Microsatellite markers for plants of the species Triticum...

Details Microsatellite markers for plants of the species Triticum... Microsatellite markers for plants of the species Triticum...

1998-05-01

2004-04-13

Benzion, Gary (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091200, C435S320100, C435S410000, C536S023100, C536S024300, C536S024330, C800S295000, C800S320300

Reexamination Certificate

active

06720137

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to novel genetic markers for wheats of the species Triticum aestivum and closely related species of the tribe Triticeae and to the use of said markers.
The most widely spread, known, DNA-based genetic markers are the so-called restriction fragment length polymorphisms (RFLP) markers. For using these markers, genomic DNA is digested with restriction enzymes, separated on agarose gels and transferred to nylon membranes (Southern Blot). Specific fragments are detected by hybridization with radioactively labeled DNA probes. When mutations occur in the region of the restriction enzymes used or when smaller deletions/insertions occur, polymorphisms between different lines are found, which are passed on stably and mostly codominantly. The use of RFLP markers in hexaploid cultivated wheat is possible only to a limited extent, since only very little polymorphism is detected in wheat in this manner.
It has already been described that microsatellite markers detect significantly more polymorphism between different wheat lines than do RFLP markers. This can be attributed particularly to the occurrence of multiple alleles per locus (Rbder et al., Mol. Gen. Genet. (1995) 246, 327-333). Moreover, it is known that microsatellite markers have the advantage that they can be detected by way of PCR and that therefore large amounts of samples can be analyzed more easily.
SUMMARY OF THE INVENTION
It is an object of the invention to provide novel microsatellite markers for the genetic analysis of plants of the
Triticum aestivum
species, which markers are distinguished by a degree of DNA polymorphism, which is higher than that of other molecular probes, that have been developed previously for the wheat genome.
This objective is accomplished as follows. The inventive markers are based on the amplification of certain hypervariable genome sections, the so-called microsatellites, with the help of their polymerase chain reaction (PCR). For specific amplification, two primers, in each to the case left and the right in the flanking sequences, are required for each microsatellite locus. On the average, these primers are 20 i 3 bases long and are defined by their sequences. In principle, a microsatellite marker is a sequence tagged site (STS), which is defined by two specific primers. These primers flank, in each case to the left and the right, a so-called microsatellite sequence. A microsatellite sequence is defined as a tandem repetitive repetition of a di-, tri- or tetranucleotide sequence, for example (GA)
n
, in which n>10. Composite microsatellite sequences also occur, such as (GT)
n
(AT)
n
, as well as imperfect sequences, in which individual bases are mutated, such as (GA)
n
CA(GA)
n
. Among various lines and varieties, there is variation in the number of repeats at a certain locus. After amplification of the microsatellites, this leads, by means of the specific primers in the flanking sequences, to PCR products of different length and, with that, to polymorphisms. These polymorphisms are passed on stably and can therefore be used as genetic markers. In some cases, null alleles (no visible fragment) also occur, when there are mutations within the binding site for the primers.
The separation and detection of the PCR products obtained can be carried out with different technical variants. For separating the fragments, highly resolving agarose gels, native polyacrylamide gels or denaturing polyacrylamide gels (=sequencing gels) can be used. Depending on the separation system, fragments are detected using ethidium bromide staining, silver staining or, after labeling the PCR fragments radioactively, using autoradiography. A further, very effective variation for separation and detection consists of the use of an automatic sequencer with dye- or fluorescence-labeled primers. For this purpose, it is necessary to synthesize a dye- or fluorescence-labeled primer from each microsatellite primer pair. PCR amplification results in a labeled product, which can be detected by the sequencing equipment. At the same time, dye- or fluorescence-labeled size standards are also separated for each sample in the same track. After that, special software enable the absolute size of each fragment, which has been separated, to be calculated and, with that, also permits fragments from different gel runs to be compared. With this method, several hundred samples can be analyzed largely automatically in a day.
DETAILED DESCRIPTION OF THE INVENTION
Pursuant to the invention, microsatellite markers are made available, which contain the following primer pairs with assigned microsatellite sequences or a number thereof and amplify the loci of all chromosomes of the wheat genome and therefore find use for gene marking.
WMS
Length (bp)
Annealing
Number
WMS Primer Left
WMS Primer Right
in “cs”)
Repeat Type
Temperature
WMS052
5′ CTA TGA GGC GGA GGT TGA AG 3′
(SEQ. ID NO. 1)
5′ TGC GGT GCT CTT CCA TTT 3′
(SEQ. ID NO. 2)
150
GTimp
60° C.
WMS055
5′ GCA TCT GGT ACA CTA GCT GCC 3′
(SEQ. ID NO. 3)
5′ TCA TGG ATG CAT CAC ATC CT 3
(SEQ. ID NO. 4)
127
GTimp
60° C.
WMS057
5′ TCG ATT CTG AAA GGT TCA TCG 3′
(SEQ. ID NO. 5)
5′ CGA TCA AGT AGT TGA AAG CGC 3′
(SEQ. ID NO. 6)
224
AAAAAimp
60° C.
WMS058
5′ TCT GAT CCC GTG AGT GTA ACA 3′
(SEQ. ID NO. 7)
5′ GAA AAA AAT TGC ATA TGA GCC C 3′
(SEQ. ID NO. 8)
118
CA
60° C.
WMS060
5′ TGT CCT ACA CGG ACC ACG T3′
(SEQ. ID NO. 9)
5′ GCA TTG ACA GAT GCA CAC G 3′
(SEQ. ID NO. 10)
211
CA
60° C.
WMS063
5′ TCG ACC TGA TCG CCC CTA 3′
(SEQ. ID NO. 11)
5′ CGC CCT GGG TGA TGA ATA GT 3′
(SEQ. ID NO. 12)
271
GAA,CA,TA
60° C.
WMS067
5′ ACC ACA CAA ACA AGG TAA GCG 3′
(SEQ. ID NO. 13)
5′ CAA CCC TCT TAA TTT TGT TGG G 3′
(SEQ. ID NO. 14)
85
CA
60° C.
WMS068
5′ AGG CCA GAA TCT GGG AAT G 3′
(SEQ. ID NO. 15)
5′ CTC CCT AGA TGG GAG AAG GG 3′
(SEQ. ID NO. 16)
182
GA
60° C.
WMS070
5′ AGT GGC TGG GAG AGT GTC AT 3′
(SEQ. ID NO. 17)
5′ GCC CAT TAC CGA GGA CAC 3′
(SEQ. ID NO. 18)
194
GT
60° C.
WMS071
5′ GGC AGA GCA GCG AGA CTC 3′
(SEQ. ID NO. 19)
5′ CAA GTG GAG CAT TAG GTA CAC G 3′
(SEQ. ID NO. 20)
128
GT
60° C.
WMS077
5′ ACA AAG GTA AGC AGC ACC TG 3′
(SEQ. ID NO. 21)
5′ ACC CTC TTG CCC GTG TTG 3′
(SEQ. ID NO. 22)
153
CA,GA
55° C.
WMS082
5′ ACG TTA GAA GGT GCA ATG GG 3′
(SEQ. ID NO. 23)
5′ AGT GGA TGC ACC GAC TTT G 3′
(SEQ. ID NO. 24)
152
GT,GAimp
60° C.
WMS088
5′ CAC TAC AAC TAT GCG CTC GC 3′
(SEQ. ID NO. 25)
5′ TCC ATT GGC TTC TCT CTC AA 3′
(SEQ. ID NO. 26)
121
GT
60° C.
WMS095
5′ GAT CAA ACA CAC ACC CCT CC 3′
(SEQ. ID NO. 27)
5′ AAT GCA AAG TGA AAA ACC CG 3′
(SEQ. ID NO. 28)
121
CA
60° C.
WMS099
5′ AAG ATG GAC GTA TGC ATC ACA 3′
(SEQ. ID NO. 29)
5′ GCC ATA TTT GAT GAC GCA TA 3′
(SEQ. ID NO. 30)
119
CA
60° C.
WMS102
5′ TCT CCC ATC CAA CGC CTC 3′
(SEQ. ID NO. 31)
5′ TGT TGG TGG CTT GAC TAT TG 3′
(SEQ. ID NO. 32)
143
CT
60° C.
WMS106
5′ CTG TTC TTG CGT GGC ATT AA 3′
(SEQ. ID NO. 33)
5′ AAT AAG GAC ACA ATT GGG ATG G 3′
(SEQ. ID NO. 34)
139
GA
60° C.
WMS107
5′ ATT AAT ACC TGA GGG AGG TGC 3′
(SEQ. ID NO. 35)
5′ GGT CTC AGG AGC AAG AAC AC 3′
(SEQ. ID NO. 36)
195
CT
60° C.
WMS108
5′ CGA CAA TGG GGT CCT AGC AT 3′
(SEQ. ID NO. 37)
5′ TGC ACA CTT AAA TTA CAT CCG C 3′
(SEQ. ID NO. 38)
132
GTimp
60° C.
WMS111
5′ TCT GTA GGC TCT CTC CGA CTG 3′
(SEQ. ID NO. 39)
5′ ACC TGA TCA GAT CCC ACT CG 3′
(SEQ. ID NO. 40)
205
CT,GT
55° C.
WMS112
5′ CTA AAC ACG ACA GCG GTG G 3′
(SEQ. ID NO. 41)
5′ GAT ATG TGA GCA GCG GTC AG 3′
(SEQ. ID NO. 42)
101
CTimp
55° C.
WMS113
5′ ATT CGA GGT TAG GAG GAA GAG G 3′
(SEQ. ID NO. 43)
5′ GAG GGT GGG CCT ATA AGA CC 3′
(SEQ. ID NO. 44)
148
GT
60° C.
WMS114
5&pri

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