Note: Descriptions are shown in the official language in which they were submitted.
2~fl08
7107~1)
MET~OD OF IDENTIFYING DNA SEQ~CES IN C~ROMOSOMES OF PLANTS
The present invantion relates to a method of identifying DNA
sequences in the nuclear genome of a plant and to a method of
plant breeding using this method.
It is often desirable to incorporate DNA sequences into the
genome of a plant from another source. Conventional plant breeding
techniques have crossed different strains of the same species or
even different species in order to introduce genes coding for
desirable properties (e.g. disease resistance, protein quality) into
the resultant plant variety. The traditional method relied on
cultivating large numbers of random hybrids between donor and
acceptor species or varieties ant selecting those hybrids which had
the desired property for further propagation and inbreeding. This
is a slow procedure. The transfer of genes relies on the transfer
of whole chromosomes from one plant to another, followed by several
generations of selection, often involving backcrossing. During that
time, recombination between chromosomes will occur. In many
varieties only small chromosome segments are transferred from the
donor species into the target plant.
It is now often possible to determine which DNA sequences are
responsible for a given characteristic in the plant having that
sequence. Recent developments in molecular biology enable more
; direct insertion of DNA segments into new varieties. In all cases,it is helpful to have a technique to follow the incorporation of
alien DNA into the genome, without relying only on field testing of
plants.
;` 2~
The methods currently used to follow and identify incorporatsd
alien DNA which may carry important genes in a target plant are
expensive and difficult to interpret. Thus transfer of DNA sequences
may be identified by the morphology of the plant, by the morphology
of the chromosome itself, by chromosome banding, by determining ths
iso-enzymes present, or by RFLP (restriction fragment length
polymorphism). All these methods have disadvantages. Another
method is to hybridize the chromosome3 in a hybrid plant cell
in situ with cloned DNA sequences as disclosed by N Lapitan et al
(J Heredity 77, 415-419, 1986). By using clones, the probe DNA
sequences will all be identical. The sequences cloned are selected
so as to be characteristic of one of the sources of the DNA in the
target plant. However, the clones are difflcult to isolate, and are
unlikely to be generally applicable.
It is disclosed by Le et al in an abstract published in
connection with the XVIth International Congress of Genetics,
Toronto (1988), that total genomic DNA has been used to distinguish
chromosomes or large chromosome segments of rye origin from the
chromosomes originating from wheat in a rye-wheat hybrid and a wheat
~0 variety carrying a rye interchange. This method relies Gn producing
a single stranded version of the chromosomes and bringing them into
contact with labelled single stranded DNA isolated from the rye
parent. The single stranded chromosomes of the hybrid which are
derived from rye will link preferentially to the labelled
complementary probe DNA from the rye source.
The metnod disclosed by Le et al (loc. cit.) will identify the
origin of whole chromosomes or large chromosome segments in plants
where donor and acceptor species differ greatly - for example, in
hybrids between parents from different genera of plants. However,
in plant breeding it is often desirable to introduce short sequencPs
of DNA (typically a few tens of thousands of base pairs), from a
related source, into the nuclear genome of a target plant. Thus
resistance to eyespot disease in one commercial wheat variety is
provided by a DNA sequence from Ae~ilops ventricosa, a wild diploid
wheat species. In producing such modîfied plants, whether by sexual
hybridization or by more direct laboratory manipulation, there is a
need to determine whether a sequence of DNA from an alien source is
present in a given plant. This enables a plant breeder to see if a
DNA sequence has been been transferred and to follow that DNA
sequence through generations to see if it is changing in length or
posi~ion on the chromosome~ and to ex~mine its pairiDg behaviour at
meiosis. If the technique described by Le et al. (loc. cit.) is
applied to the nuclear genome of a plant whose DNA is derived from
two closely related sources, then the total labelled genomic single
stranded probe DNA from one source will hybridize (combine to form
double stranded DNA) not only with the single stranded nuclear DNA
from the same source but also with the sequences from the other
source because of close homologies.
There is therefore a need to find a simple method of
identifying DNA sequences from a given source in the nuclear genome
of a target plant containing DNA sequences from more than one source
which may be close-ly related. In addition, there is a need to find
a method which can be applied to different situations, including
hybrids and plants carrying alien DNA sequences involving a wide
range of species.
According to the present invsntion a method for identifying the
origin of one or more sequences in the nuclear genome of a target
eukaryotic plant containing genetic material from at least two
different sources comprising hybridi~ing DNA from the target plant
with labelled total genomic DNA fragments selected to hybridize to
DMA from one of the sources, while preventing hybridization of the
probe to sequences common to more than one source with unlabelled
total genomic DNA fragments selected to block such common sequences
by hybridizing with them in the target plant DNA and/or in the
labelled DNA, and detecting the sites of hybridization of the
labelled probe to the DNA of the target plant.
There are various schemes for classifying living organisms.
The scheme used for the purpose of the present specification is that
given on page 33 of "Elementary Microbiology" by 0 ~yss,
0 B Williams and E W ~ardener Jr., John Wiley & Sons Tnc 19S3. In
X00~ )8
this classlfication the plant kingdom includes bacteria which are
included in a phylum identified as the ProtoPhyta (primitive plants
without intracellular membranes). Eukaryotic plants therefore
are all tbe members of the plant kingdom except the Protophyta.
The plant is preferably a vascular plant, more preferably one
of the Spermatophyta (angiosperms, gymnosperms). The plant is most
preferably monocotyledonous, e.g. one of the Gramineae. The target
plant may contain DNA from Hordeum, Secale, Triticum, or Ae~ilo~
species. Specific target plants to which the present inveniton may
be applied are those containin~ a) DNA from a Hordeum and ~rom a
Secale source, b) DNA from a Triticu or a AeRilops source and a
Secale source.
-
In this specification the "target plant" is the plant whose DNAsequences are under investigation. The tareet plant potentially
contains DNA sequences originating from more than one distinct
"source" introduced by sexual methods or genetic engineering (i.e.
using a viral vector). The "tarBet DNA sequencesl' are some of the
DNA sequences which may be genomes, chromosomes or chromosome
segements potentially included in the nuclear genome of the target
plant which are to be detected and followed through successive
generations.
The "source" of the DNA ion the target plant may ba the parents
(e.g. in the first generation hybrid H. chilense x ~k_~E19~e9~) or
be more distant ancestors of the target plant (e.g S. cereale and
T. durum Desf. ln Triticale, or T. monococcum and others in T
aestivum).
The total genomic DNA used as labelled probe or blocking DNA
can be isolates from the sources of DNA in the target plant. The
probe or blocking DNA may also be isolated from "remote sources"
which are taxa related to the sources (e.g. o~ the same family, or
the same genus).
At least one, and preferably all sources of the DNA sequences
in the target plant should be distinct. One source should
preferably have some recognizable characteristic not present in the
other sources which results from a difference in its genetic make-up
~)O~flOfil -
~e.g. disease reslstance, morphology and also including gene
expression factors).
The fragments o~ nuclear genome used to block DNA sequences in
common between the labelled probe and more than one of the sources
in the target plant will be selected according to the degree of
difference between the sources in the target plant. Where the
sources are closely related (e.~. H. vulgare and H. bulbosum) it
will be preferably to use total genomic DNA from one of the sources
as block.
~here the sources are less closely related for example Hordsum
vul~are and Secale africanum the total genomic DNA used as a
blocking agent may be from a remote source if it is less closely
related to the source of labelled DNA than to the other source or
sources of DNA in the target plant.
The term "hybridization" as used for the artificial reannealing
of single stranded forms of the DNA from plants with single stranded
labelled DNA is understood in the art (as shown by Le et al and
Lapitan et al (loc. cit.), for review see Henderson, Int.Rev.Cytol.
76, 1-46, 1982). It is, of course, distinct from the hybridization
which takes place as a consequence of sexual reproduction in
nature. The single stranded DNA from the tarBet plant may be in the
form of chromosomes prepared for microscopy. Thus the present
invention may be applied to DNA in the target plant which DNA is
present as a metaphase or interphase chromosome preparation from the
target plant. The DNA may be extracted from the tar8et plant and
immobiliæed on a membrane, optionally on restricted areas of the
membrane. The DNA from the tar8et plant may be digested ~ith one or
more reskriction enæymes and si7.e separated by electrophoresis
before transfer to the membrane. Alternatively the DNA may be
immobiliæed on the membrane by squashing target plant tissue and
transferring the exudate on to the membrane. Methods of producing
single stranded DNA and of hybridizing it with labelled DNA probes
are generally known and are for example disclosed by Lapitan et al
and Henderson (loc. cit.).
The process of the present invention makes use of labelled
408
total genomic DNA as a probe to hybridize to the target DNA
sequences, bloc~sd with unlabelled total ~enomic DNA from other
sources. The lengths of the labelled and unlabelled probe DNA
sequences are generally less than that of the total length of the
DNA sequence in the chromosome, S0 to 1000 base paira typically.
The length reduction may be achievsd by, for e~ample, the DNA
extraction process itself, autoclaving the DNA, sonication or
mechanical shearing. The DNA labelling procèdure may also produce
fragments of DNA sequences - oligo-labelling giv~s fr~gments which
10 have a length of 80-120 base pairs.
Total genomic DNA from one of the sources present in the target
plant, is labelled and referred to as "labelled DNA". Preferably
total genomic DNA ~rom one or more of the sources present in the
target plant, but not DNA from the source which supplied the
labelled DNA, is used as "blockin~ DNA" and is unlabelled. The
DNAs are m~de single-stranded before being placed under conditions
which allow hybridiæation. Before and/or during the hybridization,
the blocking DN~, which may be present in more than five times the
concentration of the labelled DNA is brought in contact with (a) the
target plant DNA, (b) the labelled DNA or (a) and (b) combined. In
case (a), the labelled sequences can hybridize with those sequences
of the target plant DNA which are ~till in single stranded form
because they do not correspond to sequences in the blocking DNA. In
case (b) many of those sequences of the labelled DNA which are not
common to the blocking DNa remain single-stranded, and they are then
the only labelled sequences remaining single-stranded and thus
available to hybridize to the target plant DNA.
The blocking DNA hybridizes with the sequences of the target
plant derived from the same souxce as the blocking DNA, and also
sequences of the target plant DNA and/or labelled DNA which are in
common with the blocking source DNA. In all cases, hybrid DNA
between unlabelled and unlabelled, between labelled and unlabelled
and between labelled and labelled will for~. Because of the higher
concentration of unlabelled DNA, the frequency of the latter will be
lower, leaving labelled, generally low copy number sequences
available to hybridize to corrssponding sequences of the DNA from
the target plant.
After hybridization, the labelled DNA is detected in some way
as being different from the unlabelled DNA sequences, for example
due to the presence of radioactive atoms or other atoms or groups,
e.g. biotin or mercury. ~ethods of labelling DNA sequences and
detection of labelled DNA after hybridization are well known.
Radioactive lab lling may be used, which may be detected by
autoradiography. A preferred method is labelling with biotin, as
described by, for example, Lapitan et al (loc. cit.), using nick
translation. The presence of biotin labelled material may be
detected by fluorescence or colorimetric techniques. An alternative
method is to label the DNA with an ~nzyme and to detect the label by
a reaction catalysed by the enzyme.
The marking of the potentially present target DNA may be
positive or negative. Positive marking would mark the target DNA by
the presence of e.8. radioactive atoms or biotin groups after the
hybridization. Alternatively, negative marking may distinguish the
tarBet DNA by the absence of label.
The invention will now be described by reference to the
following examples.
Example 1 and Comparative Test A
Plant DNAs: Total genomic DNA was isolated from leaves o~ ordeum
vulgare, H. chilense Roem ~ Schult, Secale cereale, S afri anu
Stapf and the hybrids H. vul~are x S. africanum and H. chilense x S.
africanum. Following a standard dot blotting protocol, the DNA was
denatured and transferred to a hybridization transfer membrane
(GeneScreenPlus membrane, E I du Pont de Nemours & Co Inc, Boston,
USA), applying approximately 0.08, 0.2, 0.8 and 2.0 ~g DNA of each
~pecies to different dots of the dot blotter, before alkaline
denaturation.
Labelled DNA: Total genomic DNA from S. africanum was labelled by
oligo-labelling using biotin~l1-dUTP.
Blockin~ DNA: Autoclaved total genomic DNA from H. vul~are.
The transfer membrane was pre-hybridized with 200 ~g of
denatured blocking DNA in 2 ml of a 3tandard hybridization solution
(Sharp PJ, Kreis M, Shewry PR, Gale ND. Theor. Appl. Genet.
75:289-290, 1988~. For hybridization, 0.5 ~g denatured labelled DNA
and 1 mg denatured herring carrier DNA were added to the plastic bag
and incubated overnight at 70C. Post-hybridization washes were
performed with 0.16xSSC (20xSSC: 3N sodium chloride, 0.3M sodium
citrate, pH7) at 60C. Hybridization under such conti~ions should
allow sequences with 80X homology to form hybrids.
Hybridized biotinylated DNA was visualised (made visible) using
ths streptavidin/alkaline phosphatasç colorimetric detection system
by Bethesda Research Laboratories (Maryland, USA).
Hybridization to both S. africanum and S. cereale was strongly
visible in the dots containing 2.0 and 0.8 ~g of DNA and only weakly
to 0.2 ~g DNA. The hybridization to the DNA of the two Hordeum
species was only detected in the 2.0 ~g dot. Differentiation
between S. africanum and the Hordeum species and detection of the S.
africanum DNA in the hybrids was possible (Example 1), but
impo~sible between S. afr anum and S. cereale (Test A).
Example 2
DNA from S. cere~ale, used as blocking DNA, in a hybridization
expsriment with S. afri anum as labelled DNA (carrled out as in
Example l), allo~ed discrimination between species in one g~nus, S
africanum and S. cereale.
Example 3 and Comparative Test B
A hybridization membrane was prepased and hybridized as
described in Example 1. Total genomic DNA from H. vul~are (0.5 ~g)
was labelled with biotin by nick-translation, and autoclaved total
genomic DNA (lO0 ~g) from H. chilsnse was used as blocking DNA.
The dots containing H. vul~are and H. vul~_re x S. africanum
DNA (Example 2) showed more hybridization signal than the dots
containing H. chilense and H. chilense x S. africanum DNA (TPst B).
Example 4 and Comparative Tests C and D
Plant DNAs: Total genomic DNA from H. vulgare and H. bulbosum Nevski
was digested with the restriction enzymes EcoR~ and DraI~ size
separated by 2gaross gel electrophoresis and transferred to Hybond
20~
N~ (~mersham International plc, Amersham, U.K.) support membrane
using alkaline transfer methodology.
Lab lled DNA: Tot~l genomic DNA from H. bulbosum.
Blockin~ DNA: Autoclaved total genomic DNA from H. vulgare.
For probe labelling, hybridization and detection of
hybridization s~tes, the chemiluminescence method ECL (Amersham) was
used following the manufacturer's protocol. Briefly, the transfer
membrane was pre-incubated for 30 min in the hybridization solution
and denatured blocking DNA ~6 ~g/ml). 12 ng/ml of denatured
horse-radish peroxidase labelled DNA was added and hybridization
carried out overnight at 42C. Post-hybridization washes were
adjusted by sodium ion concentration to allow s~quences with an
estimated 90% identity to remain annealed. Hybridization sites were
detected by the emission of light directly recorded on film.
Strong signal resulting from hybridization of labelled probe to
genomic DNA was observed on the tracks containing DNA rom H.
bulbosum, while little signal was detected on the tracks of ~.
vul~are DNA (Test C). In contrast, when no blocking DNA was used
(Test D) substantial crosshybridization to H. vul~are was detected.
The method of hybridization with labelled total genomic DNA together
with blocking enables the differentiation of related species within
the same genus.
Exam~e 5_and Comparative Test E
Total genomic DNA from H. ch _ense, H. vul~are, S. africanum
was digested with EcoRI and treated as in Example 4.
The "Southern" transfer was blocked with 8 ~g/ml total genomic
DNA from S. africanum and hybridized with 17 ng/ml labelled total
genomic DNA from H. chilense. Post-hybridization washes were
carried out at 80X and 90% stringency.
At both stringencies, strong hybridization signal was observed
on the DNA track of H. chilense. Cross hybridization to both DNA
from H. vul~are and S. africanum was reduced by the iddition of the
unlabelled S. aPricanum DNA (as compared with a similar blot
~.
hybridized without blocking DNA). (Test ~.
i~0~ 8
Exam~le 6
DNA from H. vul~are, used as a blocking DNA~ ln a hybridization
experlment (carried out a~ in Example 5) suppressed th~ cross
hybridization of labelled DNA ~rom H. chilense to DNA from both S.
africanum and H. vul~are.
Examples 5 and 6 showed that the DNA from a remote source can
be used to block the common sequences in hybridization experiments.
Example 7 and C~ g~ E~e~_~
DNAs: DraI restriction enzyme fragments of total genomic DNA from
hexaploid triticale (x Triticosecale Wittmark) cv. Lasko, three
different cultivars of wheat, (T. aestivum L. cv. Chinese Spring,
cv. Beaver and cv. Glenson), and rye, (S. cereale), were treated as
in Example 4.
Labelled DNA: 10 ngtml total genomic DNA from S. cereale was
labelled as in Example 4.
Blockin~ DNA: 3 ~g/ml unlabelled autoclaved total genomic DNA from
T. aestivum cv. Chinese Spring.
The DNA of _. aestivum cv. Chinese Spring (Test F) showed only
weak hybridization. Stronger hybridization was detected to DNA of
S. cereale, triticale, T. aestivum cv. Beaver and cv. Glennson.
Thus, using total genomic DNA Prom rye as a probe and appropriate
blocking, rye DNA can be discriminated in triticale (a hybrid
between wheat and rye) and in wheat varieties containing a rye
chromosome segment (cv. Beaver and Glennson). Signal quantification
showed that the hybridization was approximately proportional to the
amount of rye material present.
Example 8
DNAs: Chromosome preparations were made from fixed root tips from
H. chilense x S. africanum by placing them in a mixture of 4%
cellulase and 40% liguid pectinase in O.OlM citric acidtsodium
citrate buffer (pH 4.o) for 1-2h at 37C. They were subsequently
squashed in 45% acetic acid following standard cytological
procedures.
Labelled DNA: Total genomic DNA from S. afr_ca~num was labelled by
nick~translation using biotin-ll-dUTP.
~o~
Blocking DNA: Autoclaved total genomic DNA from H. chilense.
Hybridization was carried out using standard techniques
(Schwarzacher T, Leitch AR, Bennett MD, Heslop-Harrison JS.
Ann. Bot. 64:315-324, 1989). The chromosomes wer~ denatured in
deionized 70% formamide in 2xSSC for 2 minutes at 68-72C,
dehydrated and air dried. Overnight hybridization was carrisd out
with 0.1 ~g of denatured biotinylated DNA with 1 ~g of denatured
blocking DNA in 2~ ~1 of a solution o~ 502 formamide, lOZ dextran
sulphate, 0.1~ sodium dodecyl sulphate and 2xSSC overnight. After
hybridization, the slide was washed in 50% formamide in 2xSSC at
42~C. Hybridization under these conditions should occur at 80-85%
homology levels.
Hybridized labelled probe was detected using fluoresceinated
avidin, and amplified with biotinylated anti-avidin. Chromatin was
counterstained with propidium iodide (1-2 ~g/ml in phosphate
buffered saline).
The avidin in situ hybridization signal and propidium iodine
were excited at 450-490 nm, the former fluoresces greenish yellow,
the latter red. At metaphase the seven larger chromosomes
originating from S. afr_canum were distinctly yellow. The seven
smaller chromosomes ~rom H. chilense showed no detectable label.
Thus the tschnique clearly separated the two evolutionarily related
chromosome sets.
Example 9
Prosphases, telophases and interphases which were -treated as
described in ~xample 8 showed distinct domains belonging to either
yellow labelled chromosomes from ~S. africanum or red unlabelled
chromosomes from H. chilense.
Example 10
Chromosome preparations from root tips of H. vul~are x S.
africanum were made and treated as described in Example 8. In situ
hybridization was carried out using 0.05 ~g biotinylated total
genomic DNA from S. africanum as labelled DNA and 1.5 ~g of total
genomic DNA from H. chilense as block.
The two sets of chromosomes were clearly differentiated as
labelled and unlabelled. At metaphase, the seven larger chromosomes
from S. aEric~num were labelled and fluoresced brightly yellow,
while the ~even smaller chromosomes from H. v~l~are were orange-red
showing no detectable label. At prosphase, telophase and
interphase, distinct domains of either yellow or red chromosomes
were distinguishable.
Example 11
In situ hybridi~ation experiments were performed as described
in example 8 using chromosome preparations from the wheat variety
Beaver carrying a lB/lR translocation, biotinylated total genomic
DNA from S. cereale as a probe and unlabelled total genomic DNA from
T. durum. The Texas Red conjugated avidin used here detected the
hybridization sites by red fluorescence, while unlabelled chromatin
remained invisible. The translocated rye segment oould be
identified at both metaphase and interphase. Exact measurements of
the breakpoint of the translocation were possible.
