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VEGETAL SEQUENCES INCLUDING A POLYMORPHIC
SITE AND THEIR USES
The genomes of alI or<~anisms undergo spontaneous
mutation in the course of th~=ir continuing evolution
generating variant forms of prog~snitor sequences (Gusella,
d Ann, Rev. Biochem. 55, 831-854 (1~386)). The variant form may
confer an evolutionary advantage or disadvantage relative to
a progenitor form or may be neutral. In some instances, a
variant form confers a lethal disadvantage and is not
transmitted to subsequent generations of the organism. In
other instances, a variant form confers an evolutionary
advantage to the species and is eventually incorporated into
the DNA of many or most memf>ers of the species and
effectively becomes the progenitor form. In many instances,
both progenitor and variant forms) survive and co-exist in
a species population. The coexistence of multiple forms of a
sequence gives rise to polymorphisms.
Several different types of polymorphism have
been reported. A restriction fragment length polymorphism
(RFLP) means a variation in DNA sequence that alters the
length of a restriction fragment as described in Botstein et
al., Am. J. Hum. Genet. 32, 314-331 (1980). The restriction
fragment length polymorphism may create or delete a
restriction site, thus changing the length of the
restriction fragment. RFLPs have been widely used in human
and animal genetic analyses (see WO 90/13668; WO 90/11369;
Donis-Kelley, Cell 51, 319-337 (1987); Lander et al.,
Genetics 121, 85-99 (1989)). When a heritable trait can be
linked to a particular RFLP, the ~~resence of the RFLP in an
individual can be used to predict the likelihood that the
animal will also exhibit the trait.
Other polymorphisms take the form of short
- tandem repeats (STRs) that include tandem di-, tri- and
tetra-nucleotide repeated motifs These tandem repeats are
- also referred to as variable nurnber tandem repeat (VNTR)
polymorphisms. VNTRs have been used in identity and
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paternity analysis (US 5,075,217; Armour et al , FEBS Lett.
307, 113-115 (1992); Horn et al., LnlO 91/14003; Jeffreys,
EP 370,719), and in a large number of genetic mapping
studies.
Other polymorphisms take the form of single
nucleotide variations between individuals of the same
species. Such polymorphisms are far more frequent than
RFLPs, STRs and VNTRs. Some single nucleotide polymorphisms
occur in proteincoding sequences, in which case, one of the
polymorphic forms may give rise to the expression of a
defective or other variant protein. Other single nucleotide
polymorphisms occur in noncoding regions. Some of these
polymorphisms may also result in defective or variant
protein expression (e. g., as a result of defective
splicing). Other single nucleotide polymorphisms have no
phenotypic effects. Single nucleotide polymorphisms can be
used in the same manner as RFLPs, and VNTRs but offer
several advantages. Single nucleotide polymorphisms occur
with greater frequency and are spaced more uniformly
throughout the genome than other forms of polymorphism. The
greater frequency and uniformity of single nucleotide
polymorphisms means that there is a greater probability that
such a polymorphism will be found in close proximity to a
genetic locus of interest than would be the case for other
polymorphisms. Also, the different forms of characterised
single nucleotide polymorphisms are often easier to
distinguish that other types of polymorphism (e.g., by use
of assays employing allele-specific hybridization probes or
primers).
Despite the increased amount of nucleotide
sequence data being generated in recent years, only a minute
proportion of the total repository of polymorphisms has so
far been identified. The paucity of polymorphisms hitherto
identified is due to the large amount of work required for
their detection by conventional methods. For example, a
conventional approach to identifying polymorphisms might be
to sequence the same stretch of oligonucleotides in a
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population of individuals by didoxy sequencing. In this type
of approach, the amount of work increases in proportion to
both the length of sequence and the number of individuals in
a population and becomes impractical for large stretches of
DNA or large numbers of subject;.
SUWARY OF THE INVED1TION
The invention provides nucleic acid segments
containing at least 10, 15 or 20 contiguous bases from a
vegetal fragment including a polymorphic site notably a
single nucleotide polymorphism (SNP). In a particular
embodiment, a vegetal fragment does not belong to the
Cruciferae family.
The segments can loe DNA or RNA, and can be
double- or single-stranded. Som.=_ segments are 10-20 or 10-50
bases long. Preferred segments include a diallelic
polymorphic site. In a preferr~=d embodiment, the invention
concerns nucleic acid segments from a fragment shown in
Table I (corn).
The Invention further provides allele-specific
oligonucieotides that hybridizes to a segment of a vegetal
fragment, for example fracrment in Table I. These
oligonucleotides can be probes or primers. Also provided are
isolated nucleic acid" compris:~ng a sequence of Table I or
the complement thereto, in which the polymorphic site within
the sequence is occupied by a base other than the reference
base shown in Table I.
The invention furi~her provides a method of
analyzing a nucleic acid from a subject. The method
determines which base or bases is/are present at any one of
the polymorphic vegetal sites for example of those of Table
I. Optionally, a set of basE=s occupying a set of the
n polymorphic sites shown in Table I is determined. This type
of analysis can be performed on a plurality of subjects who
are tested for the presence of a phenotype. The presence or
absence of phenotype can then be correlated with a base or
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PCT/EP97/07134
set of bases present at the polymorphic sites in the
subjects tested.
DEFINITIONS
A nucleic acid, such an oligonucleotide,
oligonucleotide can be DNA or RNA, and single- or
double-stranded. Oligonucleotides can be naturally occurring
or synthetic, but are typically prepared by synthetic means.
Preferred nucleic acids of the invention include segments of
DNA, or their complements including any one of the
polymorphic sites shown in Table I. The segments are usually
between 5 and 100 bases, and often between 5-10, 5-20,
10-20, 10-50, 20-50 or 20-100 bases . The polymorphic site
can occur within any position of the segment. The segments
can be from any of the allelic forms of DNA shown in Table
I. Methods of synthesizing oligonucleotides are found in,
for example, 0ligonucleotide Synthesis: A Practical ApproacA
(Gait, ed., IRL Press, Oxford, 1984).
Hybridization probes are oligonucleotides
capable of binding in a base-specific manner to a
complementary strand of nucleic acid. Such probes include
peptide nucleic acids, as described in Nielsen et al.,
Science 254, 1497-1500 (1991).
The term primer refers to a single-stranded
oligonucleotide capable of acting as a point of initiation
of template-directed DNA synthesis under appropriate
conditions (i.e., in the presence of four different
nucleoside triphosphates and an agent for polymerization,
such as, DNA or RNA polymerase or reverse transcriptase) in
an appropriate buffer and at a suitable temperature. The
appropriate length of a primer depends on the intended use
of the primer but typically ranges from 15 to 30
nucleotides. Short primer molecules generally require cooler
temperatures to form sufficiently stable hybrid complexes
with the template A primer need not reflect the exact
sequence of the template but must be sufficiently
complementary to hybridize with a template. The term primer
site refers to the area of the target DNA to which a primer
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hybridizes. The term primer pair means a set of primers
including a 5' upstream primer that hybridizes with the 5'
end of the DNA sequence to be amplified and a 3', downstream
primer that hybridizes with the complement of the 3' end of
5 the sequence to be amplified.
Linkage describes the tendency of genes,
alleles, loci or genetic marker: to be inherited together as
a result of their location on vhe same chromosome, and can
be measured by percent recombination between the two genes,
alleles, loci or genetic markers..
Polymorphism refers to the occurrence of two or
more genetically determined alternative sequences or alleles
in a population. A polymorphic marker or site is the locus
at which divergence occurs. Preferred markers have at least
two alleles, each occurring at j=requency of greater than 1%,
and more preferably greater than 10°s or 20°s of a selected
population. A polymorphic locus may be a" small as one base
pair. Polymorphic markers include restriction fragment
length polymorphisms, variable number of tandem repeats
(VNTR's), hypervariable regions, minisatellites,
dinucleotide repeats, trinucleot:ide repeats, tetranucleotide
repeats, simple sequence repeats, and insertion elements
such as Alu. The first identified allelic form is
arbitrarily designated as a the reference form and other
allelic forms are designated as alternative or variant
alleles. The allelic form occurring most frequently in a
selected population is sometime: referred to as the wildtype
form. Diploid organisms may be: homozygous or heterozygous
for allelic forms. A diallelic polymorphism has two forms. A
triallelic polymorphism has three forms.
A single nucleotide polymorphism occurs at a
polymorphic site occupied by a single nucleotide, which is
the site of variation between allelic sequences. The site is
usually preceded by and followed by highly conserved
sequences of the allele (e. g., sequences that vary in 1QSS
than 1/100 or 1/1000 members of the populations).
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A single nucleotide polymorphism usually arises
due to substitution of one nucleotide for another at the
polymorphic site. A transition is the replacement of one
purine by another purine or one pyrimidine by another
pyrimidine. A transversion is the replacement of a purine by
a pyrimidine or vice versa. Single nucleotide polymorphisms
can also arisefrom a deletion of a nucleotide or an
insertion of a nucleotide relative to a reference allele.
Hybridizations are usually performed under
stringent conditions, for example, at a salt concentration
of no more than 1 M and a temperature of at least 25°C For
example, conditions of 5X SSPE (750 mM NaCl, 50 mM
NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C
are suitable for allele-specific probe hybridizations.
Nucleic acids of the invention are often in
isolated form. An isolated nucleic acid means an object
species that is the predominant species present (i.B., on a
molar basis it is more abundant than any other individual
species in the composition). Preferably, an isolated nucleic
acid comprises at least about 50, 80 or 90 percent (on a
molar basis) of all macromolecular species present. Most
preferably, the object species is purified to essential
homogeneity (contaminant specie" cannot be detected in the
composition by conventional detection methods).
DESCRIPTION OF THE PRESENT INVED~~fION
I. Novel PolvmorlJhi~ms of the Invention
The present application provides for example
oligonucleotides containing polymorphic sequences isolated
from graminae species for example maize. The invention also
includes various methods for using those novel
oligonucleotides to identify, distinguish, and determine the
relatedness of individual strains or pools of nucleic acids
from plants.
EXAMPLES
Example 1. Maize DNA ex rac ion
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DNA was extracted from maize lines as described
in Rogers and Bendich (1988 Plant Mol Biol Manual A6 . 1-
10) with modification described in Murigneux et al (1993
theo Appl Genet 86 . 837-842).
PCR amplification vuas done on six maize lines
representing a wide range of genetic variability and
including both european flint material and US dent
germplasm. Those six maize lines have been choosen to
maximize the genetic variability of cultivated maize. Doing
so, optimize the chance of finding polymorphism in the
allelic sequences. For example G1, an european flint line
and G3, an US Corn Belt Stiff: Stalk line, are completly
unrelated. Their genetic distance (coefficient of
dissimilarity} calculated with our standard approach (89
RFLP probe/enzyme combinations rind Nei-li distance) is 0.69.
This value is close to the maximum distance between two
cultivated maize lines.
Among the 15 genetics distance between couple of
these 6 lines . 8 are superior t:o 0.6, 6 superior to 0.5 and
only one inferior to 0.5. This shows that the choice of the
lines avoided as much as it was possible the potential
redudancy (or similarity) of allele at the locus sequenced.
With the same effort of sequencing we should therefore have
collected the maximum number of polyphomism.
Genotvoes
G1=flint line
G2=flint line
G3=Dent line
G4=Dent line
G5=Dent line
G6=Dent line
Example 2. Choice of the markers
The markers have been chosen with the following
criteria.
1. Selection of markers that give a single band
in southern hybridization. This is to avoid as much as
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possible the problems of duplicated sequences (very frequent
in plants). If the same (or nearly the same) sequence occurs
at several position in the genome (locus 2 and 2) and if the
primers used to type the SNP found on locus 1 allow
amplification of the sequence at the locus 2, the results of
hybridization on the chips will be the addition of two
markers pattern and therefore impossible to use.
2. Distribution on the genome . most of the
genetic analysis in plant aim to characterize the whole
genome (genetic variability evaluation, mapping quantitative
trait-locus, back-cross assisted selection). The second
criteria was therefore to choose markers nicely distributed
over the 10 chromosomes (see Table A hereunder for map
position).
3. Selection of gene coding for enzymes involved
in the Carbone metabolism. Wxl, Ael, Sh2, Brel, Btl, Ssu,
Bt2 are involved in sugar-starch metabolism. Such a choice
will allow to have a very fast characterization of the
allelic variability (possibly linked to efficiency) of gene
2d involved in this metabolism.
The following markers have been used . see
Table A.
LEGEND OF TABLE A
Probe = name of the marker
COD = in-house code.
MAP Pos - map position, given by the bin location of the
University of Missouri map (Maize Genetic Newsletter n°69
1995). Examples of reading the "MAP Pos" and "Prim" columns
. 1.01-1.02 means that it is the core probe that delimit the
bins 1 and 2 on chromosome 1
5.01 means that it is located in the bin 5.01 (on chromosome
5)
4 means that it is located on chromosome 4
SO1F is the forward primer for probe 1
SOlR is the reverse primer for probe 1
Genbank/ EMBL = Genbank/ EMBL number
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TABLE A
Csnpld (33 markers)
PROBE COD Map Pos PRIM SEQUENCES OLIGOS Genbank/EMBL
UMC157 S01 1.01-1.02 SO1F CGCACGCACATTAGCTTTCG 610822
SO1R TGCAACCGAACAGGATCTGC 610823
UMC76 S02 1.02-1.03 S02F ATTATTCGGCGTCCAGCCCC 610865
lO S02R TTACCAGCGGTGAGAGCTGC 610866
UMC67 S03 1.05-1.06 S03F CGTTCGTGTGGCATCAATCG 610864
S03R CGACATCATCATCGGCAACC 613173
UMC161 S06 1.10-1.11 S06F CAGACCTTGGTTGGAGGCAAC610824
S06R TCGCTCCCCTTCTTCCTTCC 610825
UMC53 S08 2.01-20.2 S08F CGGACGTGATGCAAGTTTCG 610851
S08R AGCGGCTCAAGCTCTCCATC 620852
UMC131 S10 2.04-2.05 S10F2 TCCTTGGCACTCACGCTACC 610816
S10R2 AGCATGGGGGGCAACAACTC 610817
UMC49 S12 2.08-2.09 S12F CAGAGAGCCGTCTCGAATCG 610845
S12R TTGATACTGCCGTCTGCCG 610846
UMC102 S14 3.04-3.05 S14F TGCTGTGCTGTCACATGGCG 610801
S14R CTGGGTCGTCGTGCTTTGAG 610802
UMC63 S16 3.08-3.09 S16F2 ACGCCCTGACAGAACCATCG 610857
S16R TTGCTCACTCGTGGTCGTGG 610857
Adh2 S17 4.03 S17F2 TGCCTGCTGCATCTCTAGCC X02915
S17R2 CAAGCCCGAAAATCGCCAC X02915
UMC66 S19 4.06-4.07 S19F TGGAGTGTCCAAAGACCGACC610862
S19R ACCAAAACGGGTGGTCTGCC 610863
UMC90 S22 5.01 S22F GCAGGTGAACAATGCTGCCC 610870
S22R CCAAAAGGCGGAGAACCGAC 610871
Ael S23 5.05 S23F TCGCTGGGGTTTTAGCATTG L08065
S23R CACTCGAACTCTGTTCAAGGCTTG
L08065
UMC59 S26 6.01-6.02 S26F TCCAAAGCGAAAGCCTGATG 610853
S26R TACGATGGCCGTGACCCTTC 610854
UMC65 S27 6.03-6.04 S27F TTCCAGCTTTCCTCGGCACC 610860
S27R AGCAGCAAGAGCAGAGCGTG 610861
UMC21 S28 6.04-6.05 S28F TGCAGATGTGCCTTTCCTGTG610830
S28R CAGTGGATTCGCTCCCTTCTC610831
UMC132 S29 6.06-6.07 S29F CGCACAGAGGCAGATGCAGC 610824
S29R CGCTAGGCAGAGGTTCGAGC 610819
UMC254 S33 7.03-7.04 S33F CCGGGCGCAAAAGAATGTG 610832
S33R AAGAAACCAGCACCAGCGGG 610833
UMC80 S34 7.04 S34F TCGCCTTTATCGGTGCAATG 610867
S34R TGGAGCAAGCATGGAGATCG 610868
BNL9-11 S38 8.01-8.02 S38F2 CGAGGGAATGTCATCAACCC 610778
S38R2 ACCAAAGCTCCTCAGCCAAG 610779
UMC109 S42 9.00-9.01 S42F GCACCGTCGTTTACCTCAAGC613177
S42R TAGCCATCATCAGCGGCGTG 610807
Wxl S43 9.02-9.03 S43F CGTGCTACCTCAAGAGCAAC X03935
S43R ACTTCACGGCGATGTACTTG X03935
UMC95 S44 9.04-9.05 S44F CACTCGGAAGTCGGAATCGC 610872
S44R ACCTTCGCAGTGTTGCGGAC 610872
CSU61 S45 9.05-9.06 S45F TCTCCACGAATCCCACCGTC T12691
S45R AAGGGAGGGAATCCTCTACCGT12691
UMC130 S48 10.02-10.03 S48F AAGGGGGAAGAAGGTCATC GI0824
S48R CGATGGCAACAACTACCAGTAG610815
CSU109 S53 2.09 S53F GCTTTCGGTTCCGGATAGCG T22721
S53R ACTGGGCCATCTCCGACCAG T12721
UAZ77 S56 5.04 S56F2 GCAACCAACTGCAACATCGC T18762
S56R2 GAAGGAGCTCAAGGCCAAGG T18762
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Sh1 S57 9.01 S57F TGCTGTTATTGCGTGCCGTG X02382
S57R2 AAGGTGGCACCAAGGCGTTC X02382
Sh2 S63 3.09 S63F TTCTTCACTGCACCCCGATG M816D3
S63R CTGCTCACTCTGCAATGCCC M81603
5 Bre1 565 6 S65F AGCAGCAGATCAGGCACACC U17897
S65R TTGAAGTTCGTTTCGGGCAC U17897
Bt1 S66 5 S66F GGCAAGGATCGGAGTTGCTC M79333
S66R TAGCGTGGAGGACGTTCTGG M79333
Ssu S67 S67F GCAAGCAAGCAAGCAGCGAG D00170
1~ S67R GACCCGAAGCAAAACCGAAC D00170
Bt2 S71 4 S71F TGCCGAAAAAGGTGGCATTC Seq (Bae et al
1990)
S71R GCCCCCAATGTCGATTCAAC
Example 3 PCR amplification
PCR amplification was done with primer designed
using the DNA sequences of the markers listed above. The
sequences for all markers/genes were available on Genbank/
EMBL.
Forward and reverse primers are given in the
table A hereabove.
PCR condition were as followed
For each reaction in 30 microliters . DNA :60
ng; Taq DNA polymerase (Amersham) . 0.9 unit; Buffer 10x . 3
microliter; dNTP's . 0.2 mM each; MgCl2 . 1.5 mM; BSA 0.8mg/
ml; primers 1.5 ng/microliter each; glycerol 50.
Polymerisation was done in a perkin Elmer 9600 .
1' at 95°C, followed by 35 cycles of (30" at 94°C, 30" at
60°C, 1'30" at 72°C) followed by 1'30" at 72°C.
The sequencing of 186 maize amplicon was then
done with the primers used for DNA allele amplification. DNA
sequences were edited and aligned. Sequence surronding
polymorphism (see table I here-under were collected from
these alignments.
LEGEND OF TABLE I (with references to the Bt2
gene for instance.)
Column 1 (Bt2) represents the name of the marker
or gene.
Column 2 (Bt2-G2/G6-1) represents .
- the name of the maker (Bt2)
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the genotype number (G2)
- the second genotype number (G6)
- and the number of the SNP (single nucleotide
polymorphism). So, in this cage, it is a SNP found on a
sequence nucleotide Bt2 between the genotypes (strains of
maize) G2 and G6 and this SNP was numbered 1 (Sometimes there
are several SNP between two genotypes for the same sequence)
Column 3 represents . similar to column 2, but
with the codification of the marl~:er/gene.
Column 4 represent~~ sequence holding the SNP_
Into brackets . [G/T] means that the sequence of G2, at this
position of Bt2 gene, is G, while for G6, it is T.
On the other hand, /G (CSU61-Gl/G5-1A) means
deletion of the base pair G in G7_ compared to G5.
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iz
TABLE I
csnpld
Ssu 8t2-G2/G6-1 S71G2IG6-tATAATACTTGATATGCCATT[G/1~TGTCCTCTTATTTTTAA
S
CAT
Ssu su- G1JG5-1 S67G1/GS-1ATGGCCTCGTCGGCCACTGC[A/C]GTCGCTCCGTTCCATG
S
GGCT
Ssu su-G11G3-1 S67G1/G3-1GCCGCTCCTCCAGAAGCCTC[G/A]GCAACGTCAGCAACG
GCGGA
Ssu Ssu-G1/G3-2 S67G11G3-2GTGTTGCCCATCCCATCCCA[A!Z]TTCCCAACCCCAA
ACGAACC
Bti Ssu-G1/G3-3 S67GiIG33 GTACCTGCCGCCGCTGTCGA[CG/AC]GGACGACCTGCTG
B
AAGCAGG
Bti tt-G2/G3-1 S66G2/G3-1AGTGAGCCCGCTTCTTATTC[/T]TAAGGTGATAGGTTTCTA
B
AA
Bt1 ti-G2/G3-2 S66Gi/G3-iAATGTAATGGTACTCCGCGC[T/C]ATGGCTCTGGTACTTA
GGAA
Bre1 Bt1-G2/G3-3 S66G1/G2-1AAATAGGCTCGGGCAATTAT[C/jCAGCTTAGGGACAGCAAGC
B
G
Bret rei-G3/G6-1 S65G3/Gtr1TCCGCCCTGCCTCCGGTTTT[M']GCCCGACCTTCGAAACATT
B
C
Br ret-G3/Gti-2S65G3IG6-2ACCACTGACGTAGCACCTCC[G/T]ACTTCTCGTTGTAA
1
AACCCC
e Bre1-G3/G5-1S65G3IG5-1GGAGGTTCGCCTCATGTTAT[C!f]GTTGACGAGCCA
B
i
CATCCACT
re Bre1-GMG6-1 S65G41G6-1GCTCCGACTTCCAATCTTGA[A/C]CCTCCACCCTGC
ASG72
CTCCGGTT
~Gi2 ASG12-G1/G3-1S64G1/G3-1CTGGTTGAAATGTGTTGAAG[C/A]TACTAGTGATGAA
CTGCTTG
ASG1 ASGi2-Gl/g3-2AS64G1/g3-2AGCTGCTCCAAGCGAGCCCGC[C!G]CCGAAAAAGG
AAAAAGGTGA
2 ASG12-GlIg3-28S64G1/g3-2BGCTGCTCCAAGCGAGCCCGC[C/G]CCGAAAAAGG
AS ~
AAAAA(
G12 ASG12-GlIg3-3AS64G11g3-3A,TTGA
ASGi2 CGCCCCGAAAAAGGAAAAAG[G/T]TGAAGGTCCTTAC
TCACCGA
ASGi ASGi2'Gi/g3~BS64G1I93-3BCGCGCCGAAAAAGGAAAAAG[G/T]TGAAGGTCCTTA
CTCACCGA
2 ASG12-Gl/gG3-4AS64G1/gG3-4AGAACCGGCCACAGTGCCTGA[TIA)T'TTGGCGGTGAG
ASG12
ACCTCTTC
Sh2 ASG12-Gl/g3-4AS64GtJg3-4AGAACCGGCCACAGTGCCTGA[T/A]TTTGGCGGTGAGAC
TTCTTC
S~ Sh2-G5/G6-1 S63G5/G6-1CAATTGTTACCTGAGCAAGA(f/]TTTTGTGTACTTGACTT
GTT
Sh2 Sh2~4/G6-1 S6:iG4/Gti-1TACTGAGAGAATGCAACATC[C/G]AGCATTCTGTGATTGGAGTC
5h2-G4IG5-1 S63GMG5-i TTTTAGTGTACTTGACTTGT[C/T]CTCCTCCACAGATGAAATAT
Sh2 A A
Sh2-G41G5-1 St~G4lGS-1TTTTTGTGTACTTGACTTGT[G7]CTCCTCCACAGATGAAATAT
Sh2 B B
Sh2-G3/G6-1 S63G3/G6-iTCTGTGATTGGAGTCTGCTC[G1A]CGTGTCAGCTCTGGATGTGA
Sh1
Sh1-G5/G6-1 S57G5JG6-1AACTACJ1AAAAGCATCTCCT[GIT]GGATTTGGCTATCTCCTTTT
Shi
Sh1-G21G5-i S57G2/G5-1TTAGCGCCAAAAAAAApCTC[/T)TTTTTTTTTGTCCTTTTACT
Shi
Sh1-G21G3-1 S57G2/G3-1TCAATCCAATCAATTTAATT[TIC]CTTCCTTTAAAAATATTATC
Sh1
Shl~l/G2-1 S57G11G2-iTTACTACGAAAAACTCTTGA[GIT]TCTAGGAATTTGAATTTGTG
Sh1
Sh1-G1/G2-2AS57G1/G2-2ACTTCTTGGATTTTGCTATCT[T/C]CTTTTACTACGAAAAACTCT
Sht
Sh1-G1IG2-2BS57G1IG2-2BCTCCTTGGATTTTGCTATCT[T/C]CTTTTACTACGAAAMCTCT
Sh1
Sh1-G1/G2~A S57G11G2~ATTTTACTACGAAAAGCATCT[TIC]CTTGGATTTTGCTATCTTCT
Sh1
Sh1-G1/G238 S57G1/G2~BTTTTACTACGAAAAGCATCT[TIC]CTTGGATTTTGCTATCTCCT
Sh1
S57G1/G2-4 S57G1JG2-dGAAGCCAAATCCTATTATTT[T/C]CTGCCTCTAGGGTCTGAATG
UAZ77
UAZ77-GNG6-iS56G4lG6-1GTACACTGTTACAATCACAC[fIG]TAGTGAAGCGCAACACAGAT
UI1Z77
UAZ77-G4/G6-2S56GNGti-2GCCTTATCATCCTCTAGGTA[f/A]TGGAGACGAGTGACCAGTCT
UAZ77
UAZ77-G4IG63S56GMGti-3CTTTTCTTCAGACCCGAGCC[CITjCCAATCGCGCCCTTCTGTGC
U
AZ77 UAZ77.G4lG63SS6G4IG63 CTTTTCTTCAGACCCGAGCC[CJT)CCAATCGCGCCCTTTTGTGC
UAZ77
UAZ77~41G5-1AS56G4/G5-1AGAGCCCCCAATCGCGCCCTT[GT]TGTGCCTTGGCCTTGAGCTC
UAZT7.G41G5-1AS56G4IG5-1AGAGCCTCCAATCGCGCCCTT[GT]TGTGCCTTGGCCTTGAGCTC
UAZ171
UAZ171G1IG3-1S55G1/G3-1GAAGGAGCAGCAGCGCAAGG(NJACGTGTTCCAAGTCAACGTC
UMC17 UMC117-G2IG3-1S54G2/G3-1GTAGAAAGTTAGCAAAAACA[T~7T-~TTAGTGAAAAAACATA
UMC17 UMC117~2Xa3-2S54G21G3-2ATTGTGGCTAGAAACTTTGG[I~TTITTITApp~TTATGGTCAT
CSU109CSU70S~SfG6-1S53G5lG&1 GCAAACCMCACCAp~TCTTC[G/C]AAATGAGCAAAGCAGAGACT
CSU109CSU1l79.G51G6-2S53G5/G6-2CAGATCGGTTGTCCTCAGAG[AJJAAGTCACCTACCTGCAAACC
CSU109CSU1 3 S53G5/Gf,-3AATTCTAG4TAGGAGTCATG[G1]ACAAGTACTTGTTTAAAGGA
CSU109CSUIt~5IG6-4S53GSIGfi-4ACAAGTACTTGTTTAAAGGA(GjCATGCCGGAATACACGCTGC
~~~A GAGCGAGATCGATCCTGTTG[T/C]CATCCATCACTGCCATAGGA
CSUt09CSU109-GSIG6.SBS53GSIGSSBGAGCGAGATCGATCCTGTTG[TI~CATCCATCA
CTGCCGTAGGA
CtaU109CSU1t79.G4/Gfr.1S53G4IG6-1~
TAGTCATAGCHA~f'.AGCATGaG//~JTCG~,ATGTAGCGTTCACCC
CSU109CSUtOA.GMG6 S53G4/G6-2CAATTGAAGAGG4~1AAAAppA(/~CT~pTTCATGTAC
CSU1092
CSU109.G4AG5-1S53G4/GS-1~G~GAGTCCACAATAOTTCGTC
CSU109
CSU109.C311p5-1S53G3K35.1CCCACCGCGG~TGGTGG[T/JTAGAAGG;GAACCACCGAGC
CSUIt~CSU109.G?hGG-1S53G2K~6-1ACTTGTTTMI~GGA~CATGCC(<i~GGApTq~CTGCCCAGGC
CSU109CSU1~IG3-1 S53G2Aa.1 OCCAGGCCTTCOCACGGCGG[AK33]GATGGTGGTTAGAAGCGGAA
CSU109CSU109-011G6-1S53G1K36-1
TCCGCAATAGT
CSU109CSU109.G1~G6-
2S53G1~Gfs.2GJ1ACI4C',AGTCCGCMTAGTT[T/~CaATCCTAATGCTi4CTTOGAGC
UMC130UMC130~31G6.1S48G3IG6-1GATTCJ1GAAACA~GTGGCGGC[AK~GATGTAGCATCAACACGCCC
CSUtiI.GS/G6-1S45ta51G6-1ATGAGTATATTC~AAGTCATA[TICjTGTGAACTAGAATGTTATTT
CSU61 CSU61-G5Ki6-2AS45GSlGfr2ACCTAGJ40GCTGACCGOC~AC~A[G//ypCGGCGG~CTGCG1AATC
CSU6I.f~G6 S45GSIG6-2BCCTAAACGCTGI1CC(~
2B CCqC
,
CSUB1 CSUB1-G5b6.3S~SGSIG6-3/~G/Np~G~CTGCCAMTC
TGAAA'.~MACCATGCGCTACC(GTjAGCTACiGTGTTTTMAC'
TM
CSU61 CSU61-G~i6-1&45G~AG6..1s
TCOGCGGJ1AACAACATCCGA(G!1'[TTCTTGAGOATAACCCAGCT
CSUB1 CSU61.G4/G5.1S45G4K35..1GGGAGGGGAMAqA~pGp~GJTTGGTTGCGGTTCAGT
CSU61 CSU61-G4fG5-2S45G4/GS2 GGCGGCTGCCAAAT~GCaG[!A)AppCGA~TtxGAGTTCTTG
CSU6i CSU61-G2IG4-1AS45G2IG4-1ACTAGMTGTTATTTCTTCAC[GA]GTTGACCATGGMAAAAACA
CSU61 CSU61-G2IG4-18S45G2/G4-1BCTAGAATGTTATTTCTTCAC[GI~GTTGACCATGGAAAGAAACA
CSU61 CSU61-G?IG4-2AS45G21G4-2ATTCACCCTTGACCATGGMA[AIIpTp,ATAAGTTCTTGT
CSU61 CSU61-G2K34-2BS45G21G4-28TTC~ACJIGTTGACCATGGAAA(NC~)I4AACAGTMTMGTTCTTGT
CSU61 CSU61-Gt/G6-1S45G1IG6-1TTCTTG~CA,GTTGACCATGG[IA~AAAAAAAG4GTMTAAGTTC
CSU61 CSU61.G1IG5.1AS45G1/G5.1AGAACCCACCOTGCCCTGOGA[/G)<3G
CSUB1 CSU61~1IG5-1BS45G11G5_iBGAACCCACCGTGCCCT
CSU61 CSU61-G11G5-2AS15G1Ki5-
2ATGGGAGGGNJIAAAqAAA~C'sAA[LiI~AGCt;',TTGGTTGCGGTTCAGT
CSU6i CSU61-G1/G53S45G1K's5~3CGTACC~1ICCTqGGMTCGTA(AK;~UIApGCCTAGACGCTGACCG
lJMC85
UMC95GSJG6-iS44G5/G6-iGCTGCGTCMTCI1TG1C1TC[T/A]CCCACI1GGCGTCAAGTACAG
U~AC85
UMC95~G3IG4-1S44G3K's4-1GACAGATTCCAAAGTAGTCG[GT)CGGCCACGTCGAAAAAGAAT
UMC95
UMC9rG2/G6-1S44G2/G6-1GGCGCTGCGTCAATCATCAC[MjTCACCCACAGGCGTCMGTA
CA 02274317 1999-06-02
WO 98/30717 PCT/EP97/07134
13
csnpld
UMC95 UMC95-G2IG4-tAS44G21G4-1ATCGGTGTCACCnCATGCATA,[TlG]TCAGGACAGATTCCAAACTA
UMC95 UMC95-G21G4-iBS44G2/G4-1BTCGGTGTCACCACATGCATA[TlG[TCAGGACAGATTCCAAAGTA
UMC95 UMC95-G?JG4-2AS44G2IG4-2AGTCGCCGGCCAGGTCGMAA(G/A]GAATACTCAGCAAAAGACCC
UMC95 UMC9SG2IG4-2BS44G2/G4-2BGTCGTCGGCCA~GGTCGAAAA[G/A]GAATACTCAGCAAAAGACCC
UMC95 UMC95-G21G3-1AS44G2/G3-1ATATTCAGGACAGATTCCAAA[GG]TAGTCGCCGGCCAGGTCGAA
UMC95 UMC95-G21G3-1S44G2/G3-1TAGTCAGGACAGATTCCAAA(GG]TAGTCGCCGGCCAGGTCGAA
B B
UMC95 UMC95-GIIG(i-1S44G1/Gti-1GCGTCAAGTACAGATACGCA[AIG]CACGCCTCAGCTTCGCCTTG
UMC95 UMC95-GllG2-1S44G1/G2-1CCTGGGACTCG:~CAAATTGC[GlA)A1GCACTCGGTGTCACCACAT
Wx1 Wx1-G?JGtr1 S43G2/G&1GCTGGTTCATTA,TCTGACC1'[GlT]GATTGCATTGCAGCTACAAG
Wx1 Wxl~?JG6-2 S43G21G6-2CTGGATTGCATTGCAGCTAC(A/G]AGMGCCCGTGGMGGCCGG
Wx1 Wx1-G2/Gt~IB S43G21G&1BGCTGGTTCA1TA,TCTGACCT'[GlT]GATTGCATTGCAGCTACGAG
Wxi Wx1-G2lGti-2BS43G2/Gtr28CTTGA1TGCATTGCAGCTAC[A/G]AGAAGCCCGTGGAAGGCCGG
Wxt Wx1-G2/Gti-3 S43GTJGti-3TCAGCCCCTACI'ACGCCGAA[G/JGAGCTCATCTCCGGCATCGC
Wxi Wx1-G2/G5-1 S43G2/G5-1TACCCGGAGCTGAACCTCCC[GG]GAGAGATTCAAGTCGTCCTT
Wx1 Wx1-G21G4-1 S43G21G4-1TGCATGTGAACATTCATGAA(f/CjGGTMCCCACAACTGTTCGC
Wx1 Wx1-G6IG1-1 S43G6/G1-1CTCCTACCAGG(~CCGGTTCG[T/]CCTTCTCCGACTACCCGGAG
Wx1 Wx1-GI/Gfr1 S43G11Gti-1TGAATGGTMCCCACAACTG[GTJTCGCGTCCTGCTGGTTCATT
Wx1 Wx1 ~11G5-1 S43G11G5-1GCCGACAGGGTCCTCACCGT[GIC]AGCCCCTACTACGCCGAAGA
Wx1 Wxi ~2lGfi-1 S43G2/G6-1GCTGGTTCATTATCTGACCT(G/T[GATTGCATTGCAGCTACAAG
Wx1 Wx1-G2/G6-1 S43G21G6-1GCTGGTTCATTATCTGACCT(G/T]GATTGCATTGCAGCTACGAG
B B
Wx1 Wx1-G2/G6-2 S43G2iGti-2CTGGATTGCATTGCAGCTAC(AJG]AGAAGCCCGTGGAAGGCCGG
Wx1 Wx1-G21G6-2B S43G21Gfr28CTTGATTGCATTGCAGCTAC[AIG]AGMGCCCGTGGAAGGCCGG
Wx1 Wx1-G2/G65 S43G21G6-5TCAGCCCCTACT'ACGCCGAA[G~GAGCTCATCTCCGGCATCGC
Wxt Wx1-G2IG4-1 S43G2JG4-1TGCATGTGMCATTCATGAA[T/C]GGTAACCCACAACTGTTCGC
Wx1 Wx1-G2IG3-1 S43G21G3-1CTGGTGGTGGTGCTTCTCTG[AAAG]TGAAACTGAAACTGACTGCA
Wx1 Wx1-G2/G3-3 S43G2IG3~3GACCATCTTCACGTACTACC(fACG JAGACCGCTTTCTGCATCCAC
Wx1 Wx1-Gl/Gti-1 S43G1/Gti-1CTGACCATCTTCACGTACTA,[CCTAI]CCAGACCGCTTTCTGCATCC
Wx1 Wx1-GtiIG1-1 S43Gti/G1-1CTCCTACCAGGGCCGGTTCG[T>jCCTTCTCCGACTACCCGGAG
Wx1 Wx1-GtilG1-1 S43G6IG1-1GAGATTCMGTCGTCCTTCG(G/jATTTCATCGACGGGTCTGTT
Wx1 Wxi-G1/G6-2 S43G1/G6-2TGAATGGTAACCCACAACTG(GT]TCGCGTCCTGCTGGTTCATT
Wx1 Wxt-G1IG6-3 S43G1/Gti3GCCGACAGGGTf:CTCACCGT[GlC]AGCCCCTACTACGCCGAAGA
Wx1 Wxl.G1/GS-1 S43G1IG5-1TCTGACCATCTT~CACGTACT[ACCTIjACCAGACCGCTTTCTGCATC
Wxi Wx1-G11G4-1 S43G1/G4-tCTTGATTGCATT~~.~CAGCTAC(GlA]AGMGCCCGTGGAAGGCCGG
Wxi Wxt-G11G4-1 S43G1IG4-1CTGGATTGCATTGCAGCTAC[G/A]AGAAGCCCGTGGAAGGCCGG
Wx1 Wxt-G1/G3-1 S43G11G3-1GCTGGTTCATTATCTGACCT(T/G]GATTGCATTGCAGCTACGAG
Wxt Wx1-GSIG6-1 S43GS1G6-1AGAGATTCMGTCGTCCTTC[G~GATTTCATCGACGGGTCTGT
UMC109 UMC109-G2IGti-1S42G2/G6-1CTCG~1TGAAAAAGGTGCCGC[/G]TACTCTCTCAGTCAGCTACT
UMCIt><J UMC109-G2/G3-1AS42G2IG3-
1ACTGCACTCCGATTGAGGGTC[C/G]GMGCAGGGCAGCGCGTGTG
UMC109 UMC109-G2IG3-1S42G2IG3-1CTGCACTCCGA'iTGAGGGTC[GGjGMGCAGGGCAGCGCGTTGT
B B
UMCIt><J UMC109-G2/G3-1CS42G21G3-
1CCTGCACTCCGATTGAGGGTC[GG]GAAGCAGGGCAGCGCGTTTG
UMC1t79 UMCIf~-G21G3-1DS42G2IG3-1DCTGCACTCCGATTGAGGGTC[CJG]GMGCAGGGCAGCGCGTTTT
UM(~.80 UMC80.G3IG5-1S34G3NG5.1CATGCCTCTGTTGATATTTT[G/C]GTGCACCTTTTGCTTGCMC
UMCt30 UMCBf>G3/G5-2S34G3/G5-2GATTTTGTAGGTTGATGCAT[Cl~jGTTTGATCTTTCTTATCTCC
UMC80 UMC80-G3/GS3S34G31G5~3TGCTTGCAAiCTAAATTMTC[AIG]TGCTCTATTTGACTMGAGT
UMC80 UMC80-G31G4-1S34G31G4-1ACATGTC.C~4GGACGCATGGIjCIJCCCAATAT'TGTTGTTGGMG
UMC80 UMC80-G31G4-2S34G31G4-2TTGATCTTTCTTATCTCCTT[!C]CGMTTTGTTCTGTGTTATA
UMCtiO UMC80-G31G4-2BS34G31G4-2BTTGATCTT'TCTTATCTCCTT[/C]CGMTTTGTTCTGTGTATAC
UMC80 UMC80.G2/G5-1S34G?aGS_1TGTAGGACTTGGAGAGCTTG(AIG]TMTTTACACATGCCTCTGT
UMC80 UMC80.G2lGS-2S34GZb5-2CATGCCTCTGTTGATATiTT(GJCjGTGCACCTTTTGCTTGCMC
UMC80 UMC8~Gr3 S34G2Ki53GATTTTGTAGGTTGATGCAT[C>nGTTTGATCTTTCTTATCTCC
UMC80 UMC804~G3-1S34G21G3-1GAGACATTTCCTACTCMT
A jCn]Ai4TTATTTGATGAMTTATT
UMC254 UMC254.G51G6-tAS33GSfG&1AAGTATCACMiACTMTCTGA[NGjTATCTGGTTGCf:AOGAAMC
UMC254 UMC254~G6.1BS33G5lGrrlBAGTATCACAQJ1CTMTCTGA[AK3]TATCTGGTTGOCACAAAAAC
UMC254 UMC254-G5fG6-2S33G51G6-2TCAAAGTGGTGCAATCGCAA[TICjCGA~CTTGGGCTTGCC(iTGGT
UMC254 UMC254-G51G6.3S33GSrGS~CCI1CTTGGGCTTGCCGTGGT(CIJCGTATCGTACGCAGGTAGCA
UMC254 UMC254-G51G6-4S33GSK36-4AGCATTTTTfGTTTTGTTTT[TJC]CCTTGGCAGACMCI1GACAG
tl~AC254 UMC254-G';~G65AS33GS/G65ACAGTCCCOAGANTCCCAAAT[GjCAQAAAAAGGTTTTGTTTTT
tlAAC254 UMC254-G51G8.58S33GSIG6~58CAGTCCCGAGMTCCCAAAT(GJCAGIUW1I1GOTTTTGTTTTA
UMC254 UMC254.G4RIG6-
1AS33G4R/GfrlAGGC;AuC~AC~1A~CA~C'~CA~CATCA[AG1CNCATGCTTGCATTTACTCCCA
UMC254 UMC254-G4RIG6-1S33G4RKi6-
1GGCAGACAACAGiACAraATCA[AG/C~CATGCTTGCATTTACTCTCA
B B
UMC254 UMC254-G3RIG6-1AS33G3RI08-1AGTGATCACAGACTMTCTGA[NGjTATCTGGTTGCCACGMMC
UMC254 UMC254G3RIG6-1S33G3RIG8-1GTGATCAf:AGACTMTCTGA(AIGjTATCTGGTTGCCACAAAAAC
B B
UMC254 UMC2S4~G3RIG6-2AS33G3WG6-
2ATGTGMTATCTGGTTGCCAC[G/AjAAAAiCCOGGACACMCiAGAG
UMC254 UMC254~i3R/Gtr2BS33G3R/G8-
2BTCTGAGTATCTGGTTGCCAC[G/AjAAAACCGGGACACMGAGAG
UMC254 UMC254.G3/G6-3S33G3~ TCAGTCAAACTCAGTCCCGA[AK3]AATCCCAAATCAGAAAMGG
UMC254 UMC254~3X35-1AS33G3IG5-1AGGTTGCCACOIV~1ACCOGGA(CIG TCAGAGT
UMCZ54 UMC254~G31G5-1S33G3JG5-
1GGTTGCCACGAiIAA~CCGGGA[GG)ACAAf"aAGAGAAACTCAAAGT
B B
UMC254 UMC254-G2R/G3-tAS33G2R1G3-
1AACGCATGCTTGCATTTACTC[CJTJCAGTCAAACTCAGTCCCGAA
UMC254 UMC254-G2R/G3-1S33G2R1G3-1ACACATGCTTGCATTTACTC[GT)CAGTCAAACTCAGTCCCGAA
B B
UMC254 UMC254-G1R/G2-1S33G1R/G2-1TATTATTCAATTTTGAATM[IGjGAAGOAAATTTTAGG1,CCTC
ASG49 ASG49-G3IG5-1S32G3IG~r1ATTMTAMTGCATCCTCTG[GGjTAAAAAAACCCATTTTGMT
ASG49 ASG49-G3/G5-2S32G31G~r2ATGMTTGMGCTCTGMTA[GITjAGMTCCACCATTCTTCCGA
ASG48 ASG43-G31G5-3S32G31G5-3GMTCG4Cf:A1TC1TCCGAA(A!G]CTGCTTCCTACAAAACTCGA
ASG49 ASG49-G3IG5-4S32G3/GS-4GAAAGGATGTGZTTTTGATA[G!A)CCTTCAGTCTTTCAGATGGA
ASGB ASGS-G3lGS-1S31G31Gr1CAATGTCTTGTTCGTTATCA(A/G)CGAAAGTTTGMTCCCCACA
ASG8 ASG8~G31G4-1S31G3IG4-1TGTATCGGCTAGTCTGGATG[GIAjTCGCACTGGCACTCAGTGCT
CA 02274317 1999-06-02
WO 98/30717 PCT/EP97/07134
14
csnpid
UMC132UMC132-G4/GS-1S29G4lGS-1
TCTATTCAGCAGTCTGAGAA[GCAICT]AGGATGGTCGGCTTCTTCAG
UMC132UMC132-G11G5-1S29G1/G5-1 CCTTACACTATTAACAGGCC[GT]GTGATCTACCTGAATGCCTG
UMC21UMC21-G5/G6-1S28GS/G&1 CAAGAAGCCTCTTCAGTGTC[A/C]GTCGTAGCTTCCTCAAGACC
UMC21UMC21-G51G6-2S28G5IG6-2 AGACCTTCCTGATGTGCGGA[T!C]GCTAATCCATGGAGCAGGGA
UMC21UMC21-GSIG6-28S28GS/G6-2BAAGACCTCCTGATGTGCGGA[f/C]GCTAATCCATGGAGCAGGGA
UMC21UMC21-G5/G6-3S28G51G6-3 CTAATCCATGGAGCAGGGAG[G/A]AAGGGGCGAGGGGCAGCAAG
UMC21UMC21-GMGS-1S28G4IG5-1 TCGTCGCGAATACAGCCGGG(G/C]GAGGGGGTGGTCGCGACTGG
UMC21UMC21-G3/G6-1528G3/G6-1 GTCGTAGCTTCCTCAAGACC[TIJTCCTGATGTGCGGACGCTAA
UMC21UMC21-G3IG4-1S28G3/G4-1 GAGTCGTCGCGAATACAGCC[A/G]GGGGAGGGGGTGGTCGCGAC
UMC21UMC21-G31G4-2S28G3IG4-2 AGGGGGTGGTCGCGACTGGA[T1G]CGCCCGAGCAGCGAGCAAGC
UMC21UMC21-G3/G4-3528G3IG4-3 AAGCACATGTTTTAACCTTT[T/G]ATTCAAACTTTCCAGCCGTT
UMC21UMC21-G31G4-3BS28G3/G4-3BAAGCACATGTTTTAACCTTT[T/G]ATTCAAACTTTCCAGCGTTA
UMC21UMC21-G2/G6-1S28G2IG6-1 GAATGTTGCTGTTATATTAC[TIC]CGTAGGTGACAAAGGGTTCA
UMC21UMC21-G21G4-1S28G21G4-1 AGAAAAATTTACATAAAAAA[G/C]CACACTCCATGATTGTTAAA
UMC21UMC21-G2/G4-1S28G2/G4-1 AGAAAAATTTACATAAAAAA[GIC)CACACTCCATGATTGTTTAA
B B
UMC21UMC21-G2IG3-1S28G21G3-1 CTTTTATTCAAHCTTTCCAG[/C]CGTTAATTTGTTATCCGTTG
UMC21UMC21-G6IG1-1S28G6/G1-1 TGTTGAACATGCTCTCAGGA[/CC]CCCCCTATTGTGACACAGCA
UMC21UMC21-G1IG3-1S28G1IG3-1 TACATCTTAACAAGCACATG[TGIT'11 jTMCCTTTTATTCAAACTTT
UMC65UMC65-G3IGfi-1AS27G3/G&1A AGTAATGTGTGACTGTGGGC[GG]CGTGTGACAGCTTTTACGTA
UMC65UMC65-G3/G6-1S27G3IG6-1 AGTAGTGTGTGACTGTGGGC[GG]CGTGTGACAGCTTTTACGTA
B B
UMC65UMC65-G3/G6-2S27G3/G6-2 TTCGCTTGGTAGCCGTAGCA[G/A]TATACTTTTACCGGCCACAG
UMC65UMC65-G3/GB-3S27G3/G6-3 GGGCTTTGGGTTGTGAACTT[CCAIC TTTCCC
UMC59UMC59-GSIG6-1UMC59-GSIGfr1CCAAGAAAGATTAATGCTGG[/T]TAAAATATTGTTTCCAGTCT
UMC59UMC59-G51G6-2UMC59-G5/G6-2AAAATCAGGACTGCGAAAAA(A/C]CCAAGAAAGATTAATGCTGG
UMC59UMC59~5/G6-2BUMC59-GS/G6-2BAAAATG~GGACTGCGAAAAA[A!C]CCAAGMGATTAATGCTGGT
UMC59UMC59-G5/G6-3UMC59-G51G&3AAAGTGTGTGTTGTTGCCCA[GIA]ATGATTCCATTCCACACAAG
UMC59UMC59-G4IG5-1UMC59-G4/G5-1AGGACTGCGAAAAAACCAAG[lA]AAGATTAATGCTGGTAAAAT
UMC59UMC59-G4/G5-2UMG59-G4IG5-2ATGCTGGTAAAATATTGTTT[/C]CAGTCTTTCACAAAGTGTGT
UMC59UMC59-G3IG4-1UMC59-G3/G41CTACAAAAATCAGGACTGCG[lA]AAAMCCMGAAGATTAATG
UMC59UMC59-G31G4-2UMC59-G3/G4-2TTGTTTCAGTCTTTCACAAA[/GT]GTGTGTGTGCCAGATGATTC
UMC59UMC59-G3/G4-3UMC59~31G4-3TCACACACCGACCTGCCTGG[/T]TATCAGGAACCATCCTCCTG
Ae1 Ael~4/G5-1 S23G4JG5-1 GGTGMTTGGTGATGCATGC[TIG]GGGGGTGCTCGAGTTGGATG
Ae1 Ae1-G4IG5-2 S23G4lGS-2 TTCCAGTCGGATGAACTGGA[TlG)GTTCGTCATCCACTCGTG~C
Ae1 Ae1-G3JG6-1 S23G3IG6-1 GGTGAATTGGTGATGCATGC[AIT]GGGGGTGCTCGAGTTGGATG
Ae1 Aei-G51G3-t S23G51G3-1 TTAAGTGAACATGCCCAAAC[GG]GTTAAACTTTCCATGGAACT
Ae1 Ae1-G5IG3-1 S23G5/G3-18ATTAATGAAGATGCCCAAAC(GG]GTTAAACTTTCCATGGAACT
B
Ae1 Aet-G1JG6-1 S23G11G6-1 TGATTCGGGTCTGTATGCGA[G/nTGTTGTGGTGGTGAACTGGT
Ae1 Ae1-G1/G5-1 S23G1/G5-1 CGGGTCTGTATGCGAGTGTT[G/A]TGGTGGTGAACTGGTGAATT
Aet Ae1-G1/G4-1 S23G1/G4-1 GTTCGCGGTTTCTGGGGCCG[GIT]GGGCGGTGCTCGGTGGGGCC
UMC90UMC90-GS/G6-1S22G5/G6-1 CAGATTGGTGTCGTTTACTA[A!G]AATTCAGTTCTGTCCATTTG
UMC90UMC90.G5lG6-2S22G~2 AAGTAAGCATTCTTTATATG[ITjTACTTCCCATGATAAACTTT
UMC90UMC90~5IG6-3S22GSIG63 CAAAGGGCTTACTGTACTTT[IC]CATCTTATTGGCAGGGCACC
UMC66UMC66-GS/G6-1S19G~1 ACTTGGCCGGGGACGTCGAC[GIA]ATCGTCGTAGCACTACTGGT
UMC66UMC66GSIG62 S19GSIG6-2 AGTACATGGCGAGCGTTGTA[G/C]CAGCTGCTTAGGTGATGTGG
Adh2 Adh2-G4IG6-1S17G4IG6-1 CTATTTCG1AGCTAACAACC[C!G]CTCTTGGTCCCAACATCCTG
Adh2 Adh2-G3IG6-1S17G31G6-1 GGTTCTAAACATAGCTCGTC[GI1JATTCATGATTCATCTCGAGC
UMC63UMC63.G4IG6-1S16G4/G6-1 TCAGCAAGCCTCCAAGGCTC[GA)AATGGTCCAGTTACTTGGTT
UMC63UMC63G2/G&1 S16G21G6-1 GTGTGTAGCTTCATTCGCAA[TGIAT]TTTGAA~CAGCCTCTGCAAGT
UMC63UMC63-G21G6-2AS16G21G6-2AGTGCTTTCGTAMCCTAGAG[TIC]TGAiCCAGCTGTGATTTCGGT
UMC63UMC63-G2IG6-2BS16G2IG6-2BGTGCTTTCGTAAA~CCTAGAG[TKxTGA~OCA,GCTGTGATTTCGAT
UMC63UMC63~21Grr3AS16G2~G63A GCTGACCAGCTGTGATTTCG(GJI~]1'GTATTCCAOGACGA~CGAGT
UMC63UMC63-G1IG6-1S16G11G6-1 TGTGTAGCTTCATTCGCA~AjGtTJTTTGJ1A~CAGCCTCTGC.AAGT
UMC63UMC63.G1IG3-2AS16G1JG3-2AGTGCTTCCGTAAACCTAGAO[TfC]TGACCAGCTGTGATTTCGAT
UMC63UMC63-G1/G3-2BS16G11G3-2BGTGCTTCCGTAMCCTAGAG[T1C]TGA~7CAGCTGTGATTTCGGT
UMC63UMC63-G1IG2-1S16G1IG2-1 GTGTGTAGCTTCATTaGCAA[[NT]GTnG~C;CCTCTGCMG
UMC102UMC102-G5IG6-1S14G51G6-1 GCTCAGCTGCCGGAGTA~CG
T(NT]GGCTTGCTCTCCGGCCGGCC
UMC102UMC102-GSIG6-1BS14G51G6-18ATAGCTCTGCCGGAGTACG
T(AITJGGCTTGCTCTCCGGCCrGCC
ASG24ASG24-GS1G6-1Sl3GSlG6-1 TTTCACAACTCAAiCTGATTG(M]CTTGCTTTGATGTQOATTCT
ASG24ASG24-GZ/G6.1S13G2IG6-1 TTGGTMTTTCAGA,CCTAGA[CI~GjMCTTACTGTGGTACAOGCC
UMC49UMC49~,41G6-1S12G4lG6-1 ACCTTTGCTGTGTTT1T11'T[TlrajGTATTOGMTGGA~GGGAGTA
UMC49UMC49-G2/Gr1S12G2JG5-1 AAAACAGCCAAGOTGGTGOT[CK3]AA~UCsGAAGGTGTCAQ~AAGGT
UMC49UMC49-G21G5-2S12G2IGr2 TCTGTTCGTTCCATCTCTTT[AIGJCI1GTAMTATCCGTMTTAC
UMC49UMC49-G2lGS-3S12G2IG53 CGTMTTACTTTGTTACTAC(TAfC]I4GTMTTTTATATATATCCT
UMC49UMC49-G21G5-4S12G2IG5.4 TATATATATCCTCATTTCAA[AfnGr4A~CAGTCAAAGTTAGTTTT
UMC49UMC49-G2/G5-4BS12G21G~r48TATATATATCCTCATTTCAPv(NT]GAA~CAGTCAAAGTAGTTTTG
UMC49UMC49-G2/G4-1S12G2IG4-1 TATTTCTTATCCAGGATTGT[TIC)CT1TGGCCAAAGCATGGTAC
UMC49UMC49-G2IG4-2S12G2JG4-2 CGTTCCATCTCTTTACAGTA[ING]ATATCCOTMTTACTTTGTT
UMC49UMC49-G21G4-3S12G2/G43 ATCCGTMTTACTTTGTTA~TA/ACjCTMGTMTTTTATATATAT
UMC49UMC49-G2IG3-4S12G21G3-4 GTAATTACT'iTGTfACTACT[AIJAOTMTTTTATATATATCCT
UMC49UMC49-GllG6-1S12G1IG6-1 CTGTGTTTTTTT1TGGTATT[G>C)GMTOGJ4CiGGAGTATTATTT
UMC49UMC49-G1/G6-1BS12G1/G6-1BGCTGTG1ZT11nTGGTATT[OIC]OMTGGAGGGAGTATTATTT
UMC49UMC49-G1IG5-1S12G11G5-1 ACTTAGATGATGACCAGGTG
[N/AGAGTTTGGCACCITTGCTG
UMC49UMC49-G1/G5-2S12G1/G5-2 AGTTTGGCACCTTTGCTGTG(TQTTTTTTTTGGTATTGGAATG
UMC49UMC49-G11G5-3Sl2GtIG5-3 CTTTACTGATTGGGTTACAA(AIG]AGGTTATTTCTTATTCAGGC
UMC49UMC49-G1IG5-4S12G11G5-4 AATTACTTTGTTACTACCAG[Tl]TMTTTTATATATATCCTCC
UMC131UMC131-G41G6-iS10G4JG6-1 AGCGACAGGGATGTCGAGCA[GfT]CTACGGAAGGCAATAATGAG
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UMC131 UMC131-G4IG6-2 S10G4IG6-
2AATTTGGGAAAATCAATGCA[GAA/CAC]ATCAGTGATTAATCCACATA
UMC131 UMC131-G3/G6-1 S10G31G6-1GCATGGCGGAGTGAGGGAGG[TGQTGTGTGTGTGTGGCTCCACA
UMC131 UMC131-G31G6-2A S10G3/G6-
2AGGCCGCTAGGCCATTTAGCG[G!A]ATTTGGGAAAATCAATGCAG
UMC131 UMC131-G3/G6-28 S10G31G6-
2BGGCCGCTACGCCATTTAGCG[G/A]ATTTGGGAAAATCAATGCAC
UMC131 UMC131-G1IG6-1 S10G1/G6-1CATCCCCGCCGGCAGAACAA[GG]GTACGAGAAGGATGGAATGC
UMC53 UMC53~5JG6-1 UMC53-G5IG6-1GTCCCAGATCAGGTCCACGT[1/C]CGAGCTCGCTGTTCCCGCTT
UMC53 UMC53-GSIG6-2 UMC53~SIG6-2TGGTTCTTCACCfvCCACCGC[GG]CCGGGCGCGCCCAGCGCCTC
UMC53 UMC53-GMG6-1 UMC53-GMG6-1GCAGCCTCAGGT~4CACGGGG[/A]AAGTCGGAGTGGTTCT1CAC
UMC53 UMC53-G4JG6-2 UMC53-G4IG6-2GCCGGGCGCGCC;CAGCGCCT[/C]GGTCCCAGATCAGGTCCACG
UMC53 UMC53-G3/G6-1 UMC53-G3/G6-tGCACGTCGTTGG'f
GAAGAAG[AGCA]GCGGTACGGGTGCTTGTCGA
UMC53 UMC53.G3lG5-1 UMC53-G31G5-
1AGGTACACGGGG~4AGTCGGA[G/T]TGGTTCTTCACCACCACCGC
UMC53 UMC53-G3/G5-2 UMC53-G3/G5-2CGACGGCGTCCAGCAC;CGAC[G/JCCTCCGCCTTCACCCCGCGC
UMC:53 UMC53-G3/G4-1 UMC53-G31G4-
1GTCCACGTCGAGf:TCGCTGT[G1]CCC;GCTGCCCACGACGGCGT
UMC53 UMC53-G1/G4-1 UMC53-G1/G4-1GCACGTCGTTGG'fGAAGMG[A/CJAGCGGTACGGGTGCTTGTCG
UMC161 UMC161-G2/G3-1 SO6G21G3-
1NAACCAAACCCTG'~qCTATTA[TIC]AGGTAGATTAGACTAGACAC
UMC161 UMCt61-G21G3-2 SO6G2/G3-2ACGGTGAGGAGT<aGCACATG[A/C]GATGGAAAGTTCCTGTAGAC
UMC161 UMC161-G21G3-2B S06G2/G3-
28ACGGTAAGGAGTC~GCACATG[A/C]GATGGAAAGTTCCTGTAGAC
UMC107 UMC107G21G4-1 S05G21G4-1TATGCTTGGAAAGTGGGAAA[G/JGGGAACATACGATGGAGGAC
UMC67 UMC67-GS/G6-1 S03GSIG6-1AAACAATAATTTTTACACAG[ITJTGCTMGGTTTTACTGTTTT
UMC67 UMC67-G21G6-1 S03G21G6-1ATATCCATGTTGTI:GCCTGC[/TG]TGTGCGCTTGCTTGCCGCTA
UMC76 UMC76~4/G6-1 S02G4lG6-1TTGCTGCTATGTTTACTGGG[ITjTGTAGAAAAAAAAATAATAT
UMC76 UMC76-G21G6-1 S02G2/G6-1GCTCGGTAATAATTCTGGCT[GG]CGATGGCACCCATATTCCTC
UMC76 UMC76-G?JG6-1 B S02G2/G6-1GCTCGGTAATAATTCTGGCTjGG]CGATGGCACCCATATTCCTG
B
UMC76 UMC76-G2/G5-1 S02G21G5-1AAAACACGTGGTGTTTGTTA[GIA]GAAAGACCTAGTTTCTCGGC
UMC76 UMC76-G2/GS-1 B S02G2IG5-1AAATCACGTGGTGTTTGTTA[G/A]GAAAGACCTAGTTTCTC;GGC
B
UMC76 UMC76-G2/G5.1 S02G21G5-1TAGTTTCTCGGCAATTGGCA[G/T]TGTGGAATGACGATCTCGTG
UMC76 UMC76-G2/G5-1 B S02G21G5-1TAGTTTCTCGGCAATTGGCA[GIT]TGTGGAATGACCATCTCGTC
B
UMC76 UMC76~2/G5-2 S02G2/G5-2GTGTGGAATGAC:CATCTCGT[G/C]GTGATGCCAGCATGCTGTTA
UMC76 UMC76~2/GS-2B S02G2IG5-28GTGTGGAATGACCATCTC:GT[GlC]GTGATGCCAGCATGCTACTA
UMC76 UMC76-G2lGS-3 S02G21G5-3ACCCTGTCAGGCTTCCACAG[AIC]TATAATAT1TGTTGTGGTGT
UMC76 UMC76-G2/G5-3B S02G2IGS-3BACTCTGTCAGGCTTCCACAG[A/C]TATAATATTTGTTGTGGTGT
UMC76 UMC76-G2/G5-3C S02G2/G53CACTCTGTCAGGCTTCCACAG[A/C]TATAATATTTGTTGTGTGTG
UMC76 UMC76-G2/GS3D S02G21G5-3DACCCTGTCAGGCTTCCACAG[A/C]TATMTATTTGTTGTGTGTG
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Example 4 Analysis of Polvmor hi ms
A. Preparation of Samples
Polymorphisms are detected in a target nucleic
acid from a plant being analyzed. Target nucleic acids can be
genomic or cDNA. Many of the methods described below require
amplification of DNA from target samples. This can be
accomplished by e.g., PCR. See generally PCR Technology:
Principles and Applications (or DNA Amplification (ed. H.A.
Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide
to Methods and Applications (eds. Innis, et al., Academic
Press, San Diego, CA, 1990); Mattila et al., Nucleic Acids
Res. 19, 4967 (1991); Eckert et al., PCR Methods and
Applications 1, 17 (1991); PCR (eds. McPherson et al., IRL
Press, Oxford); and U.S. Patent 4,683,202 (each of which is
incorporated by reference for all purposes}.
Other suitable amplification methods include the
ligase chain reaction {LCR) (see Wu and Wallace, Genomics 4,
560 (1989), Landegren et al., Science 241, 1077 (1988),
transcription amplification (Kwoh et al., Proc. Natl. Acad.
Sci. USA 86, 1173 (1989)), and self-sustained sequence
replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87,
1874 (1990)) and nucleic acid based sequence amplification
(NASBA}. The latter two amplification methods involve
isothermal reactions based on isothermal transcription, which
produce both single stranded RNA (ssRNA) and double stranded
DNA (dSDNA} as the amplification products in a ratio of
about 30 or 100 to 1, respectively.
B. Detection of Polvmort~hisms in Taraet DNA
There are two distinct types of analysis
dependinq whether a polymorphism in question has already been
characterized. The first type of analysis is sometimes
referred to as de novo characterization. This analysis
compares target sequences in different individual plants to
identify points of variation, i.e., polymorphic sites. The de
novo identification of the polymorphisms of the invention is
described in the Examples section, The second type of
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analysis is determining which forms) of a characterized
polymorphism is (are) present in plants under test. There are
a variety of suitable procedures, which are discussed in
turn.
1. Allele-Specific Pro es
The design and use of allele-specific probes for
analyzing polymorphisms is described by e.g., Saiki et al.,
Nature 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO
89/11548. Allele-specific probes can be designed that
hybridize to a segment of target DNA from one member of a
species but do not hybridize t~~ the corresponding segment
from another member due to the presence of different
polymorphic forms in the respe~~tive segments from the two
members. Hybridization conditions should be sufficiently
stringent that there is a significant difference in
hybridization intensity between alleles, and preferably an
essentially binary response, whereby a probe hybridizes to
only one of the alleles. Some probes are designed to
hybridize to a segment of target DNA such that the
polymorphic site aligns with a central position (e.g., in a
15 mer at the 7 position; in a .L6 mer, at either the 8 or 9
position) of the probe. This design of probe achieves good
discrimination in hybridization between different allelic
forms.
Allele-specific probes are often used in pairs,
one member of a pair showing a perfect match to a reference
form of a target sequence and the other member showing a
perfect match to a variant form. Several pairs of probes can
then be immobilized on the same support for simultaneous
analysis of multiple polymorphisms within the same target
sequence.
2. Tiling Arravs
The polymorphisms can also be identified by
hybridization to nucleic acid arrays, some example of which
are described by Wo 95/11995 (i.ncorporated by reference in
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its entirety for all purposes). One form of such arrays is
described in the Examples section in connection with de novo
identification of polymorphisms. The same array or a
different array can be used for analysis of characterized
polymorphisms. WO 95/11995 also describes subarrays that are
optimized for detection of a variant forms of a
precharacterized polymorphism. Such a subarray contains
probes designed to be complementary to a second reference
sequence, which is an allelic variant of the first reference
sequence. The second group of probes is designed by the same
principles as described in the Examples except that the
probe" exhibit complementarity to the second reference
sequence. The inclusion of a second group (or further groups)
can be particular useful for analysing short subsequences of
the primary reference sequence in which multiple mutations
are expected to occur within a short distance commensurate
with the length of the probes (i.e., two or more mutations
within 9 to 21 bases).
3. Allele-Specific Primers
An allele-specific primer hybridizes to a site
on target DNA overlapping a polymorphism and only primes
amplification of an allelic form to which the primer exhibits
perfect complementarity. See Gibbs, Nucleic Acld Res . 17 ,
2427-2448 (1989). This primer is used in conjunction with a
second primer which hybridizes at a distal site.
Amplification proceeds from the two primers leading to a
detectable product signifying the particular allelic form is
present. A control is usually performed with a second pair of
primers, one of which shows a single base mismatch at the
polymorphic site and the other of which exhibits perfect
complementarity to a distal site. The single-base mismatch
prevents amplification and no detectable product is formed.
The method works best when the mismatch is included in the
3'-most position of the oligonucleotide aligned with the
polymorphism because this position is most destabilizing to
elongation from the primer. See, e.g., WO 93/22456.
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4. Direct-Sequencing:
The direct analysis of the sequence of
polymorphisms of the present invention can bo accomplished
using either the dideoxy chain termination method or the
Maxam Gilbert method (see Sambrook et al., Molecular Cloning,
A Laboratory Manual (2nd Ed., C;SHP, New York 1989); Zyskind
et al., Recombinant DNA Laboratory Manual, (Acad. Press,
1988)).
5. Denaturincr Gradient Gel Electrot~horesis
Amplification products generated using the
polymerase chain reaction can be analyzed by the use of
denaturing gradient gel electrophoresis. Different alleles
can be identified based on the different sequence-dependent
melting properties and electrophoretic migration of DNA in
solution, Erlich, ed., PCR 5~echnology, Principles and
Applications for DNA Amplification, (W. H. Freeman and Co,
New York, 1992), Chapter 7.
6. Finale-Strand Conformation Polymorphism
Analysis
Alleles of target sequences can be
differantiated using single-strand conformation polymorphism
analysis, which identifies base differences by alteration in
electrophoretic migration of sinc3le stranded PCR products, as
described in Orita et al . , ~~roc, Nat . Acad. Sci . 8 6 ,
2766-2770 (1989). Amplified PCR products can be generated as
described above, and heated or otherwise denatured, to form
single stranded amplification products. Single-stranded
nucleic acids may refold or form secondary structures which
are partially dependent on the base sequence. The different
electrophoretic mobilities of single-stranded amplification
products can be related to base-sequence difference between
alleles of target sequences.
Example 5 Methods of Use
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After determining polymorphic forms) present in
a subject plant at one or more polymorphic sites, this
information can be used in a number of methods.
A. Fingerprint Analvsis
Analysis of which polymorphisms are present in a
plant is useful in determining of which strain the plant is a
member and in distinguishing one strain from another. A
genetic fingerprint for an individual strain can be made by
10 determining the nucleic acid sequence possessed by that
individual strain that corresponds to a region of the genome
known to contain polymorphisms. For a discussion of genetic
fingerprinting in the animal kingdom, see, for example,
Stokening et.al., Am. J. Hum. Genet. 48:370-382 (1991). The
15 probability that one or more polymorphisms in an individual
strain is the same as that in any other individual strain
decreases as the number of polymorphic sites is increased.
The comparison of the nucleic acid sequences
from two strains at one or multiple polymorphic sites can
20 also demonstrate common or disparate ancestry. Since the
polymorphic sites are within a large region in the genome,
the probability of recombination between these polymorphic
sites is low. That low probability means the haplotype (the
set of all the disclosed polymorphic sites) set forth in this
application should be inherited without change for at least
several generations. Knowledge of plant strain or ancestry is
useful, for example, in a plant breeding program or in
tracing progeny of a proprietary plant. Fingerprints are also
used to identify an individual strain and to distinguish or
determine the relatedness of one individual strain to
another. Genetic fingerprinting can also be useful in hybrid
certification, the certification of seed lots, and the
assertion of plant breeders rights under the laws of various
countries.
B. Correlation of Polvmorphisms with Phenotv~ic
Tr i
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The polymorphism;s of the invention may
contribute to the phenotype of a plant in different ways.
Some polymorphisms occur within a protein coding sequence and
contribute to phenotype by affecting protein structure. The
effect may be neutral, beneficial or detrimental, or both
beneficial and detrimental, depending on the circumstances.
Other polymorphisms occur in noncoding regions but may exert
phenotypic effects indirectly via influence on replication,
transcription, and translation. A single polymorphism may
affect more than one phenotypic trait. Likewise, a single
phenotypic trait may be affected by polymorphisms in
different genes. Further, some polymorphisms predispose a
plant to a distinct mutation that is causally related to a
certain phenotype.
Phenotypic traits _~nclude characteristics such
as growth rate, crop yield, crop quality, resistance to
pathogens, herbicides, and other toxins, nutrient
requirements, resistance to high temperature, freezing,
drought, requirements for light= and soil type, aesthetics,
and height. Other phenotypic traits include susceptibility or
resistance to diseases, such as plant cancers. Often
polymorphisms occurring within the same gene correlate with
the same phenotype.
Correlation is performed for a population of
plants, which have been tested for the presence or absence of
a phenotypic trait of interest and for polymorphic markers
sets. To perform such analysis, the presence or absence of a
set of polymorphisms (i.e. a polymorphic set) is determined
for a set of the plants, some ~~f whom exhibit a particular
trait, and some of which exhibit lack of the trait. The
alleles of each polymorphism of the set are then reviewed to
determine whether the presence or absence of a particular
allele is associated with the trait of interest. Correlation
can be performed by standard statistical methods such as a
K-squared test and statistica7.ly significant correlations
between polymorphic forms) and phenotypic characteristics
are noted.
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Correlations between characteristics and
phenotype are useful for breeding for desired
characteristics. By analogy, Beitz et al., US 5,292,639
discuss use of bovine mitochondrial polymorphisms in a
breeding program to improve milk production in cows. To
evaluate the effect of mtDNA D-loop sequence polymorphism on
milk production, each cow was assigned a value of 1 if
variant or O if wildtype with respect to a prototypical
mitochondrial DNA sequence at each of 17 locations
considered. Each production trait was analyzed individually
with the following animal model:
Yijkpn = ~ + YSi + Pj; + X~; ~ 131 + ... 1317 + PE11 + a" +ep
where Yijgpnis the milk, fat, fat percentage, SNF, SNF
percentage, energy concentration, or lactation energy record;
~ is an overall mean; YSi is the effect common to all cows
calving in year-season; X~ is the effect common to cows in
either the high or average selection line; (31 to 131 are the
binomial regressions of production record on mtDNA D-loop
sequence polymorphisms; PEn is permanent environmental effect
common to all records of cow n; an is effect of animal n and
is composed of the additive genetic contribution of sire and
dam breeding values and a Mendelian sampling effect; and ep
is a random residual. It was found that eleven of seventeen
polymorphisms tested influenced at least one production
trait. Bovines having the best polymorphic forms for milk
production at these eleven loci are used as parents for
breeding the next generation of the herd.
One can test at least several hundreds of
markers simultaneously in order to identify those linked to a
gene or chromosomal region. For example, to identify markers
linked to a gene conferring disease resistance, a DNA pool is
constructed from plants of a segregating population that are
resistant and another pool is constructed from plants that
are sensitive to the disease. Those two DNA pools are
identical except for the DNA sequences at the resistance gene
locus and in the surrounding genomic area. Hybridization of
such DNA pools to the DNA sequences listed in Table 1 allows
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the simultaneous testing of several hundreds of loci for
polymorphisms. Allelic polymorphism-detecting sequences that
show differences in hybridization patterns between such DNA
pools will represent loci linked to the disease resistance
gene.
The method just described can also be applied to
rapidly identify rare alleles in large populations of plants.
For example, nucleic acid pools are constructed from several
individuals of a large population. The nucleic acid pools are
hybridized to nucleic acids having the polymorphism-detecting
sequences listed in Table I. The detection of a rare
hybridization profile will indicate the presence of a rare
allele in a specific nucleic acid pool. RNA pools are
particularly suited to identify differences in gene
expression.
C. Marker assisted hack-cross
The markers are used to select, in back-cross
populations, the plant that have the higher percentage of
recurrent parent, while still :remaining the genes given by
the donor plant.
Example 6. Modified Polvaeptides and Gene
~eauences
The invention further provides variant forms of
nucleic acids and corresponding proteins. The nucleic acids
comprise at least 10 contiguous amino acids of one of the
sequences for example as described in Table I, in any of the
allelic forms shown. Some nucleic acid encode full-length
proteins.
Genes can be expre:~sed in an expression vector
in which a gene is operably 7. inked to a native or other
promoter. Usually, the promoter is an eukaryotic promoter for
expression in a eukaryotic cell. The transcription regulation
sequences typically include an heterologous promoter and
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optionally an enhancer which is recognized by the host. The
selection of an appropriate promoter, for example trp, lac,
phage promoters, glycolytic enzyme promoters and tRNA
promoters, depends on the host selected. Commercially
available expression vectors can be used. Vectors can include
host-recognized replication systems, amplifiable genes,
selectable markers, host sequences useful for insertion into
the host genome, and the like.
The means of introducing the expression
construct into a host cell varies depending upon the
particular construction and the target host. Suitable means
include fusion, conjugation, transfection, transduction,
electroporation or injection, as described in Sambrook,
supra.A wide variety of host cells can be employed for
expression of the variant gene, both prokaryotic and
eukaryotic. Suitable host cells include bacteria such as E.
coli, yeast, filamentous fungi, insect cells, mammalian
cells, typically immortalized, e.g., mouse, CHO, human and
monkey cell lines and derivatives thereof, and plant cells.
Preferred host cells are able to process the variant gene
product to produce an appropriate mature polypeptide.
Processing includes glycosylation, ubiquitination, disulfide
bond formation, general post-translational modification, and
the like.
The DNA fragments are introduced into cultured
plant cells by standard methods including electroporation
(From et al., Proc. Nat1 Acad. Sci, USA 82, 5824 (19853,
infection by viral vectors such as cauliflower mosaic virus
(CaMV) (Hohn et a1. , Molecular Biology of Plant Tumors,
(Academic Press, New York, 1982) pp. 549-560; Howell, US
4,407,956), high velocity ballistic penetration by small
particles with the nucleic acid either within the matrix of
small beads or particles, or on the surface (Klein et al.,
Nature 327, 70-73 (1987) ), USQ of pollen as vector (WO
85/01856), or use of Agrobacterium tumefaciens transformed
with a Ti plasmid in which DNA fragments are cloned. The Ti
plasmid is transmitted to plant cells upon infection by
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Agrobacterium tumefaciens, and is stably integrated into the
plant genome (Horsch et al., Science, 233, 496-498 (2984);
Fraley et al., Proc. Natl. Acad. Sci. USA 80, 4803 (1983)).
The protein may be isolated by conventional
5 means of protein biochemistry and purification to obtain a
substantially pure product, i.e., 80, 95 or 99% free of cell
component contaminants, as described in Jacoby, Methods in
Enzymology Volume 104, Academic Press, New York (1984);
Scopes, Protein Purification, ~~rinciples and Practice', 2nd
10 Edition, Springer-Verlag, New York (1987); and Deutscher
(ed), Guide to Protein Purification' Methods in Enzymology,
Vol. 182 (1990). If the protein is secreted, it can be
isolated from the supernatant in which the host cell is
grown. If not secreted, the protein can be isolated from a
15 lysate of the host cells.
The invention further provides transgenic plants
capable of expressing an exogenous variant gene and/or having
one or both alleles of an endogenous variant gene
inactivated. Plant regeneration from cultural protoplasts is
20 described in Evans et al., "Protoplasts Isolation and
Culture," Handbook of Plant Cell Cultures .2 , 124-176
(MacMillan Publishing Co., New York, 1983); Davey, "Recent
Developments in the Culture and Regeneration of Plant
Protoplasts," Protoplasts, (1983) - pp. 12-29, (Birkhauser,
25 Basal 1983); Dale, "Protoplast Culture and Plant Regeneration
of Cereals and Other Recalcitrar.~t Crops," Protoplasts (1983)
- pp. 31-41, (Birkhauser, Basel :1983); Binding, "Regeneration
of Plants," Plant ProtopLasts, pp. 22-73, (CRC Press, Boca
Baton, 1985). For example, a variant gene responsible for a
disease-resistant phenotype can be introduced into the plant
to simulate that phenotype. Expression of an exogenous
variant gene is usually achieved by operably linking the qene
to a promoter and optionally an enhancer. Inactivation of an
exogenous variant genes can be achieved by forming a
transgene in which a cloned variant genes is inactivated by
insertion of a positive selection marker. See Capecchi,
Science 244, 1288-1292 (1989). Such transgenic plant are
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useful in a variety of screening assays. For example, the
transgenic plant can then be treated with compounds of
interest and the effect of those compounds on the disease
resistance can be monitored. In another example, the
transgenic plant can be exposed to a variety of environmental
conditions to determine the effect of those conditions on the
resistance to the disease.
In addition to substantially full-length
polypeptides, the present invention includes blologically
active fragments of the polypeptides, or analogs thereof,
including organic molecules which simulate the interactions
of the peptides. Biologically active fragments include any
portion of the full-length polypeptide which confers a
biological function on the variant gene product, including
ligand binding, and antibody binding. Ligand binding includes
binding by nucleic acids, proteins or polypeptides, small
biologically active molecules, or large cellular structures.
Polyclonal and/or monoclonal antibodies that
specifically bind to one allelic gene products but not to a
second allelic gene product are also provided. Antibodies can
be made by injecting mice or other animals with the variant
gene product or synthetic peptide fragments thereof.
Monoclonal antibodies are screened as are described, for
example, in Harlow & Lane, Antibodies, A Laboratory Manual,
Cold Spring Harbor Press, New York (1988); Goding, Monoclonal
antibodies, Pr~incip.Ies and Practice (2d ed.) Academic Press,
New York (1986). Monoclonal antibodies are tested for
specific immunoreactivity with a variant gene product and
lack of immunoreactivity to the corresponding prototypical
gene product. These antibodies are useful in diagnostic
assays for detection of the variant form, or as an active
ingredient in a pharmaceutical composition.
Example 7. Kits
The invention further provides kits comprising
at least one allele-specific oligonucleotide as described
above. Often, the kits contain one or more pairs of
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allele-specific oligonucleotide~s hybridizing to different
forms of a polymorphism. In some kits, the allele-specific
oligonucleotides are provided immobilized to a substrate. For
example, the same substrate ca.n comprise allele-specific
oligonucleotide probes for detecting at least 10, 100 or all
of the polymorphisms shown in Table I. Optional additional
components of the kit include; for example, restriction
enzymes, reverse-transcriptase or polymerase, the substrate
nucleoside triphosphates, means used to label (for example,
an avidin-enzyme conjugate and enzyme substrate and chromogen
if the label is biotin), and t:he appropriate-buffers for
reverse transcription, PCR, or hybridization reactions.
Usually, the kit also contains instructions for carrying out
the methods.
