A rapid assisted breeding method for high-amylose durum wheat based on SNP markers

Abstract

The present invention discloses a method for rapid, assisted breeding of high-amylose durum wheat based on SNP markers. The method employs highly specific SNP markers based on allele-specific PCR using unequal-length primers. Under a defined PCR reaction system and PCR amplification program, mutation sites and non-mutation sites are distinguished and identified based on agarose gel electrophoresis-stained bands. Single nucleotide polymorphisms at one or more specific SNP sites in the tested plants are measured to enable rapid, assisted breeding of high-amylose durum wheat. The present invention successfully develops co-dominant SNP markers based on allele-specific PCR using unequal-length primers for efficient molecular marker-assisted breeding of high-amylose durum wheat, addressing the need for rapid breeding of high-amylose durum wheat.

Description

Technical Field

[0001] The present invention relates to the field of wheat molecular breeding, and in particular to the technical field related to functional wheat molecular breeding. Background Art

[0002] Resistant starch has the potential to prevent type 2 diabetes, obesity, cardiovascular disease, and intestinal diseases. As people become more aware of their health, the demand for foods rich in resistant starch continues to grow. The amylose content in wheat flour is significantly positively correlated with the resistant starch content. While high-amylose wheat has been extensively researched in the United States, the United Kingdom, Australia, and Japan, China lacks high-amylose wheat germplasm resources, and there are no reports on the innovation of high-amylose wheat germplasm or the breeding of new varieties.

[0003] Durum wheat accounts for approximately 10% of the global wheat planting area and is primarily used for making pasta, couscous, and flatbread. Compared to common wheat, durum wheat exhibits improved stress tolerance and is high in protein, lysine, carotene, and trace elements. Therefore, increasing the amylose and resistant starch content of durum wheat, creating new high-amylose durum wheat germplasm, and breeding new durum wheat varieties are of great significance for enriching people's diets, increasing wheat production enthusiasm, and improving public health.

[0004] Uauy et al. (2009) screened out a mutant from the spring durum wheat (Kronos) EMS mutant library. SBEIIa-A (G401A), SBEIIa-B (G1347A), SBEIIb-A (G308A) and SBEIIb-B (C1290T) mutant. Each mutant SBEIIa or SBEIIb Only one SNP site in the gene mutated. Through backcrossing and direct sequencing technology, Uauy et al. created a high-amylose durum wheat (PI670160) with mutations in all four SNP sites of the gene. When using durum wheat (PI670160) as a parent, high-amylose durum wheat breeding was carried out because its high amylose trait was caused by SBEIIa-A 、 SBEIIa-B 、 SBEIIb-A and SBEIIb-B The point mutations of the four genes determine SBEIIa and SBEIIbThe development of SNP molecular markers for SNP mutation sites has become crucial for breeding high-amylose durum wheat. Currently, SNP detection methods primarily include classic gel electrophoresis-based methods, such as single-strand conformation polymorphism (SSCP), allele-specific PCR (AS-PCR), and enzyme-amplified polymorphism markers (CAPS), and high-throughput, highly automated methods, such as denaturing high-performance liquid chromatography (DHPLC), mass spectrometry (MS), high-resolution melting analysis (HRM), and competitive allele-specific PCR (KASP). The former are complex, costly, and inaccurate, while the latter require expensive equipment and reagents.

[0005] Unequal length primer allele-specific PCR primers are an improved SNP marker based on the principle of allele-specific PCR. They only require a common PCR instrument and agarose gel electrophoresis and are suitable for small and medium-sized laboratories. Unequal length primer allele-specific PCR is a co-dominant marker with the characteristics of simple operation, low cost and easy differentiation. Therefore, a novel allele-specific PCR primer based on durum wheat (PI670160) was developed. SBEIIa-A 、 SBEIIa-B 、 SBEIIb-A and SBEIIb-B Allele-specific PCR primers (SNP markers) with unequal lengths targeting point mutations in four genes have high application value for small and medium-sized laboratories in breeding high-amylose durum wheat, thereby promoting high-amylose durum wheat breeding in my country. However, durum wheat is a partially homeotic allotetraploid, and the development of SNP markers for homeotic genes in durum wheat, particularly those suitable for small and medium-sized laboratories, is highly challenging and has yet to be reported. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide SNP marker related materials and a rapid assisted breeding method for high-amylose durum wheat based on SNP markers.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows.

[0008] A kit for rapid assisted breeding of high-amylose durum wheat is disclosed. The kit performs rapid assisted breeding of high-amylose durum wheat by detecting single nucleotide polymorphisms at one or more specific SNP sites in test plants. The kit comprises a primer pair consisting of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in the sequence listing, and / or a primer pair consisting of SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 in the sequence listing, and / or a primer pair consisting of SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9 in the sequence listing, and / or a primer pair consisting of SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12 in the sequence listing.

[0009] A co-dominant primer pair is used for rapid assisted breeding of high-amylose durum wheat, comprising a primer pair consisting of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in a sequence listing.

[0010] A co-dominant primer pair is used for rapid assisted breeding of high-amylose durum wheat, comprising a primer pair consisting of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing.

[0011] A co-dominant primer pair is used for rapid assisted breeding of high-amylose durum wheat, comprising a primer pair consisting of SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 in the sequence listing.

[0012] A co-dominant primer pair is used for rapid assisted breeding of high-amylose durum wheat, comprising a primer pair consisting of SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing.

[0013] The above kit and primer pair are used to identify or assist in identifying the amylose content trait of durum wheat.

[0014] A molecular marker-assisted breeding method for high-amylose durum wheat uses highly specific SNP markers based on allele-specific PCR using unequal-length primers. Under a set PCR reaction system and PCR amplification program, mutation sites and non-mutation sites are distinguished and identified based on agarose gel electrophoresis staining bands. The single nucleotide polymorphisms of one or more groups of specific SNP sites in the test plants are measured to conduct rapid assisted breeding of high-amylose durum wheat.

[0015] As a preferred technical solution of the present invention, the PCR reaction system and PCR amplification procedure include:

[0016] (1) SBEIIa-A For SNP sites, 10 μL of 2× Taq PCR StarMix (GenStar) was used, along with 1 μL of each of the primers SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 (10 μmol / L), 1 μL of template genomic DNA (50–100 ng), and 6 μL of ddH2O. The PCR amplification program was as follows: 94°C initial denaturation for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 20 s, followed by extension at 72°C for 5 min. The PCR amplification products were detected by 3% agarose gel electrophoresis in 1× TAE buffer.

[0017] (2) SBEIIa-B For SNP sites, 10 μL of 2× Taq PCR StarMix (GenStar) was used, along with 1 μL of each of the primers SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 (10 μmol / L), 1 μL of template genomic DNA (50-100 ng), and 6 μL of ddH2O. The PCR amplification program was as follows: 94°C initial denaturation for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 20 s, followed by extension at 72°C for 5 min. The PCR amplification products were detected by 3% agarose gel electrophoresis in 1× TAE buffer.

[0018] (3) SBEIIb-A SNP sites: 2× Taq PCR StarMix (GenStar) 10 μL, primers SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9 (10 μmol / L), 1 μL each, template genomic DNA 1 μL (50-100 ng), ddH2O 6 μL; PCR amplification program: 94°C pre-denaturation 5 min; 94°C denaturation 30 s, 55°C annealing 30 s, 72°C extension 20 s, 30 cycles; 72°C extension 5 min; PCR amplification products were detected by 3% agarose gel electrophoresis in 1× TAE buffer.

[0019] (4) SBEIIb-BFor SNP sites, 10 μL of 2× Taq PCR StarMix (GenStar) was added, 1 μL of each of the primers SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 (10 μmol / L), 1 μL of template genomic DNA (50-100 ng), and 6 μL of ddH2O. The PCR amplification program was as follows: pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 20 s; and extension at 72°C for 5 min. PCR amplification products were detected by 3% agarose gel electrophoresis in 1× TAE buffer.

[0020] As a preferred technical solution of the present invention, the PCR amplification products are stained with ethidium bromide to identify four groups of co-dominant amplification bands:

[0021] (1) SBEIIa-A SNP sites: 164 bp and 143 bp;

[0022] (2) SBEIIa-B SNP sites: 180 bp and 159 bp;

[0023] (3) SBEIIb-A SNP sites: 193 bp and 172 bp;

[0024] (4) SBEIIb-B SNP sites: 250 bp and 229 bp;

[0025] This high specificity is achieved SBEIIa and SBEIIb The four SNP sites were identified by differentiating homozygous mutations, wild-type homozygous mutations, and heterozygous sites, and rapid molecular marker-assisted breeding of high-amylose durum wheat was carried out.

[0026] The beneficial effect of the above technical solution is that the present invention has successfully developed a co-dominant SNP marker based on unequal length primer allele-specific PCR for efficient molecular marker-assisted breeding of high-amylose durum wheat. SBEIIa and SBEIIb Four SNPs were identified to differentiate homozygous mutations, wild-type homozygosity, and heterozygous sites. Based on this, four pairs of SNP markers can be used to efficiently carry out molecular marker-assisted breeding of high-amylose durum wheat, providing a new breeding tool for the rapid selection of high-amylose durum wheat and addressing the need for rapid breeding of high-amylose durum wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Schematic diagram of SNP marker design based on allele-specific PCR with unequal-length primers for high-amylose durum wheat (PI670160) in Example 1; in the figure: A, SBEIIa-A SNP marker; B, SBEIIa-B SNP marker; C, SBEIIb-A SNP marker; D, SBEIIb-B SNP markers; * bold capital letters indicate point mutation sites; lowercase letters indicate mismatched bases introduced by primer design.

[0028] Figure 2 The high amylose durum wheat (PI670160) in Example 1 SBEIIa and SBEIIb Allele PCR amplification map; in the figure: A, [[ID= SNP marker; B, ​ SNP marker; C, ​ SNP marker; D, ​ SNP marker; M, 50 bp DNA marker (band lengths from top to bottom are 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp, and 50 bp); 1, Shiluan 02-1; 2, Jimai 22; 3, Heng 4399; 4, Kronos; 5, AS2363; 6, Shijiazhuang No. 11; 7-9, PI670160; 7, ddH2O.

[0029] ​ This is a verification diagram of the SNP marker of the present invention in Example 2 in the F2 population; in the figure: A, ​ SNP marker; B, ​ SNP marker; C, ​ SNP marker; D, ​ SNP marker; M, 50 bp DNA marker (band lengths from top to bottom are 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp, and 50 bp); 1-17, F2 generation individual plants of Shijiazhuang No. 11 × PI670160; 18, PI670160; 19, ddH2O. DETAILED DESCRIPTION

[0030] The present invention is described in detail in the following examples. The various raw materials and equipment used in the present invention are conventional commercial products and can be directly purchased from the market.

[0031] In the following description of the embodiments, specific details such as specific system structures and technologies are provided for illustration rather than limitation to facilitate a thorough understanding of the embodiments of the present application. However, it should be clear to those skilled in the art that the present application may be implemented in other embodiments without these specific details. In other cases, detailed descriptions of well-known systems, devices, and methods are omitted to avoid obscuring the description of the present application with unnecessary details.

[0032] It should be understood that when used in the present specification and the appended claims, the term "comprising" indicates the presence of the described features, wholes, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components and / or their collections. It should also be understood that the term "and / or" used in the present specification and the appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations. In addition, in the description of the present specification and the appended claims, the terms "first", "second", "third", etc. are only used to distinguish the description and are not to be understood as indicating or implying relative importance.

[0033] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in one or more embodiments of the present application. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in other embodiments" appearing in various places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more but not all embodiments," unless otherwise specifically emphasized. The terms "including," "comprising," "having," and variations thereof all mean "including but not limited to," unless otherwise specifically emphasized.

[0034] Example 1. Development of SNP markers based on allele-specific PCR using unequal-length primers in high-amylose durum wheat

[0035] 1.1 Test materials

[0036] The test materials included three durum wheat lines: Kronos, AS2363, and Shijiazhuang No. 11; high-amylose durum wheat lines (PI670160): Selection 1, Selection 2, and Selection 3; and three common wheat lines: Shiluan 02-1, Jimai 22, and Heng 4399. The test materials were collected and preserved by the Molecular Breeding Laboratory of the Shijiazhuang Academy of Agriculture and Forestry Sciences.

[0037] 1.2 Development of SNP marker ASA

[0038] based on ​A co-dominant SNP marker ASA based on allele-specific PCR with unequal-length primers was developed for the SNP locus.

[0039] Primer sequences: ASA-F 5'-CAACTAGCTTACTGGACTTACAACT-3' (SEQ ID NO. 1), ASA-TR 5'- ​ TCATAGTATCTGCATTCGGATTGT-3' (SEQ ID NO. 2), ASA-CR 5'-TCATAGTATCTGCATTCGGATACC-3' (SEQ ID NO. 3).

[0040] Primers were synthesized by Invitrogen. PCR reactions were performed on a Biometra T-Gradient Thermoblock. The PCR reaction system (20 μL) consisted of 10 μL of 2× Taq PCR StarMix (GenStar), 1 μL of each of the primers SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 (10 μmol / L), 1 μL of template genomic DNA (50–100 ng), and 6 μL of ddH2O. The PCR amplification protocol was as follows: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 20 s; and extension at 72°C for 5 min. PCR products were detected by electrophoresis on 3% agarose gels in 1× TAE buffer and visualized with ethidium bromide staining.

[0041] PCR amplification results showed that the SNP marker ASA was a single band in the wild type durum wheat (Kronos), Shijiazhuang No. 11 and the durum wheat mutant (PI670160), with high specificity ( ​ A). The results of PCR product cloning and sequencing showed that the amplified fragment of SNP marker ASA in durum wheat wild type (Kronos) and Shijiazhuang No. 11 was 143 bp long and had a close relationship with gene TraesCS2A02G293400 ( ​ ). The amplified fragment in the durum wheat mutant (PI670160) was 164 bp long, consistent with the expected sequence. These results indicate that the SNP marker ASA can effectively distinguish durum wheat from the durum wheat mutant (PI670160).

[0042] 1.3 Development of SNP-marked ASB

[0043] based on ​ A co-dominant SNP marker ASB based on allele-specific PCR using unequal-length primers was developed for the SNP loci.

[0044] Primer sequences: ASB-F 5'-CAACCAACCATGGTGATGTG-3' (SEQ ID NO. 4), ASB-TR 5'- ​ ​ GGATGTTGGAAGACATACTTCTGT-3' (SEQ ID NO. 5), ASB-CR 5'-GGATGTTGGAAGACATACTTCACC-3' (SEQ ID NO. 6).

[0045] Primers were synthesized by Invitrogen. PCR reactions were performed on a Biometra T-Gradient Thermoblock. The PCR reaction system (20 μL) consisted of 10 μL of 2× Taq PCR StarMix (GenStar), 1 μL of each of the primers SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 (10 μmol / L), 1 μL of template genomic DNA (50–100 ng), and 6 μL of ddH2O. The PCR amplification protocol was as follows: 94°C initial denaturation for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 20 s; and extension at 72°C for 5 min. PCR products were detected by electrophoresis on 3% agarose gels in 1× TAE buffer and visualized with ethidium bromide staining.

[0046] PCR amplification results showed that the SNP marker BSA was a single band in the wild type durum wheat (Kronos), Shijiazhuang No. 11 and the durum wheat mutant (PI670160), with high specificity ( ​ B). The results of PCR product cloning and sequencing showed that the amplified fragment of SNP marker BSA in wild type durum wheat (Kronos) and Shijiazhuang No. 11 was 159 bp long and had a close affinity with gene TraesCS2B02G309500 ( ​ ). The amplified fragment in the durum wheat mutant (PI670160) was 180 bp long, consistent with the expected sequence. These results indicate that the SNP marker BSA can effectively distinguish durum wheat from the durum wheat mutant (PI670160).

[0047] 1.4 Development of SNP-labeled BSA

[0048] based on ​ A co-dominant SNP marker BSA based on allele-specific PCR using primers of unequal lengths was developed for the SNP loci.

[0049] Primer sequences: BSA-AF 5'-AAGGTAACGCCAGGGTTTTCCGTTTGGATT TGTGCGCAGA-3' (SEQ ID NO.7), BSA-GF 5'-GTTTGGATTTGTGCGCTGG-3' (SEQ ID NO.8), BSA-R 5'-TACTTAGGTAAACGATCAGACAGTC-3' (SEQ ID NO.9).

[0050] Primers were synthesized by Invitrogen. PCR reactions were performed on a Biometra T-Gradient Thermoblock. The PCR reaction system (20 μL) consisted of 10 μL of 2× Taq PCR StarMix (GenStar), 1 μL of each of the primers SEQ ID NO. 7, SEQ ID NO. 8, and SEQ ID NO. 9 (10 μmol / L), 1 μL of template genomic DNA (50–100 ng), and 6 μL of ddH2O. The PCR amplification protocol was as follows: initial denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 20 s; and extension at 72°C for 5 min. PCR products were detected by electrophoresis on 3% agarose gels in 1× TAE buffer and visualized with ethidium bromide staining.

[0051] PCR amplification results showed that the SNP marker BSA was a single band in the wild type durum wheat (Kronos), Shijiazhuang No. 11 and the durum wheat mutant (PI670160), with high specificity ( ​ C). The results of PCR product cloning and sequencing showed that the amplified fragment of SNP marker BSA in wild type durum wheat (Kronos) and Shijiazhuang No. 11 was 172 bp long and had a close affinity with gene TraesCS2A02G310300 ( ​ ). The amplified fragment in the durum wheat mutant (PI670160) was 193 bp long, consistent with the expected sequence. These results indicate that the SNP marker BSA can effectively distinguish durum wheat from the durum wheat mutant (PI670160).

[0052] 1.5 Development of SNP-marked BSBs

[0053] based on ​ A co-dominant SNP marker BSB was developed based on allele-specific PCR using primers of unequal lengths.

[0054] Primer sequences: BSB-F 5'-GTTTTCAGAATGACCTCGGTG-3' (SEQ ID NO. 10), BSB-AR 5'- A ​ CGGAGTACTTGATCCAAGCTA-3' (SEQ ID NO. 11), BSB-GR 5'-CGGAGTACTTGATCCAAGGAG-3' (SEQ ID NO. 12).

[0055] Primers were synthesized by Invitrogen. PCR reactions were performed on a Biometra T-Gradient Thermoblock. The PCR reaction system (20 μL) consisted of 10 μL of 2× Taq PCR StarMix (GenStar), 1 μL of each of the primers SEQ ID NO. 10, SEQ ID NO. 11, and SEQ ID NO. 12 (10 μmol / L), 1 μL of template genomic DNA (50–100 ng), and 6 μL of ddH2O. The PCR amplification protocol was as follows: 94°C initial denaturation for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 20 s; and extension at 72°C for 5 min. PCR products were detected by electrophoresis on 3% agarose gels in 1× TAE buffer and visualized with ethidium bromide staining.

[0056] PCR amplification results showed that the SNP marker BSB was a single band in the wild type durum wheat (Kronos), Shijiazhuang No. 11 and the durum wheat mutant (PI670160), with high specificity ( ​ D). The results of PCR product cloning and sequencing showed that the amplified fragment of SNP marker BSB in durum wheat wild type (Kronos) and Shijiazhuang No. 11 was 229 bp long and had a close relationship with gene TraesCS2A02G327300 ( ​ ). The amplified fragment in the durum wheat mutant (PI670160) was 250 bp long, consistent with the expected sequence. These results indicate that the SNP marker BSB can effectively distinguish durum wheat from the durum wheat mutant (PI670160).

[0057] Example 2: Validation of SNP Markers

[0058] In natural populations, SNP markers ASA, ASB, BSA, and BSB can effectively distinguish hexaploid wheat, tetraploid durum wheat, and high-amylose durum wheat PI670160 ( ​). To test the effectiveness of SNP markers ASA, ASB, BSA and BSB, we performed PCR detection on F2 plants of the hybrid combination of durum wheat Shijiazhuang No. 11 (wild type) and durum wheat PI670160 (mutant). PCR amplification results showed that SNP markers ASA, ASB, BSA and BSB can effectively distinguish wild type, wild mutant heterozygous type and mutant type in the F2 segregating population of Shijiazhuang No. 11 and durum wheat PI670160 ( ​ In the F2 progeny population (90 plants), the segregation ratios for wild-type plants, wild-mutation heterozygous plants, and mutant plants were: 23:40:27 for markers ASA and BSA (P=0.738), 18:45:27 for marker ASB (P=0.630), and 17:42:31 for marker BSB (P=0.336). The chi-square test confirmed the segregation pattern of single genes (1:2:1). These results indicate that markers ASA, ASB, BSA, and BSB are codominant SNP markers and can be used for molecular marker-assisted breeding of high-amylose durum wheat.

[0059] In the above embodiments, the description of each embodiment has its own focus. For parts that are not described or recorded in detail in a certain embodiment, reference can be made to the relevant description of other embodiments.

[0060] Based on the above examples, it can be seen that the present invention provides a method for breeding high-amylose durum wheat as a breeding parent, and provides four pairs of ​ and ​ SNP markers (co-dominant) based on allele-specific PCR with unequal-length primers were developed at four SNP mutation sites, and a method for molecular marker-assisted breeding of high-amylose durum wheat based on the four SNP markers.

[0061] The embodiments described above are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention, and should all be included in the scope of protection of the present invention. Sequence Listing <110> Shijiazhuang Academy of Agriculture and Forestry Sciences <120> A rapid assisted breeding method for high-amylose durum wheat based on SNP markers <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 25 <212> DNA <213> Common wheat (Triticum aestivum L) <400> 1 caactagctt actggactta caact 25 <210> 2 <211> 45 <212> DNA <213> Artificial Sequence <400> 2 aaggtaacgc cagggttttc ctcatagtat ctgcattcgg attgt 45 <210> 3 <211> twenty four <212> DNA <213> Artificial Sequence <400> 3 tcatagtatc tgcattcgga tacc 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 caaccaacca tggtgatgtg 20 <210> 5 <211> 45 <212> DNA <213> Artificial Sequence <400> 5 aaggtaacgc cagggttttc cggatgttgg aagacatact tctgt 45 <210> 6 <211> twenty four <212> DNA <213> Artificial Sequence <400> 6 ggatgttgga agacatactt cacc 24 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <400> 7 aaggtaacgc cagggttttc cgtttggatt tgtgcgcaga 40 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <400> 8 gtttggattt gtgcgctgg 19 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <400> 9 tacttaggta aacgatcaga cagtc 25 <210> 10 <211> twenty one <212> DNA <213> Artificial Sequence <400> 10 gttttcagaa tgacctcggt g 21 <210> 11 <211> 42 <212> DNA <213> Artificial Sequence <400> 11 aaggtaacgc cagggttttc ccggagtact tgatccaagc ta 42 <210> 12 <211> twenty one <212> DNA <213> Artificial Sequence <400> 12 cggagtactt gatccaagga g 21

Claims

1. A kit for rapid assisted breeding of high-amylose durum wheat, wherein the kit detects one or more groups of single nucleotide polymorphisms at specific SNP sites in test plants to perform rapid assisted breeding of high-amylose durum wheat; the kit comprises a primer pair consisting of SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 in the sequence listing, a primer pair consisting of SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 in the sequence listing, a primer pair consisting of SEQ ID NO.7, SEQ ID NO.8, and SEQ ID NO.9 in the sequence listing, and a primer pair consisting of SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12 in the sequence listing; the high-amylose durum wheat is durum wheat PI670160; and the kit is used to distinguish durum wheat from a durum wheat mutant PI670160.

2. A co-dominant primer pair, used for rapid assisted breeding of high-amylose durum wheat, comprising a primer pair consisting of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 in the sequence listing; the high-amylose durum wheat is durum wheat PI670160; the primer pair is used to distinguish durum wheat from the durum wheat mutant PI670160.

3. A co-dominant primer pair, used for rapid assisted breeding of high-amylose durum wheat, comprising a primer pair consisting of SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 in the sequence listing; the high-amylose durum wheat is durum wheat PI670160; the primer pair is used to distinguish durum wheat from the durum wheat mutant PI670160.

4. A co-dominant primer pair, used for rapid assisted breeding of high-amylose durum wheat, comprising a primer pair consisting of SEQ ID NO.7, SEQ ID NO.8 and SEQ ID NO.9 in the sequence listing; the high-amylose durum wheat is durum wheat PI670160; the primer pair is used to distinguish durum wheat from the durum wheat mutant PI670160.

5. A co-dominant primer pair, used for rapid assisted breeding of high-amylose durum wheat, comprising a primer pair consisting of SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 in the sequence listing; the high-amylose durum wheat is durum wheat PI670160; the primer pair is used to distinguish durum wheat from the durum wheat mutant PI670160.

Images