Molecular markers tightly linked to the effective tiller number QTL QPTN.sau-4B in wheat and their application

Abstract

The present invention discloses a molecular marker tightly linked to the wheat effective tiller number QTL QPTN.sau‑4B and its application, belonging to the technical field of wheat molecular breeding. The molecular marker KASP‑7 and the wheat effective tiller number QTL QPTN.sau‑4B are co-located on the short arm of wheat chromosome 4B and are located within the QPTN.sau‑4B interval; the molecular marker KASP‑7 is located at position 32030698 of chromosome 4B using the Chinese Spring v2.1 genome as a reference genome, and its polymorphism is C / T. The SNP molecular marker KASP‑7 linked to the wheat effective tiller number QTL QPTN.sau‑4B of the present invention is tightly linked to the wheat effective tiller number QTL, can be used to assist in the detection of QTL and molecular breeding of multi-tillering and high-yield wheat, and improve detection efficiency and breeding efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of wheat molecular breeding, in particular to a molecular marker tightly linked to wheat effective tiller number QTL QPTN.sau-4B and an application thereof. Background Art

[0002] Common wheat (Triticum aestivum L.) is a major grass crop that provides approximately 20% of human dietary calories. Wheat yield has steadily increased in recent years, while sown area has decreased. This trend has heightened the urgency of understanding the genetic mechanisms controlling wheat yield. Wheat yield is determined by the number of spikelets per unit area, the number of spikelets per spikelet, and 1000-grain weight. These quantitative traits are difficult to accurately select for in traditional breeding, resulting in slow breeding progress. However, the application of molecular marker-assisted selection (MAS) has significantly accelerated breeding efforts. Quantitative traits are influenced by both genetic and environmental factors, but under stable environmental conditions, genetic factors play a dominant role. Because quantitative traits exhibit dosage effects—that is, gene dosage is proportional to phenotypic expression—enriching for target trait loci related to yield is a key strategy for improving yield in wheat breeding. Utilizing molecular markers linked to target traits can significantly improve selection efficiency, thereby achieving both yield and quality improvements in wheat breeding.

[0003] Tillering, a branching phenomenon unique to monocotyledons, is of great importance to many crops, including wheat and rice. In wheat, tillering is also a key agronomic trait that influences adaptability to the growing environment. The number of effective tillers, which refers to the number of spikes that ultimately form ears, directly affects the number of spikes per hectare, ultimately impacting wheat grain yield. Furthermore, effective tiller number plays a role in shaping plant architecture by influencing other agronomic traits, such as plant height and flowering time. Therefore, the use of molecular markers linked to effective tiller number is of great significance for optimizing wheat plant architecture and increasing grain yield.

[0004] Crops exhibit polymorphism across varieties. Genotypic polymorphism in plants primarily includes single nucleotide polymorphisms (SNPs), microsatellites (Simple Sequence Repeats, SSRs), and insertion / deletion polymorphisms (InDels), with SNPs comprising the largest proportion. Comparing polymorphisms between wheat varieties based on resequencing data and Sanger sequencing results and developing molecular markers linked to phenotypes can help more quickly locate candidate genes for effective tiller number, improve breeding efficiency, and more effectively increase yield. The advantages of SNP markers are their large number and widest distribution across the genome, as well as their uneven distribution across the genome, making SNP allele frequencies easy to estimate. Based on SNPs, KASP, CAPS and InDel markers can be developed, among which KASP markers have the following advantages: high accuracy: KASP technology typing results are accurate, with an accuracy greater than 99.8%; low cost: compared with other technologies, KASP technology has a lower cost; low sample usage: KASP technology requires a small amount of DNA sample; high detection throughput: KASP technology can perform high-throughput genotyping; rapid detection cycle: KASP technology has a short detection cycle and can obtain results quickly; flexibility: KASP technology is flexible and can meet the requirements of low-, medium- and high-throughput genotyping based on ordinary laboratory operations; universal probes: KASP technology uses universal probes, which do not require probe synthesis for each specific site, reducing the reagent cost of the experiment; wide application: KASP technology is not limited to a single SNP, but is applicable to small segments with polymorphisms.

[0005] Previous studies have mapped QTLs for effective tiller number, and QTLs controlling effective tiller number have been detected on multiple chromosomes in wheat. However, there are currently very few molecular markers that are tightly linked to the effective tiller number trait and can be used in practical molecular breeding. Therefore, identifying exploitable QTLs or genes for effective tiller number and using molecular biology techniques to increase effective tiller number and, consequently, yield per unit area, ultimately achieving the goal of breeding new, higher-yielding wheat varieties, is of great significance in wheat breeding. Summary of the Invention

[0006] The present invention aims to provide a molecular marker tightly linked to the wheat effective tiller number QTL QPTN.sau-4B and its application to address the problems of the prior art. The present invention designs a single-nucleotide polymorphism (SNP) molecular marker KASP-7, located on chromosome 4B and tightly linked to the wheat effective tiller number QTL QPTN.sau-4B, that can be used to assist in QTL detection and molecular breeding of high-tillering, high-yield wheat, improving both detection and breeding efficiency.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides a SNP molecular marker KASP-7 linked to the wheat effective tiller number QTL QPTN.sau-4B. The molecular marker KASP-7 and the wheat effective tiller number QTL QPTN.sau-4B are co-located on the short arm of wheat chromosome 4B and located within the QPTN.sau-4B interval.

[0009] The molecular marker KASP-7 is located at position 32030698 of chromosome 4B using the Chinese Spring v2.1 genome as a reference genome, and its polymorphism is C / T.

[0010] The present invention also provides a use of the molecular marker KASP-7 in any of the following:

[0011] A1. Detection of the wheat effective tiller number QTL QPTN.sau-4B;

[0012] A2. Early identification or screening of high-tillering, high-yielding wheat varieties;

[0013] A3. Wheat molecular genetics and breeding;

[0014] A4. Improvement of wheat germplasm resources;

[0015] A5. Conduct genetic analysis and fine mapping of genes related to the effective tiller number in wheat.

[0016] The present invention also provides a KASP primer set targeting the molecular marker KASP-7, comprising an upstream primer 1 as shown in SEQ ID NO.19, an upstream primer 2 as shown in SEQ ID NO.20, and a downstream primer as shown in SEQ ID NO.21.

[0017] Furthermore, the upstream primer 1 and the upstream primer 2 are respectively labeled with different fluorescent modification groups. Optionally, the fluorescent modification groups include but are not limited to FIFC, FAM, TET, HEX, JOE, TAMRA, and BHQ.

[0018] The present invention also provides a use of the KASP primer set described above in any of the following:

[0019] B1. Detection of the wheat effective tiller number QTL QPTN.sau-4B;

[0020] B2. Early identification or screening of high-tillering, high-yielding wheat varieties;

[0021] B3. Wheat molecular genetics and breeding;

[0022] B4. Improvement of wheat germplasm resources;

[0023] B5. Conduct genetic analysis and fine mapping of genes related to effective tiller number in wheat;

[0024] B6. Prepare reagents, kits or chips for detecting the wheat effective tiller number QTL QPTN.sau-4B.

[0025] The present invention also provides a kit for detecting the wheat effective tiller number QTL QPTN.sau-4B, wherein the kit comprises the KASP primer set.

[0026] The present invention also provides a method for detecting the wheat effective tiller number QTL QPTN.sau-4B, comprising the following steps:

[0027] Using the genomic DNA of the plant to be tested as a template, the KASP primer set was used for fluorescence quantitative PCR amplification, and the results were judged based on the fluorescence readings;

[0028] Plants in which the fluorescent modification group marked by SEQ ID NO.20 was read were identified as plants containing the wheat effective tiller number QTL QPTN.sau-4B; plants in which the fluorescent modification group marked by SEQ ID NO.19 was read were identified as plants not containing the wheat effective tiller number QTL QPTN.sau-4B.

[0029] Optionally, the reaction system of the fluorescent quantitative PCR is: 5 μL of 2×PACE Master Mix, 0.2 μL each of upstream primer 1 and upstream primer 2, 0.5 μL of downstream universal primer, and 4.1 μL of 1-10 ng / μL DNA template;

[0030] The concentrations of the upstream primer 1, upstream primer 2 and downstream primer were all 10 μM.

[0031] Optionally, the fluorescent quantitative PCR reaction procedure is: pre-denaturation at 4°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 60°C for 60 s, for a total of 15 cycles; denaturation at 94°C for 20 s, annealing / extension at 57°C for 60 s, for a total of 27 cycles.

[0032] The present invention discloses the following technical effects:

[0033] (1) The present invention discloses a molecular marker KASP-7 located on wheat chromosome 4B and tightly linked to the effective tiller number QTL of wheat. The molecular marker is a molecular marker within the effective tiller number QTL QPTN.sau-4B interval on the short arm of wheat chromosome 4B and has a high degree of linkage.

[0034] (2) The present invention discloses a molecular marker, KASP-7, for accurately detecting the wheat effective tiller number QTL, QPTN.sau-4B, based on a fluorescent quantitative PCR platform. This codominant marker offers accurate and efficient detection and convenient and stable amplification. This marker can be used to rapidly screen plants harboring this locus, facilitating molecular-assisted breeding for high effective tiller numbers and high-yield wheat.

[0035] (3) The molecular marker KASP-7 provided by the present invention is closely linked to the effective tiller number QTL QPTN.sau-4B locus on wheat chromosome 4B, and can be used for the precise positioning and discovery of genes regulating the effective tiller number in wheat.

[0036] (4) The fluorescent quantitative molecular marker KASP-7 disclosed in the present invention exhibits co-segregation characteristics with the effective tiller number of wheat. This improves the efficiency of selecting and identifying wheat varieties with high effective tiller numbers that are adaptable to different environments, with a high success rate. This allows the elimination of plants with low effective tiller numbers during the breeding process, improving breeding efficiency and providing a basis for the study of wheat effective tiller number genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 KASP-7 was used to detect NIL in Example 1. 川农16 '×'NIL 20828 'The fluorescence reading results of the secondary population plants; Among them, HEX (square) fluorescence is the genotype and 'NIL 川农16 'Consistent plants; FAM (circle) fluorescence is the genotype and 'NIL 20828 'Consistent plants; black fluorescence (diamond) is the negative control;

[0039] Figure 2 Figure 2 shows the fluorescence readings of 22 advanced lines or varieties with a CN16 background detected using KASP-7 in Example 2; HEX (square) fluorescence represents plants containing the QPTN.sau-4B synergistic site; FAM (circle) fluorescence represents plants without the QPTN.sau-4B synergistic site; black fluorescence (diamond) represents the negative control. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] The technical solutions of the present invention are as follows:

[0046] Based on resequencing data and Sanger next-generation sequencing results from '20828' and 'Chuannong 16', polymorphisms between the parents were identified. Further, using genomic and bioinformatics techniques, sequence extraction, alignment, and analysis were performed on reference genomes of multiple published wheat varieties to identify a specific nucleic acid sequence on chromosome 4B. This polymorphism between the parents was further verified to improve the reliability of the polymorphic locus. Furthermore, the polymorphisms between multiple varieties and direct polymorphisms in homologous sequences were used to design specific primers to amplify the target region. Ultimately, the polymorphism in this region was developed into a KASP molecular marker, which was then genotyped and validated against the population's effective tiller number phenotypic data.

[0047] There are several challenges that need to be overcome before developing markers in wheat. The following are some of the main reasons why it is difficult to design practical specific primers:

[0048] (1) Polyploidy of wheat: Common wheat is an allohexaploid, which means that its genome contains multiple homologous and paralogous genomic sequences, which makes the design of specific and useful PCR primers time-consuming and labor-intensive.

[0049] (2) Many repetitive sequences: The wheat genome contains many repetitive sequences, which increases the difficulty of designing specific primers. Repetitive sequences may cause primers to bind nonspecifically to non-target regions, thereby affecting the specificity and efficiency of PCR.

[0050] (3) Genome complexity: The wheat genome is highly complex, with a large number of gene families and homologous genes, which makes it more difficult to design specific primers.

[0051] (4) Variation of genome sequence: There are a large number of SNPs in the wheat genome. These variations may cause the designed primers to be unable to completely match the genome sequences of all wheat varieties, thereby affecting the specificity of the primers.

[0052] In order to develop molecular markers quickly and effectively, the present invention overcomes the difficulties in primer design through the following scheme.

[0053] By utilizing resequencing data and second-generation Sanger sequencing results, we identified polymorphisms between parents with significant differences in effective tiller number. We used previously reported wheat genome data (http: / / 202.194.139.32) to specifically align the nucleic acid sequences within the mapped region and verify the authenticity of the polymorphic sites. Based on the SNP variants at the identified polymorphic sites, we designed two forward KASP primers and one reverse-directed primer to label the identified sites.

[0054] The original KASP primers were screened for specificity to identify candidate KASP primers. Genome-specific alignment or in silico genome amplification methods can be used. Primers should be between 21 and 28 bases in length and have a GC content between 40% and 60%. Primers should contain no more than three consecutive G or C bases, minimizing hybridization between primers and the formation of dimers or trimers. Ultimately, six pairs of KASP primers were designed (see Table 1), with their sequences shown in SEQ ID NOs. 4-21.

[0055] The heterozygous self-cross family method was used to construct near-isogenic lines. That is, the KASP marker developed earlier, which was closely linked to QPTN.sau-4B, was used to screen heterozygous plants from the '20828' and 'Chuannong 16' construction populations to construct tillering near-isogenic lines. The remaining heterozygous plants in the above populations were grown in the greenhouse for generations, and heterozygous plants were retained in each generation until F 10 Generation, F 10 In the next generation, two homozygous genotypes of each heterozygous plant were retained and planted in the Wenjiang experimental field for preliminary phenotypic identification. A pair of materials with no differences in other agronomic traits (flowering period, plant height, number of spikelets, spike length, and length and width of flag leaf) except for the number of effective tillers were obtained. Finally, a pair of near-isogenic lines with the same effective tiller number, 'NIL 20828 '(fewer effective tillers) and 'NILchuannong 16'(more effective tillers).

[0056] Using near-isogenic lines with multiple effective tiller numbers (NIL 川农16 ) as the male parent, a near-isogenic line with a small number of effective tillers (NIL 20828 ) as the female parent, and obtained hybrid F1, self-pollination to obtain 157 F2 generation secondary populations. The effective tiller number phenotype of the secondary population in the field was identified, and the parent'NIL 川农16 ' and 'NIL 20828 ', as well as DNA from its F2:3 generation secondary population plants, were accurately genotyped and genetically analyzed using the KASP primers listed in Table 1. The marker KASP-7 was ultimately found to be tightly linked to the effective tiller number QTL, QPTN.sau-4B. Compared to the previously discovered KASP-1 molecular marker (Ma Jian, Lan Xiujin, Liu Jiajun, et al. SNP molecular markers linked to the effective tiller number QTL for wheat and their applications [P]. Sichuan Province: CN201910652002.1, 2020-12-25. Primer information is shown in Table 1), KASP-7 is even more tightly linked to the effective tiller number QTL, QPTN.sau-4B.

[0057] Example 1

[0058] The development process of the molecular marker KASP-7, which is tightly linked to the wheat effective tiller number QTL QPTN.sau-4B, is as follows:

[0059] Using resequencing data from '20828' and 'Chuannong 16' and second-generation Sanger sequencing results, we identified polymorphisms between parents with significant differences in effective tiller number. We used previously reported wheat genome data (http: / / 202.194.139.32) to specifically align the nucleic acid sequences within the mapped region and verify the authenticity of the polymorphic sites. Based on the SNP variants at the identified polymorphic sites, we designed two forward KASP primers and one reverse-directed primer. Six pairs of KASP primers (numbered KASP-2 to KASP-7) were designed, as shown in Table 1.

[0060] Table 1 Molecular markers and KASP primer sequences

[0061]

[0062]

[0063] To confirm the practicality of molecular markers, this paper used a population related to the number of effective tillers in wheat to verify its linkage with the phenotype, laying the foundation for the application of molecular markers in the breeding of wheat varieties with high effective tiller numbers. The process is as follows:

[0064] (1) The wheat variety '20828' with a small number of effective tillers was used as the female parent and the wheat variety 'Chuannong 16' with a large number of effective tillers was used as the male parent to construct a recombinant inbred line population. Based on the self-pollination breeding method of heterozygous individuals at the wheat effective tiller number QTL QPTN.sau-4B locus, a near-isogenic line with high tillering was constructed. 10 After the background is homozygous, it is constructed into 'NIL 川农16 ' and 'NIL 20828 'Strain.

[0065] (2) Use the near-isogenic lines with high genetic similarity to construct secondary populations, and use the near-isogenic lines with a large number of effective tillers as the 川农16 'NIL is a near-isogenic line with a small number of effective tillers as the male parent 20828 ' was used as the female parent to obtain hybrid F1, and a secondary population of F2 generation of 157 lines was obtained (numbered NIL-1 to NIL-157).

[0066] (3) The effective tiller number phenotype of the secondary population in the field was identified. When the wheat grew to maturity, the effective tiller number of the secondary population of the near-isogenic line was identified and analyzed. The single plants at both ends of each row were removed, and 10 single plants with consistent growth were collected. The effective tiller number was calculated and the average value was obtained to represent the effective tiller number of the line.

[0067] (4) Using the KASP primers in Table 1, we accurately performed genotyping and genetic analysis on some secondary populations. 川农16 '、'NIL 20828 ' and F 2：3 The DNA of the secondary population plants of the family was extracted for quality inspection using an ultra-micro spectrophotometer. After passing the inspection, the primers listed in Table 1 were used for fluorescence quantitative PCR amplification.

[0068] The amplification system for fluorescent quantitative PCR amplification is: 10 μL PCR amplification reaction system: 5 μL 2×PACE MasterMix, 0.2 μL each of upstream primer 1 (10 μM) and upstream primer 2 (10 μM), 0.5 μL of downstream universal primer (10 μM), 4.3 μL of 1-10 ng / μL DNA template, and 3 independent blanks with double-distilled water instead of DNA template were added as negative controls.

[0069] Fluorescence quantitative PCR program: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 60°C for 60 s, for a total of 15 cycles; denaturation at 94°C for 20 s, annealing / extension at 57°C for 60 s, for a total of 27 cycles; after completion, fluorescence signal collection was performed.

[0070] (5) In order to densify the genetic map and obtain a molecular marker tightly linked to the effective tiller number QTL QPTN.sau-4B, after multiple rounds of KASP marker development, primer design and amplification, the marker KASP-7 (polymorphism is T / C) was finally obtained, which was tightly linked to the effective tiller number of wheat.

[0071] The fluorescence readings of some secondary populations detected by KASP-7 are shown in Figure 1 , will detect the same as 'NIL 20828 'Consistent FAM(circle, 'NIL 20828 The genotype of the fluorescent plants was recorded as A, which was a line with few effective tillers, the same as NIL. 川农16 'Consistent HEX(square, 'NIL 川农16 Plants with fluorescent ') genotypes were designated B, indicating a high number of effective tillers. Using KASP-1 (primers in Table 1) as a control, the genotypes and field phenotypic values ​​of effective tiller numbers for each line tested with KASP-7 are shown in Table 2.

[0072] Table 2 Genotypes and effective tiller numbers of each line in the field

[0073]

[0074]

[0075]

[0076] The results in Table 2 show that the molecular marker KASP-1 was used for typing and found to be related to 'NIL 川农16 The average effective tiller number of plants of the same type was 7.85, which was significantly higher than that of plants of the NIL 20828 The average number of effective tillers of plants of the same type was 4.28. Using molecular marker KASP-7 for typing, it was found that 'NIL 川农16 The average effective tiller number of plants of the same type was 8.15, which was significantly higher than that of plants of the NIL 20828 The number of effective tillers of the 'type' plants was 4.30 (mean). KASP-1 and KASP-7 were both able to significantly genotype the population, but the genotyping results of KASP-7 were more accurate. 20828 There was basically no difference in the mean number of effective tillers among the 'types, and the 'NIL 川农16 The average number of effective tillers was higher than that of KASP-1. Among them, NIL-40, NIL-62 and NIL-70 were the key strains for distinguishing the accuracy of KASP-1 and KASP-7. These three strains were 'NIL 20828 ' genotype, while in KASP-1 typing it is 'NIL 川农16 'Genotype, through the corresponding effective tiller number phenotype, analysis found that these three key strains have a phenotype of low effective tiller number, which is more linked to the phenotype of KASP-7.

[0077] The above results indicate that the molecular marker KASP-7 of the present invention is closely linked to the effective tiller number of wheat. Compared with the molecular marker KASP-1 invented earlier, the molecular marker KASP-7 is more accurate and can be used to screen wheat varieties with a high effective tiller number.

[0078] Example 2

[0079] In order to verify the molecular marker KASP-7 that is closely linked to the effective tiller number QPTN.sau-4B of wheat, and to further explore whether QPTN.sau-4B has the potential to increase the effective tiller number or the number of ears per mu, 22 high-generation lines or varieties with Sichuan Agricultural University 16 background (provided by the Wheat Research Institute of Sichuan Agricultural University) were genotyped. The fluorescence quantitative PCR process was the same as in Example 1. The fluorescence reading results are shown in Figure 2Plants with HEX fluorescence (squares) consistent with the presence of the QPTN.sau-4B synergistic locus were designated as B, indicating a high effective tiller number. Plants with FAM fluorescence (circles) consistent with the absence of the QPTN.sau-4B synergistic locus were designated as A, indicating a low effective tiller number. The genotypes of each line (identified using the molecular marker method of the present invention) and the field phenotypic values ​​for effective tiller number are shown in Table 3.

[0080] Table 3 22 strains / variety materials with Sichuan Agricultural University 16 background

[0081] Material name genotype Number of effective tillers Material name genotype Number of effective tillers Shumai 415 A 3.93 L2979-2980 B 5.73 D3211 A 4.80 P120 B 6.03 D4354 A 5.00 P121 B 5.87 D4702 A 4.67 Sichuan wheat 1603 B 8.83 D5146 A 4.47 D505 B 5.43 K7429-7433 A 4.73 D532 B 6.07 Sichuan wheat 1580 A 4.70 K8324-8328 B 5.73 Sichuan wheat 98 A 4.50 Sichuan wheat 96 B 6.63 Shumai 1746 A 3.37 Neimai 561 B 5.03 20S-2429-2 B 5.87 Shumai 1679 B 5.23 D484 B 5.53 D493 B 5.37

[0082] The results in Table 3 show that 13 accessions contained the QPTN.sau-4B synergistic locus, while 9 accessions did not, indicating that this locus could be selected for in wheat breeding. Phenotypic analysis of effective tiller number revealed that lines containing the QPTN.sau-4B synergistic locus had significantly higher effective tiller numbers (mean, 5.95) than lines without the QPTN.sau-4B synergistic locus (mean, 4.46), indicating that this locus can indeed increase effective tiller number. Therefore, KASP-7 has potential application in wheat breeding.

[0083] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of a single-nucleotide polymorphism (SNP) molecular marker KASP-7 linked to the wheat effective tiller number QTL QPTN.sau-4B in the following A1 or A2: A1. Detection of the wheat effective tiller number QTL QPTN.sau-4B; A2. Early identification or screening of high-tillering, high-yielding wheat varieties; The molecular marker KASP-7 and the wheat effective tiller number QTL QPTN.sau-4B are co-located on the short arm of wheat chromosome 4B and are located within the QPTN.sau-4B interval; The molecular marker KASP-7 is located at position 32030698 of chromosome 4B using the Chinese Spring v2.1 genome as a reference genome, and its polymorphism is C / T.

2. A KASP primer set targeting the SNP molecular marker KASP-7 linked to the wheat effective tiller number QTL QPTN.sau-4B, characterized in that: The KASP primer set includes an upstream primer 1 as shown in SEQ ID NO.19, an upstream primer 2 as shown in SEQ ID NO.20, and a downstream primer as shown in SEQ ID NO.21; The molecular marker KASP-7 and the wheat effective tiller number QTL QPTN.sau-4B are co-located on the short arm of wheat chromosome 4B and are located within the QPTN.sau-4B interval; The molecular marker KASP-7 is located at position 32030698 of chromosome 4B using the Chinese Spring v2.1 genome as a reference genome, and its polymorphism is C / T.

3. The KASP primer set according to claim 2, characterized in that The upstream primer 1 and the upstream primer 2 are respectively labeled with different fluorescent modification groups.

4. Use of the KASP primer set according to claim 2 or 3 in any of the following: B1. Detection of the wheat effective tiller number QTL QPTN.sau-4B; B2. Early identification or screening of high-tillering, high-yielding wheat varieties; B3. Prepare a kit or chip for detecting the wheat effective tiller number QTL QPTN.sau-4B.

5. A kit for detecting the wheat effective tiller number QTL QPTN.sau-4B, characterized in that: The kit comprises the KASP primer set according to claim 2 or 3.

6. A method for detecting the effective tiller number QTL QPTN.sau-4B in wheat, characterized in that: The steps include: Using the genomic DNA of the plant to be tested as a template, performing fluorescence quantitative PCR amplification using the KASP primer set described in claim 3, and making a determination based on the fluorescence reading result; Plants in which the fluorescent modification group marked by SEQ ID NO.20 was read were identified as plants containing the wheat effective tiller number QTL QPTN.sau-4B; plants in which the fluorescent modification group marked by SEQ ID NO.19 was read were identified as plants not containing the wheat effective tiller number QTL QPTN.sau-4B.

7. The method according to claim 6, characterized in that The reaction system of the fluorescent quantitative PCR is as follows: 5 μL of 2×PACE Master Mix, 0.2 μL each of upstream primer 1 and upstream primer 2, 0.5 μL of downstream universal primer, and 4.1 μL of 1-10 ng / μL DNA template; The concentrations of the upstream primer 1, upstream primer 2 and downstream primer were all 10 μM.

8. The method according to claim 6, characterized in that The fluorescence quantitative PCR reaction program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing / extension at 60°C for 60 s, for a total of 15 cycles; denaturation at 94°C for 20 s, annealing / extension at 57°C for 60 s, for a total of 27 cycles.

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