JPWO2007020983A1 - Wheat breeding method using diploid wheat genetic map prepared using barley EST sequence - Google Patents

Abstract

Provided is a method for breeding wheat using a diploid wheat genetic map prepared using a barley EST sequence. Breeding wheat using a primer set designed based on a barley EST sequence, and amplifying the DNA using wheat genomic DNA as a template, and a polymorphism detecting step for detecting the polymorphism of the amplified DNA fragment Is the method. Here, the DNA fragment is a DNA marker mapped on a genetic map covering the entire genome of diploid wheat, and at least 50 or more DNA markers mapped on the genetic map are used. .

Description

  The present invention relates to a method for breeding wheat using a diploid wheat genetic map prepared using a barley EST sequence.

  Conventionally, for breeding crops, it is necessary to cross cultivated varieties or wild varieties with a target trait, actually cultivate many individuals, select individuals with the target trait, and genetically fix the target trait First of all, it required a vast field, a lot of human power, and considerable years. Further, if the target trait is a trait that is easily affected by the cultivation environment factor, it is difficult to determine whether or not the target trait expressed by the selected individual is a gene phenotype.

  Therefore, in recent years, breeding methods by selection using genetic markers as indices have been used in order to shorten the breeding period, reduce labor and field area, and reliably select useful genes. In breeding with such a genetic marker, selection can be made at the seedling stage depending on the genotype of the marker, and the presence or absence of the target trait can be easily confirmed. Efficient breeding can be realized by using genetic markers, but in order to realize breeding using genetic markers, it is essential to develop genetic markers that are strongly linked to the target trait.

  By the way, many of the traits important in agriculture are many that show continuous mutations in hybrid progeny. Such traits are called quantitative traits because they are measured on a quantitative scale such as time and length. In general, quantitative traits are often determined by the action of multiple genes, rather than a single dominant gene-dominated trait. Many traits that are targeted for improvement in crop breeding, such as yield, quality, and taste, are often quantitative traits.

  A genetic position occupied by such a gene having a quantitative trait on a chromosome is referred to as QTL (Quantitative Trait Loci). As a method of estimating QTL, QTL analysis using a genetic marker existing in the vicinity of QTL is used. With the advent of DNA markers in the late 1980s, detailed linkage map creation using DNA markers has greatly progressed, and QTL analysis has been performed on many organisms based on the maps.

  As described above, the development of genetic markers linked to the target trait is enabled by high-accuracy QTL analysis based on a detailed linkage map that can cover the entire chromosome, and by using the obtained genetic markers, it is efficient. It can be said that simple breeding can be realized.

  Regarding a wheat genetic map based on a DNA marker, it is first prepared by an RFLP marker obtained by hybridization (see, for example, Non-Patent Document 1), and thereafter, PCR-based markers have been used for genetic map construction. It was. For these markers, RAPDs (for example, see Non-Patent Document 2), AFLPs (for example, see Non-Patent Document 3), and SSRs (for example, see Non-Patent Document 4) have been mainly used. Recently, the development of EST markers has progressed also in wheat, and the positioning of ESTs on a linkage map has been reported (for example, see Non-Patent Document 5).

  On the other hand, since a genetic linkage map using RFLP has been constructed in barley, STSs, RAPDs, SSRs, AFLPs, EST-SSR, and the like have been used for map construction. In addition, the inventors of the present invention have obtained a double d from a cross of a doubling of a barley “Haruna Nijo” (Hordeum vulgare ssp. Haploid) population, about 3,000 barley EST markers are mapped, and a high-density linkage map (genetic map) of barley covering 1,362.7 cM (centiorgan) is constructed (Non-Patent Document) 6).

By genetic map construction, the genome structure of wheat and barley can be partially compared, and it has been reported that there is a homology of markers between these species (see, for example, Non-Patent Document 7).
[Non-Patent Document 1]
Chao S, Sharp PJ, World AJ, Koebner RMD, Gale MD (1989) RFLP-based tented map of wheat homologous group-7 chromosomes. Theor Appl Gene 78: 493-504
[Non-Patent Document 2]
Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbiter primers are used as an assemblage. Nucleic Acids Res. 18 (22): 6531-5
[Non-Patent Document 3]
Lolli C, Salvi S, Pasqualone A, Toberosa R, Blanco A (2000) Integration of AFLP markers into an RFLP-based map of durum heat. Plant Breed. 119: 393-401
[Non-Patent Document 4]
Roder MS, Korzon V, Wendehake K, Plaschke J, Tixier MH, Leroy P, Ganal MW (1998) A microsatellite map of heat. Genetics Soc America 149: 2007-2023
[Non-Patent Document 5]
Hossain KG, Kalavacharla V, Lazo GR, Hegstad J, Wentz MJ, Kianian PM, Simons K, Gehlhar L, OberoriK, Bhamidah. , Gill BS, Linkiewicz AM, Ratnasiri A, Dubkovsky J, Akhunov ED, Dvorak J, Miftahudin, Ross K, Gustafson JP, Radha HS, DivhaS. E, Sorells ME, Feril O, Pathan MS, Nguyen HT, Gonzalez-Hernandez JL, Conley EJ, Anderson JA, Choi DW, Fenton D, Close TJ, QuGi PE, McGuire PE, McGure PE 2148 expressed sequence tag loci of wheat homoelogous group 7. Genetics. 168 (2): 687-99
[Non-Patent Document 6]
Nankuku N. Motoi Y., et al. Ueki H .; , Sato K. and Takeda K.K. (2005) High density SNP based EST map in barley. Proc. Plant & Animal Genomes XIII Conference, p317.
[Non-Patent Document 7]
Varshney RK, Graner A, Sorells ME (2005) Genomic microsatellite markers in plants: features and applications. Trends Biotechnol. 23 (1): 48-55
As described above, the genome structure of wheat and barley can be partially compared even with the genetic map constructed in the past, but in order to make a more detailed comparison, it is necessary to construct a new genetic map. It was. In particular, it is considered that a more detailed comparison can be made by using a wheat genetic map and a barley genetic map constructed with a common marker.

  Since the inventors have a high-density genetic map by the above-mentioned barley EST marker and have a lot of useful information obtained from the high-density genetic map, the inventors have used wheat EST sequences to produce wheat. Inspired to construct a genetic map. If a genetic map of wheat can be constructed using the barley EST sequence, it will be possible to effectively utilize the genome information of the barley that the inventors have in breeding wheat.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a method for breeding wheat using a diploid wheat genetic map prepared using a barley EST sequence.

  In order to solve the above-mentioned problems, the present inventors have used a primer based on a barley EST sequence that was originally developed, and a polymorphism between varieties into an amplified DNA fragment amplified using diploid wheat genomic DNA as a template. Having DNA was selected as a DNA marker. A diploid wheat genetic map was completed by mapping these DNA markers onto the diploid wheat chromosome (A genome). The inventors further conducted QTL analysis on 16 types of agricultural traits using the genetic map of the diploid wheat, found QTLs for all traits, and found the genetic map of the diploid wheat and barley height. It was found that there is a sequence of DNA markers in the density genetic map. As a result, the genetic map of the diploid wheat was confirmed to be very useful for wheat breeding, and the present invention was completed.

That is, the wheat breeding method according to the present invention uses a primer set designed based on a barley EST sequence, an amplification step of amplifying DNA using wheat genomic DNA as a template, and a polymorphism of the amplified DNA fragment. A wheat breeding method comprising a polymorphism detection step of detecting comprising:
The DNA fragment is a DNA marker mapped on a genetic map covering the entire genome of diploid wheat, and at least 50 or more DNA markers mapped on the genetic map are used. It is said.

  The barley EST sequence is preferably selected from the base sequences shown in SEQ ID NOs: 1 to 486.

  The primer set comprises a primer consisting of the base sequence shown in SEQ ID NO: n (n is an odd number) and a primer consisting of the base sequence shown in SEQ ID NO: n + 1 among the base sequences shown in SEQ ID NOS: 487 to 1458. A combined primer set is preferred.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment described in at least one of the following (a) to (g) among the at least 50 DNA markers, It may include a step of determining a genotype of a locus involved in panicle length.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 587 and a primer consisting of the base sequence shown in SEQ ID NO: 588 are combined, or from the base sequence shown in SEQ ID NO: 589 A DNA fragment which is amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 590 and a primer consisting of the base sequence shown in SEQ ID NO: 590 are combined (b) a primer consisting of the base sequence shown in SEQ ID NO: 591 and the base shown in SEQ ID NO: 592 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 593 and a primer consisting of a base sequence shown in SEQ ID NO: 594 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 759 and a primer consisting of the base sequence shown in SEQ ID NO: 760 or the base sequence shown in SEQ ID NO: 761 A DNA fragment that is amplified using a primer set in which a primer comprising the nucleotide sequence shown in SEQ ID NO: 762 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 762 are combined (d) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 763 and SEQ ID NO: 764 A DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer set consisting of a primer consisting of a base sequence shown in SEQ ID NO: 765 and a primer consisting of a base sequence shown in SEQ ID NO: 766 Amplified using DNA fragment (e) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 or the base shown in SEQ ID NO: 949 A DNA fragment amplified using a primer set in which a primer consisting of the sequence and a primer consisting of the base sequence shown in SEQ ID NO: 950 are combined (f) a primer consisting of the base sequence shown in SEQ ID NO: 995 and shown in SEQ ID NO: 996 Primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence to be synthesized or a primer consisting of a base sequence shown in SEQ ID NO: 997 and a primer consisting of a base sequence shown in SEQ ID NO: 998 Amplify using DNA fragment (g) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 999 and a primer consisting of the base sequence shown in SEQ ID NO: 1000 or shown in SEQ ID NO: 1001 A DNA fragment that is amplified using a primer set that combines a primer consisting of a base sequence and a primer consisting of a base sequence shown in SEQ ID NO: 1002.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) to (d) among the at least 50 DNA markers, It may include a step of determining the genotype of a locus involved in culm length.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 759 and a primer consisting of the base sequence shown in SEQ ID NO: 760 are combined, or from the base sequence shown in SEQ ID NO: 761 A DNA fragment that is amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 762 and a primer consisting of the base sequence shown in SEQ ID NO: 762 are (b) a primer consisting of the base sequence shown in SEQ ID NO: 763 and the base shown in SEQ ID NO: 764 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 765 and a primer consisting of a base sequence shown in SEQ ID NO: 766 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1235 and a primer consisting of the base sequence shown in SEQ ID NO: 1236 or the base sequence shown in SEQ ID NO: 1237 A DNA fragment (d) amplified using a primer set in which a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 1238 are combined (d) a primer consisting of the base sequence shown in SEQ ID NO: 1239 and SEQ ID NO: 1240 A primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer consisting of a base sequence shown in SEQ ID NO: 1241 and a primer consisting of a base sequence shown in SEQ ID NO: 1242 DNA fragments amplified using.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment described in the following (a) among the at least 50 or more DNA markers, and further a gene locus involved in the number of spikelets. It may include a step of determining the genotype.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 are combined, or from the base sequence shown in SEQ ID NO: 949 A DNA fragment that is amplified using a primer set in which a primer consisting of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 950 is combined.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers, It may include the step of determining the genotype of the locus involved in the first internode length.
(A) From a DNA fragment amplified using a primer set that combines a primer consisting of the base sequence shown in SEQ ID NO: 939 and a primer consisting of the base sequence shown in SEQ ID NO: 940 or the base sequence shown in SEQ ID NO: 941 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 942 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 942 (b) A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 945 and a primer consisting of a base sequence shown in SEQ ID NO: 946 Amplified DN Fragment.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment described in at least one of the following (a) to (h) among the at least 50 DNA markers, It may include a step of determining the genotype of a locus related to the intercob length.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 503 and a primer consisting of the base sequence shown in SEQ ID NO: 504 are combined, or from the base sequence shown in SEQ ID NO: 505 A DNA fragment that is amplified using a primer set in which a primer comprising the nucleotide sequence represented by SEQ ID NO: 506 is combined with a primer comprising the nucleotide sequence represented by SEQ ID NO: 507 and the base represented by SEQ ID NO: 508 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 509 and a primer consisting of a base sequence shown in SEQ ID NO: 510 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1203 and a primer consisting of the base sequence shown in SEQ ID NO: 1204 or a base sequence shown in SEQ ID NO: 1205 A DNA fragment that is amplified using a primer set that combines a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 1206 (d) a primer consisting of the base sequence shown in SEQ ID NO: 1207 and that shown in SEQ ID NO: 1208 A primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer consisting of a base sequence shown in SEQ ID NO: 1209 and a primer consisting of a base sequence shown in SEQ ID NO: 1210 DNA fragment to be amplified using (e) DNA fragment or SEQ ID NO: amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1355 and a primer consisting of the base sequence shown in SEQ ID NO: 1356 A DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in 1357 and a primer consisting of the base sequence shown in SEQ ID NO: 1358 (f) a primer consisting of the base sequence shown in SEQ ID NO: 1359; A DNA fragment amplified using a primer set in combination with a primer consisting of the base sequence shown in SEQ ID NO: 1360, or a primer consisting of the base sequence shown in SEQ ID NO: 1361 and a primer consisting of the base sequence shown in SEQ ID NO: 1362 Combine DNA fragment amplified using the primer set (g) Amplified using a primer set that combines a primer consisting of the base sequence shown in SEQ ID NO: 1395 and a primer consisting of the base sequence shown in SEQ ID NO: 1396 DNA fragment or DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1397 and a primer consisting of the base sequence shown in SEQ ID NO: 1398 (h) a base shown in SEQ ID NO: 1399 A DNA fragment amplified using a primer set in which a primer consisting of a sequence and a primer consisting of a base sequence shown in SEQ ID NO: 1400 are combined, or a primer consisting of a base sequence shown in SEQ ID NO: 1401 and a base shown in SEQ ID NO: 1402 An array DNA fragments amplified using the primer set of a combination of a primer.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment described in the following (a) among the at least 50 or more DNA markers, and further comprises a gene locus involved in the heading date. It may include a step of determining a genotype.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 are combined, or from the base sequence shown in SEQ ID NO: 949 A DNA fragment that is amplified using a primer set in which a primer consisting of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 950 is combined.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) to (d) among the at least 50 DNA markers, It may include a step of determining the genotype of a locus involved in culm length.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1115 and a primer consisting of the base sequence shown in SEQ ID NO: 1116 are combined, or from the base sequence shown in SEQ ID NO: 1117 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1118 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1118 (b) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1119 and a base shown in SEQ ID NO: 1120 A DNA fragment amplified using a primer set combined with a primer composed of a sequence or a primer set composed of a primer composed of a base sequence represented by SEQ ID NO: 1121 and a primer composed of a base sequence represented by SEQ ID NO: 1122 DNA fragment to be amplified (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1407 and a primer consisting of the base sequence shown in SEQ ID NO: 1408 or SEQ ID NO: 1409 A DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown and a primer consisting of the base sequence shown in SEQ ID NO: 1410; and (d) a primer consisting of the base sequence shown in SEQ ID NO: 1411 and SEQ ID NO: A DNA fragment amplified using a primer set in combination with a primer consisting of the base sequence shown in 1412 or a primer consisting of the base sequence shown in SEQ ID NO: 1413 and a primer consisting of the base sequence shown in SEQ ID NO: 1414 The DNA fragments amplified using the line-mer set.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) to (d) among the at least 50 DNA markers, It may include the step of determining the genotype of the locus involved in the armor guard.
(A) From a DNA fragment amplified using a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: 943 and a primer consisting of the base sequence shown in SEQ ID NO: 944 or the base sequence shown in SEQ ID NO: 945 A DNA fragment that is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 946 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 946 (b) a primer consisting of the base sequence shown in SEQ ID NO: 947 and the base shown in SEQ ID NO: 948 A DNA fragment amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 949 and a primer consisting of a base sequence shown in SEQ ID NO: 950 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1371 and a primer consisting of the base sequence shown in SEQ ID NO: 1372 or a base sequence shown in SEQ ID NO: 1373 A DNA fragment which is amplified using a primer set in which a primer comprising the nucleotide sequence shown in SEQ ID NO: 1374 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1374 are combined (d) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1375 and SEQ ID NO: 1376 A primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer consisting of a base sequence shown in SEQ ID NO: 1377 and a primer consisting of a base sequence shown in SEQ ID NO: 1378 DNA fragments amplified using.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) to (c) among the at least 50 DNA markers, It may include a step of determining the genotype of a locus involved in the plant type.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 are combined, or from the base sequence shown in SEQ ID NO: 949 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 950 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 950 are combined (b) a primer consisting of the base sequence shown in SEQ ID NO: 1259 and the base shown in SEQ ID NO: 1260 A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 1261 and a primer consisting of a base sequence shown in SEQ ID NO: 1262 Amplified DNA fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1263 and a primer consisting of the base sequence shown in SEQ ID NO: 1264 or shown in SEQ ID NO: 1265 A DNA fragment amplified using a primer set in which a primer consisting of a base sequence and a primer consisting of a base sequence shown in SEQ ID NO: 1266 are combined.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers, It may include a step of determining a genotype of a locus involved in 1000 grain weight.
(A) From a DNA fragment amplified using a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: 735 and a primer consisting of the base sequence shown in SEQ ID NO: 736, or from the base sequence shown in SEQ ID NO: 737 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 738 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 738 and a base consisting of the nucleotide sequence shown in SEQ ID NO: 740 A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 741 and a primer consisting of a base sequence shown in SEQ ID NO: 742 Amplified DN Fragment.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) to (d) among the at least 50 DNA markers, It may include a step of determining the genotype of a locus involved in cobs detachment.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1071 and a primer consisting of the base sequence shown in SEQ ID NO: 1072 are combined, or from the base sequence shown in SEQ ID NO: 1073 A DNA fragment that is amplified using a primer set in which a primer comprising the nucleotide sequence shown in SEQ ID NO: 1074 is combined (b) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1075 and the nucleotide sequence shown in SEQ ID NO: 1076 Using a primer set that combines a DNA fragment that is amplified using a primer set that is combined with a primer consisting of or a primer that consists of the base sequence shown in SEQ ID NO: 1077 and a primer that consists of the base sequence shown in SEQ ID NO: 1078 A DNA fragment to be amplified (c) a DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1371 and a primer consisting of the base sequence shown in SEQ ID NO: 1372, or SEQ ID NO: 1373 A DNA fragment amplified using a primer set comprising a primer consisting of the nucleotide sequence shown and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1374; and (d) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1375 and SEQ ID NO: A DNA fragment amplified using a primer set in combination with a primer consisting of the base sequence shown in 1376 or a primer consisting of the base sequence shown in SEQ ID NO: 1377 and a primer consisting of the base sequence shown in SEQ ID NO: 1378 Tap DNA fragments amplified using the timer set.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment described in at least one of the following (a) to (f) among the at least 50 DNA markers, It may include a step of determining a genotype of a locus involved in dormancy (after 0 weeks).
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 575 and a primer consisting of the base sequence shown in SEQ ID NO: 576 are combined, or from the base sequence shown in SEQ ID NO: 577 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 578 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 578 are combined (b) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 579 and the base shown in SEQ ID NO: 580 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 581 and a primer consisting of a base sequence shown in SEQ ID NO: 582 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 835 and a primer consisting of the base sequence shown in SEQ ID NO: 836 or the base sequence shown in SEQ ID NO: 837 A DNA fragment that is amplified using a primer set that combines a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 838 (d) a primer consisting of the base sequence shown in SEQ ID NO: 839 and that shown in SEQ ID NO: 840 A DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer set consisting of a primer consisting of a base sequence shown in SEQ ID NO: 841 and a primer consisting of a base sequence shown in SEQ ID NO: 842 Amplified using DNA fragment (e) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1123 and a primer consisting of the base sequence shown in SEQ ID NO: 1124 or a base shown in SEQ ID NO: 1125 A DNA fragment amplified using a primer set in which a primer consisting of the sequence and a primer consisting of the base sequence shown in SEQ ID NO: 1126 are combined (f) a primer consisting of the base sequence shown in SEQ ID NO: 1127 and a primer consisting of the base sequence shown in SEQ ID NO: 1128 A DNA fragment that is amplified using a primer set that is combined with a primer consisting of a base sequence that is combined with a primer that consists of a base sequence shown in SEQ ID NO: 1129 and a primer that consists of a base sequence shown in SEQ ID NO: 1130 DNA fragment amplified using

Further, among the at least 50 or more DNA markers, a DNA marker comprising the DNA fragment described in at least one of the following (a) to (f) is included, and further involved in dormancy (after 3 weeks): The method for breeding wheat according to the present invention including the step of determining the genotype of a locus may be any.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 591 and a primer consisting of the base sequence shown in SEQ ID NO: 592 are combined, or from the base sequence shown in SEQ ID NO: 593 A DNA fragment that is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 594 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 594 (b) a primer consisting of the base sequence shown in SEQ ID NO: 595 and the base shown in SEQ ID NO: 596 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 597 and a primer consisting of a base sequence shown in SEQ ID NO: 598 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 627 and a primer consisting of the base sequence shown in SEQ ID NO: 628 or the base sequence shown in SEQ ID NO: 629 A DNA fragment amplified using a primer set in which a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 630 are combined (d) a primer consisting of the base sequence shown in SEQ ID NO: 631 and shown in SEQ ID NO: 632 A primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer consisting of a base sequence shown in SEQ ID NO: 633 and a primer consisting of a base sequence shown in SEQ ID NO: 634 Amplified using DNA fragment (e) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 903 and a primer consisting of the base sequence shown in SEQ ID NO: 904 or the base shown in SEQ ID NO: 905 A DNA fragment amplified using a primer set in which a primer consisting of a sequence and a primer consisting of a base sequence shown in SEQ ID NO: 906 are combined (f) a primer consisting of a base sequence shown in SEQ ID NO: 907 and shown in SEQ ID NO: 908 Primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence to be synthesized or a primer consisting of a base sequence shown in SEQ ID NO: 909 and a primer consisting of a base sequence shown in SEQ ID NO: 910 Amplify using DNA fragment.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers, It may include a step of determining a genotype of a gene that controls the paste powder layer color.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1055 and a primer consisting of the base sequence shown in SEQ ID NO: 1056 are combined, or from the base sequence shown in SEQ ID NO: 1057 A DNA fragment that is amplified using a primer set in which a primer comprising the nucleotide sequence represented by SEQ ID NO: 1058 is combined with a primer comprising the nucleotide sequence represented by SEQ ID NO: 1059 and a base represented by SEQ ID NO: 1060 A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 1061 and a primer consisting of a base sequence shown in SEQ ID NO: 1062 DNA fragments amplified.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers, The method may include a step of determining the genotype of a gene that controls leaf blades.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1247 and a primer consisting of the base sequence shown in SEQ ID NO: 1248 are combined, or from the base sequence shown in SEQ ID NO: 1249 A DNA fragment which is amplified using a primer set in which a primer comprising the nucleotide sequence represented by SEQ ID NO: 1250 is combined with a primer comprising the nucleotide sequence represented by SEQ ID NO: 1250; and (b) a primer comprising the nucleotide sequence represented by SEQ ID NO: 1251 and the base represented by SEQ ID NO: 1252 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 1253 and a primer consisting of a base sequence shown in SEQ ID NO: 1254 DNA fragments amplified.

The wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers, It may include a step of determining the genotype of a gene that controls the degree of autumn sowing.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1255 and a primer consisting of the base sequence shown in SEQ ID NO: 1256 is combined, or from the base sequence shown in SEQ ID NO: 1257 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1258 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1258 are combined. A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 1261 and a primer consisting of a base sequence shown in SEQ ID NO: 1262 DNA fragments amplified.

Furthermore, the wheat breeding method according to the present invention includes a DNA marker comprising the DNA fragments described in the following (a) to (g) among the at least 50 or more DNA markers, and further comprises a dormancy (0 Preferably, the method includes a step of determining genotypes of loci involved in weekly), dormancy (after 3 weeks), heading date, thousand grain weight, panicle length, number of spikelets, and plant type.
(A) From a DNA fragment amplified using a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: 735 and a primer consisting of the base sequence shown in SEQ ID NO: 736, or from the base sequence shown in SEQ ID NO: 737 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 738 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 738 and a base consisting of the nucleotide sequence shown in SEQ ID NO: 740 A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 741 and a primer consisting of a base sequence shown in SEQ ID NO: 742 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 903 and a primer consisting of the base sequence shown in SEQ ID NO: 904 or the base sequence shown in SEQ ID NO: 905 A DNA fragment (d) amplified using a primer set in which a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 906 are combined, and a primer consisting of the base sequence shown in SEQ ID NO: 907 and shown in SEQ ID NO: 908 A DNA fragment amplified using a primer set combined with a primer consisting of a base sequence, or a primer set consisting of a primer consisting of a base sequence shown in SEQ ID NO: 909 and a primer consisting of a base sequence shown in SEQ ID NO: 910 Amplified using DNA fragment (e) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 or the base shown in SEQ ID NO: 949 A DNA fragment amplified using a primer set in which a primer consisting of the sequence and a primer consisting of the base sequence shown in SEQ ID NO: 950 are combined (f) a primer consisting of the base sequence shown in SEQ ID NO: 1123 and shown in SEQ ID NO: 1124 Primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence to be synthesized or a primer consisting of a base sequence shown in SEQ ID NO: 1125 and a primer consisting of a base sequence shown in SEQ ID NO: 1126 For DNA fragment to be amplified (g) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1127 and a primer consisting of the base sequence shown in SEQ ID NO: 1128 or SEQ ID NO: 1129 A DNA fragment that is amplified using a primer set that combines a primer consisting of the base sequence shown in FIG. 1 and a primer consisting of the base sequence shown in SEQ ID NO: 1130.

  Other objects, features, and advantages of the present invention will be fully understood from the following description. The benefits of the present invention will become apparent from the following description with reference to the accompanying drawings.

It is a figure which shows the diploid wheat genetic map produced using the barley EST arrangement | sequence. It is a figure which shows a response | compatibility with the marker seated on the genetic map of the 4A ^m chromosome of a diploid wheat, and the 5A ^m chromosome, and the marker seated on a barley genetic map. It is a figure which shows the result of having determined the typing which which part of the genome originates from which parent | strain using one DNA of the recombinant inbred line | system | group of a diploid wheat using 96 DNA markers. Determines which part of the genome is derived from which parent using 96, 50, 25, or 14 DNA markers in a single inbred line of diploid wheat It is a figure which shows the result. It is a figure which shows the result of having examined the selection effect of QTL in connection with dormancy after 0 weeks using 96 DNA markers. It is a figure which shows the result of having examined the selection effect of QTL in connection with dormancy after 0 weeks using 50 DNA markers. It is a figure which shows the result of having examined the selection effect of QTL which is related to dormancy after 0 weeks using 25 DNA markers. It is a figure which shows the result of having examined the selection effect of QTL in connection with dormancy after 0 weeks using 14 DNA markers.

  The wheat breeding method according to the present invention uses a primer set designed based on a barley EST sequence, an amplification step of amplifying DNA using wheat genomic DNA as a template, and detects a polymorphism of the amplified DNA fragment A polymorphism detection step is included. Further, the DNA fragment is a DNA marker mapped on the genetic map of diploid wheat. In other words, the breeding method of the present invention is a method for breeding wheat using a diploid wheat genetic map prepared using a barley EST sequence. Therefore, first, a barley EST sequence and a primer set designed based on the sequence will be described, and then a diploid wheat genetic map prepared using the barley EST sequence will be described.

[Barley EST sequence and primer set]
In the present invention, the barley EST sequence used for designing the primer set may be a part of the base sequence of the barley cDNA. The barley EST sequence, that is, the base sequence of barley cDNA can be obtained by using a known molecular biological technique.

  For example, the inventors prepared mRNA from leaves of several kinds of barley varieties, and prepared a cDNA library by a known method. Using the plasmid with these cDNAs as a template, using the base sequence of the plasmid part outside the insertion site as a primer, adjusting the quality of the base sequence read in one sequence analysis on both sides, removing the vector sequence The EST sequence is acquired. However, it is not limited to this.

  The inventors currently have a database of about 240,000 barley EST sequences. The barley EST sequence obtained by the inventors corresponds to two base sequences read from the 5 'side and the 3' side for one cDNA clone.

  The primer set used in the present invention only needs to be designed based on the barley EST sequence. The primer set designed based on the barley EST sequence is intended to be a pair of a forward primer composed of a part of the barley EST sequence and a reverse primer composed of a part of the complementary sequence of the barley EST sequence. The

  For example, the inventors have designed a primer set for each EST using the primer design software Primer3 based on the barley EST sequence obtained independently. These primer sets are designed so that DNA fragments are amplified by PCR under the same annealing temperature conditions. As a result, it is not necessary to optimize PCR conditions for each individual primer set, and PCR can be performed simultaneously under the same conditions, which is very useful.

[Diploid wheat genetic map prepared using barley EST sequence]
A diploid wheat genetic map can be generated using the barley EST sequence. Specifically, for example, it can be produced as follows.

  The inventors have determined that the diploid wheat wild species Triticum booticum Boiss. (Accession No. KT1-1) and diploid wheat cultivar Triticum monococcum L. A genetic map of diploid wheat was produced using 93 lines of recombinant inbred lines (hereinafter referred to as “RIL”) and parents bred from crossing with (accession No. KT3-5) (implementation) See Example 1).

  First, PCR was performed using a primer set designed based on the barley EST sequence, using the genomic DNAs of parents (T. booticum Boiss. And T. monococcum L.) as templates. And the primer set which can detect a polymorphism of parents was selected. Next, PCR was performed using the selected primer set with the genomic DNA of the RIL population as a template to find 243 pairs of primer sets that amplify a DNA fragment that could serve as a DNA marker. By mapping 243 DNA markers amplified by these 243 pairs of primer sets and 96 known wheat RFLP (restriction fragment length polymorphism) markers onto the chromosomes of diploid wheat, A diploid wheat genetic map (linkage map) using the barley EST sequence was prepared.

  In the wheat breeding method according to the present invention, the barley EST sequence used for designing the primer set is not particularly limited, but is at least one base sequence selected from the base sequences shown in SEQ ID NOs: 1 to 486. It is preferable. These base sequences are the barley EST sequences actually used by the inventors for the genetic map creation of the above-mentioned diploid wheat. Here, SEQ ID NO: n (n is an odd number) and SEQ ID NO: n + 1 are a base sequence (n) obtained by reading the same cDNA from the 5 'side and a base sequence (n + 1) read from the 3' side. Therefore, any one EST sequence may be used for the same cDNA, and the inventors usually create a genetic map using the 3'-side barley EST sequence. However, those skilled in the art will readily understand that the same genetic map can be prepared using the 5'-side EST sequence.

  In addition, the primer set used in the wheat breeding method according to the present invention is not particularly limited as long as it is designed based on the barley EST sequence. Among the nucleotide sequences shown in SEQ ID NOs: 487 to 1458, SEQ ID NO: The primer set is preferably a combination of a primer having a base sequence represented by n (n is an odd number) and a primer having a base sequence represented by SEQ ID NO: n + 1. These primer sets are the primer sets actually used by the inventors for the genetic mapping of the above-described diploid wheat.

  Here, the primer set designed based on the barley EST sequence shown in SEQ ID NO: 1 is a combination of primers each consisting of the base sequences shown in SEQ ID NO: 487 and SEQ ID NO: 488. That is, the sequence number indicating the base sequence of the primer based on the individual base sequence (barley EST sequence) shown in SEQ ID NOs: 1 to 486 is (2), where m is the sequence number of the barley EST sequence on which primer design is based. × m−1) +486 and (2 × m) +486.

  If there is a polymorphism in a DNA fragment amplified using wheat genomic DNA as a template and a primer set designed based on the barley EST sequence, the DNA fragment becomes a DNA marker. In the wheat breeding method according to the present invention, it is sufficient that the DNA fragment that can serve as the DNA marker is mapped on the genetic map of diploid wheat.

  Amplified by a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: n (n is an odd number) and a primer consisting of the base sequence shown in SEQ ID NO: n + 1 among the base sequences shown in SEQ ID NOS: 487 to 1458 The inventors have found a polymorphism between “Triticum boeoticum Bois. KT1-1” and “Triticum monococcum L. KT3-5” by the inventors, and further, inherited diploid wheat. Since it is mapped on the map, it is suitable as a primer set used in the wheat breeding method according to the present invention.

  Hereinafter, the polymorphisms found in the barley EST sequence, primer set and amplified DNA fragment (DNA marker) used by the inventors for the preparation of the diploid wheat genetic map will be described in detail.

Tables 1 and 2 show the DNA markers mapped to the 1A ^m chromosome of diploid wheat in the order of distance from the short arm. Similarly, 2A ^m chromosomes in Tables 3 and 4, the 3A ^m chromosomes in Tables 5 and 6, the 4A ^m chromosome in Tables 7 and 8, the 5A ^m chromosomes in Table 9 and Table 10, 6A ^m chromosome Are shown in Table 11 and 7A ^m chromosome is shown in Table 12. In each table, the barley EST clone name, which is the basis of the primer set design used for amplification of the DNA marker, is described as the marker name, and the corresponding sequence number is the base sequence of the barley EST clone. The corresponding sequence number. Those without a sequence number are known RFLP markers.

Tables 13 and 14 show the SEQ ID NOs of primers used for amplification of DNA markers mapped to the 1A ^m chromosome of diploid wheat and the types of polymorphisms found in the DNA markers. Similarly, 2A ^m chromosomes in Table 15 and Table 16, the 3A ^m chromosomes in Table 17 and Table 18, the 4A ^m chromosomes in Table 19 and Table 20, the 5A ^m chromosome in Table 21 and Table 22, 6A ^m chromosome Are shown in Table 23 and 7A ^m chromosome is shown in Table 24.

  Tables 13 to 24 show four types of polymorphism: CAPS, SNP, size_poly, and RFLP. Among these, RFLP uses a known marker, and polymorphisms found in DNA markers of diploid wheat obtained by the inventors using barley EST sequences are classified into three types: CAPS, SNP, and size_poly. It is classified. size_poly is a polymorphism having a difference in length of the amplified fragment. Among the sizes_poly, the codominant is amplified in DNA using either “Triticum booticum Boys. KT1-1” or “Triticum monococcum L. KT3-5” as a template, but the length is different. Dominant is one in which only one of the DNAs is amplified. size_poly can detect the polymorphism by subjecting the amplified DNA fragment to electrophoresis and the difference in the position of the band or the presence or absence of the band.

  CAPS is a polymorphism depending on the presence or absence of a restriction enzyme recognition sequence. Most are based on single nucleotide polymorphisms in restriction enzyme recognition sequences, but also include those based on insertion deletions of one or more bases. A marker whose type is described as SNP is a marker in which no single nucleotide polymorphism or insertion deletion exists in the restriction enzyme recognition sequence. CAPS can be detected by subjecting the amplified DNA fragment to a restriction enzyme treatment, followed by electrophoresis, and the number of bands or the position of the band. SNPs other than CAPS can be detected by SNP typing, that is, by actually confirming the base at the single nucleotide polymorphism site or insertion deletion site. However, it is needless to say that polymorphism can also be detected for CAPS by SNP typing.

In Tables 25 to 38, among the DNA markers obtained by the inventors, single nucleotide polymorphisms in the CAPS marker “Triticum booticum Boys. KT1-1” and “Triticum monococcum L. KT3-5” or near insertion deletion The base sequence, restriction enzyme name, and recognition sequence of the restriction enzyme are shown. Tables 25 and 26 are for the 1A ^m chromosome, Tables 27 to 29 are for the 2A ^m chromosome, Tables 30 and 31 are for the 3A ^m chromosome, Tables 32 and 33 are for the 4A ^m chromosome, Tables 34 and 35 are Regarding the 5A ^m chromosome, Table 36 and Table 37 show the 6A ^m chromosome, and Table 38 shows the 7A ^m chromosome. Among these, one having a plurality of polymorphisms in one fragment is included.

Tables 39 to 50 show the base sequences near single nucleotide polymorphisms of SNP markers (other than CAPS) among the DNA markers obtained by the inventors. Tables 39 and 40 are for the 1A ^m chromosome, Tables 41 and 42 are for the 2A ^m chromosome, Tables 43 and 44 are for the 3A ^m chromosome, Tables 45 and 46 are for the 4A ^m chromosome, Tables 47 and 48 are For the 5A ^m chromosome, Table 49 shows the 6A ^m chromosome, and Table 50 shows the 7A ^m chromosome. Among these, one having a plurality of polymorphisms in one fragment is included.

  In Tables 25 to 50, bases indicating single nucleotide polymorphisms or insertion deletions are underlined. In the sequence listing, the base of the single nucleotide polymorphism site is indicated by “universal code”. In addition, the restriction enzyme recognition sequences described in Tables 9 to 16 were used for the bases that can be represented by the “universal code”.

"Universal code" is defined as follows.
m: A or C
r: G or A
w: A or T
s: G or C
y: T or C
k: G or T
v: A or G or C
h: A or C or T
d: A or G or T
b: G or C or T
n: (A or C or G or T) or (unknown or other base)

〔コムギの育種方法〕
本発明に係るコムギの育種方法は、オオムギＥＳＴ配列に基づいて設計されるプライマーセットを用い、コムギのゲノムＤＮＡを鋳型としてＤＮＡを増幅する増幅工程、および増幅されたＤＮＡ断片の多型を検出する多型検出工程を包含するものであって、上記ＤＮＡ断片は、二倍体コムギのゲノム全体を対象とする遺伝地図上にマップされているＤＮＡマーカーであり、当該遺伝地図上にマップされている少なくとも５０個以上のＤＮＡマーカーを用いるものであればよい。

[Wheat breeding method]
The wheat breeding method according to the present invention uses a primer set designed based on a barley EST sequence, an amplification step of amplifying DNA using wheat genomic DNA as a template, and detects a polymorphism of the amplified DNA fragment Including a polymorphism detection step, wherein the DNA fragment is a DNA marker mapped on a genetic map covering the entire genome of diploid wheat, and is mapped on the genetic map What is necessary is just to use at least 50 or more DNA markers.

  Since the diploid wheat is a wheat composed of an A genome, the present method can be applied to a wheat having an A genome. That is, it can be used for breeding diploid wheat composed of A genome, tetraploid wheat composed of A genome and B genome, and hexaploid wheat composed of A genome, B genome and D genome. Especially, it is suitable to use for the breeding of the diploid wheat which consists of A genomes. Especially, the diploid wheat which consists of wild-type or cultivated species A genome in the diploid wheat which consists of cultivated species A-genome. It is optimal to use this method in the breeding process in which useful traits are introduced by crossing. Furthermore, a useful trait of diploid wheat can be introduced into a highly polyploid wheat by crossing a diploid wheat into which a useful trait has been introduced and a tetraploid wheat or a hexaploid wheat.

  In order to obtain a wide range of marker information in a single trial, it is preferable to use a large number of primer sets simultaneously in the amplification step and simultaneously detect a polymorphism of a large number of DNA markers in the subsequent polymorphism detection step. Therefore, at least 50 or more DNA markers are used in this method. The 50 or more DNA markers are preferably selected with an appropriate inter-marker distance so as to cover the entire A genome. In the case of a marker mapped to the diploid wheat genetic map prepared by the present inventors, it is preferable that the appropriate inter-marker distance is selected such that the average inter-marker distance is 25 cM or less. Is 20 cM or less, more preferably 15 cM or less, particularly preferably 10 cM or less, and most preferably 5 cM or less. Needless to say, when 243 total DNA markers mapped to the genetic map of diploid wheat are used, the average inter-marker distance is 5 cM or less, which is most preferred.

  In the amplification step, the method for amplifying DNA is not particularly limited, and a known nucleic acid amplification method can be appropriately selected and used. Of these, the PCR method is suitable as a nucleic acid amplification method used in this step. In particular, among the base sequences shown in SEQ ID NOs: 487 to 1458, a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: n (n is an odd number) and a primer consisting of the base sequence shown in SEQ ID NO: n + 1 is combined. When a plurality of primer sets are used simultaneously, it is optimal to use the PCR method because these primer sets are designed so that DNA fragments are amplified by PCR under the same annealing temperature conditions.

  In the amplification step, the method for extracting genomic DNA from the test wheat individual is not particularly limited. For example, the CTAB method (see Murray HG et al. Nuc Acid Res (1980) 8: 4321-4325.), Murray et al. A known genomic DNA extraction method such as a method (see Nucleic Acids Res., 8: p4321-4325, 1980) can be used.

  The primer can be synthesized by a known method. For example, a commercially available DNA synthesizer may be used according to the instruction manual.

  In the polymorphism detection step, a method for detecting a polymorphism may be appropriately selected according to the type of polymorphism to be detected. As described above, of the base sequences shown in SEQ ID NOs: 487 to 1458, the primer consisting of the base sequence shown in SEQ ID NO: n (n is an odd number) and the primer consisting of the base sequence shown in SEQ ID NO: n + 1 are combined. Polymorphisms found in DNA fragments amplified by the primer set can be classified into three types: CAPS, SNP, and size_poly.

  For example, size_poly can be subjected to electrophoresis of an amplified DNA fragment (PCR product), and a polymorphism can be detected by the difference in the position of the band or the presence or absence of the band. CAPS can be detected, for example, by subjecting an amplified DNA fragment to a restriction enzyme treatment, followed by electrophoresis, and the number of bands or the position of the band. SNP can be detected by SNP typing, that is, by actually confirming the base at the single nucleotide polymorphism site or insertion deletion site. For example, SNP typing can be easily performed by using a commercially available SNP detection kit or the like. Note that the method for detecting a polymorphism is not limited to the above example. In addition, polymorphisms other than those described above can also be detected using a known detection method.

  By using the breeding method according to the present invention, it is possible to greatly increase the efficiency of wheat breeding, particularly diploid wheat breeding. Specifically, since DNA markers located on all chromosomes can be amplified comprehensively and polymorphisms thereof can be detected, introduction of a plurality of useful traits can be confirmed at a time. In addition, breeding of wheat using information of barley based on the correspondence between the diploid wheat genetic map prepared using the barley EST sequence and the barley genetic map prepared using the barley EST marker. Is also possible. For example, the presence or absence of the QTL can be estimated by detecting a wheat DNA marker corresponding to a DNA marker in the vicinity of QTL, which is apparent in barley.

  The breeding method according to the present invention may include steps other than the amplification step and the polymorphism detection step. For example, it is preferable to include a step of identifying a DNA marker that sits closest to a gene locus involved in a specific trait and determining the genotype of the DNA marker.

  As described above, the inventors prepared a diploid wheat genetic map in which 243 DNA markers amplified with a primer set designed based on the barley EST sequence and 96 known RFLP markers were mapped. Using the genetic map, QTL analysis was performed on 16 types of agricultural traits (see Example 2). As a result, a DNA marker located closest to each QTL or gene is found. Therefore, in the breeding method according to the present invention, subsequent to the amplification step and the polymorphism detection step, a step of determining the genotype of a locus (gene) involved in a specific trait (hereinafter referred to as “genotype determination step”). This makes it possible to determine the introduction of the target character.

  In the genotyping step, each of the QTLs or the DNA markers sandwiching the gene is selected based on the polymorphism (genotype) of the DNA marker on either one (short arm side or long arm side). Although genotype can be determined, it is preferable to determine based on polymorphism (genotype) of DNA markers on both sides.

  Hereinafter, the traits that can be determined in the breeding method according to the present invention will be described.

(1) Ear length
The head length indicates the length from the head of the head to the tip of the head, and the longer head length does not hinder the enhancement of the grains, and therefore the grain shape tends to be good. Therefore, the modification of panicle length is one of the important traits in agriculture and one of breeding goals. The panicle length is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors have found QTLs involved in four panicle lengths by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 1A ^m chromosome is amplified by the primer set of SEQ ID NOs: 587 and 588 or the primer set of SEQ ID NOs: 589 and 590. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 591 and 592 or the primer set of SEQ ID NOs: 593 and 594.

The DNA marker on the short arm side of QTL present in the 2A ^m chromosome is amplified by the primer set of SEQ ID NOs: 759 and 760 or the primer set of SEQ ID NOs: 761 and 762. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 763 and 764 or the primer set of SEQ ID NOs: 765 and 766.

The DNA marker on the short arm side of QTL present in the 3A ^m chromosome is amplified by the primer set of SEQ ID NOs: 947 and 948 or the primer set of SEQ ID NOs: 949 and 950. In addition, since the QTL sits on the long arm end of the 3A ^m chromosome, there is no DNA marker amplified by the primer set designed based on the barley EST sequence on the long arm side of the QTL. However, a known RFLP marker (marker name: JIP) is present on the long arm side.

The DNA marker on the short arm side of QTL present on the 4A ^m chromosome is amplified by the primer set of SEQ ID NOs: 995 and 996, or the primer set of SEQ ID NOs: 997 and 998. The long arm side DNA marker is amplified by the primer set of SEQ ID NOs: 999 and 1000 or the primer set of SEQ ID NOs: 1001 and 1002.

(2) Cum length
The chief is the length from the border to the head. A long cocoon is susceptible to wind resistance and tends to fall as a result of raindrops adhering to the ear and increasing weight during rain. Therefore, it can be said that it is a preferable trait for a cultivar having a moderately short culm length. If the culm length is too short, the leaves developed based on the culm cannot receive enough light, and the ear portion cannot be efficiently harvested during mechanical harvesting. Therefore, when breeding varieties having excellent traits, it is necessary to adjust the culm length to an appropriate length. Note that culm length is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors have found QTLs involved in two culm lengths by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 2A ^m chromosome is amplified by the primer set of SEQ ID NOs: 759 and 760 or the primer set of SEQ ID NOs: 761 and 762. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 763 and 764 or the primer set of SEQ ID NOs: 765 and 766.

The DNA marker on the short arm side of QTL present on the 5A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1235 and 1236, or the primer set of SEQ ID NOs: 1237 and 1238. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 1239 and 1240 or the primer set of SEQ ID NOs: 1241 and 1242.

(3) Number of spikelets
The number of spikelets is the number of grains attached to the spike, which is a guideline for estimating the yield. It can be said that a cultivar having a large number of spikelets has a higher yield, and a cultivar having a large number of spikelets is a preferable trait. The number of spikelets is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors found a QTL involved in the number of spikelets by QTL analysis using a diploid wheat genetic map prepared using a barley EST sequence. The QTL genotype is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO. That is, the genotype of the DNA marker is “Triticum boeeticum Boiss. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 3A ^m chromosome is amplified by the primer set of SEQ ID NOs: 947 and 948 or the primer set of SEQ ID NOs: 949 and 950. In addition, since the QTL sits on the long arm end of the 3A ^m chromosome, there is no DNA marker amplified by the primer set designed based on the barley EST sequence on the long arm side of the QTL. However, a known RFLP marker (marker name: JIP) is present on the long arm side.

(4) Top-internode length
The first internode length indicates the length from the head to the upper first node, and the longer the first internode length, the easier it is to fall. Therefore, it can be said that a shorter trait length is a preferable trait in cultivars. The first internode length is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors found a QTL involved in one first internode length by QTL analysis using a diploid wheat genetic map prepared using a barley EST sequence. The QTL genotype is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO. That is, the genotype of the DNA marker is “Triticum boeeticum Boiss. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present on the 3A ^m chromosome is amplified by the primer set of SEQ ID NOs: 939 and 940 or the primer set of SEQ ID NOs: 941 and 942. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 943 and 944 or the primer set of SEQ ID NOs: 945 and 946.

(5) Rachis-internode length
The intercob internode length indicates the length per cob and is an indicator of the density of grains on the cob. If the length of the intercob node is too short, the grains become small or thin. Therefore, it is necessary to select a breed having an appropriate intercob length. Therefore, the modification of the intercob length is one of the important breeding goals. In addition, the length between cob nodes is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors found QTLs involved in four intercobicular lengths by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 1A ^m chromosome is amplified by the primer set of SEQ ID NOs: 503 and 504 or the primer set of SEQ ID NOs: 505 and 506. The long arm side DNA marker is amplified by the primer set of SEQ ID NOs: 507 and 508 or the primer set of SEQ ID NOs: 509 and 510.

The DNA marker on the short arm side of QTL present on the 5A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1203 and 1204 or the primer set of SEQ ID NOs: 1205 and 1206. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 1207 and 1208 or the primer set of SEQ ID NOs: 1209 and 1210.

The DNA marker on the short arm side of the first QTL present on the 7A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1355 and 1356 or the primer set of SEQ ID NOs: 1357 and 1358. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOS: 1359 and 1360 or the primer set of SEQ ID NOS: 1361 and 1362.

The DNA marker on the short arm side of the second QTL present on the 7A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1395 and 1396 or the primer set of SEQ ID NOs: 1397 and 1398. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 1399 and 1400 or the primer set of SEQ ID NOs: 1401 and 1402.

(6) Heading date (day from April)
Heading date is a trait that is an indicator of early and late life. The modification of heading date is an important breeding target for regional adaptability and decentralization of harvesting work. For example, if cultivars with slightly different heading dates are cultivated, the harvesting work can be dispersed, making it possible to improve the efficiency of farming work and avoid danger to obstacles. In Japan, wheat growing south of Honshu is cultivated as a rice paddy, so it must be harvested before the rainy season or planting. Therefore, the development of early varieties is one of the important breeding goals of wheat in Japan. The heading date is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors found a QTL involved in one heading date by QTL analysis using a diploid wheat genetic map prepared using a barley EST sequence. The QTL genotype is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO. That is, the genotype of the DNA marker is “Triticum boeeticum Boiss. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 3A ^m chromosome is amplified by the primer set of SEQ ID NOs: 947 and 948 or the primer set of SEQ ID NOs: 949 and 950. In addition, since the QTL sits on the long arm end of the 3A ^m chromosome, there is no DNA marker amplified by the primer set designed based on the barley EST sequence on the long arm side of the QTL. However, a known RFLP marker (marker name: JIP) is present on the long arm side.

(7) Awn length
The length of the kite indicates the length of the kite, and the longer the kite is, the more advantageous it is for photosynthesis. However, long persimmons can interfere with manual harvesting, and varieties without persimmons have been bred. On the other hand, straw is not a problem for machine harvesting. Therefore, it is necessary to adjust the length of the cocoon according to the cultivation conditions. Note that culm length is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors have found QTLs involved in two culm lengths by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present on the 5A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1115 and 1116 or the primer set of SEQ ID NOs: 1117 and 1118. The long arm side DNA marker is amplified by the primer set of SEQ ID NOs: 1119 and 1120 or the primer set of SEQ ID NOs: 1121 and 1122.

The DNA marker on the short arm side of QTL present on the 7A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1407 and 1408 or the primer set of SEQ ID NOs: 1409 and 1410. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 1411 and 1412 or the primer set of SEQ ID NOs: 1413 and 1414.

(8) Glum length
The guard is the length of the guard attached to the side of the floret. The longer the protection, the higher the humidity on the surface of the ears due to raindrops and dew, etc., leading to the propagation of pathogenic bacteria and the occurrence of ear germination. It is also known that the length varies depending on the species and variety. Therefore, it is possible to impart resistance to obstacles by cultivating varieties with short protective tempura, and easily cultivating varieties with different culmination lengths from other varieties to easily identify different varieties. can do. Therefore, the modification of the chief guard is one of the important breeding goals. Note that the guardian is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors found QTLs involved in two guardian lengths by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 3A ^m chromosome is amplified by the primer set of SEQ ID NOs: 943 and 944 or the primer set of SEQ ID NOs: 945 and 946. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 947 and 948 or the primer set of SEQ ID NOs: 949 and 950.

The DNA marker on the short arm side of QTL present in the 7A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1371 and 1372 or the primer set of SEQ ID NOs: 1373 and 1374. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 1375 and 1376 or the primer set of SEQ ID NOs: 1377 and 1378.

(9) Plant type
The plant type shows the shape of the plant during wintering, and in cold regions, the varieties with the plant type tend to have higher wintering ability. Also, grass-type upright varieties are generally premature. Therefore, in the cultivar, it is necessary to change the grass type depending on the cultivation area. The plant type is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors found QTLs involved in two grass types by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 3A ^m chromosome is amplified by the primer set of SEQ ID NOs: 947 and 948 or the primer set of SEQ ID NOs: 949 and 950. In addition, since the QTL sits on the long arm end of the 3A ^m chromosome, there is no DNA marker amplified by the primer set designed based on the barley EST sequence on the long arm side of the QTL. However, a known RFLP marker (marker name: JIP) is present on the long arm side.

The DNA marker on the short arm side of QTL present in the 5A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1259 and 1260 or the primer set of SEQ ID NOs: 1261 and 1262. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOS: 1263 and 1264 or the primer set of SEQ ID NOS: 1265 and 1266.

(10) Thousand-kenel weight
Thousand grain weight is a trait that measures the weight of the grain after ripening and converts it into a thousand grains, and serves as an index of the weight of the grain. In wheat, when the weight of a thousand grains is too small, the wheat flour recovery rate decreases, and conversely, when it is too large, the protein (gluten) content required for bread tends to decrease. For this reason, it is necessary to select individuals with an appropriate thousand grain weight, and modification of the thousand grain weight is one of important breeding goals. Thousand grain weight is a quantitative trait and is a trait determined by a plurality of loci.

  The inventors have found a QTL involved in one thousand grain weight by QTL analysis using a diploid wheat genetic map prepared using a barley EST sequence. The QTL genotype is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO. That is, the genotype of the DNA marker is “Triticum boeeticum Boiss. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 2A ^m chromosome is amplified by the primer set of SEQ ID NOs: 735 and 736 or the primer set of SEQ ID NOs: 737 and 738. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 739 and 740 or the primer set of SEQ ID NOs: 741 and 742.

(11) COB detachment (Rachis brittleness)
Cob detachment is a trait that expresses a phenotype in which the cob becomes brittle after ripening and the seed falls (cob detachment), or a phenotype in which the seed does not fall after ripening (cob non-dropping) . In the wild species, it is important that the seeds fall naturally from the ear in order to leave the next generation. On the other hand, although the cultivated species is basically non-cobbing, the hybrid is coccal shed in the cross between the cultivated species and the wild species, and the cob shed hybrid is separated from the cross between the cultivated species. There are things to do. In the case of a cob declining individual, seeds fall naturally from the panicle and the yield cannot be evaluated. Therefore, it is important to be non-cobbing for the evaluation of agricultural traits. Cob detachment is a quantitative trait that is determined by a plurality of loci.

  The inventors have found two QTLs that are involved in cob shedding by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present on the 4A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1071 and 1072 or the primer set of SEQ ID NOs: 1073 and 1074. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 1075 and 1076 or the primer set of SEQ ID NOs: 1077 and 1078.

The DNA marker on the short arm side of QTL present in the 7A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1371 and 1372 or the primer set of SEQ ID NOs: 1373 and 1374. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 1375 and 1376 or the primer set of SEQ ID NOs: 1377 and 1378.

(12) Dormancy at 0 weeks (Dormancy at 0 week)
Freshly harvested seeds are dormant and are known not to germinate immediately. This tendency is strong in wild species, and some dormancy is also observed in cultivated species. If there is no dormancy, there will be a problem of ear germination where seeds will germinate due to rain before harvest. That is, dormancy is a trait that acts as a resistance factor to ear germination. However, if the dormancy is too deep, problems such as weeding without germination until the next year will occur. Therefore, it has become an important breeding goal to modify it so as to have moderate dormancy. Note that dormancy is a quantitative trait and is a trait determined by a plurality of loci.

  For investigation of dormancy after 0 weeks, seeds immediately after yellowing were harvested, dried, and immediately stored in a -20 ° C freezer. The stored seeds were allowed to absorb water without awakening from dormancy, and the germination rate after 4 days at 25 ° C. was investigated.

  The inventors found QTLs involved in three dormancy (after 0 weeks) by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of QTL present in the 1A ^m chromosome is amplified by the primer set of SEQ ID NOs: 575 and 576 or the primer set of SEQ ID NOs: 577 and 578. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 579 and 580 or the primer set of SEQ ID NOs: 581 and 582.

The DNA marker on the short arm side of QTL present on the 3A ^m chromosome is amplified by the primer set of SEQ ID NOs: 835 and 836 or the primer set of SEQ ID NOs: 837 and 838. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 839 and 840 or the primer set of SEQ ID NOs: 841 and 842.

The DNA marker on the short arm side of QTL present on the 5A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1123 and 1124 or the primer set of SEQ ID NOs: 1125 and 1126. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 1127 and 1128 or the primer set of SEQ ID NOs: 1129 and 1130.

(13) Dormancy at 3 weeks (Dormancy at 3 weeks)
Freshly harvested seeds are dormant and are known not to germinate immediately. This tendency is strong in wild species, and some dormancy is also observed in cultivated species. If there is no dormancy, there will be a problem of ear germination where seeds will germinate due to rain before harvest. That is, dormancy is a trait that acts as a resistance factor to ear germination. However, if the dormancy is too deep, problems such as weeding without germination until the next year will occur. Therefore, it has become an important breeding goal to modify it so as to have moderate dormancy. Note that dormancy is a quantitative trait and is a trait determined by a plurality of loci.

  For investigation of dormancy after 3 weeks, seeds immediately after yellowing were harvested, dried, and immediately stored in a -20 ° C freezer. The stored seeds were awakened by dormancy at 25 ° C. for 3 weeks, then water-absorbed, and the germination rate after 4 days at 25 ° C. was examined.

  The inventors found QTLs involved in three dormancy (after 3 weeks) by QTL analysis using a diploid wheat genetic map prepared using barley EST sequences. The genotype of each QTL is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum boeuticum Boys. KT1-1. Or “Triticum monococcum L.KT3-5” type can be detected and determined based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of the first QTL present in the 1A ^m chromosome is amplified by the primer set of SEQ ID NOs: 591 and 592 or the primer set of SEQ ID NOs: 593 and 594. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 595 and 596 or the primer set of SEQ ID NOs: 597 and 598.

The DNA marker on the short arm side of the second QTL present in the 1A ^m chromosome is amplified by the primer set of SEQ ID NOs: 627 and 628 or the primer set of SEQ ID NOs: 629 and 630. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 631 and 632 or the primer set of SEQ ID NOs: 633 and 634.

The DNA marker on the short arm side of QTL present in the 3A ^m chromosome is amplified by the primer set of SEQ ID NOs: 903 and 904 or the primer set of SEQ ID NOs: 905 and 906. Further, the DNA marker on the long arm side is amplified by the primer set of SEQ ID NOs: 907 and 908 or the primer set of SEQ ID NOs: 909 and 910.

(14) Aleurone layer color
The paste powder layer color can be determined by visually examining the paste powder layer color of each individual before ripening. That is, it can be judged by whether or not the film portion called the paste powder layer existing on the surface of the fruit inside the spikelets is colored.

  Paste layer color is a trait governed by a single dominant gene and is inherited according to Mendel's law. There are two alleles in the gene locus that controls the color of the glue layer, that is, a gene that colors the glue layer and a gene that expresses a non-colored light brown glue layer, and coloring is dominant. For example, when a useful trait is selected from a wild type that represents a colored paste layer by hybrid breeding to a variety that represents a light brown paste layer, the gene that the target trait controls the paste layer color As long as it is on the chromosome on which it sits, not only the desired trait but also the gene that controls the paste layer color may be introduced at the same time. Therefore, if a variety having a colored paste powder layer is selected, it can be said that there is a high possibility that it will be a variety introduced with the desired character. In addition, the paste powder layer color can be used as an index for identifying the mixing of different varieties, so that it is possible to easily perform varieties inspection and the like.

  The inventors have found a DNA marker that sandwiches a gene that controls the color of the starch layer and sits in the vicinity by QTL analysis using a diploid wheat genetic map prepared using a barley EST sequence. It was. The genotype of the gene that controls the paste layer color is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO. That is, the genotype of the DNA marker is “Triticum It is possible to detect whether it is “bototicum Boiss. KT1-1” type or “Triticum monococcum L. KT3-5” type, and determine based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of the gene that controls the glue layer color present in the 4A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1055 and 1056 or the primer set of SEQ ID NOs: 1057 and 1058. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 1059 and 1060 or the primer set of SEQ ID NOs: 1061 and 1062.

(15) Leaf hairline
The leaf ridge can be confirmed by touching the ridge on the surface of the leaf.

  Leaf blade is a trait governed by a single dominant gene and is inherited according to Mendel's law. There are two alleles at the locus that controls the leaf trichomes: the gene that expresses the leaves with hair follicles and the gene that expresses the leaves without follicles, and the leaves with the follicles are dominant. For example, when trying to select a useful trait from wild varieties with hair follicles by cross breeding for varieties without hair follicles, the chromosome on which the gene that controls the leaf blade is located If it is above, not only the desired trait but also a gene that controls leaf blades may be introduced at the same time. If varieties with hair flies are selected, it can be said that there is a high possibility that the varieties will have the target trait introduced. In addition, leaf curly can be used as an index for identifying the mixing of different varieties, and varieties can be easily verified.

  The inventors have found a DNA marker that sandwiches the gene that controls leaf blades and sits in the vicinity by QTL analysis using a diploid wheat genetic map prepared using a barley EST sequence. It was. The genotype of the gene that governs leafy hair folds is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum It is possible to detect whether it is “bototicum Boiss. KT1-1” type or “Triticum monococcum L. KT3-5” type, and determine based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of the gene that controls leaf blades present on the 5A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1247 and 1248 or the primer set of SEQ ID NOs: 1249 and 1250. The long arm DNA marker is amplified by the primer set of SEQ ID NOs: 1251 and 1252 or the primer set of SEQ ID NOs: 1253 and 1254.

(16) Vernalization requirement
The degree of autumn sowing can be judged by the period of low temperature and short days necessary for flowering heading. The degree of autumn sowing is investigated by the number of days until the leaf leaves develop under constant temperature conditions (usually 20 ° C.) under continuous illumination. Varieties with a high degree of autumn sowing do not reach flowering without leading to leaf development, whereas varieties with a low degree of autumn sowing (spring sowing) lead to the development of flag leaves in a shorter period of time. Varieties with a high autumn sowing property do not produce ears even when cultivated in spring, but are suitable for sowing in winter and cultivating over winter. On the other hand, when cultivating varieties with low autumn sowing ability overwintering, the growth progresses during autumn, and it may wither in the cold. Thus, in breeding, it is important to change the degree of autumn sowing depending on the cultivation environment.

  The inventors have found a DNA marker that sandwiches a gene that controls the degree of autumn dissemination and sits in the vicinity by QTL analysis using a diploid wheat genetic map prepared using a barley EST sequence. It was. The genotype of the gene governing the degree of autumn seeding is a polymorphism of a DNA marker amplified by a primer set in combination with a primer consisting of the base sequence shown in the following SEQ ID NO, that is, the genotype of the DNA marker is “Triticum It is possible to detect whether it is “bototicum Boiss. KT1-1” type or “Triticum monococcum L. KT3-5” type, and determine based on the genotype of the DNA marker. Please refer to the corresponding marker in the above table for the types of polymorphism and the base sequence of the polymorphic site.

The DNA marker on the short arm side of the gene that controls the degree of autumn dissemination on the 5A ^m chromosome is amplified by the primer set of SEQ ID NOs: 1255 and 1256, or the primer set of SEQ ID NOs: 1257 and 1258. The long arm side DNA marker is amplified by the primer set of SEQ ID NOs: 1259 and 1260 or the primer set of SEQ ID NOs: 1261 and 1262.

  In the breeding method according to the present invention, it is preferable to include, as an essential marker, a DNA marker comprising the DNA fragments described in the following (a) to (g) among at least 50 or more DNA markers to be used. By including these markers, it is a very important trait for wheat breeding, which is dormancy after 0 weeks, dormancy after 3 weeks, heading date, grain weight, panicle length, number of spikelets and plant type. It is possible to simultaneously determine the genotype of the locus involved in a single trial.

  (A) From a DNA fragment amplified using a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: 735 and a primer consisting of the base sequence shown in SEQ ID NO: 736, or from the base sequence shown in SEQ ID NO: 737 A DNA fragment that is amplified using a primer set that combines a primer that comprises a primer comprising the nucleotide sequence represented by SEQ ID NO: 738. These are DNA markers linked to QTL involved in 1000 grain weight.

  (B) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 739 and a primer consisting of the base sequence shown in SEQ ID NO: 740 are combined, or from the base sequence shown in SEQ ID NO: 741 A DNA fragment that is amplified using a primer set that combines a primer that comprises a primer comprising the base sequence represented by SEQ ID NO: 742. These are DNA markers linked to QTL involved in 1000 grain weight.

(C) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 903 and a primer consisting of the base sequence shown in SEQ ID NO: 904 are combined, or from the base sequence shown in SEQ ID NO: 905 A DNA fragment that is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 906 is combined. These are DNA markers linked to QTL involved in dormancy after 3 weeks.
(D) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 907 and a primer consisting of the base sequence shown in SEQ ID NO: 908 are combined, or from the base sequence shown in SEQ ID NO: 909 A DNA fragment that is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 910 is combined. These are DNA markers linked to QTL involved in dormancy after 3 weeks.
(E) from a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 are combined, or from the base sequence shown in SEQ ID NO: 949 A DNA fragment that is amplified using a primer set in which a primer consisting of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 950 is combined. These are DNA markers linked to QTL related to heading date, head length, number of spikelets and plant type.
(F) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1123 and a primer consisting of the base sequence shown in SEQ ID NO: 1124 are combined, or from the base sequence shown in SEQ ID NO: 1125 A DNA fragment that is amplified using a primer set in which a primer consisting of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1126 is combined. These are DNA markers linked to QTL that are involved in dormancy after 0 weeks.
(G) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1127 and a primer consisting of the base sequence shown in SEQ ID NO: 1128 are combined, or from the base sequence shown in SEQ ID NO: 1129 A DNA fragment that is amplified using a primer set in which a primer consisting of a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1130 is combined. These are DNA markers linked to QTL that are involved in dormancy after 0 weeks.

A DNA marker other than the DNA markers described in the above (a) to (g) may be arbitrarily selected from the remaining 243 DNA markers, but is preferably selected preferentially As a DNA marker linked to QTL present in the 1A ^m chromosome involved in dormancy after 0 weeks (the short arm amplified by the primer set of SEQ ID NOs: 575 and 576 or the primer set of SEQ ID NOs: 577 and 578) DNA marker and the long arm DNA marker amplified by the primer set of SEQ ID NOs: 579 and 580 or the primer set of SEQ ID NOs: 581 and 582), present in the 3A ^m chromosome involved in dormancy after 0 weeks DNA markers linked to QTL (SEQ ID NOs: 835 and 836) The short arm DNA marker amplified by the primer set of SEQ ID NOS: 837 and 838, and the long arm amplified by the primer set of SEQ ID NOS: 839 and 840, or the primer set of SEQ ID NOS: 841 and 842 A DNA marker linked to the first QTL present in the 1A ^m chromosome involved in dormancy after 3 weeks (by the primer set of SEQ ID NOs: 591 and 592 or the primer set of SEQ ID NOs: 593 and 594) Amplified short arm DNA marker and long arm DNA marker amplified by primer set of SEQ ID NOs: 595 and 596 or SEQ ID NO: 597 and 598) Involved in dormancy after 3 weeks present in 1A ^m chromosome DNA markers linked to the second QTL (a short arm DNA marker amplified by the primer set of SEQ ID NO: 627 and 628 or the primer set of SEQ ID NO: 629 and 630, and the primer of SEQ ID NO: 631 and 632) Set, or a DNA marker on the long arm side amplified by the primer set of SEQ ID NOS: 633 and 634).

  Furthermore, if at least one DNA marker linked to the QTL or main gene of the above traits (1) to (16) is selected for each trait, the genotypes of 16 types of traits useful for wheat breeding can be simultaneously selected. It is possible to determine. More preferably, all DNA markers linked to all QTLs and main genes of 16 types of traits are selected.

[Equipment used to implement wheat breeding method according to the present invention]
The wheat breeding method according to the present invention can be more efficiently carried out by using an instrument. For example, an array type polymorphism detection instrument can be mentioned. Specifically, “a device used for breeding wheat, wherein a DNA fragment or a part thereof amplified by a primer set designed based on a barley EST sequence using a wheat genomic DNA as a template is placed on a support. An instrument that is fixed and capable of detecting a polymorphism present in the DNA fragment ”. However, it is not limited to this. Hereinafter, the appliances exemplified above will be described.

In the above apparatus, a DNA fragment amplified by a primer set designed based on a barley EST sequence using a wheat genomic DNA as a template is mapped onto a genetic map of diploid wheat according to the present invention. It is a DNA marker. That is, the instrument may be an instrument in which a DNA marker mapped on a genetic map of diploid wheat is immobilized on a support. The DNA fragment to be immobilized may be the entire amplified DNA fragment or a part thereof as long as it contains a polymorphic site (for example, SNP site, deletion / insertion site, etc.).
Polymorphisms such as SNPs present in the amplified DNA fragment by hybridizing the DNA immobilized on the instrument to the DNA fragment amplified in the amplification step in the wheat breeding method according to the present invention Can be detected.

  Preferably, DNA fragments derived from 50 or more DNA markers are immobilized on the support, and all of the DNA markers mapped to the genetic map of diploid wheat are immobilized. Particularly preferred. Furthermore, the DNA immobilized on the support is preferably arranged in the same order as that mapped on the genetic map, that is, the order on the chromosome.

  The support is not particularly limited as long as it can immobilize the polynucleotide, and may have any shape or material. In general, for example, inorganic materials such as glass and silicon wafers, natural polymers such as paper, synthetic polymers such as nitrocellulose and nylon, synthetic polymers and natural polymers are used as the support material. And the like. Further, the shape of the support is not particularly limited as long as it has a sufficient area for immobilizing the polynucleotide. In general, a substrate having little or little flexibility, flexible A film having a two-dimensional extent such as a film having a membrane (membrane) and a flexible substrate in the middle of the film can be preferably used. The thickness of the substrate or film is not particularly limited, and may be set as appropriate depending on the material and application. Furthermore, various beads can be used as the support.

[Kit used for carrying out wheat breeding method according to the present invention]
The method for breeding wheat according to the present invention can be more efficiently carried out by using a kit. Specifically, for example, “a kit used for breeding wheat and having a primer set designed based on a barley EST sequence” can be mentioned. However, it is not limited to this. The kit exemplified above will be described below.

  As used herein, a “kit” is intended to be a package including a container (eg, bottle, plate, tube, dish, etc.) containing a specific material. Preferably, an instruction manual for using the material is provided. The instructions for use may be written or printed on paper or other media, or may be affixed to electronic media such as magnetic tape, computer readable disk or tape, CD-ROM, and the like.

  The primer set provided in the above exemplified kit includes a primer consisting of the base sequence shown in SEQ ID NO: n (n is an odd number) among the base sequences shown in SEQ ID NO: 487 to 1458 and the base sequence shown in SEQ ID NO: n + 1 The primer set which combined the primer which consists of is preferable. In addition, the primer set provided in the kit is preferably plural, more preferably at least 50 or more selected from the entire genome of diploid wheat, based on the nucleotide sequence shown in SEQ ID NOs: 487 to 1458. It is particularly preferable that all the primers are provided.

  In addition, the kit may include a primer set other than the primer set designed based on the barley EST sequence. The specific configuration other than the primer set is not particularly limited, and a necessary reagent, instrument, or the like may be appropriately selected to form a kit configuration. For example, a polymerase, a buffer, a reaction tube, etc. can be mentioned. Further, the above-described polymorphism detection instrument may be configured as a kit. The kit having the configuration including the polymorphism detection instrument makes it possible to carry out the wheat breeding method according to the present invention more quickly and efficiently.

  Those skilled in the art who read this specification will easily understand that the method of using the kit may be in accordance with the above-described method for breeding wheat according to the present invention.

  It should be noted that the specific embodiments and the following examples made in the section of the best mode for carrying out the invention are intended to clarify the technical contents of the present invention, and to such specific examples. It is not to be construed as limiting in any way whatsoever, and those skilled in the art can implement the invention within the spirit of the invention and the scope of the appended claims.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

  EXAMPLES Hereinafter, although this invention is demonstrated concretely using an Example, this invention is not limited to this.

[Example 1: Construction of diploid wheat genetic map using barley EST sequence]
(1) Creation of map populations Diploid wheat wild species Triticum booticum Boiss. (Accession No. KT1-1) and diploid wheat cultivar Triticum monococcum L. Recombinant inbred line (RIL) 93 line grown from crossing with (accession No. KT3-5) and parents, and RFLP information (Shindo C., Sasakuma T., Watanabe N. and Noda K. (2002) Two-gene systems of vernalization requirement and narrow-sense earliness in einkorn heat. Genome 45: 563-569.).

(2) DNA extraction About 10 cm of RIL population and parents' leaf blades were collected, placed in a 1.5 ml Eppendorf tube, frozen in liquid nitrogen, and crushed with a plastic pestle. Genomic DNA (1-5 μg) was obtained by the method of Palotta et al. (Palotta MA, Graham RD, Langridge P., Sparrow DHB and Barker S. J. 2000. RFLP mapping of machinery efficiencies. barley.Theor.Appl.Genet.101: 1100-1108.), but no RNase was added to the final dissolved TE buffer.

(3) Detection of polymorphism of PCR product The PCR reaction was performed in a 96-well plate or a 384-well plate using GeneAmp PCR System (Applied Biosystems). The PCR reaction system exceeded 1 × Ex-Taqbuffer (Takara), forward / reverse primer 1.0 μM, dNTPs 2.0 mM, MgCl ₂ 0.5 mM, template DNA 10 ng and Ex-Taq DNA polymerase (Takara) 0.035 U Pure water was added to make 10 μl. PCR conditions were 94 ° C for 2 minutes, 94 ° C for 30 seconds, 65 ° C for 30 seconds, 72 ° C for 1 minute for 5 cycles, followed by 94 ° C for 30 seconds, 65 ° C for 30 seconds, 72 ° C for 1 minute for 35 cycles, 72 It was carried out at 7 ° C. 2 μl of loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 30% Glycerol) is added to 10 μl of the PCR product, 6 μl of which is added to 0.5 × TBE buffer (45 mM Tris-acetate, 45 mM Boric acid, 1 mM (PH 8.0)) was separated on a 2% (w / v) agarose gel in which ethidium bromide (Sigma) was added.

  Parents' genomic DNA was amplified by PCR using 2,695 pairs of barley EST-derived primers, and agarose gel electrophoresis showed a band only on one parent (dominant marker), and parents had different band sizes (size) Polymorphic marker) was selected.

  With respect to those in which a band with the same size (a band whose size difference cannot be recognized on the agarose gel) appeared with both parents, the nucleotide sequence was analyzed and a single nucleotide polymorphism (SNP) was detected. Among SNP markers, those having restriction enzyme sites at any polymorphic site were selected as CAPS markers, and those without restriction enzyme sites were selected as SNP typing markers.

  In order to purify the PCR product, Sephadex G-50 (Amesham Biosciences) was filled into a column loader, flattened with a glass plate, and then reversed with an HV plate (MILLIPORE) on the column loader. Sephadex G-50 was transferred to. 300 μl of ultrapure water was added to the HV plate, left at room temperature for 3 hours, centrifuged at 900 × G for 2 minutes, 150 μl of ultrapure water was added, and centrifugation at 900 × G for 2 minutes was repeated three times or more. . After putting the PCR product in the center of each well, layer the titer plate (PS-microplate; Greiner), centrifuge adapter, HV plate, HV plate lid in this order, centrifuge at 900 x G for 3 minutes, and further change the direction And further centrifuged for 3 minutes.

  For the sequencing reaction, BioDye Terminator v1.1 Cycle Sequence RR-100 (Applied Biosystems) 1.0 μl, 5 × sequence buffer (Applied Biosystems) 1.0 μl, and reverse PCR primer 5 mM were added. The total volume was made up to 10.0 μl with ultrapure water. PCR conditions were 96 ° C. for 2 minutes, followed by 25 cycles of 96 ° C. for 10 seconds, 50 ° C. for 5 seconds, and 60 ° C. for 4 minutes. For the sequence product, the plate was centrifuged for several seconds, 2.5 μl of 125 mM EDTA and 30 μl of 100% ethanol were added, sealed with a seal, mixed by inversion several times, covered with aluminum foil, and left at room temperature for 15 minutes. Furthermore, after centrifuging at 3,100 g × G for 30 minutes, immediately reverse the plate and remove the supernatant, add 30 μl of 70% ethanol, centrifuge at 1,700 g × G for 15 minutes, remove the supernatant again, 10 μl of Hi-Di Formatide (Applied Biosystems) was added, and after reaction with GeneAmp PCR System at 95 ° C. for 5 minutes, the plate was immediately transferred onto ice and protected from light. The purified sequence product was analyzed with ABI Prism 310 genetic analyzer (PE Applied Biosystems) or 3100 Genetic Analyzer (Applied Biosystems). For analysis of sequence data, commercially available package software DNA SIS Pro Ver. 2.0 (Hitachi Soft) was used for alignment, and a cleated amplified polymorphism sequence (CAPS) was detected from the difference in the base sequences of the parents.

(4) Construction of genetic map (linkage map) A barley EST-derived marker in which polymorphism was observed in parents was used for polymorphism analysis of the RIL population. That is, PCR was performed using the selected primer set using the genomic DNA of the RIL population as a template. As a result, 243 barley EST-derived markers were PCR-amplified with the RIL population.

  In the RIL population, 243 barley EST-derived markers and 96 known RFLP marker separation data were analyzed using MAPMKER / EXP ver. 3.0 (Lander ES, Green P, Abrahamson J, Barlow A, Dalley MJ (1987) MAPMKER: an interactive computer package for urinary priming pharmapology of the linking of the symposium. In addition to the (2002) RFLP information, a genetic map was created. The threshold of marker linkage in each linkage group was LOD 3.0, and the Kosambi map function was used for map distance (Kosambi DD (1994) The estimation of map distances from recombination values. Anals of Eugens 75 12;

As a result, 49 in 1A ^m chromosome (derived from EST: 38, RFLP: 11), 57 in 2A ^m chromosome (derived from EST: 42, RFLP: 15), 49 in 3A ^m chromosome (derived from EST: 36, RFLP) 13) 54 on 4A ^m chromosome (derived from EST: 40, RFLP: 14), 67 on 5A ^m chromosome (derived from EST: 39, RFLP: 28), 27 on 6A ^m chromosome (derived from EST: 19, RFLP: 8), 36 (EST-derived: 38, RFLP: 11) were seated on the 7A ^m chromosome.

  The total map distance was 1,08.5 cM and the average marker density was 3.0 cM / locus.

  In addition, the diploid comb genetic map produced in FIG. 1 was shown. In FIG. 1, the top of each chromosome is the short arm end. The right side of each chromosome represents the marker name, and the left side represents the distance (cM) from the short arm end.

[Example 2: Analysis of agricultural trait loci of diploid wheat]
Using the diploid wheat genetic map prepared in Example 1 above, QTL analysis was performed on 16 types of agricultural traits.

(1) Target traits Thirteen types of quantitative traits and three types of qualitative traits were targeted.

  Specifically, the quantitative traits include ear length, culm length, number of spikelets, top-internode length, cob length ( Rachis-internode length, heading date (day from April), chief length (Glen length), plant type (Plant type), Thousand-head It is a total of 13 traits: dropout (Rachis brittleness), dormancy after 0 weeks (Dormancy at 0 week), and dormancy after 3 weeks (Dormancy at 3 weeks). The qualitative traits are three traits: glue layer color, leaf hairline, and vernalization requirement.

(2) Plant cultivation 114 RIL populations and their parents were sown and cultivated in the network room of Okayama University Institute of Resource and Biological Science in mid-November 2004. Each strain and parents were cultivated in two pieces each by placing culture soil (Fujino Jitsugyo) in a plastic growth pot with a diameter of 24 cm and a depth of 21 cm. Of these, one individual from each line was transplanted to an experimental field of the Institute for Resource Biology, Okayama University in January 2005, and cultivated with a culm width of 90 cm and a strain of 30 cm.

(3) Characteristic evaluation Date of heading, head length, head length, number of spikelets, first internode length, intercob length, leaf curl, paste layer color and plant type are measured before harvesting Axial shedding, culm length, 1000-grain weight and gall length were measured in the harvested ears.

  The heading date was defined as the number of days from April 1 until the first head of the head appeared. The head length was measured from the ground to the base of the ear, and the head length was measured from the base of the head to the tip (cm), 3 ears for each strain. As for the number of spikelets, the number of spikelets from the base of the spike to the tip of the spike was counted for three spikelets of each line. For the first internode length, the length (cm) from the neck to the upper first node was measured for 3 lines in each line. The intercob node length was measured by measuring the length of the cob between 10 spikelets in the center of the panicle (mm), and the culm length was measured by measuring the length of the cocoon in the center of the panicle (cm) by 3 ears. Leaf blades were confirmed by touching the hair ears on the surface of the leaves. The cob shedding ability was divided into four stages, with the non-falling ability being 0 and the natural shedding ability being 3. The paste powder layer color was classified into four stages, with 0 being the complete white color and 3 being the complete blue color. In the grass type, the angle of the entire leaf with respect to the ground was indexed in five steps from substantially vertical (0) to wiping (4).

  The test for the degree of autumn sowing is to allow seeds to absorb water at 4 ° C for 4 days, sow 2 individual plants in 5 pots in a pot, and keep them for 24 hours in a greenhouse at a minimum room temperature of 15 ° C or more. Cultivated and recorded the leaf expansion stage and the number of leaves. The survey period was 120 days.

  In order to investigate dormancy, the ears immediately after yellowing were harvested, dried, and immediately stored in a -20 ° C freezer. In addition, 20 grains of each line were used for the 0th week of dormancy, and 50 grains of each line were tested for the 3rd week. The 0th week sample was germinated at 25 ° C. for 4 days after water absorption with a petri dish, and the 3rd week was awakened by dormancy at 25 ° C. for 3 weeks.

(4) QTL analysis The software for QTL analysis includes MAPMAKER / QTL ver. 1.1b (Lander ES, Botstein D (1989) Mapping Mendellian factors underlying quantitative traits using RFLP linkage maps. Genetics 121: 185-199). Simple interval mapping (SIM) was used for the algorithm of QTL analysis. The determination of the presence or absence of QTL was performed using LOD 2.0 as a threshold and the QTL estimation interval being 0.1 cM.

(5) Results Table 51 shows the results of QTL analysis of 13 types of quantitative traits. As apparent from Table 51, QTL was detected in all 13 quantitative traits.

In Table 51, for example, for ear length,
(1) Four QTLs related to panicle length were detected (2) Four QTLs sit on chromosomes 1A ^m , 2A ^m , 3A ^m and 4A ^m , respectively (3) Loci on chromosome 1A ^m The QTL to ride is a DNA marker amplified by a primer set designed based on the barley EST clone name k04016 (SEQ ID NO: 589 and 590) and a primer set designed based on the barley EST clone name k00958 (SEQ ID NO: 589 and 590) is present (interval: 24.6 cM) (4) a QTL peak is present at 14.3 cM from the DNA marker based on k04016 (5) peak LOD Score is 2.2 (6) 13.5 of variance of phenotype in this QTL % (7) This QTL can be explained by Triticum monococcum L. It has been shown that having an (accession No. KT3-5) type allele shortens the ear length by 17.6 mm. Other traits can be read in the same manner.

３種類の質的形質のＱＴＬ解析結果を表５２に示した。表５２から明らかなように、３種類の質的形質の座乗位置が特定された。

Table 52 shows the QTL analysis results of the three types of qualitative traits. As is clear from Table 52, the sitting positions of three types of qualitative traits were identified.

In Table 52, for example, for the glue layer color (Aleurone layer color),
(1) sit on chromosome 4A ^m (2) DNA marker amplified based on the primer set designed based on barley EST clone name k04722 (SEQ ID NOs: 1057 and 1058) and design based on barley EST clone name k09478 (3) a QTL peak at a position of 2.3 cM from the DNA marker based on k04722 (interval: 3.9 cM) between the DNA markers amplified by the prepared primer set (SEQ ID NOs: 1061 and 1062) It has been shown to exist. Other traits can be read in the same manner.

なお、両親（Ｔ．ｂｏｅｏｔｉｃｕｍ Ｂｏｉｓｓ．およびＴ．ｍｏｎｏｃｏｃｃｕｍ Ｌ．）を比較した場合の各形質の表現型は、表５３に示したとおりである。

Table 53 shows the phenotype of each trait when parents (T. booticum Boys. And T. monococcum L.) are compared.

〔実施例３：オオムギＥＳＴ配列を利用した二倍体コムギ遺伝地図とオオムギ遺伝地図との比較〕
上記実施例１で構築した二倍体コムギ遺伝地図（連鎖地図）とオオムギＥＳＴマーカーが高密度にマップされたオオムギ遺伝地図（連鎖地図）との間で、マーカーの並びを比較した。

[Example 3: Comparison of diploid wheat genetic map and barley genetic map using barley EST sequence]
The alignment of the markers was compared between the diploid wheat genetic map (linkage map) constructed in Example 1 above and the barley genetic map (linkage map) to which barley EST markers were mapped at high density.

(1) Barley genetic map The barley genetic map used for the comparison was obtained from the brewing barley “Haruna Nijo” (Hordeum vulgare varieti Harunanijo) and wild barley “H602” (Hordeum vulgare ssp. About 3,000 barley EST markers are mapped using the obtained doubled haploid population, and the total distance of the map is 1,362.7 cM (Non-Patent Document 5: Nankuku N. et al. , Motoi Y., Ueki H., Sato K. and Takeda K. (2005) High density SNP based EST map in barley.Proc.Plant & Anima Genomes XIII Conference, p317.).

(2) Results In many linked groups of diploid wheat and barley congeners, the corresponding marker sequences were often the same. However, DNA markers corresponding to the DNA markers seated at the long arm ends of barley 5H and 4H chromosomes were seated at the long arm ends of 4A ^m chromosome and 5A ^m chromosome, respectively.

FIG. 2 shows the correspondence between markers seated on the genetic map of 4A ^m chromosome and 5A ^m chromosome of diploid wheat and markers seated on the barley genetic map. In FIG. 2, the top of each chromosome is the short arm end. The right side of each chromosome represents the marker name, and the left side represents the distance (cM) from the short arm end. From this result, as Devos et al. (Devos KM and Gale MD (2000) Genome relations: the glass model in current research. Plant Cell 12: 637-646.) Reported in the past, In diploid wheat and barley, it was clearly shown that an inversion translocation exists in homologous groups 4 and 5.

[Example 4: Examination of comprehensive genome typing]
We examined how many markers should be used to obtain highly accurate analysis results in comprehensive genome typing.

(1) Identification of a part derived from parents For one line of the recombinant inbred line (RIL) prepared in Example 1 described above, which part of the genome was derived from which parent was typed.

FIG. 3 shows which part is derived from which parent based on the genotype of each marker using 96 DNA markers selected from the entire range of the diploid wheat genetic map constructed in Example 1 above. The judgment result was shown. In FIG. 3, the top of each chromosome is the short arm end, and the distance (cM) from the short arm end of the DNA marker used is shown on the right side of each chromosome. Three to 1A ^m chromosome, four places in 2A ^m chromosome, four places in 3A ^m chromosome, three to 4A ^m chromosome, two places 5A ^m chromosome, 0 points to 6A ^m chromosome, the one place 7A ^m chromosome pairs There was a change.

FIG. 4 shows the results when the number of DNA markers used is 50, 25, and 14, compared with the case of using 96 DNA markers. In FIG. 4, the top of each chromosome is the short arm end, and the distance (cM) from the short arm end of the DNA marker used is shown on the right side of each chromosome. When the number of DNA markers was 50, recombination of a narrow region of the 1A ^m chromosome and 3A ^m chromosome could not be detected, but the same result was obtained as when 96 other recombination sites were used. On the other hand, when the number of DNA markers was 25, 3A ^m chromosome recombination could not be detected at all, and a decrease in accuracy was observed for other chromosomes. Moreover, when the number of DNA markers is 14, genotype identification is impossible.

(2) Examination of selection effect of QTL involved in dormancy after 0 weeks 1A Recombinant inbred strain prepared in Example 1 above for selection effect of QTL involved in dormancy after 0 weeks existing in ^m chromosome The number of DNA markers used was 96, 50, 25, and 14 for 40 lines of the line (RIL). Specifically, for each individual of 40 strains, the genotype of the used DNA marker was determined, and the dormancy of seeds obtained from each individual immediately after harvesting was investigated by the method described in Example 2 above. . Based on these results, the effect of selecting dormancy due to the genotype of QTL involved in dormancy after 0 weeks existing in the 1A ^m chromosome was evaluated. The genotype of the QTL was the DNA marker genotype closest to the QTL.

The results of 96, 50, 25 and 14 DNA markers are shown in FIGS. 5, 6, 7 and 8, respectively. FIG. 5 shows that when 96 DNA markers are used, an individual whose QTL genotype involved in dormancy after 0 weeks existing in 1A ^m chromosome is A has a deep seed dormancy of 17.1% and is selected. The effect was great. As shown in FIG. 6, when 50 DNA markers are used, an individual whose QTL genotype involved in dormancy after 0 weeks existing in 1A ^m chromosome is A has a deep seed dormancy of 10.9% and is selected. The effect was recognized. FIG. 7 shows that when 25 DNA markers are used, an individual whose QTL genotype involved in dormancy after 0 weeks existing in the 1A ^m chromosome is A has a deep seed dormancy of 5.9% and is selected. The effect was small. From FIG. 8, when 14 DNA markers are used, an individual whose QTL genotype involved in dormancy after 0 weeks existing in 1A ^m chromosome is A has a seed dormancy of -3.9% deeper, There was no selection effect. Therefore, it was confirmed that the selection effect was recognized for a specific trait by using 50 or more DNA markers.

  As described above, by selecting and using at least 50 DNA markers at an appropriate inter-marker distance from a diploid wheat genetic map covering the entire genome, it is possible to obtain highly accurate analysis results in comprehensive genome typing. It was confirmed that it can be obtained. Therefore, it was proved that the breeding target trait can be efficiently selected by the wheat breeding method according to the present invention. Furthermore, from the above results, according to the wheat breeding method according to the present invention, it is necessary to distinguish a genomic region necessary for breeding from a genomic region unnecessary for breeding (genomic region that is preferably excluded). It can be easily understood that selective breeding of an individual having a region and not having an unnecessary region can be performed efficiently.

  It should be noted that the specific embodiments or examples made in the best mode for carrying out the invention are merely to clarify the technical contents of the present invention, and are limited to such specific examples. The present invention should not be interpreted in a narrow sense, and various modifications can be made within the spirit of the present invention and the following claims.

  The wheat breeding method according to the present invention uses a diploid wheat genetic map prepared using a barley EST sequence. In the diploid wheat genetic map, 243 barley EST markers sit on the entire wheat A genome, and the distance between markers is 0 to 33.4 cM (average marker density: 3.0 cM / locus). Therefore, if a plurality of markers are selected from the entire genome at an appropriate distance between the markers, and the polymorphism of each marker is detected at the same time, the recombination information of the entire A genome can be obtained in one trial. .

  In addition, since the method for breeding wheat according to the present invention uses a diploid wheat genetic map prepared using a barley EST sequence, it is obtained by a high-density genetic map of barley constructed using a barley EST marker. The barley genome information can be efficiently used for wheat breeding. In addition, a highly efficient breeding system such as a DNA array developed based on the barley gene sequence can be used as it is.

  Furthermore, since the diploid wheat genetic map prepared by using the barley EST sequence is mapped with 16 kinds of useful agricultural traits QTL or main genes, 16 kinds of agriculturally useful traits are selected. The target wheat can be bred quickly and easily, and useful wheat varieties can be bred efficiently in a short period of time.

  According to the present invention, varieties useful for each agricultural trait can be bred by selecting traits of diploid wheat. In addition, since the genome of hexaploid wheat used to produce bread and noodles is highly homologous to the genome of diploid wheat, the traits and selection system identified in the present invention are also useful for breeding whole wheat. Can be used. In addition, a highly efficient breeding system such as a DNA array developed based on the barley gene sequence can be used as it is, and wheat can be bred based on the genome information of the barley. Therefore, it can be used for agriculture in general including crop production. Furthermore, it is effective in the food industry using wheat as a raw material.

Claims (20)

Breeding wheat including a step of amplifying DNA using a primer set designed based on barley EST sequence and using the genomic DNA of wheat as a template, and a step of detecting a polymorphism of the amplified DNA fragment A method,
The DNA fragment is a DNA marker mapped on a genetic map covering the entire genome of diploid wheat, and at least 50 or more DNA markers mapped on the genetic map are used. Wheat breeding method.   The breeding method according to claim 1, wherein the barley EST sequence is selected from the nucleotide sequences shown in SEQ ID NOs: 1 to 486.   The primer set is a combination of a primer consisting of the base sequence shown in SEQ ID NO: n (n is an odd number) and a primer consisting of the base sequence shown in SEQ ID NO: n + 1 among the base sequences shown in SEQ ID NOs: 487 to 1458. It is a primer set, The breeding method of Claim 1 characterized by the above-mentioned. The DNA marker comprising the DNA fragment according to at least one of the following (a) to (g) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the locus which concerns on a panicle length.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 587 and a primer consisting of the base sequence shown in SEQ ID NO: 588 are combined, or from the base sequence shown in SEQ ID NO: 589 A DNA fragment which is amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 590 and a primer consisting of the base sequence shown in SEQ ID NO: 590 are combined (b) a primer consisting of the base sequence shown in SEQ ID NO: 591 and the base shown in SEQ ID NO: 592 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 593 and a primer consisting of a base sequence shown in SEQ ID NO: 594 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 759 and a primer consisting of the base sequence shown in SEQ ID NO: 760 or the base sequence shown in SEQ ID NO: 761 A DNA fragment that is amplified using a primer set in which a primer comprising the nucleotide sequence shown in SEQ ID NO: 762 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 762 are combined (d) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 763 and SEQ ID NO: 764 A DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer set consisting of a primer consisting of a base sequence shown in SEQ ID NO: 765 and a primer consisting of a base sequence shown in SEQ ID NO: 766 Amplified using DNA fragment (e) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 or the base shown in SEQ ID NO: 949 A DNA fragment amplified using a primer set in which a primer consisting of the sequence and a primer consisting of the base sequence shown in SEQ ID NO: 950 are combined (f) a primer consisting of the base sequence shown in SEQ ID NO: 995 and shown in SEQ ID NO: 996 Primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence to be synthesized or a primer consisting of a base sequence shown in SEQ ID NO: 997 and a primer consisting of a base sequence shown in SEQ ID NO: 998 Amplify using DNA fragment (g) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 999 and a primer consisting of the base sequence shown in SEQ ID NO: 1000 or shown in SEQ ID NO: 1001 DNA fragment amplified using a primer set in which a primer comprising a base sequence to be combined with a primer comprising a base sequence represented by SEQ ID NO: 1002 The DNA marker comprising the DNA fragment according to at least one of the following (a) to (d) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the gene locus which concerns on a cocoon length.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 759 and a primer consisting of the base sequence shown in SEQ ID NO: 760 are combined, or from the base sequence shown in SEQ ID NO: 761 A DNA fragment that is amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 762 and a primer consisting of the base sequence shown in SEQ ID NO: 762 are (b) a primer consisting of the base sequence shown in SEQ ID NO: 763 and the base shown in SEQ ID NO: 764 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 765 and a primer consisting of a base sequence shown in SEQ ID NO: 766 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1235 and a primer consisting of the base sequence shown in SEQ ID NO: 1236 or the base sequence shown in SEQ ID NO: 1237 A DNA fragment (d) amplified using a primer set in which a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 1238 are combined (d) a primer consisting of the base sequence shown in SEQ ID NO: 1239 and SEQ ID NO: 1240 A primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer consisting of a base sequence shown in SEQ ID NO: 1241 and a primer consisting of a base sequence shown in SEQ ID NO: 1242 DNA fragments amplified using The DNA marker comprising the DNA fragment described in the following (a) in the at least 50 or more DNA markers,
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the locus which concerns on the number of spikelets.
(A) From a DNA fragment amplified using a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 or the base sequence shown in SEQ ID NO: 949 DNA fragment amplified using a primer set in which a primer comprising the nucleotide sequence shown in SEQ ID NO: 950 is combined The DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the locus which concerns on 1st internode length.
(A) From a DNA fragment amplified using a primer set that combines a primer consisting of the base sequence shown in SEQ ID NO: 939 and a primer consisting of the base sequence shown in SEQ ID NO: 940 or the base sequence shown in SEQ ID NO: 941 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 942 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 942 (b) A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 945 and a primer consisting of a base sequence shown in SEQ ID NO: 946 Amplified DN Fragment Among the at least 50 or more DNA markers, including a DNA marker comprising a DNA fragment according to at least one of the following (a) to (h):
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the locus which concerns on the length between cob nodes.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 503 and a primer consisting of the base sequence shown in SEQ ID NO: 504 are combined, or from the base sequence shown in SEQ ID NO: 505 A DNA fragment that is amplified using a primer set in which a primer comprising the nucleotide sequence represented by SEQ ID NO: 506 is combined with a primer comprising the nucleotide sequence represented by SEQ ID NO: 507 and the base represented by SEQ ID NO: 508 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 509 and a primer consisting of a base sequence shown in SEQ ID NO: 510 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1203 and a primer consisting of the base sequence shown in SEQ ID NO: 1204 or a base sequence shown in SEQ ID NO: 1205 A DNA fragment that is amplified using a primer set that combines a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 1206 (d) a primer consisting of the base sequence shown in SEQ ID NO: 1207 and that shown in SEQ ID NO: 1208 A primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer consisting of a base sequence shown in SEQ ID NO: 1209 and a primer consisting of a base sequence shown in SEQ ID NO: 1210 DNA fragment to be amplified using (e) DNA fragment or SEQ ID NO: amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1355 and a primer consisting of the base sequence shown in SEQ ID NO: 1356 A DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in 1357 and a primer consisting of the base sequence shown in SEQ ID NO: 1358 (f) a primer consisting of the base sequence shown in SEQ ID NO: 1359; A DNA fragment amplified using a primer set in combination with a primer consisting of the base sequence shown in SEQ ID NO: 1360, or a primer consisting of the base sequence shown in SEQ ID NO: 1361 and a primer consisting of the base sequence shown in SEQ ID NO: 1362 Combine DNA fragment amplified using the primer set (g) Amplified using a primer set that combines a primer consisting of the base sequence shown in SEQ ID NO: 1395 and a primer consisting of the base sequence shown in SEQ ID NO: 1396 DNA fragment or DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1397 and a primer consisting of the base sequence shown in SEQ ID NO: 1398 (h) a base shown in SEQ ID NO: 1399 A DNA fragment amplified using a primer set in which a primer consisting of a sequence and a primer consisting of a base sequence shown in SEQ ID NO: 1400 are combined, or a primer consisting of a base sequence shown in SEQ ID NO: 1401 and a base shown in SEQ ID NO: 1402 An array DNA fragments amplified using the primer set of a combination of a primer The DNA marker comprising the DNA fragment described in the following (a) among the at least 50 DNA markers,
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the gene locus which concerns on a heading date.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 are combined, or from the base sequence shown in SEQ ID NO: 949 DNA fragment amplified using a primer set in which a primer comprising the nucleotide sequence shown in SEQ ID NO: 950 is combined The DNA marker comprising the DNA fragment according to at least one of the following (a) to (d) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the gene locus which concerns on a cocoon length.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1115 and a primer consisting of the base sequence shown in SEQ ID NO: 1116 are combined, or from the base sequence shown in SEQ ID NO: 1117 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1118 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1118 (b) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1119 and a base shown in SEQ ID NO: 1120 A DNA fragment amplified using a primer set combined with a primer composed of a sequence or a primer set composed of a primer composed of a base sequence represented by SEQ ID NO: 1121 and a primer composed of a base sequence represented by SEQ ID NO: 1122 DNA fragment to be amplified (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1407 and a primer consisting of the base sequence shown in SEQ ID NO: 1408 or SEQ ID NO: 1409 A DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown and a primer consisting of the base sequence shown in SEQ ID NO: 1410; and (d) a primer consisting of the base sequence shown in SEQ ID NO: 1411 and SEQ ID NO: A DNA fragment amplified using a primer set in combination with a primer consisting of the base sequence shown in 1412 or a primer consisting of the base sequence shown in SEQ ID NO: 1413 and a primer consisting of the base sequence shown in SEQ ID NO: 1414 The DNA fragments amplified using the line-mer sets The DNA marker comprising the DNA fragment according to at least one of the following (a) to (d) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the gene locus which concerns on a guardian chief.
(A) From a DNA fragment amplified using a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: 943 and a primer consisting of the base sequence shown in SEQ ID NO: 944 or the base sequence shown in SEQ ID NO: 945 A DNA fragment that is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 946 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 946 (b) a primer consisting of the base sequence shown in SEQ ID NO: 947 and the base shown in SEQ ID NO: 948 A DNA fragment amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 949 and a primer consisting of a base sequence shown in SEQ ID NO: 950 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1371 and a primer consisting of the base sequence shown in SEQ ID NO: 1372 or a base sequence shown in SEQ ID NO: 1373 A DNA fragment which is amplified using a primer set in which a primer comprising the nucleotide sequence shown in SEQ ID NO: 1374 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1374 are combined (d) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1375 and SEQ ID NO: 1376 A primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer consisting of a base sequence shown in SEQ ID NO: 1377 and a primer consisting of a base sequence shown in SEQ ID NO: 1378 DNA fragments amplified using The DNA marker comprising the DNA fragment according to at least one of the following (a) to (c) among the at least 50 or more DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the locus which concerns on a grass type | mold.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 are combined, or from the base sequence shown in SEQ ID NO: 949 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 950 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 950 are combined (b) a primer consisting of the base sequence shown in SEQ ID NO: 1259 and the base shown in SEQ ID NO: 1260 A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 1261 and a primer consisting of a base sequence shown in SEQ ID NO: 1262 Amplified DNA fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1263 and a primer consisting of the base sequence shown in SEQ ID NO: 1264 or shown in SEQ ID NO: 1265 DNA fragment amplified using a primer set in which a primer consisting of a base sequence and a primer consisting of a base sequence shown in SEQ ID NO: 1266 are combined The DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the gene locus which concerns on a thousand grain weight.
(A) From a DNA fragment amplified using a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: 735 and a primer consisting of the base sequence shown in SEQ ID NO: 736, or from the base sequence shown in SEQ ID NO: 737 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 738 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 738 and a base consisting of the nucleotide sequence shown in SEQ ID NO: 740 A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 741 and a primer consisting of a base sequence shown in SEQ ID NO: 742 Amplified DN Fragment The DNA marker comprising the DNA fragment according to at least one of the following (a) to (d) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the locus which concerns on a cob fallenness.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1071 and a primer consisting of the base sequence shown in SEQ ID NO: 1072 are combined, or from the base sequence shown in SEQ ID NO: 1073 A DNA fragment that is amplified using a primer set in which a primer comprising the nucleotide sequence shown in SEQ ID NO: 1074 is combined (b) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1075 and the nucleotide sequence shown in SEQ ID NO: 1076 Using a primer set that combines a DNA fragment that is amplified using a primer set that is combined with a primer consisting of or a primer that consists of the base sequence shown in SEQ ID NO: 1077 and a primer that consists of the base sequence shown in SEQ ID NO: 1078 A DNA fragment to be amplified (c) a DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1371 and a primer consisting of the base sequence shown in SEQ ID NO: 1372, or SEQ ID NO: 1373 A DNA fragment amplified using a primer set comprising a primer consisting of the nucleotide sequence shown and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1374; and (d) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1375 and SEQ ID NO: A DNA fragment amplified using a primer set in combination with a primer consisting of the base sequence shown in 1376 or a primer consisting of the base sequence shown in SEQ ID NO: 1377 and a primer consisting of the base sequence shown in SEQ ID NO: 1378 Tap DNA fragments amplified using the timer set The DNA marker comprising the DNA fragment according to at least one of the following (a) to (f) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the locus which concerns on dormancy (after 0 weeks).
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 575 and a primer consisting of the base sequence shown in SEQ ID NO: 576 are combined, or from the base sequence shown in SEQ ID NO: 577 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 578 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 578 are combined (b) a primer consisting of the nucleotide sequence shown in SEQ ID NO: 579 and the base shown in SEQ ID NO: 580 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 581 and a primer consisting of a base sequence shown in SEQ ID NO: 582 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 835 and a primer consisting of the base sequence shown in SEQ ID NO: 836 or the base sequence shown in SEQ ID NO: 837 A DNA fragment that is amplified using a primer set that combines a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 838 (d) a primer consisting of the base sequence shown in SEQ ID NO: 839 and that shown in SEQ ID NO: 840 A DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer set consisting of a primer consisting of a base sequence shown in SEQ ID NO: 841 and a primer consisting of a base sequence shown in SEQ ID NO: 842 Amplified using DNA fragment (e) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1123 and a primer consisting of the base sequence shown in SEQ ID NO: 1124 or a base shown in SEQ ID NO: 1125 A DNA fragment amplified using a primer set in which a primer consisting of the sequence and a primer consisting of the base sequence shown in SEQ ID NO: 1126 are combined (f) a primer consisting of the base sequence shown in SEQ ID NO: 1127 and a primer consisting of the base sequence shown in SEQ ID NO: 1128 A DNA fragment that is amplified using a primer set that is combined with a primer consisting of a base sequence that is combined with a primer that consists of a base sequence shown in SEQ ID NO: 1129 and a primer that consists of a base sequence shown in SEQ ID NO: 1130 DNA fragments amplified using The DNA marker comprising the DNA fragment according to at least one of the following (a) to (f) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the locus which concerns on dormancy (after 3 weeks).
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 591 and a primer consisting of the base sequence shown in SEQ ID NO: 592 are combined, or from the base sequence shown in SEQ ID NO: 593 A DNA fragment that is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 594 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 594 (b) a primer consisting of the base sequence shown in SEQ ID NO: 595 and the base shown in SEQ ID NO: 596 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 597 and a primer consisting of a base sequence shown in SEQ ID NO: 598 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 627 and a primer consisting of the base sequence shown in SEQ ID NO: 628 or the base sequence shown in SEQ ID NO: 629 A DNA fragment amplified using a primer set in which a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 630 are combined (d) a primer consisting of the base sequence shown in SEQ ID NO: 631 and shown in SEQ ID NO: 632 A primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence or a primer consisting of a base sequence shown in SEQ ID NO: 633 and a primer consisting of a base sequence shown in SEQ ID NO: 634 Amplified using DNA fragment (e) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 903 and a primer consisting of the base sequence shown in SEQ ID NO: 904 or the base shown in SEQ ID NO: 905 A DNA fragment amplified using a primer set in which a primer consisting of a sequence and a primer consisting of a base sequence shown in SEQ ID NO: 906 are combined (f) a primer consisting of a base sequence shown in SEQ ID NO: 907 and shown in SEQ ID NO: 908 Primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence to be synthesized or a primer consisting of a base sequence shown in SEQ ID NO: 909 and a primer consisting of a base sequence shown in SEQ ID NO: 910 Amplify using DNA fragment The DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the gene which controls glue paste layer color.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1055 and a primer consisting of the base sequence shown in SEQ ID NO: 1056 are combined, or from the base sequence shown in SEQ ID NO: 1057 A DNA fragment that is amplified using a primer set in which a primer comprising the nucleotide sequence represented by SEQ ID NO: 1058 is combined with a primer comprising the nucleotide sequence represented by SEQ ID NO: 1059 and a base represented by SEQ ID NO: 1060 A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 1061 and a primer consisting of a base sequence shown in SEQ ID NO: 1062 Amplified the DNA fragments The DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the gene which controls a leaf blade.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1247 and a primer consisting of the base sequence shown in SEQ ID NO: 1248 are combined, or from the base sequence shown in SEQ ID NO: 1249 A DNA fragment which is amplified using a primer set in which a primer comprising the nucleotide sequence represented by SEQ ID NO: 1250 is combined with a primer comprising the nucleotide sequence represented by SEQ ID NO: 1250; and (b) a primer comprising the nucleotide sequence represented by SEQ ID NO: 1251 and the base represented by SEQ ID NO: 1252 A DNA fragment that is amplified using a primer set that combines a primer consisting of a sequence or a primer set that combines a primer consisting of a base sequence shown in SEQ ID NO: 1253 and a primer consisting of a base sequence shown in SEQ ID NO: 1254 Amplified the DNA fragments The DNA marker comprising the DNA fragment according to at least one of the following (a) and (b) among the at least 50 DNA markers:
Furthermore, the breeding method of Claim 1 including the process of determining the genotype of the gene which controls the autumn seeding degree.
(A) From a DNA fragment amplified using a primer set in which a primer consisting of the base sequence shown in SEQ ID NO: 1255 and a primer consisting of the base sequence shown in SEQ ID NO: 1256 is combined, or from the base sequence shown in SEQ ID NO: 1257 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1258 and a primer consisting of the nucleotide sequence shown in SEQ ID NO: 1258 are combined. A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 1261 and a primer consisting of a base sequence shown in SEQ ID NO: 1262 Amplified the DNA fragments Among the at least 50 or more DNA markers, including a DNA marker comprising a DNA fragment described in the following (a) to (g),
Further, the method includes a step of determining genotypes of loci involved in dormancy (after 0 weeks), dormancy (after 3 weeks), heading date, 1000 grain weight, panicle length, number of spikelets and plant type. The breeding method according to claim 3.
(A) From a DNA fragment amplified using a primer set combining a primer consisting of the base sequence shown in SEQ ID NO: 735 and a primer consisting of the base sequence shown in SEQ ID NO: 736, or from the base sequence shown in SEQ ID NO: 737 A DNA fragment which is amplified using a primer set in which a primer consisting of the nucleotide sequence shown in SEQ ID NO: 738 is combined with a primer consisting of the nucleotide sequence shown in SEQ ID NO: 738 and a base consisting of the nucleotide sequence shown in SEQ ID NO: 740 A DNA fragment amplified using a primer set combined with a primer consisting of a sequence or a primer set combining a primer consisting of a base sequence shown in SEQ ID NO: 741 and a primer consisting of a base sequence shown in SEQ ID NO: 742 Amplified DN Fragment (c) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 903 and a primer consisting of the base sequence shown in SEQ ID NO: 904 or the base sequence shown in SEQ ID NO: 905 A DNA fragment (d) amplified using a primer set in which a primer consisting of the above and a primer consisting of the base sequence shown in SEQ ID NO: 906 are combined, and a primer consisting of the base sequence shown in SEQ ID NO: 907 and shown in SEQ ID NO: 908 A DNA fragment amplified using a primer set combined with a primer consisting of a base sequence, or a primer set consisting of a primer consisting of a base sequence shown in SEQ ID NO: 909 and a primer consisting of a base sequence shown in SEQ ID NO: 910 Amplified using DNA fragment (e) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 947 and a primer consisting of the base sequence shown in SEQ ID NO: 948 or the base shown in SEQ ID NO: 949 A DNA fragment amplified using a primer set in which a primer consisting of the sequence and a primer consisting of the base sequence shown in SEQ ID NO: 950 are combined (f) a primer consisting of the base sequence shown in SEQ ID NO: 1123 and shown in SEQ ID NO: 1124 Primer set combining a DNA fragment amplified using a primer set combined with a primer consisting of a base sequence to be synthesized or a primer consisting of a base sequence shown in SEQ ID NO: 1125 and a primer consisting of a base sequence shown in SEQ ID NO: 1126 For DNA fragment to be amplified (g) DNA fragment amplified using a primer set comprising a primer consisting of the base sequence shown in SEQ ID NO: 1127 and a primer consisting of the base sequence shown in SEQ ID NO: 1128 or SEQ ID NO: 1129 A DNA fragment amplified using a primer set comprising a primer consisting of the nucleotide sequence shown in FIG.

Images