Molecular marker closely linked with effective tiller number QTL QPTN.sau-4B of wheat and application of molecular marker

Abstract

The invention discloses a molecular marker closely linked with the effective tiller number QTL QPTN.sau-4B of wheat and application of the molecular marker, and belongs to the technical field of molecular breeding of wheat. The molecular marker KASP-7 and the wheat available tiller number QTL QPTN.sau-4B are co-located on a wheat 4B chromosome short arm, and the molecular marker KASP-7 and the wheat available tiller number QTL QPTN.sau-4B are located in the QPTN.sau-4B interval; the molecular marker KASP-7 takes a Chinese spring v2.1 genome as a reference genome and is located at the 32030698th site of a 4B chromosome, and the polymorphism of the molecular marker KASP-7 is C / T. The SNP molecular marker KASP-7 linked with the effective tillering number QTL QPTN.sau-4B of the wheat is closely linked with the effective tillering number QTL of the wheat and can be used for auxiliary detection of the QTL and multi-tillering and high-yield wheat molecular breeding, and the detection efficiency and the breeding efficiency are improved.

Description

Technical Field

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

[0002] Common wheat (Triticum aestivum L.) is a major grass crop that provides about 20% of dietary calories for humans. In recent years, wheat yield has increased steadily, while the sown area has decreased. This trend has increased the urgency of the need to better understand the genetic mechanisms that control wheat yield. Wheat yield is determined by the number of spikelets per unit area, the number of spikelets per spikelet, and the thousand-grain weight. These quantitative traits are difficult to accurately select in traditional breeding, resulting in slow breeding progress. However, the application of molecular marker-assisted selection technology has significantly accelerated the breeding process. Quantitative traits are affected by both genetic and environmental factors, but genetic factors play a decisive role under stable environmental conditions. Since quantitative traits have a dosage effect, that is, the gene dosage is proportional to the phenotypic performance, enrichment of target trait loci related to yield is a key strategy to increase yield in breeding. The use of molecular markers linked to the target trait can significantly improve the selection efficiency, thereby achieving a dual increase in yield and quality in wheat breeding.

[0003] Tillering is a branching phenomenon unique to monocotyledons and is of great value to many crops, including wheat and rice. In wheat, tillering is also an important agronomic trait that affects adaptability to the planting environment. The effective tiller number refers to the number of ears that eventually form ears, which directly affects the number of ears per hectare and ultimately affects the wheat grain yield. In addition, the effective tiller number plays a role in shaping the plant structure by affecting other agronomic traits such as plant height and flowering period. Therefore, the use of molecular markers linked to the effective tiller number is of great significance for optimizing wheat plant structure and increasing grain yield.

[0004] There are polymorphisms between different varieties of crops. The genotype polymorphisms in plants mainly include single nucleotide polymorphisms (SNPs), microsatellites (Simple Sequence Repeats, SSRs) and insertion / deletion polymorphisms (InDels), among which SNPs account for the largest proportion. Comparing the polymorphisms between wheat varieties based on resequencing data and the results of Sanger second-generation sequencing, and developing molecular markers linked to phenotypes can help locate candidate genes for the number of effective tillers more quickly, and help improve breeding efficiency, which can more effectively increase yields. The advantages of SNP markers are that they are numerous in the genome, are most widely distributed, are unevenly distributed throughout the genome, and the SNP allele frequency is easy to estimate. Based on SNP, 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 of more 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; fast detection cycle: KASP technology has a short detection cycle and can get results quickly; flexibility: KASP technology is flexible and can meet the requirements of low-, medium- and high-throughput genotyping on the basis of 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 polymorphism.

[0005] Previously, some scholars have located the QTL for the number of effective tillers, and QTLs controlling the number of effective tillers have been detected on multiple chromosomes in wheat. However, there are currently very few tightly linked molecular markers associated with the trait of the number of effective tillers in wheat that can be used in actual molecular breeding. Therefore, it is of great significance in wheat breeding to study and obtain available QTLs or genes for the number of effective tillers, use molecular biology techniques to increase the number of effective tillers, and then increase the yield per unit area, and ultimately achieve the goal of breeding new wheat varieties with increased yields. Summary of the invention

[0006] The purpose of the present invention is to provide a molecular marker closely linked to the wheat effective tiller number QTL QPTN.sau-4B and its application, so as to solve the problems existing in the above-mentioned prior art. The present invention designs a SNP molecular marker KASP-7 linked to the wheat effective tiller number QTL QPTN.sau-4B, which is located on chromosome 4B and closely linked to the wheat effective tiller number QTL, and can be used to assist in the detection of QTL and molecular breeding of multi-tillering and high-yield wheat, thereby improving the detection efficiency 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 a wheat effective tiller number QTL QPTN.sau-4B, wherein the molecular marker KASP-7 and the wheat effective tiller number QTL QPTN.sau-4B are co-localized on the short arm of wheat chromosome 4B and are located within the QPTN.sau-4B interval;

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

[0010] The present invention also provides an application of the molecular marker KASP-7 in any of the following items:

[0011] A1. Detection of 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 breeding;

[0014] A4. Improvement of wheat germplasm resources;

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

[0016] The present invention also provides a KASP primer set for the molecular marker KASP-7, the KASP primer set 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] Further, 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 an application of the KASP primer set in any of the following items:

[0019] B1. Detection of 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 breeding;

[0022] B4. Improvement of wheat germplasm resources;

[0023] B5. Conduct genetic analysis and fine positioning 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 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 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 is used to perform fluorescence quantitative PCR amplification, and the determination is made based on the fluorescence reading results;

[0028] The 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; the 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 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, the upstream primer 2 and the downstream primer are all 10 μM.

[0031] Optionally, the fluorescence quantitative PCR reaction program 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 wheat effective tiller number QTL. 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, and is a co-dominant marker with accurate and efficient detection and convenient and stable amplification. The marker can be used to quickly screen plants with the locus, thereby facilitating molecular-assisted breeding of 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 of wheat.

[0036] (4) The fluorescent quantitative molecular marker KASP-7 disclosed in the present invention presents a co-segregation marker characteristic with the effective tiller number of wheat. The selection and identification efficiency of wheat varieties with multiple effective tiller numbers that are adapted to different environments is improved, and the success rate is high. Thus, plants with fewer effective tiller numbers are eliminated during the breeding process, the breeding efficiency is improved, and a basis is provided 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 drawings required for use in the embodiments will be briefly introduced below. 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 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 genotype and 'NIL 20828 'Consistent plants; black fluorescence (diamond) is the negative control;

[0039] Figure 2 The fluorescence reading results of 22 high-generation lines or varieties with CN16 background detected by KASP-7 in Example 2; HEX (square) fluorescence represents plants containing the QPTN.sau-4B enhancement site; FAM (circle) fluorescence represents plants without the QPTN.sau-4B enhancement site; black fluorescence (diamond) is 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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 solution of the present invention is as follows:

[0046] Based on the resequencing data and Sanger second-generation sequencing results of '20828' and 'Chuannong 16', the polymorphism between the parents was obtained. Further through genomics technology and bioinformatics technology, the reference genomes of multiple published wheat varieties were sequenced, aligned and analyzed to obtain specific nucleic acid sequences on chromosome 4B, and the polymorphism between the parents was further verified to improve the reliability of the polymorphic sites. The polymorphism between multiple varieties and the direct polymorphism of homologous sequences were used to design specific primers for amplifying the target region. Finally, the polymorphism of this segment was developed into a KASP molecular marker, and the developed molecular markers and the phenotypic data of the effective tillering number of the population were genotyped and verified.

[0047] There are some challenges that need to be overcome before developing markers. Designing primers in wheat may face some challenges. The following are some of the 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 it time-consuming and laborious to design specific and useful PCR primers.

[0049] (2) Many repetitive sequences: There are many repetitive sequences in the wheat genome, which increases the difficulty of designing specific primers. Repetitive sequences may cause nonspecific binding of primers to non-target regions, thus 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, thus affecting the specificity of the primers.

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

[0053] By using resequencing data and the results of the second-generation Sanger, the polymorphism between parents with significant differences in the number of effective tillers was determined. The nucleic acid sequences in the positioning segment were specifically compared using the reported wheat genome data (http: / / 202.194.139.32) to verify the authenticity of the polymorphic site. At the determined polymorphic site, two forward original KASP primers and one reverse specific primer were designed based on the variation of the SNP.

[0054] The original KASP primers were specifically screened to obtain candidate KASP primers. The genome-specific alignment method or the genome electronic simulation amplification method can be used. The primer length should be between 21 and 28 bases, and the GC content should be between 40% and 60%. The primers should be selected without more than 3 consecutive G or C bases, and at the same time reduce the hybridization between primers and the formation of dimer or trimer structures. Finally, 6 pairs of KASP primers were designed (see Table 1), and their sequences are shown in SEQ ID NO.4-21.

[0055] The heterozygous self-pollination family method was used to construct near-isogenic lines, that is, the KASP markers that were developed earlier and closely linked to QPTN.sau-4B were used to screen heterozygous plants in 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, spikelet number, spike length, and flag leaf length and width) except for the number of effective tillers were obtained. Finally, a pair of near-isogenic lines with the number of effective tillers, 'NIL 20828 '(less effective tillering number) and 'NILchuannong16'(more effective tillering number).

[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 the hybrid F 1 , 157 F lines were obtained by self-pollination 2 The effective tillering number phenotype of the secondary population in the field was identified and the parent'NIL 川农16 ' and 'NIL 20828 ', and its F 2 : DNA of plants of the 3rd generation secondary population was used to accurately perform genotyping and genetic analysis on the secondary population using the KASP primers in Table 1. Finally, the marker KASP-7 was found to be closely linked to the effective tiller number QTL QPTN.sau-4B. Compared with the previously invented KASP-1 molecular marker (Ma Jian, Lan Xiujin, Liu Jiajun, et al. SNP molecular markers linked to the effective tiller number QTL of wheat and their applications [P]. Sichuan Province: CN201910652002.1, 2020-12-25. Primer information is shown in Table 1), KASP-7 is more closely 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 the resequencing data of '20828' and 'Chuannong 16' and the results of the second-generation Sanger, the polymorphism between parents with significant differences in the number of effective tillers was determined. The nucleic acid sequences in the positioning segment were specifically compared using the reported wheat genome data (http: / / 202.194.139.32) to verify the authenticity of the polymorphic sites. Two forward original KASP primers and one reverse specific primer were designed based on the variation of SNP at the determined polymorphic sites. A total of 6 pairs of KASP primers (numbered KASP-2-KASP-7) were designed, see Table 1.

[0060] Table 1 Molecular markers and KASP primer sequences

[0061]

[0062]

[0063] In order to confirm the practicality of molecular markers, the present invention uses a population related to the effective tiller number of wheat to verify its linkage with the phenotype, laying a foundation for the application of molecular markers in breeding wheat varieties with more effective tiller numbers. The process is as follows:

[0064] (1) The wheat line '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. The method of self-pollination breeding of heterozygous individuals at the QTL QPTN.sau-4B locus for the number of effective tillers in wheat was used to construct a near-isogenic line with high tillering. 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 select the near-isogenic lines with a large number of effective tillers, 'NIL 川农16 'NIL is the male parent, a near-isogenic line with a small number of effective tillers 20828 ' is the female parent, and the hybrid F 1 157 strains of F were obtained. 2 Generation of secondary populations (numbered NIL-1 to NIL-157).

[0066] (3) The phenotype of the effective tiller number 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, which represented the effective tiller number of the strain.

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

[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 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, we finally obtained the marker KASP-7 (polymorphism is T / C) 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(round, 'NIL 20828 The genotype of the plants with fluorescence 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 The genotype of the plant with fluorescence was recorded as B, which was a line with multiple effective tiller numbers. Using KASP-1 (primers in Table 1) as a control, the genotypes of each line detected by KASP-7 and the field phenotypic values ​​of effective tiller numbers are shown in Table 2.

[0072] Table 2 Genotypes and field phenotypes of each strain and effective tiller number

[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 '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 'NIL 20828 The number of effective tillers of the plants of the 'type' was 4.30 (mean). Both KASP-1 and KASP-7 were 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 KASP-7 type of '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 the key strains for distinguishing the accuracy of KASP-1 and KASP-7 in KASP-7. 20828 ' genotype, while in KASP-1 typing it is 'NIL 川农16 'Genotype, through the phenotype corresponding to the number of effective tillers, analysis found that the three key strains had a phenotype of low effective tiller number, which was more linked to the phenotype of KASP-7.

[0077] The above results show 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 for the screening of wheat varieties with more effective tiller numbers.

[0078] Example 2

[0079] In order to verify the molecular marker KASP-7 closely linked to the effective tiller number QPTN.sau-4B of wheat, and 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, and the fluorescence quantitative PCR process was the same as in Example 1. The fluorescence reading results are shown in Figure 2The plant genotypes with HEX (square) fluorescence consistent with the QPTN.sau-4B synergistic site were recorded as B, which was a line with more effective tillers, and the plant genotypes with FAM (circle) fluorescence consistent with the QPTN.sau-4B synergistic site were recorded as A, which was a line with less effective tillers. The genotypes of each line (genotype identified by the molecular marker method of the present invention) and the field phenotypic values ​​of the 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 materials contain the QPTN.sau-4B enhancement site, and 9 materials do not contain the QPTN.sau-4B enhancement site, which indicates that the site can be selected in breeding. Phenotypic identification of the effective tiller number found that the effective tiller number of the strain containing the QPTN.sau-4B enhancement site (mean 5.95) was significantly higher than that of the strain without the QPTN.sau-4B enhancement site (mean 4.46), indicating that the site can indeed increase the effective tiller number. Therefore, KASP-7 has utilization value in wheat breeding.

[0083] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A SNP molecular marker KASP-7 linked to the wheat effective tiller number QTL QPTN.sau-4B, characterized in that: The molecular marker KASP-7 and the wheat effective tiller number QTL QPTN.sau-4B are co-localized on the short arm of wheat chromosome 4B and are located within the QPTN.sau-4B interval; The molecular marker KASP-7 uses the Chinese Spring v2.1 genome as a reference genome, is located at position 32030698 of chromosome 4B, and has a polymorphism of C / T.

2. Use of the molecular marker KASP-7 according to claim 1 in any of the following: A1. Detection of wheat effective tiller number QTL QPTN.sau-4B; A2. Early identification or screening of high-tillering, high-yielding wheat varieties; A3. Wheat molecular genetics breeding; A4. Improvement of wheat germplasm resources; A5. Conduct genetic analysis and fine positioning of genes related to the effective tiller number of wheat.

3. A KASP primer set for the molecular marker KASP-7 according to claim 1, 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.

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

5. Use of the KASP primer set according to claim 3 or 4 in any of the following: B1. Detection of wheat effective tiller number QTL QPTN.sau-4B; B2. Early identification or screening of high-tillering, high-yielding wheat varieties; B3. Wheat molecular genetics breeding; B4. Improvement of wheat germplasm resources; B5. Conduct genetic analysis and fine positioning of genes related to effective tiller number in wheat; B6. Prepare reagents, kits or chips for detecting the wheat effective tiller number QTL QPTN.sau-4B.

6. 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 3 or 4.

7. A method for detecting wheat effective tiller number QTL QPTN.sau-4B, 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 or 4, and making a determination based on the fluorescence reading result; The 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; the 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.

8. The method according to claim 7, characterized in that The reaction system of the fluorescent quantitative PCR is: 5 μL of 2×PACE Master Mix, 0.2 μL 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, the upstream primer 2 and the downstream primer are all 10 μM.

9. The method according to claim 7, characterized in that: The fluorescence 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.

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