., less than
about 30%
identity); in the absence of non-specific binding the probe will not hybridize
to the
second non-complementary target.
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The art knows well that numerous equivalent conditions may be employed to
comprise either low or high stringency conditions; factors such as the length
and
nature (DNA, RNA, base composition) of the probe and nature of the target
(DNA,
RNA, base composition, present in solution or immobilized, etc.) and the
concentration
of the salts and other components (e.g., the presence or absence of formamide,
dextran
sulfate, polyethylene glycol) are considered and the hybridization solution
may be
varied to generate conditions of either low or high stringency hybridization
different
from, but equivalent to, the above listed conditions. The term "hybridization"
as used
herein includes "any process by which a strand of nucleic acid joins with a
complementary strand through base pairing" (Coombs, Dictionary of
Biotechnology,
Stockton Press, New York NY [ 1994].
"Stringency" typically occurs in a range from about Tm 5°C (5°C
below the Tm
of the probe) to about 20°C to 25°C below Tm. As will be
understood by those of
skill in the art, a stringent hybridization can be used to identify or detect
identical
polynucleotide sequences or to identify or detect similar or related
polynucleotide
sequences.
As used herein, the term "Tm" is used in reference to the "melting
temperature."
The melting temperature is the temperature at which a population of double-
stranded
nucleic acid molecules becomes half dissociated into single strands. The
equation for
calculating the Tm of nucleic acids is well known in the art. As indicated by
standard
references, a simple estimate of the Tm value may be calculated by the
equation: Tm =
81.5 + 0.41 (% G + C), when a nucleic acid is in aqueous solution at 1 M NaCI
(See
e.g., Anderson and Young, Quantitative Filter Hybridisation, in Nucleic Acid
Hybridisation [1985]). Other references include more sophisticated
computations
which take structural as well as sequence characteristics into account for the
calculation of Tm.
As used herein the term "hybridization complex" refers to a complex formed
between two nucleic acid sequences by virtue of the formation of hydrogen
bounds
between complementary G and C bases and between complementary A and T bases;
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these hydrogen bonds may be further stabilized by base stacking interactions.
The twa
complementary nucleic acid sequences hydrogen bond in an antiparallel
configuration.
A hybridization complex may be formed in solution (e.g., Cot or Rot analysis)
or
between one nucleic acid sequence present in solution and another nucleic acid
sequence immobilized to a solid support (e.g., a nylon membrane or a
nitrocellulose
filter as employed in Southern and Northern blotting, dot blotting or a glass
slide as
employed in in situ hybridization, including FISH [fluorescent in situ
hybridization]).
As used herein, the term "antisense" is used in reference to RNA sequences
which are complementary to a specific RNA sequence (e.g., mRNA). Antisense RNA
may be produced by any method, including synthesis by splicing the genes) of
interest
in a reverse orientation to a viral promoter which permits the synthesis of a
coding
strand. Once introduced into a cell, this transcribed strand combines with
natural
mRNA produced by the cell to form duplexes. These duplexes then block either
the
further transcription of the mRNA or its translation. In this manner, mutant
phenotypes may be generated. The term "antisense strand" is used in reference
to a
nucleic acid strand that is complementary to the "sense" strand. The
designation (-)
(i.e., "negative") is sometimes used in reference to the antisense strand,
with the
designation (+) sometimes used in reference to the sense (i.e., "positive")
strand.
The term "antigenic determinant" as used herein refers to that portion of an
antigen that makes contact with a particular antibody (i. e., an "epitope").
When a
protein or fragment of a protein is used to immunize a host animal, numerous
regions
of the protein may induce the production of antibodies which bind specifically
to a
given region or three-dimensional structure on the protein; these regions or
structures
are referred to as antigenic determinants. An antigenic determinant may
compete with
the intact antigen (i. e., the immunogen used to elicit the immune response)
for binding
to an antibody.
The terms "specific binding" or specifically binding" when used in reference
to
the interaction of an antibody and a protein or peptide means that the
interaction is
dependent upon the presence of a particular structure (i. e., the antigenic
determinant or
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epitope) on the protein; in other words the antibody is recognizing and
binding to a
specific protein structure rather than to proteins in general. For example, if
an
antibody is specific for epitope "A," the presence of a protein containing
epitope "A"
(or free, unlabelled "A") in a reaction containing labelled "A" and the
antibody will
reduce the amount of labelled "A" bound to the antibody.
As used herein, the term "immunogen" refers to a substance, compound,
molecule, or other moiety which stimulates the production of an immune
response.
The term "antigen" refers to a substance, compound, molecule, or.other moiety
that is
capable of reacting with products of the immune response. For example, BN28 or
other proteins associated with freezing tolerance may be used as immunogens to
elicit
an immune response in an animal to produce antibodies directed against the
protein
used as an immunogen. The protein may then be used as an antigen in an assay
to
detect the presence of antibodies directed against the protein in the serum of
the
immunized animal.
"Alternations in the polynucleotide" as used herein comprise any alteration in
the sequence of polynucleotides encoding any protein, in, including deletions,
insertions, and point mutations that may be detected using hybridization
assays.
Included within this definition is the detection of alterations to the genomic
DNA
sequence which encodes (e.g., by alterations in pattern of restriction enzyme
fragments
capable of hybridizing to any sequence (e.g., by RFLP analysis), the inability
of a
selected fragment of any sequence to hybridize to a sample of genomic DNA
(e.g.,
using allele-specific nucleotide probes), improper or unexpected
hybridization, such as
hybridization to a locus other than the normal chromosomal locus for genes of
interest
(e.g., using FISH to metaphase chromosomes spreads, etc.).
A "variant" in regard to amino acid sequences is used to indicate an amino
acid
sequence that differs by one or more amino acids from another, usually related
amino
acid. The variant may have "conservative" changes, wherein a substituted amino
acid
has similar structural or chemical properties (e.g., replacement of leucine
with
isoleucine). More rarely, a variant may have "non-conservative" changes, e.g.,
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replacement of a glycine with a tryptophan. Similar minor variations may also
include
amino acid deletions or insertions (i. e., additions), or both. Guidance in
determining
which and how many amino acid residues may be substituted, inserted or deleted
without abolishing biological or immunological activity may be found using
computer
programs well known in the art, for example, DNAStar software. Thus, it is
contemplated that this definition will encompass variants of any gene of
interest. Such
variants can be tested in functional assays or by other means.
The term "sample" as used herein is used in its broadest sense. For example,
it
refers to any type of material obtained from plants, plant cells or tissue
cultures, cell
lines.
Brassica Types
Winter types of Brassica cultivars are planted in the fall, overwinter, and
complete their development the following spring. During the overwintering
period, the
plants must meet a vernalization requirement in order to flower. This
requirement is
met by extended exposure to low, non-freezing temperatures. For example, B.
napus
cv Cascade requires at least 6 weeks of exposure to 4°C, in order to
meet its
vernalization requirement. Prior to vernalization, plant development is
arrested at the
rosette stage Once the requirement is met, the plants bolt, and become
reproductive,
first forming inflorescence meristems, followed shortly thereafter by flower
meristems.
Subsequent steps in floral development are analogous in winter and spring
types.
The novel spring type B. napus with a high degree of freezing tolerance
provided by the present invention (e.g., VERN-) resulted from significant
genetic
changes that led to the loss of the vernalization requirement of its parent
plants.
Freezing tolerance was assessed in a segregating double haploid population
(DH,)
produced using microspore culture. Individual DH, lines provided a homozygous
non-
segregating source of plant material. The relationship between inherent and
acclimation-specific freezing tolerance was assessed using phenotypic
characterization.
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Genotypic characterization was used to examine the segregation of acclimation-
specific-
freezing tolerance.
The observed polymorphism between the parents and the novel cultivar of the
present invention can be detected with a number of restriction enzymes in VERN-
, but
never in the parent plants, suggesting that there has been a rearrangement or
deletion
of more than a few base pairs. The 600:0 ratio for vernalization in the F,s,
followed
by the 85:1 ratio in the DH,s indicated that the change arose between these
generation.
The VERN- line of the present invention differs significantly from other
described vernalization mutants (See, Koomeef et al., Mol. Gen. Genet., 229:57-
66
[1991]; Chandler et al., Plant J., 10:637-644 [1996]; and Martinez-Zapatre and
Somerville, Plant Physiol., 92:770-776 [1995]). First, it has a high degree of
freezing
tolerance. Second, it appears to be on the vernalization-dependent pathway,
rather the
vernalization-independent pathway. Finally, it is recessive for the spring
growth habit.
The latter two properties are characteristic of the late-flowering ecotypes of
Arabidopsis (Dennis et al., Cell. Develop. Biol., 7:441-448 [1996]; Lee et
al., Mol.
Gen. Genet., 237:171-176 [1993]; Clarke and Dean, Mol. Gen. Genet., 242:81-89
[1994]; and Lee and Amasino, Plant Physiol., 108:157-162 [1995]).
In addition, comparisons between VERN-, vernalized VERN+, and spring
cultivars demonstrated that the vemalization response was ameliorated in VERN-
, as it
flowered in the same time-frame as the spring types. All of the other known
vernalization mutants simply delay or accelerate flowering.
Earliness is a function of the length of both the vegetative phase and the
reproductive phase. In VERN-, it was noticed that both phases were generally
shorter,
relative to the spring cultivars. The fact that VERN- reached the 4-leaf stage
in
advance of the winter parents suggested that rapid vegetative growth is a
function of
genetic alteration(s). In addition, once flowering commenced, it was more
prolific and
proceeded more quickly in VERN-.
The freezing tolerance of VERN- was significantly greater than that of the
tolerant parent, Cascade. This result is significant, as a lack of adequate
freezing
tolerance in winter canola is consistently and repeatedly cited as a major
constraint to
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expansion of the area for production of canola. Although an understanding of
the
mechanism is not necessary to use the present invention, the improvement was
noted
in the F, generation, indicating that it may have occurred because of
heterosis or the
genetic similarity between the parents. Teutonico et al. (Teutonico et al.,
Mol. Breed.,
1:329-339 [1995]) reported transgressive segregants in their population of
canola.
However, the highest tolerance obtained by these authors was only -
13°C, well below
the -18°C suggested with the present invention. Thus, the VERN- line of
the present
invention will find use in areas where it is desirable to have a canola line
with
increased freezing tolerance.
Freezing also severely limits the acreage available to grow spring canola.
Spring frosts can kill or set back rosette-stage plants, resulting in yield
reductions at
the end of the growing season. Fall frosts adversely affect quality (Johnson-
Flanagan
et al., Plant Physiol., 81:301-308 [1990]). These problems associated with
freezing are
overcome by the VERN- plant line of the present invention, as it is a spring
type with
1 S an inherent tolerance of -8.5°C expressed at both the vegetative
and reproductive
stages. These plants will not be adversely affected by frosts that rarely
exceed -5°C.
Breeding Strategies in Brassica Development
Production of new Brassica cultivars has generally made use of traditional
methods of plant breeding, coupled with phenotypic assessment. The phenotype
is
measured using visible parameters such as plant vigor and general appearance,
or
quantifiable traits, such as yield, oil and protein composition and production
(See e.g.,
Rafalski et al., RAPD Markers--A New Technology for Genetic Mapping and Plant
Breeding, CAB Int'1, pp. 645-648 [1991]). Although traditional methods have
been
commonly used to date, in complex systems, such as low temperature responses,
this
selection process is difficult.
The pedigree method of breeding is used in populations of self and cross-
pollinated species for the development of desirable homogenous lines (See
e.g., Fehr,
Principles of Cultivar Development, vol. 1, Macmillan Publishing, Ames, Iowa
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[1993]). Pedigree breeding produces F,s by hybridization of two parental
lines, which-
are then grown to maturity for FZ seed (Snape, Doubled Haploid Breeding:
Theoretical Basis and Practical Applications, Second Symposium on Genetic
Manipulations in Crops, CIMMYT and IRRI, [1982]). Thus, F2 individuals are
recombinant products of the original parent, and are maintained in a
heterozygous
state. In order to stabilize the genotype, successive rounds of selfing and
selection are
required. Generally, sufficient homozygosity can be attained after five or six
generations (Fehr, supra). One advantage of the pedigree method is that
recombination occurs during each meiotic generation allowing for expression
and
selection of favorable traits. However, the time needed to complete the
selfing process
is a major limitation to this approach, especially where environmental
interactions
require seasonal field assessment and selection of individual generations.
The double haploid breeding method makes use of the ability to develop
individuals from gametes without fertilization. In order to be successful,
tissue culture
1 S technology must be able to produce large numbers of embryos representing
the genetic
diversity of the parents. An advantage of this system is that whole plants are
regenerated from individual cells, with each cell having an unique genotype.
Haploid embryo production in the Brassicaceae is achieved by the isolation of
microspores from the pollen sac. At this stage, the microspore is uninucleate,
and has
not yet undergone the first mitotic division (Fan et al., Protoplasma 147:191-
199
[1988]). In vitro culture of microspores under sterile conditions leads to the
formation
of haploid embryos. These embryos undergo cell divisions and grow in a manner
similar to zygotic embryos. When placed in appropriate media, the embryos form
roots
and shoots, similar to germinating seeds. Unless spontaneous doubling occurs,
the
resulting DH, plants remain sterile, and must have their chromosome number
doubled
with colchicine. Colchicine is an alkaloid drug that inhibits spindle fiber
formation
during metaphase and anaphase of mitosis, preventing separation of the paired
chromatids after splitting of the centromere. The result is a single cell that
is
homozygous at all loci. One advantage of double haploid technology is that it
fixes
recombinant gametes directly as homozygous lines in a single generation,
whereas five
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or six generations of selfing and selection are needed in conventional
pedigree
programs. However, a disadvantage of double haploid breeding observed in
previous
work, is the inability to combine favorable loci through generations of
recombination.
The DH, lines produced during the development of the present invention are
S stable and no longer segregate for freezing tolerance. In order to complete
the
required requisites, several plants had to be grown, spanning four generations
of self
pollination. Statistical assessment of inherent freezing tolerance and
acclimation-
specific freezing tolerance of DH, lines demonstrated a lack of segregation
within
individual lines. The stability of the population was important in order to
allow
characterization of the lines.
Vernalization and Freezing Tolerance
As indicated above, exposure to low temperatures not only induces freezing
tolerance and adjustment to low temperature, it is also required for
vennalization in
winter species. Fulfillment of vernalization requirements results in the
transition from
vegetative to reproductive growth (Dennis et al., Cell Develop. Biol., 7:441-
448
[1996]).
There is a broad range of vernalization responses, ranging from an absolute
requirement to a very weak requirement. Those plants with a weak requirement
eventually flower under favorable conditions in the absence of vernalization.
Further,
exposure to extended photoperiods and/or high light intensity overcomes the
low
temperature requirement. This weak requirement is referred to as a
quantitative
response. For example, this is the response demonstrated by most of the
Arabidopsis
ecotypes studied (See, Lee et al., Mol. Gen. Genet., 237:171-176 [1993]; and
Napp-
Zinn, On the genetic basis of vernalization requirement in Arabidopsis
thaliana, in
2S Champagnat and Jacques (eds.), La Phsiologie de la floraison, Coll. Int.
CN1ZS, Paris,
pp. 217-220 (1985]). In these plants, exposure to vernalization conditions
serves to
hasten floral induction. On the other hand, those plants with a strong
vernalization
requirement must be exposed to low temperature in order to flower. This is
referred
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to as a qualitative response, and is the response observed in winter Brassica
sp.
(See,Hodgson, Aus. J. Agric. Sci. Cambridge 84:693-710 [1978]; and Tommey and
Evans, Ann. Appl. Biol., 118:201-208 [1991]).
However, little is known about the genetics of vernalization in Brassica.
Early
work on B. napes suggested that vernalization is controlled by two recessive
genes
(Thurling and Das, Aust. J. Agr. Res., 30:261-269 [1979]). Similarly, studies
on B.
oleracea indicate poIygenic inheritance (Pelofske and Baggett, [1979];
Teutonico and
Osborn, Theor. Appl. Genet., 90:727-732 [1994]), with the annual growth habit
being
dominant (Baggett and Kean, Hort. Sci., 24:262-264 [1989]; Teutonico and
Osborn,
Theor. Appl. Genet., 90:727-732 [1994]). More recently, linkage maps have
shown
one region that is strongly linked to vernalization and days to flowering and
two to
other regions with smaller effects on days to flowering (Ferreira et al.
Theor. Appl.
Genet., 89:885-894 [1995]).
In winter cereals, vernalization has been reported to be linked, either
pleotropically or genetically, to freezing tolerance (Brute-Babel and Fowler,
Crop Sci.,
28:879-884 [1988]). Recent reports demonstrate an obligate relationship
between
vernalization and freezing tolerance, with tolerance decreasing significantly
once the
vernalization requirement is met (Fowler et al., Theor. Appl. Genet., 93:554-
559
[1996]; and Fowler et al., Can. J. Plant Sci., 76:37-42 [1995]).
There is limited evidence which suggests that freezing tolerance and
vernalization are not linked in Brassica. For example, Markowski and Rapacz
(Markowski and Rapacz, J. Agron. Crop Sci., 173:184-192 [1994]) indicated that
there
was no relationship between the degree of freezing tolerance and the degree of
vernalization required in B. napes lines. In addition, genetic analysis showed
that
separate linkage groups exist in both B. napes and B. raga for the capacity to
attain
freezing tolerance and vernalization (Ferreira et al., Theor. Appl. Genet.,
90:727-732
[1995]; and Teutonico et al., Mol. Breed., 1:329-339 [1995]). However,
although the
lines developed in these studies expressed considerable differences in their
ability to
acclimate and in their vernalization requirements, no lines were found that
failed to
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express either trait. Consequently, it has been generally assumed by those in
the art, -
that freezing tolerance and vennalization cannot be inherited separately,
despite the lack
of statistical correlation between the traits.
The complexity of the genetics of freezing tolerance has further hindered the
determination of the relationship between freezing tolerance and
vernalization. For
example, Teutonico et al. (Teutonico et al.,Mol. Breed., 1:329-339 [1995])
showed
that freezing tolerance in the Brassicas may be controlled by a number of
linkage
groups spread throughout the genome. They also observed that regions linked to
freezing tolerance in B. rapa were not linked with tolerance in B. napes. The
complexity of the trait is compounded by the fact that plants have both
inherent
tolerance and acclimation-specific tolerance (Stone et al. Proc. Natl. Acad.
Sci.,
90:7869-7873 [1993]; and Teutonico et al., Mol. Breed., 1:329-339 [1995]).
These
two traits are under separate genetic control (Stone et al., supra).
The present invention also provides means for determining the relationship
between freezing tolerance and vernalization. Vernalization has been reported
to be
linked, either pleotropically or genetically to freezing tolerance (Brute-
Babel and
Fowler, Crop Sci., 28:879-884 [1988]). Early work suggested that vernalization
is
controlled by two recessive genes (Thurling and Das, Aust. J. Agr. Res.,
30:261-269
[1979]), although more recent reports indicate that there is a genetic region
that is
strongly associated with vernalization requirements and days required to
flowering
(Ferreira et al., Theor. Appl. Genet., 90:727-732 [1995]). In addition, other
reports
indicate that separate linkage groups exist for the capacity to attain a level
of freezing
tolerance.
To date, the only loci that appear to be true vernalization loci are FRI (Lee
et
al., [1993], supra; Clarke and Dean, [1994], supra; and Lee and Amasino
[1995],
supra), and FLC (Lee et al., Plant J., 6:903-909 [1994]). These are found on
the
vernalization-dependent pathway (Dennis et al., [1996], supra). Other loci,
such as
vrn 1 and 2 (Chandler et al., [ 1996], supra), and fca, fy, and fve (Burns et
al., Proc.
Natl. Acad. Sci., 90:287-291 [1993]), have arisen as recessive mutations of
the spring
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ecotypes. These mutations would be expected to confer a vernalization
requirement by
blocking the vernalization-independent pathway (Martinez-Zapater and
Somerville,
[1990], supra; and Dennis et al., [1996], supra).
However, prior to the development of the present invention, the relationship
between vernalization and freezing tolerance remained unclear. Recent work on
Arabidopsis suggests that freezing tolerance and vernalization are controlled
by
completely separate pathways (Chandler et al., [1996], supra). However, it is
difficult
to draw a conclusion from this report, as the mutants were generated from the
annual
ecotype Landsberg erects, in which flowering is largely dictated by light
conditions,
with vernalization playing a secondary role. In addition, this ecotype only
attains a
moderate degree of freezing tolerance following exposure to low, non-freezing
temperatures (Chandler et aL, [1996], supra). Furthermore, analysis of the
inheritance
of BN28 throughout the Brassicaceae revealed that species-specific loci exist.
The results obtained during the development of the present invention clearly
demonstrate that freezing tolerance and vernalization can be inherited
separately. For
example, VERN- has lost the vernalization requirement, while expressing a
higher
degree of freezing tolerance than that expressed by either parent, both in the
absence
of acclimation and following acclimation. As such, the linkage between
vernalization
and freezing tolerance has been broken in VERN-. Thus, the methods used to
produce
VERN- will find use in producing superior plants with advantages such as high
freezing tolerance and earlier maturation. Unlike previously used methods to
breed for
improved tolerance to low temperature, the methods of the present invention
provide
the means to successfully accomplish the production of plants with high
freezing
tolerance levels and other highly desirable characteristics.
DETAILED DESCRIPTION OF THE INVENTION
In order to identify the difference between the requirements for vernalization
and freezing tolerance, an isogenic line was developed from doubled haploid
progeny
of reciprocal crosses between B. napus cv Rebel and B. napus cv Cascade. Both
of
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these parent cultivars are winter types, with strong vernalization responses.
Rebel was
found to have little capacity for acquisition of freezing tolerance, while
Cascade is
highly freezing tolerant. Analysis of approximately 100 isogenic lines
revealed
random segregation of freezing tolerance. A single DH, line ("6-200" or "VERN-
")
was scored as "vern-," indicating that it lacked a vernalization requirement,
yet
retained a high degree of freezing tolerance.
As both parents have similar lineages, the Cascade x Rebel cross provided
maximal selection pressure for freezing tolerance, and limited other genetic
differences. Microspore culture methods used in conjunction with colchicine
treatment
yielded approximately 100 isogenic doubled lines (DH,). Spontaneous doubling
accounted for 3% of the doubling that occurred. The remainder of the lines
were
doubled with colchicine. Aside from fertility, no other phenotypic differences
were
apparent between any of the colchicine-treated material and the spontaneous
diploids.
Derived lines were tested for acquisition of freezing tolerance and
vernalization
requirements. Freezing tolerance measurements were completed on original
materials,
as well as on selfed (DHZ) progeny, in order to ensure that no further
segregation had
occurred. Selfed progeny performed similarly to the original plant line. Based
on the
data obtained during the development of the present invention, the segregation
of
freezing tolerance was determined.
DH, plants were grouped into 2°C intervals and the distribution
was
determined. Three major groups were apparent, at a ratio of approximately
1:1:1. The
lack of a genetic trend further indicates that freezing tolerance is a
multigenetic trait.
Although an understanding of the degree of transgressive segregation appearing
in
isogenic plants is not necessary to use the present invention, isogenic plants
may
contain QTLs with more tolerant phenotypes than Cascade.
During the development of the present invention, phenotypic and genotypic
analyses of isogenic DH, lines indicated that freezing tolerance and
vernalization are
not linked, although both occur under similar environmental conditions.
Furthermore,
on the basis of the segregation ratio of BC,F, and BC,F2, it was concluded
that vern-
is a homozygous recessive locus. Reciprocal backcrosses to Rebel and Cascade
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resulted in the complementation of the vern- phenotype, and reinstatement of
the -
vernalization requirement in 100% of 600 BC,F, plants. In the BC,F2, the
phenotypic
segregation ratio for 80 plants was 6I : I9 (i. e., almost exactly a ratio of
3:1 ). These
results confirmed the homozygous recessive nature of VERN-.
Molecular analysis of VERN- revealed a distinct polymorphism at a
vernalization locus. Complementation analysis and FZ segregation using
parental
backcrosses suggested that a single recessive gene controls vernalization
requirements
at this locus, and has no influence over freezing tolerance. These results
indicated that
the linkage between freezing tolerance and vernalization can be broken in
winter types
of B. napes, as well as other cultivars with the same phenotypes.
By comparing spring and winter cultivars, it was possible to identify the
spring
and winter type alleles at each marker or gene locus. The data indicate that
VERN-
carries spring type alleles at all the loci examined. So, too, does Rebel,
with the
exception of one marker, wg7b3 in LGI2. At this locus, Rebel carries a winter
type
allele and Cascade has the spring type allele. Thus, VERN- appears to have
inherited
spring type alleles from both Cascade and Rebel.
In additional experiments, B. napes homologous clones of two flowering time
genes of Arabidopsis (Co and FCA) were used as probes to determine the
genotypes of
VERN- and its parents. Clone FCA#17 covered the B. napes FCA gene from within
intron 3 into the RRM2 (RNA Recognition Motif 2). One polymorphism between
Cascade and Rebel was detected within the clone. VERN- and Rebel shared the
same
allele at this locus.
Two genomic Co clones from linkage groups NIO and N12 in the map of
Parkin et al. (Parkin et al., Genome 38:1122-1131 [1995]; corresponding to
LGsl2 and
11 in the map of Osborn et al. [Osborn et al., Genetics 146:1123-1129 [1997],
respectively) were also used. These clones detected polymorphisms between
Cascade
and Rebel. Again, VERN- had Rebel's alleles at the two Co loci.
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EXPERIMENTAL
The following examples are provided in order to demonstrate and further
illustrate certain preferred embodiments and aspects of the present invention
and are
not to be construed as limiting the scope thereof.
In the experimental disclosure which follows, the following abbreviations
apply: eq (equivalents); M (Molar); pM (micromolar); N (Normal); mol (moles);
mmol (miIlimoles); pmol (micromoles); nmol {nanomoles); g (grams); mg
(milligrams); pg (micrograms); ng (nanograms); 1 or L (liters); ml
(milliliters); p.l
(microliters); cm (centimeters); mm (millimeters); ~.m (micrometers); nm
(nanometers); ~E (microeinstein);°C (degrees Centigrade); rpm
(revolutions per
minute); Kd or KDa (kilodalton); Kb (kilobase); MW (molecular weight); PCR
(polymerise chain reaction); RAPD (randomly amplified polymorphic DNA); RFLP
(restriction fragment length polymorphism); Rot or R°t (the product of
RNA
concentration and the time of incubation in an RNA-driven hybridization; the
analog
of Cot values used to describe DNA-driven hybridization reactions); BSA
(bovine
serum albumin); PBS (phosphate buffered saline); Tris (Tris(hydroxymethyl)
methylamine); TBS (Tris buffered saline); TAE (10 mM Tris, 1% acetic acid, 1
mM
EDTA); TE (10 mM Tris, 1 mM EDTA); SSC (salt, sodium citrate buffer); DEPC
(diethy pyrocarbonate); EDTA (ethylenediamine tetraacetic acid); ddH20 (double
distilled deionized water); SDS (sodium dodecyl sulfate); SDS-PAGE (sodium
dodecyl
sulfate polyacrylamide gel electrophoresis); QTL (quantitative trait loci); F1
or F, (first
generation offspring); F2 or FZ (second generation offspring); DH 1 or DH,
(double
haploid, first generation); BC, (backcross first generation); cv (cultivar);
FT or ft.
(freezing tolerance); LTSO or LTS° (temperature at which 50% death
occurs (i.e., 50%
of the population dies); vern+ (vernalization required); vern- (no
vernalization
requirement; VERN-); reb (Rebel); cas (Cascade); SAS (statistical analysis
software);
GLM (general linear model); S.E. (standard error); BBL (Becton Dickinson
Microbiology Systems, Cockeysville, MD); DIFCO or Difco (Difco Laboratories,
Detroit, MI); Remel (Remel, Lenexa, KS); Scientific Products (McGaw Park, IL);
Fisher (Fisher Scientific, New York, NY)U.S. Biochemical (U.S. Biochemical
Corp.,
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Cleveland, OH); Scientific Products (McGraw Park, IL); Sigma (Sigma Chemical
Co., -
St. Louis, MO.); Biorad (BioRad Laboratories, Richmond, CA); Complete Plant
Products (Complete Plant Products, Brampton, Ontario); Conviron (Conviron,
Winnipeg, Manitoba); Bach-Simpson (Bach-Simpson Ltd., London, Ontario,
Canada);
Schieicher and Schuell {Schleicher and Schuell, Keene, NH); Calbiochem
(Calbiochem, San Diego, CA); Pharmacia (Pharmacia Biotech, Piscataway, N~;
Boehringer-Mannheim (Boehringer-Mannheim Corp., Concord, CA); Amersham
(Amersham, Inc., Arlington Heights, IL); NEB (New England Biolabs, Beverly,
MA);
Pierce (Pierce Chemical Co., Rockford, IL); Eppendorf (Eppendorf Scientific,
Madison, WI); and Molecular Dynamics (Molecular Dynamics, Sunnyvale, CA);
ATCC (American Type Culture Collection, Rockville, MD); U.S. Biochemical {U.S.
Biochemical Corp., Cleveland, OH); Scientific Products (McGraw Park, IL);
Oxoid
(Oxoid, Basingstoke, England); BBL (Becton Dickinson Microbiology Systems,
Cockeysville, MD); DIFCO (Difco Laboratories, Detroit, MI); Life Technologies,
BRL, and Gibco-BRL (Life Technologies, Gaithersburg, MD); New England Nuclear
(New England Nuclear Research Products, Boston, MA); and SAS (SAS Institute,
Inc.,
Cary, North Carolina).
EXAMPLE 1
Plant Material and Growth Conditions
Seeds of B. napus cv Cascade, cv Rebel, F,s, DH,s, BC,F,, and BC,F2, as well
as other cultivars used in the following experiments (e.g., the spring
cultivars tested in
these experiments included Westar, Legend, Excel and Quantum, as well as
winter-
type sister line to VERN-, "VERN+") were planted in 15 cm pots containing a
soil
mixture of sand, peat and vermiculite ( 1:1:1 ), and grown under greenhouse
conditions,
with a 16 hour photoperiod (16 hours of "day" and 8 hours of "night"),
augmented
with high intensity discharge (HID, Sylvania, Canada) sodium vapor lights, to
maintain
a minimum light intensity of 275-300 p,E s''. The temperature regime was
20°C day,
and 16°C night. Unless otherwise indicated, the light and temperature
regimes were
maintained for the entire experiment. For example, VERN- was also grown under
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greenhouse conditions with a non-inductive photoperiod of 10 hours, using the
same
temperature and light intensity as described above. The plants were watered
daily to
field capacity, and fertilized every 14 days using 20:20:20 (N-P-K) (Complete
Plant
Products).
The main reason for choosing the parent cultivars (Cascade and Rebel) was
their difference in freezing tolerance (the TLs° for Cascade is -
15.5°C, while the TLso
for Rebel is -7.5°C. Although some common parentage is shared between
the
cultivars, they are considered to be genetically dissimilar. Both cultivars
are canola
quality and were developed at the Agricultural Experimental Station in Moscow,
Idaho.
The Cascade used in the development of the present invention was selected in
the F6 generation from crosses between Indore and three edible oil lines,
Sipal,
WW 827, and Liraglu. Segregating generations were advanced by single seed
descent
and F3 to F6 generations were screened for low levels of glucosinolate in
mature seed
(<8% seed moisture; Auld et al., Crop Sci., 27:1309-1310 [1987]).
Rebel is an F6 line derived by a cross between OAC Triton and WRE 17.
OAC Triton is a triazine-tolerant spring type from the University of Guelph
(Beversdorf et al., Sci., 64:1007-1009 [1984]). WRE 17 is a line derived by
single
seed descent from a cross between Sipal and Indore. Rebel was selected using
the
same methods that were used with Cascade.
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EXAMPLE 2
Development of Homozygous Lines
Reciprocal Crosses
At the 4-leaf stage, the parent plants were transferred to a controlled
environment chamber at 4°C for six weeks to complete vernalization with
a 16 hour
photoperiod of 400-450 p,Elri Zs', and then transferred to a programmable
growth
cabinet (Conviron PGW 36), under 10°C (day) and 6°C (night)
conditions to
synchronize bud development. Buds that started to mature and change color from
dark
green to yellow (indicating impending anthesis) were chosen for crossing.
All crosses were completed in reciprocal fashion between vernalized winter
cultivars Cascade (freezing tolerant; TLS° _ -15°C) and Rebel
(freezing sensitive;
TLS° _ -6.5°C). A total of nine (9) reciprocal crosses were
completed, to produce 18
plants. Individual buds were opened using ethanol-sterilized forceps. Sepals,
petals,
and anthers were removed from the female flower prior to dehiscence of pollen.
Stigmas were pollinated with mature pollen from the appropriate donor parent,
and
enclosed in pollination bags until silique (pod) elongation was apparent
(indicating
embryo fertilization). This helped to ensure that contamination from any
airborne
pollen did not occur. Once all reciprocal crosses had been completed, the
plants were
returned to the greenhouse to allow the F, seeds to mature and be harvested.
Microspore Culture
F, seed was planted and grown as described above. Plants used for microspore
culture were maintained in the growth cabinet after vernalization at
10°C (day) and
6°C (night) conditions, with a 16 hour photoperiod of 275-300 ~E m
Zs'', to
synchronize bud development. Buds to be used for microspore embryo production
were 3-5 mm in length and were collected from healthy plants just prior to
anthesis.
Buds were placed into a wire strainer and surface sterilized in 7% sodium
hypochlorite
(100 ml) for 15 minutes, then rinsed three times in 150 ml of sterile water
for 5
minutes each rinse. All subsequent work was conducted in a laminar flow hood,
using
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autoclaved solutions and instruments, to help ensure that sterile conditions
were
maintained.
Buds were transferred to a mortar and gently crushed in 1-3 ml of BS medium
containing 13% sucrose, as described by Coventry et al. (Coventry et al.,
Manual for
Microspore Culture Techniques in Brassica napus,OAC Publ. 0489, University of
Guelph, Guelph, Ont., [1988]), to release the microspore cells. The suspension
was
filtered through a 0.44 pm nylon filter into a 15 ml Falcon tube (Fisher), and
the
nylon filter was rinsed twice with 1 ml aliquots of BS medium. The volume in
all of
the tubes was then adjusted to 10 ml BS medium prior to centrifugation in a
tabletop
centrifuge (Model IEC-HN-SII, Needam, Mass.), at 1000 rpm for 3 minutes. The
supernatant was removed and the pellet was resuspended in 5 ml of BS medium
containing 13% sucrose. Three more washes were completed with centrifugation
at
800 rpm, 500 rpm, and S00 rpm, respectively: The final pellet was suspended in
NLN
medium containing 13% sucrose (Lichter, Z. Pflanzephysiol., 105:285-291
[1982]).
The volume of the suspension was adjusted to 1 ml for every original bud used
(approximately 20 per isolation). The suspension was then poured into sterile
petri
plates (60 x 15 mm; Fisher), and sealed with Parafilm~. The samples in the
plates
were heat shocked for 3 days at 37°C, in the dark, with no agitation.
The plates were
then transferred to, and kept on a shaker {55-60 rpm), at room temperature in
the dark,
for 4-6 weeks. Embryos were selected and plated onto solid BS medium
containing
3% agar (Difco-Bacto agar) supplemented with 3% sucrose, and placed on a light
bench with a light intensity of 150 pmole rri zs'', at room temperature, until
roots and
true leaves were apparent. DH, plantlets were transferred to sterile soil, and
placed in
a mist chamber under high humidity for 14 days to reduce desiccation and
excessive
stress, and were then transferred to the greenhouse, and allowed to grow until
3-S
mature leaves were apparent, at which time they were transferred to 4°C
for a 6 week
vernalization period.
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Fertility Assessment and Colchicine Treatment
Vemalized DH, plants were moved to the greenhouse and assessed for fertility.
Bolting plants were analyzed for the presence of pollen as an indicator of
spontaneous
doubling (i.e., spontaneous diploidization). Where chromosome doubling had not
occurred, up to 5 cuttings were taken from a single DH, plant, dipped in
powdered
rooting compound (0.2% indole-3-butyric acid)(Plant Products Co.), and rooted
in
moist soil in a misting chamber. Rooted cuttings were trimmed to 6 cm, and
foliage
was trimmed from the shoots to approximately 20 cm. Plants were then placed in
an
aqueous colchicine solution (3.14 g/1), as described by Coventry et al.
(Coventry et al.,
supra), and placed under high intensity light (300 ,mole m Zs'), for 2 hours
to
maximize transpiration and colchicine uptake. The plants were thoroughly
rinsed in
water, potted in 15 cm pots, and placed in the greenhouse. A single doubled
shoot
from each DH, line was maintained and bagged to obtain selfed seed.
Approximately 100 DH, lines were derived from tissue culture. Spontaneous
doubling accounted for 3% of the doubling that occurred. The remainder of the
lines
were doubled with colchicine. Aside from fertility, no other phenotypic
differences
were apparent between any of the colchicine-treated material and the
spontaneous
diploids.
EXAMPLE 3
Vernalization and Acclimation
In order that the flowering response of VERN+ could be compared to that of
Westar and VERN-, it was necessary to determine suitable vernalization
conditions. In
these experiments, two parameters were checked: a) the stage at which the
seedlings
were vernalized (i. e., seed, cotyledons, 2-leaf and 4-leaf) and; b) length of
cold
treatment (i. e., 3, 4, 5, and 6 weeks). Seed was allowed to imbibe for 24
hours prior
to vernalization. Seedlings were grown in the greenhouse as described above,
transferred to the growth chamber for vernalization, and then returned to the
greenhouse. The final leaf number of 10 plants was determined and the results
were
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analyzed using one-way ANOVA (p<0.05) using MSExcel. Based on these data, it
was determined that 4 leaf staged plants should be vernalized for 6 weeks. The
relationship between leaf number and flowering time was also determined.
In experiments to determine the flowering responses of the cultivars under
S different growth conditions, VERN-, Westar and VERN+ plants were grown in
growth
chambers with 22°C (day) and 17°C (night) temperatures, under 1
of 2 photoperiods of
400-4S0 pE M'Z s'; a 16 hour inductive photoperiod and an 8 hour non-inductive
photoperiod, with and without vernalization. The same photoperiod was
maintained
throughout vernalization. It was determined that VERN- and Westar seedlings
had to
be vernalized at 17 DAP (Days After Planting) when grown under a 16 hour
photoperiod, as this was the latest time at which the seedlings were in the
vegetative
stage of development. The maximum time to assessment was 1 SO DAP or 1 SO DAV
(Days After Vernalization).
For vernalization experiments, with the exception of the experiment designed
to
1 S determine the minimum leaf number needed on VERN+ in order to flower,
plants
were moved, at the 4-leaf stage, to 4°C for six weeks. Minimum leaf
number was
determined by moving plants at the cotyledonary stage, and first, second,
third, and
fourth leaf stage to 4°C for 3, 4, 5, and 6 weeks. The light intensity
and photoperiod
were maintained as described in Example 1.
Following six weeks of acclimation, VERN- expressed a high degree of
freezing tolerance, with a TLso of >_-18°C, as compared to a TLso of -
1S°C for
Cascade, and only -7.S°C for Rebel. In addition, the inherent tolerance
of VERN- was
high, compared with the tolerant parent (TLso of -8.S°C for VERN). This
tolerance
was expressed during both vegetative and reproductive development. Figure 1
2S provides a graph showing the frequency distribution of ion leakage (TLso),
for eight
lines.
The loss of the vernalization requirement was confirmed in both greenhouse
and field trials of original and selfed seed. As shown in Table 1 below, VERN-
flowered 3-S days earlier than the 4 spring cultivars in the growth chamber.
In the
greenhouse, flowering was comparable to that of the spring cultivars, while in
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field, VERN- again flowered earlier. In Table 1, the asterisk (*) indicates
that the
values were significant at the 5% level.
Table 1. Flowering Times of VERN- and Registered Spring Cultivars
Flowering time
(Days)
Culhvar ~ Gm~h Chamber... Greenhouse Field
VEItN- 45.7 1.5* 40.8 1.6* 46 1.2
Quantum 48.8 2.0* 39.0 1.3* 49 t 1.0
Westar 48.7 t 2.3* 41.8 f 2.0* Not Tested
Excel 50.6 t 1.8* 42.2 1.8* 48 1.4
Legend 50.0 t 2.0* 38.0 f 2.3* Not Tested
In comparison with three commonly used spring types (B. napus cv Altex, B.
napus cv Legend, and B. napus cv. Alto), VEItN- reached the 4-leaf stage and
then
bolted significantly faster. In addition, VERN- reached the 4-leaf stage at a
more
rapid rate than either of the parents. VERN- then continued to rapidly produce
leaves,
resulting in more leaves at flowering than were present on the spring
cultivars.
Flowering in both VEIZN- and Legend began at 35 days, and ceased at 50 days, a
full
5 days earlier than either Altex or Alto. Both the number of siliques and seed
yield
were high in VEItN- and Alto. These results indicated that more flowers were
produced in a given time frame (i. e., 15 days) in these plants, as compared
to Legend.
Further phenotypic characterization involved field assessments that compared
VERN- with Excel and Quantum at two locations (one in Alberta and one in
Saskatchewan). These cultivars were studied as blackleg tolerance is required
in the
field. VERN- was found to be developmentally faster and provided a higher
yield
than either cultivar. Excel (the early cultivar), took 5 days longer than
VEItN- to
reach the 4-leaf stage; 2 days later, initiation of flowering and maturation
occurred.
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Quantum was approximately 5 days slower in comparison with VERN-. Once again,
the yields of VERN- were superior to the spring cultivars.
The following tables summarize the results obtained in the greenhouse and
field
assessments of VERN-. Table 2 provides a comparison of the gross morphological
characters associated with growth and development from germination through
maturation. The data are expressed as the mean +/- S.E. (n=12), divided into
three
replicates, with each having 4 randomized pots per line; each pot contained 1
plant.
Tables 3 and 4 provide comparison of gross field and greenhouse characters of
growth
and development of VERN-, Excel, and Quantum. In Table 4, the values represent
the
mean ~ the standard error with n=10; these values are significant at the 5%
level. The
field data were collected from 3 locations (1 in Alberta, and 2 in
Saskatchewan), from
10 plants in each plot.
Table 2. Greenhouse Assessment of VERN-
Fourth Bolting First Last' Maturity
Line Leaf (days)(days) Flower Flow~e~ (days)
(days) (days)
VERN- 21 24 35 50 90
Rebel 23 n/a n/a n/a n/a
Cascade 24 n/a n/a n/a n/a
Altex 26 32 42 55 95
Legend 22 28 35 50 92
Alto 22 32 40 55 95
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Table 3. Field Assessment of VERN-
Fourth Leaf First Flower ty ~ y )
Line (days) ''(days) ; Maturi da s
VERN- 21 +/- 0.75 46 +/- 1.2 102 +/- 1.6
Excel 26 +/- 0.60 48 +/- 1.4 104 +/- 2.0
Quantum 27 +/- 0.70 49 +/- 1.0 107 +/- 2.1
Table 4. Greenhouse Assessment of VERN-
Line ~ Fourth Leaf (days) Maturity (days)
VERN- 21 +/- 2.2 90 +/- 1.75
Excel 26 +/- 1.0 95 +/- 1.5
Quantum 27 +/- 1.5 96 +/- 1.0
The results from greenhouse, field, and gross morphological assessments
suggested that VERN- produced more vegetative growth, despite an earlier
transition
to reproductive development. In order to determine whether this was a function
of the
mutation or was a trait of winter types, additional information was required.
To make
this determination, the effect of vernalization time and seedling stage on
leaf number
at flowering and time to flowering was determined for the full sib, VERN+. It
was
determined that four-leaf staged seedlings vernalized for 6 weeks had the
least leaves
and flowered the fastest. Data from VERN+ vernalized for 6 weeks at the 4-leaf
stage, and from VERN-, indicated that rapid vegetative growth is a winter
trait.
Furthermore, the results showed that time to flowering is not compromised in
VERN-.
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EXAMPLE 4
Assessment of Freezing Tolerance
Freezing tolerance was determined on non-acclimated plants, and plants that
had been acclimated for 6 weeks at 4°C, using an electrolyte leakage
assay. Inherent
freezing tolerance was assessed on non-acclimated material that was maintained
in the
greenhouse. Twenty-four randomly selected DH, lines were assayed. Acclimation-
specific freezing tolerance was determined on the two parent lines (Cascade
and
Rebel), six F, plants, and 70 DH, lines that had been exposed to 4°C
for six weeks, as
described for vernalization. Assessments were completed on all 78 lines. The
IO experiments were replicated three times, with five plants per replicate.
The second and third leaves of 4-leaf stage plants and apical leaves of
bolting
plants were harvested and washed in distilled water. Discs of leaf tissue ( 1
cm) were
removed with a cork borer, being careful to avoid any veins, and placed on
moistened
filter paper (Whatman) in small petri dishes (2 discs per plate). The plates
were
placed in a programmable freezer, and the temperature was lowered to
0°C over a 1
hour period. A set of plates was removed at this temperature to act as a non-
frozen
control. The temperature was then lowered to -2.5°C, and nucleation was
initiated by
touching the filter paper with a metal probe cooled in liquid nitrogen.
Samples were
maintained overnight at -2.5°C. The temperature was then lowered at a
constant rate
of -2.5°C per hour, and samples were removed at 2.5°C intervals
between -2.5°C and -
18.5°C. Samples removed from the freezer were allowed to thaw at
4°C for at least
12 hours.
Electrolyte leakage was determined by placing the thawed discs (including the
filter paper) in tubes containing 10 ml of double distilled water, and shaking
(45 rpm)
overnight at room temperature. Freeze-induced leakage was determined by using
a
radiometer (Model CAM 83, Bach-Simpson) to measure the conductivity of the
samples. Total leakage was determined after boiling the samples for 3 minutes,
cooling them to room temperature, and shaking for at least 1 hour at 45 rpm.
Ion
leakage was measured in each case and injury was expressed as a percentage of
total
(boiled). The temperature at which 50% leakage occurred, termed "TLS°,"
was used as
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a measure of plant viability at that particular temperature, given that 50%
leakage is
equivalent to 99% cell death and consequent plant mortality (Sukumaran and
Weiser,
Hort. Sci., 7:467-468 (1972]; and Boothe et al., Plant Physiol., 108:795-803
[1995]).
The developmental age of plant material selected for freeze testing has been
determined to be important in order to attain accurate ion leakage results,
with older
leaves showing reduced levels of freezing tolerance relative to younger
leaves. Thus,
second and third leaves from a four-leaf stage plant were used. These leaves
were
initiated at the same time, and were of similar physiological and
developmental age.
These leaves also provided sufficient plant material for an accurate
assessment of
freezing tolerance from individual plants, as well as plant lines.
Acclimation capacity was determined as the difference in freezing tolerance
between non-acclimated and acclimated material (i. e., the difference between
the
inherent and acclimation-specific freezing tolerance represents the
acclimation capacity
of the line). The acclimation capacity of these 24 randomly selected lines
ranged from
a low of 1.0°C to a high of 14°C. The results also indicated
that there are three
distinct aspect of freezing tolerance present in these DH, lines--inherent
freezing
tolerance, acclimation-specific tolerance, and acclimation capacity.
Figures lA-C show the inherent and acclimation-specific freezing tolerance in
representative three DH, lines. The values represent the mean +/- standard
error, with
n=10 and n=I S for inherent and acclimation-specific tolerance, respectively.
Figure
lA shows that inherent freezing tolerance and acclimation-specific freezing
tolerance
are not correlated. Likewise, inherent freezing tolerance is not correlated
with
acclimation capacity, as shown in Figure 1B. Figures 1D and E are graphs
showing
the inherent and acclimation specific-freezing tolerance in Cascade, Rebel,
F,, and the
DH, line, VERN-.
Thus, as indicated above and in Figure 1, VERN- expressed a high degree of
freezing tolerance, with a TLso of > -18°C, as compared to a TLso of -
15°C for
Cascade and only -7.5°C for Rebel. In addition, inherent tolerance was
high compared
with the tolerant parent (i.e., TLso of -8.5°C for VERN- and TLso of -
5.5°C for
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Cascade). This tolerance was expressed during both vegetative and reproductive
development.
In addition, thirty-two DH lines were assessed for acclimation-specific
freezing
tolerance as above. RFLP data were collected and used to determine possible
associations using clones ec3 e5, wg 1 g6, wg 1 g3, wg 1 f6, wg4h3, ec4h3,
tg5b2, and
tglc8, which map to the freezing tolerance associated (FTA) linkage groups in
Brassica rapa, as outlined by Teutonico et al. (Teutonico et al., Theor. Appl.
Genet.,
89:885-894 [1995]). As indicated below in Example 8, low-temperature induced
cDNA clones BN28, BN115, cor 6.6 and cor 15 were also tested. Statistical
analysis
for association with acclimation-specific freezing tolerance was completed
based on XZ
values derived using SAS ver 6.03 (SAS Institute Inc. 1988), as described in
Example
5, below. As indicated in the following Table (Table 5), strong associations
between
acclimation-specific freezing tolerance and loci wglg6 of LG4 and ec2e5 of LG7
were
identified. At these loci, VERN- inherited freezing tolerance alleles from
Cascade. At
other loci, there was either no association or no polymorphism detected
between the
parents.
Table 5. Association of RFLP Marker Loci for
Freezing Tolerance Linkage Groups
Marker Loci Xi Probability Significance
.
ec3e5 12.42 0.007 Significant
wglg6 8.66 < 0.01 Significant
glg3 14.43 0.6 Not Significant
wglf6 2.22 0.2 Not Significant
wg4h3 0.025 0.1 Not Significant
Analysis of a backcross between VERN- to Cascade (vern+), showed total
complementation by re-establishment of the vernalization requirement in all of
the F,
progeny tested. Assessment of FZ segregation showed that the vernalization
loci
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mapping with WG6B 10 segregated as a homozygous recessive, as shown by the 3
:1
vern+:VERN- ratio observed. Preliminary genotype analysis of the FZ population
confirmed the observed phenotype.
EXAMPLE 5
Statistical Analysis of Freezing Tolerance Data
SAS version 6.03 was used to assess the significance of segregation of
freezing
tolerance in the DH, lines at the level of inherent freezing tolerance and
acclimation-
specific freezing tolerance. From the DH, population, 24 lines were randomly
selected
for determination of inherent freezing tolerance. Analysis of inherent
tolerance was
completed using two replicates. Each replicate contained 5 plants per line
(n=10). For
each plant, leakage measurements were averaged from 3 discs at each of 5
different
temperatures (0, -2.5, -5, -7.5, and -10°C). Analysis of acclimation-
specific freezing
tolerance was completed using three replicates. Each replicate contained 5
plants per
line (n=15). For each plant, leakage measurements were averaged from S discs,
taken
at each of 5 different temperatures (0, -7.5, -12.5, -15, and -17.5°C).
The resulting
means were used to generate a graph, from which the TLsos were determined. The
difference between inherent (TLso) and acclimation-specific freezing tolerance
(TLso)
relationship was the acclimation capacity. The design used was ANOVA
univariate
analysis using the general linear models (GLM) procedure on replicate by line
to
examine consistency within each replicate. The line by line analysis was
completed in
the same manner and used to compare differences between individual DH, lines
as
seen by differences in freezing tolerance. Coefficient of variance was
calculated using
(root mean squared/mean) as a second measure of significance in the test.
Cascade was found to be freezing tolerant (-15.5°C), while Rebel was
freezing
sensitive (-7.5°C). Segregation of acclimation-specific freezing
tolerance in the DH,
lines showed a trimodal distribution that ranged from -3.0°C to greater
than -18°C, as
shown in Figure 3. There appeared to be a large proportion of transgressive
segregation, which favored a level of freezing tolerance greater than that of
the tolerant
parent, Cascade. The distribution curve did not conform to any Mendelian
ratio.
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Figure 3 shows the segregation of freezing tolerance based on the
TLS°s of 78
individual lines of B, napus, including the 2 parent lines, six F,, and 70 DH~
lines. In
comparing reciprocal crosses, differences in the level of freezing tolerance
could not
be attributed to maternal effects. Freezing tolerance appeared to segregate
randomly
throughout the DH, lines, regardless of the direction of the cross.
Comparison of REPLICATE X LINE using ANOVA univariate analysis
revealed no significant difference between the replicates. However, when
comparing
LINE X LINE, highly significant differences were apparent. A correlation of
variance
value of 5.8% (LINE X LINE) confirmed that significant levels of variation
existed
between segregating lines.
Correlation analysis was completed between non-acclimated and acclimated
plant tissue, to examine the possibility that inherent freezing tolerance
could be used as
a tool in predicting a line's acclimation capacity. The results showed that
neither
acclimation-specific freezing tolerance nor acclimation capacity can be
predicted using
inherent freezing tolerance as an indicator.
EXAMPLE 6
Phenotypic Characterization
Both vernalized and non-vernalized plants were grown under greenhouse
conditions and assessed on the basis of the following parameters: (1) days to
fourth
leaf, bolting, transition from vegetative to reproductive meristem, first
flower,
completion of flowering, and maturity; (2) number of expanded leaves; and (3)
yield.
Field testing of the non-vernalized plants was conducted at two locations.
In experiments to analyze plant development, Westar and VERN- were grown
in a growth chamber under a 16 hour photoperiod of 400-450 wE rri 2 s'', at
22°C (day)
and 17°C (night). VERN+ was vernalized as above, then moved to the
growth
chamber. Sampling of Westar and VERN- began at 14 days after planting and
continued up to the time that the first floral buds in the primary
inflorescence opened.
VERN+ sample collection began immediately after vernalization. Shoot tips were
collected and leaves carefully removed to expose the apical meristem; meristem
tissue
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was fixed and processed for scanning electron microscopy (SEM). Floral
development
of VERN- and the spring cultivar, Westar, was then followed by SEM, according
to
the morphological landmarks of Smyth et al. (Smyth et al., Plant Cell., 2:755-
767
[990]). The sample size was at least 10 meristems for each collection time.
Comparisons were made between VERN- and VERN+, in order to determine whether
vegetative and reproductive development in VERN- was more "winter-like" or
"spring-
like." The results indicated that unlike Arabidopsis (Bagnall, [1993]), there
is not a
linear relationship between flowering time and leaf number, (as indicated in
Table 6,
below). These observations necessitated recording both chronological time and
leaf
number (developmental time).
Table 6. Correlation Analysis Between Leaf Number and
Flowering Time in VERN+
Seedling Stage Correlation Value
Seed 0.078
Cotyledon 0.491
2-Leaf 0.044
4-Leaf 0.545
In addition, non-vernalized material was grown in the greenhouse for a
minimum of i 80 days, in order to determine the absolute vernalization
requirements of
the parents, F,s, and DH, lines. Phenotypic analysis of vernalized and non-
vernalized
DH, lines revealed that all but one line (VERN-) required six weeks of
vernalization,
in order to complete development and set viable seed.
As the vernalization requirement of some plants can be replaced by extended
photoperiods, the possibility that the photoperiod in the greenhouse could
promote
flowering was addressed. Two approaches were taken to make this determination.
First, VERN- was grown under a short (10 hour) photoperiod, and the winter
parents
{Cascade and Rebel) were allowed to grow for over 180 days. These results
indicated
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that the winter types failed to flower, even after an extended growth period,
and
confirmed that VERN- had lost the vernalization requirement present in both
parents.
Thus, since VERN- flowered, the photoperiod in the greenhouse did not replace
the
vernalization requirement.
To confirm the phenotype of the parents, Cascade and Rebel were allowed to
grow for up to 300 days under greenhouse and growth chamber conditions, with
and
without vernalization. Cascade failed to flower unless vernalized, indicating
that it has
an absolute vernalization requirement. On the other hand, Rebel flowered,
albeit
abnormally and very late, in the absence of vernalization. The flowers were
pale,
malformed and had very little pollen. Following vernalization, flowering time
was
significantly reduced and the transition to reproductive development was
normal (See,
Tables 7 and 8, below). In Table 7, the values represent the mean ~ standard
error,
with n = 10). In Tabie 8, ali of the values given (i. e., other than "Did Not
Flower")
were significant at the 5% level. As such, Rebel would be considered to have a
vernalization requirement. These results confirmed that VERN- had lost the
vernaiization requirement that was present in the parents.
Table 7. Vernalization Response of Cascade and Rebel
Flowering Time (days}
Cultivar
No Vernalization Vernalization
Cascade Did Not Flower 27.4 t 2.9
Rebel 83.5 2.2 26.3 2.5
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Table 8. Mean Final Leaf Number Under Inductive and
Non-Inductive Growth Conditions
16 Hour Photoperiod 8 Hour P6otoperiod
. .
Cultivar N' Vernalization'~ ' Vernalization
Vernalization Vernalization
:
Westar 14.2 13.3 26.8 18.6
VERN- 16.5 14.7 23.5 18.8
Did Not Did Not
VERN+ 17.4 1$.0
Flower Flower
Rebel 28.8 16 Did Not 24.0
8
. Flower
Under a 16 hour photoperiod, both Westar and VERN- underwent the transition
from vegetative to reproductive growth at 20 days after planting, as indicated
by the
formation of the inflorescence meristem. Subsequent floral meristem
development was
slightly slower in VERN-. However, this did not result in a significant
difference in
time to anthesis. Comparisons between VERN- and VERN+ showed that floral
development was faster by as much as 4 days in VERN+. Again, however, this did
_ not translate into a significant difference in time to anthesis.
Assessment of reproductive development in terms of leaf number showed that
VERN- was not developmentally compromised by a lack of vernalization (See
e.g.,
Table 8). Under a 16 hour photoperiod, VERN- had less leaves than did the
vernalized winter types, Rebel and VERN+. Admittedly, vernalization reduced
the leaf
number in VERN-, but the same response was seen in the spring type, Westar.
This
reduction in leaf number in response to vernalization in spring types is
generally
accepted (Dennis et al., supra).
VERN-'s response to photoperiod and vernalization was intermediate between
the winter and spring types (See, Table 8). The winter type VERN+, did not
flower
without vernalization and was insensitive to photoperiod. In the absence of
vernalization, Rebel was very slow to flower under a long photoperiod and
simply did
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not flower under a short photoperiod. The spring type, Westar, was photoperiod
-
sensitive; under an 8 hour photoperiod, leaf number was nearly double that
produced
under a 16 hour photoperiod. Westar was also responsive to vernalization, and
this
was most apparent under the short photoperiod. In comparison to Westar, VERN-
had
a weaker response to photoperiod, and a weaker response to vernalization under
the
short photoperiod, but a stronger response under the long photoperiod.
EXAMPLE 7
Segregation Analysis
In these experiments, the genetic behavior of the lack of a vernalization
requirement in VERN- was investigated. VERN- was backcrossed with Cascade, to
produce the BC,F, generation. This was then self pollinated to produce the
BC,FZ
generation. The vernalization requirement was determined as described above
(Example 3). Vernalization requirement assessments for BC,F, and BC,FZ
individuals
were made at 100 and 300 days after planting. One hundred days generally
represents
the complete life cycle of a spring type B. napus cultivar. If a line did not
flower
within the 100 day time frame, it was scored as a winter type (i. e.,
requiring
vernalization); the 300 day period represented an extremity.
BC,FZ were assessed for the appearance of bolting, and first anthesis, as
evidence for the lack of the vernalization requirement. Absolute vernalization
was
gauged as the presence versus the absence of bolting after 95 days of growth.
Analysis of association was completed based on Xz values derived from
orthogonal
functions according to Mather ( 1951 ) using 61 BC,FZ plants. Linkage group
analysis
was completed using MAPMAItER/QTL vl.l program (Lincoln et al., Whitehead
Institute Technical Report, Cambridge, MA [ 1992]). A LOD score of 2.0 was
chosen
as the threshold for declaring linkage. As shown in Table 9, four of the five
markers
showed significant association with the vernalization requirement. Marker
tg6a12a
had the strongest association (XZ=18.25, p<0.001 ). Interval mapping analysis
was also
performed to examine the association of these markers with the flowering time
variation in the BC,F2 population. As shown in Figure 2, the LOD plot produced
by
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MAPMAICER/QTL displayed 1 score peak, which was located in the interval
between
tg6al2a and wg5a5, close to marker tg6al2a. In this Figure, the marker loci
are
indicated on the horizontal axis and the LOD score is indicated on the
vertical axis. In
this Figure, "TTF" refers to the time to flower (in days).
Table 9. Association of RFLP Marker Loci for
Vernalization Linkage Groups in BC,FZ Progeny
Marker Loci X=. Probability Significance
wg5a5 0.016 0.8 ~ 0.9 Not Significant
tg6a12a 18.25 < 0.001 Significant
wg8glb 14.43 < 0.001 Significant
wg6b10 5.71 0.01 ~ 0.02 Significant
wg7f3a 7.93 < 0.01 Signif cant
wg2dllb 1.98 0.1 ~ 0.2 Not Significant
When assessed at 100 days after planting, all BC,F, flowered, while in the
BC1F2, 192 individuals flowered and 84 did not. This segregation pattern
indicates
that the lack of vernalization requirement in VERN- is a dominant
characteristic.
When evaluated for 300 days, 1 out of 69 and 3 out of 135 BC,FZ individuals
had not
bolted or shown any sign of flowering. The ratios were close to the expected,
if 3 or
4 loci with additive effects are assumed (XZ=0.003-0.05, 0.8 < p < 0.99).
As LG9 has been reported as having strong association with the vernalization
requirement and major effects on flowering time in the F,-derived DH
population from
'Major' x 'Stellar' (Ferreira et al., Theor. Appl. Genet., 90:727-732 [1995]),
a subset
of the above BC,FZ population was analyzed with the 9 RFLP markers in LG9 of
the
map described by Ferreira et al. (Ferreira et al., supra). Polymorphisms were
detected
between VERN- and Cascade at 6 of the 9 marker loci. Segregation data for
these 6
markers were collected from 62 individuals of the population and analyzed for
linkage
relationship. Five marker loci were grouped together; 3 loci retained their
order as in
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the original map of Ferreira et al. {Ferreira et al., supra); and the other 2
loci, tg6al2a -
and wgSaS, inverted their positions on the partial linkage map. Marker locus
wg2d11 b
was originally mapped to the bottom of LG9 (Ferreira et aL, supra), but was
placed
into another group by MAPMAKER using the segregation data in the BC,F2.
Although an understanding of the mechanism is not necessary in order to use
the
present invention, it is not known whether this is a result of a structural
rearrangement
or scarcity of markers in the neighboring region.
EXAMPLE 8
DNA Extraction
At the 4-leaf stage, leaf tissue was taken, flash frozen in liquid nitrogen
and
stored at -80°C for genotypic assessment. DNA was extracted from
approximately 5 g
of young leaf tissue using the method of Dellaporta (Dellaporta, Plant Mol.
Biol.
Reptr., 1:19-21 [1983]), with modifications according to Hawkins et al.
(Hawkins et
al., Genome 39:704-710 [1996]).
Briefly, the plant tissue was ground to a fine powder with a mortar and pestle
under liquid nitrogen, and then ground in 10 ml of extraction buffer (100 mM
Tris-
HCI, pH 8.0, 500 mM NaCI, 50 mM EDTA, pH 8.0, to which 7 ~1 of 144 mM of 2-
mercaptoethanol was added fresh). To the slurry, 1 ml of 20% SDS was added and
the sample was mixed thoroughly, then 70 ~l of 50 mg/ml proteinase K was added
and
mixed, and the sample was incubated at 65°C for 1 hour. To this, 2 ml
of S M
potassium acetate was added and sample mixed gently, but thoroughly, then
placed on
ice for 15 minutes. Samples were then centrifuged for 10 minutes at 10,000xg,
and
the supernatant filtered through a layer of Miracloth (CalBiochem). Samples
were
extracted with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1),
and
centrifuged for 10 minutes at 10,000xg. The aqueous phase was removed, placed
in a
clean tube, and precipitated with 0.6 volumes of isopropanol. DNA was then
spooled
out of solution, rinsed three times in 70% ethanol, air dried, and dissolved
in TE8 (10
mM Tris-HCI, pH 8.0, 1 ml EDTA, pH 8.0). Samples were treated with RNAse as
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known in the art (See e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual,
2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, [1989]),
then 5
M NaCI was added to a final concentration of 2.5 M (Fang et al., BioTech.,
13:52-55
[1992]). DNA was precipitated with 2 volumes of 95% ethanol, placed at -
20°C for 30
minutes, pelleted at 4,SOOxg for 10 minutes at 4°C, rinsed three times
in 70% ethanol,
and dried. Extracted DNA was resuspended in a minimal volume of TE8 (10 mM
Tris-
HCI, 1 mM EDTA, pH 8), and quantified using ethidium bromide fluorometry
(Karsten
and Wallenberger, Anal. Biochem., 77:464-470 [1972]). Briefly, 2 pl of DNA
were
mixed with 2.4 ml PBS (170 mM NaCI, 3.3 mM KCI, 10 mM NaZHP04, 1.8 mM
KHZP04, pH 7.2), and 0.1 ml ethidium bromide (100 ug/ml). Samples were read on
a
Varian SF-330 (Varian) spectrofluorometer, with excitation at 360 nm, and
absorbance at
580 nm, and were compared with a calibration curve constructed using known
quantities
of lambda DNA.
Use of low temperature-induced clones (BN28, BNI I5, and cor6.6) revealed no
polymorphism within the parents, or any of the DH, progeny. Figures 4A and 4B
show
the RFLP analysis of parent and DH, lines. These are representative blots
probed with
the cDNA clone BN28 from B. napus. In this Figure, "(cas)" indicates Cascade,
"(reb)"
indicates Rebel, "(F,)" indicates F,, and "(DH,)" indicates DH, lines.
Acclimation-
specific freezing tolerance was denoted as: (+) freezing tolerant; and (-)
freezing
sensitive. Only one cDNA (cor6.6) was previously shown to be associated with
freezing
tolerance, and this was in B. rapa (Teutonico et al., [1995], supra). However,
it was not
found to be linked nor associated with the population identified in this
Example.
Genomic screening showed that 2 of 8 genomic clones, identified by Teutonico
et
al. (Teutonico et al., supra) as being linked to freezing tolerance had
polymorphic
differences between the parents. The polymorphism was complemented in the F,,
and
segregated within the DH, lines analyzed, as shown in Figures 5 and 6. Figure
5 is a
representative blot probed with genomic clones A) EC2E5 and B) WGIF6. The same
designations for the lines as used in Figure 4 are used in Figure 5. Figure 6
is a table
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showing the recombination analysis of DH, lines. In this Figure, the
recombination
frequency is scored against the freezing tolerance phenotype. WG1G6 did not
appear
to be associated with freezing tolerance, whereas EC2E5 appeared to be
associated
with freezing tolerance.
EXAMPLE 9
Southern Blotting
As described in this Example, the genetic basis for the loss of the
vernalization
requirement in VERN- was explored by probing Southern blots of VERN-, the
parent
' cultivars, as well as a number of spring cultivars with clones that had been
shown to
map to vernalization and flowering time loci (designated VRNI to VRN3)
(Ferreira et
al. supra; and Osborn et al., Genetics 146:1123-1129 [1997]). Figure 12
provides a
graph showing the marker loci of Cascade, Rebel, and VERN-, using the marker
order
described by Ferreira et al. (Ferreira et al., supra).
Probe Preparation
The genomic clones were isolated from a genomic library of B. napus, and
were a gift from Dr. T.C. Osborn. BN115 and BN28 are clones from a low
temperature cDNA library of B. napus cv Jet Neuf. The BN clones were a gift
from
Dr. J. Singh. The cold-regulated clone (cor6.6) came from a cDNA library of
cold-
acclimated Arabidopsis thaliana, and was a gift from Dr. M. Thomashow. All of
the
clones had T7 and T3 priming sites adjacent to the multiple cloning site of
the clone.
This allowed for amplification of the inserts using PCR with Tag DNA
polymerise.
For each amplification, 0.1 pg of template was used. T7 and T3 primers were
purchased from Promega. PCR was completed using a Techne PHC-2 thermocycler,
using the fastest possible ramping times. DNA was initially denatured at
95°C for 5
minutes, followed by 35 cycles of 95°C for 1 minute, 39°C for 1
minute, and 72°C for
2 minutes. A final elongation at 72°C for 10 minutes was used to ensure
that all
products would be of equal length. The purity of the amplification products
was
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assessed by electrophoresis of 2 p.l of sample in 0.8% TAE agarose gel, and
visualized
by ethidium bromide staining as known in the art (See e.g., Sambrook et al.,
supra).
Samples were purified using G-50 Sephadex in a spun column as known in the art
(See, Sambrook et al., supra). Samples were quantified by fluorometry as
described
above, and 75-100 ng were used for each labelling reaction in the Southern
blot
procedure.
Blotting
Genomic DNA (20 or 30 fig) of VERN-, Cascade, Rebel, other DH, lines, and
a spring control (Westar) were restricted according to the manufacturer's
instructions
using EcoRI or HindIII (Boeringer Mannheim). Digested samples were separated
on
an 0.8% agarose gel in lx TAE at 80 volts for 3-5 hours (Sambrook et al.
supra).
Equal loading of restricted samples was assessed using ethidium bromide
fluorescence
of separated samples. Gels were capillary blotted onto Zeta-probe nylon
membranes
(Bio-Rad), using the semi-dry method of Rutledge et al. (Rutledge et al., Mol.
Gen.
Genet., 229:31-40 [1985).
DNA was cross-linked to the membrane by illumination at 254 nm, then baked
at 80°C for 30 minutes under vacuum. Blots were pre-hybridized, and
hybridized
according to the membrane manufacturer's instructions, using 50% formamide at
43°C.
Membranes were hybridized with '2Pa dCTP (Amersham) labeled probe (Random
Priming Kit, BRL #55567656), and rinsed at room temperature as follows--once
for 2
minutes in 2x SSC and 0.1% SDS; once for 10 minutes in lx SSC and 0.1% SDS,
once for 10 minutes in O.Sx SSC and 0.1% SDS; and a final wash at 65°C
for 10
minutes in O.lx SSC and 0.1% SDS. The membranes were then exposed to XAR-5
film (Kodak) with enhancing screens at -80°C, for 2-5 days.
Individual lines were scored as being freezing tolerant (+) or scored as being
freezing sensitive (-). Freezing tolerant (+) lines had TLS°s greater
than or equal to
-14.5°C. Freezing sensitive (-) lines had TLS°s less than or
equal to -9°C. All plants
falling between "tolerant" and "sensitive" were scored as "intermediates."
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Figure 4 is a Southern blot of EcoRI digests probed with WG6B10. In this
Figure, "Cas" indicates the lane containing Cascade, "Reb" indicates the lane
containing Rebel, and "Wes" indicates the lane containing "Westar." The row
indicated as "vern" shows whether the particular samples were vern+ or vern-.
Also,
"bcF~," indicates two lanes loaded with BCF~.
The EcoRI digests of genomic DNA probed with WG6B10 showed that VERN-
is polymorphic to Cascade and Rebel. Figure 7A is a representative blot probed
with
WG6B 10. The lanes containing Cascade (cas), Rebel (reb), and F, are
indicated. The
same polymorphic difference was observed between Westar and the winter
cultivars.
The spring types both lacked a band at 3.0 kb. A different polymorphism was
also
noted between Cascade and Rebel at this locus. Although this suggests that
there may
be more than one gene responsible for the phenotype, the bands always
segregated
with each other, indicated that they are closely linked. The results clearly
indicate that
there is no polymorphism in the winter type parents, but a polymorphic
difference
exists in Westar and the DH, line (6-200; VERN-), which also shows a spring
type
genotype. The polymorphism detected in VERN- is clearly not linked to freezing
tolerance, as this line maintains good levels of both inherent and acclimated
freezing
tolerance, yet it no longer needs vernalization.
It was determined that the band at approximately 2.8 kb was found to be
necessary, while the band at approximately 3.0 kb was lacking for the vern-
phenotype. As shown in Figures 7B and 7C, VERN- and Rebel both have bands at
"x
kb," while Cascade has a band at "n kb." In Figure 7B and 7C, the "x kb"
indicates
the band at approximately 2.8 kb, while the "n kb" indicates the band at
approximately
3.0 kb. In Figure 7C, the asterisked band in the VERN- lane (as well as the
faint band
at the same location in the F, lane) indicates a band that was apparently
incompletely
digested. In other blots, this band did not appear, and the last band in the
lane was
much larger (i.e., when the substrate is sufficiently digested, the DNA is
present in the
last band of the blot).
The approximately 3.0 kb band appears to be dominant, as plants that contain
this band required vernalization (indicated by a "+" in Figure 7B). Plants
that only
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have the approximately 2.8 kb band lacked the vernalization requirement
(indicated by
a "-" in Figwe 7B). This makes the VERN- and Rebel cultivars, as well as the
other
lines containing the 2.8 kb band, but not the 3.0 kb band valuable as parent
lines for
development of spring-type cultivars (i. e., lacking the vernalization
requirement), from
S winter type lines.
The wildtype genotype was restored when VERN- was backcrossed to either
Cascade or Rebel, as predicted based on the phenotype of the BC,F, generation.
As
phenotypic and genotypic complementation between the winter cultivars and VERN-
was demonstrated in the BC,F, lines using the clone WG6B10, the relative
linkage was
IO investigated. An example of a Southern blot used in this investigation is
shown in
Figwe 8. In this Figwe, the vernalization requirement was denoted as "(+)"
indicating
lines with a requirement for vernalization, and "(-)" indicating lines that
did not require
vernalization.
Of 80 individual BC,Fi plants, 23 had the VERN- polymorphism. As the
15 phenotypic ratio was 61:19, this indicated a recombination frequency of
approximately
8%. In comparison, flanking clones WG7F3 and WG8G1 had recombination
frequencies of 27.9% and 25%, respectively, indicating that VERN- may more
closely
associated with WG6B 10. The following Table (Table 10) shows the results of
recombination analysis of backcross progeny of Cascade x VERN-. Probes from
20 genomic clones were used. Recombination frequency was scored against the
vernalization phenotype. Phenotypic assessment of vernalization requirement
was
based on the presence of reproductive bud formation was done at 35 and 70 days
after
planting.
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Table 10. Recombination Analysis of Backcross Progeny
Cascade x VERN- Cross
Recomb
Clone Source Poly Size SaW ple
: inafion
morphic (Kb) ; >Size (n)
. . ! : ' ,; ; Frequency ,-
WG7F3 B. napus yes 2.3 27.9 42
WG6B 10 B. napus yes 3, 2.8 8 42
WG8G10 B. napus yes 4.2 25 42
The marker interval wg7fa-wg6b10 in LG9 was found to be strongly associated
with the major flowering time locus VRNI (Ferreira et al. supra); this
interval and its
adjacent regions (i.e., from wg7f3a to wg5a5) in VERN- appear to have been
inherited
from Rebel. The genomic region around marker locus ec3g3c in LG12 contains
VRN2
(Osborn et al. supra). It was also determined that VERN- carries Rebel's
alleles at
this locus as well. Comparison of the genotypes at ec3g3c, and its two
adjacent loci
(i.e., wg7b3 and wglg4), revealed that 2 crossing over events may have
occurred in
the F, plant that produced the VERN- line. This was the only locus at which
VERN-
carries alleles from Cascade. All other loci examined showed polymorphic
differences
between Cascade and Rebel, with Rebel as the contributor to the VERN-
genotype.
Thus, with regard to the marker interval wg9c7-wg6b2 in LG16 (associated with
VFN3), VERN- and Rebel shared the same alleles, and Cascade had different
alleles.
EXAMPLE 10
Occurrence and Inheritance of BN28
While molecular markers have been used in the search for cold-responsive loci
(Cai et aL, Theor. Appl. Genet., 89:606-614 [1994]), difficulties with this
approach
have been encountered due to the fact that genes for quantitative traits can
act
separately or pleotropically. Furthermore, inheritance patterns can be very
complex.
Searches for low temperature-induced genes resulted in the identification of
such a
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gene in B. napus cv Jet Neuf, termed "BN28." Recently, a homologous gene was
isolated from B. raps (brkinl).
In this Example, the occurrence and inheritance of BN28 within the
Brassicaceae family. As B. napus is an allotetraploid, it was thought that the
gene
encoding BN28 might be present in some form within all of the Brassica
species.
This was investigated in this Example.
PCR and Southern Blots
DNA from various lines was prepared as described above and PCR was
performed using 1 mg of target DNA and Tag DNA polymerase as describe above.
Primers specific to the 5' and 3' ends of the coding region of BN28 were used
(BN28
forward primer 5'-ATGTCAGAGACCAACAAGAAT-3' (SEQ ID NO:1), and BN28
reverse primer S'-GTCTTGTCCTTCACGAAGTT-3' (SEQ ID N0:2). Amplified
products were separated on 1.4% agarose gels in 1 x TAE at 5 volts/min., and
visualized by ethidium bromide staining, as described above.
The PCR results indicated that a single fragment was amplified in all diploid
species tested. The size of the fragments were: B. nigra, 465 bp; B. oleracea,
425 bp;
and B. rapa, 450 bp. Electrophoresis of a mixture of amplification products
from each
of the diploid species into resolvable fragments indicated that each fragment
is of
unique size. B. napus is the only allotetraploid that contained representative
fragments
from both of its original parents (B. oleracea and B. rapa). B. carinata
contained the
same sized fragment as B. rapa, and B. juncea contained the same sized
fragment as
B. oleracea. S. alba and S. arvensis amplified the same sized fragment as B.
nigra.
Southern blots of 20 ~g genomic DNA were digested as described in Example
9, above, with the exception that EcoRI and Pvu2 (Boehringer Mannheim) were
used.
The blot results indicated that each of the diploid parents contained a unique
homologue of BN28, as shown in Figure 9. Diploid Brassicas are considered to
be
secondary aneuploids derived by duplication of chromosomes from an extinct
common
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ancestor having six chromosomes. Duplication and extensive redistribution then
gave
rise to the three diploids outlined by U ((1; Japan J. Bot., 7:389-452
[1935]).
Figure 9A shows the results obtained with the EcoRI-digested samples, and
Figure 9B shows the results obtained with the PvuII-digested samples. In
Figure 9,
"(Bn)" indicates B. nigra, "(Bo)" indicates B. oleracea, "(Br)" indicates B.
raga, while
the B. napus cultivars were "(c)" Cascade, "(w)" Westar, "(Bc)" indicates B.
carinata,
"(Bj)" indicates B. juncea, "(ar)" indicates S. arvensis, and "(al)"
represents S. alba.
The results indicate that specific homologous of BN28 are present in each of
the
diploids, and these homologues are transferred to individual allotetraploids,
with B.
napus being the only one to acquire homologous from both original parents.
Again, B. napus was the only allotetraploid to contain restriction fragments
from both diploid parents. The other species tested, B. carinata, B. juncea,
and S.
arvensis, contained a gene from one diploid parent, in the same manner as the
PCR
analysis. S. alba contained a unique profile from all of the species
represented.
Coupled to PCR data, the Southern blots provided unambiguous identification of
all
genomes represented herein.
RNA Extraction
Total RNA was extracted from control and acclimated plant tissue (4°C
for 14
days), using the hot phenol method of Verwoerd et al. (Verwoerd et al., Nucl.
Acid
Res., 17:2362-2366 [1989]). RNA was quantified using ethidium bromide
fluorometry
as described above in Example 8. Fluorometric values attained for RNA were
multiplied by a DNA to RNA conversion factor of 2.17. Then, 30 mg of total RNA
were separated on 1.4% agarose gels as known in the art. Gels were rinsed
briefly in
SSC and transferred to Zeta-Probe membranes as described in Example 9. RNA was
cross-linked to the membrane by illumination, and hybridized with a 32PadCT-
labelled
BN28 cDNA probe as described above, washed at high stringency twice for 10
minutes in O.lx SSC and 0.1% SDS at 60 °C, and exposed to XAR-5 film
(Kodak) for
6-8 hours
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BN28 hybridizes to an mRNA transcript of approximately 0.5 kB in cold-
acclimated tissues of B. napus. Analysis of acclimated and non-acclimated
Brassicas
was accomplished by probing equally loaded blots of total RNA for the presence
of
BN28 transcript using radiolabelled BN28 cDNA. No message was detected in any
of
the non-acclimated samples. After 10 days of low temperature acclimation, all
of the
tested species showed high levels of expression of BN28 mRNA with the
exception of
B. carinata, which showed a slightly lower level of accumulation. No
significant
difference in the size of the transcripts was detected in the different
species examined.
These Northern blot results are shown in Figure 10. In this Figure, the non-
acclimating conditions are indicated with an "0" (i.e., 0 days), and low
temperature
conditions are indicated with a "14" (i.e., 14 days). The species
abbreviations used in
this Figure are the same as those described for Figure 9. These Northern
analyses
confirmed that each species expresses a BN28-like gene that was induced in
response
to low temperature. Even though different forms of BN28 were present, the
coding
regions appear to be sufficiently homologous to hybridize to a common cDNA
probe
under high stringency conditions. Although such an understanding is not
necessary for
practicing the present invention, similar patterns of stress induction may
suggest that
each of the three homologues present in the Brassicaceae contain conserved
regulatory
regions.
In addition, although all of the Brassicaceae examined had low temperature-
induced homologues of BN28, differences in protein expression were
apparent.(See,
Figure 11 ). Although such an understanding is not necessary to successfully
practice
the present invention, the lack of protein accumulation in B. nigra and S.
arvensis
suggests that gene silencing has occurred in these cultivars.
Protein Extraction and Immunoblotting
Total SDS-soluble proteins were extracted from approximately 0.1 g of plant
tissue in 50 mM Tris-HCI, pH 8.0, 50 mM NaCI, 1% SDS, and 10 mM PMSF, as
known in the art. Protein concentrations were determined using the BCA method
of
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Smith et al. (Smith et al., 150:76-85 [1985]). Then, 30 mg aliquots were mixed
with
an equal volume of 2x SDS loading buffer, and boiled for 5 minutes prior to
loading
Samples were separated on a 15% SDS-PAGE gel using a modified Tris-Tricine
running buffer (Shagger and von Jagow, Anal. Biochem., 166:368-379 [1987)), at
30
milliamps (mA), until the tracking dye ran off the end of the gel.
Proteins were transferred to 0.22 pm supported nitrocellulose membranes
(Schleicher and Schuell) in carbonate buffer, at 300 mA for 2 hours at
4°C. Blots
were then reacted with BN28 specific antibody (Boothe et al., Plant Physiol.,
108:795-
803 [1995]). The antibody was detected using alkaline phosphatase conjugated
secondary antibody (Sigma) and visualized using the 5-bromo-4-chloro-3-indolyl
phosphate-p-toluidine salt (BCIP)/p-nitroblue tetrazolium chloride salt (NBT)
reagent
system (Sigma).
The calculated molecular mass of BN28 protein is 6.6 kD, although the
polypeptide migrates at a slightly lower apparent molecular mass. No
accumulation of
BN28 protein was detected in the non-acclimated samples. However, after 14
days of
low temperature acclimation, all but two of the species tested showed protein
accumulation. The results are shown in Figure 11. The same abbreviations as
used in
Figure 10 are used in the immunoblot in Figure 11. Again, there was no
significant
difference in apparent size in any of the species showing accumulation of BN28
protein. There was no detectable signal in B. nigra and S. arvensis. Testing
several
samples at several developmental stages, including very young and mature
leaves, or
increasing the protein concentration to 200 ~g failed to produce any
detectable signal.
It is clear from the above that the present invention provides compositions
and
methods for the production and use of improved Brassica, as well as methods
and
compositions for the development of additional strains and/or cultivars of
agricultural
importance.
All publications and patents mentioned in the above specification are herein
incorporated by reference. Various modifications and variations of the
described
method and system of the invention will be apparent to those skilled in the
art without
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departing from the scope and spirit of the invention. Although the invention
has been
described in connection with specific preferred embodiments, it should be
understood
that the invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes for carrying
out
the invention which are obvious to those skilled in plant biology, molecular
biology,
plant genetics, or related fields are intended to be within the scope of the
present
invention.
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