KASP primer set for QTLs for co-enhancing grain length and grain weight in wheat and its application

Abstract

The invention discloses a KASP primer set for a QTL for co-enhancing wheat grain length and grain weight and an application thereof. The co-enhancing QTL is named QgWT.L_2D, is located on wheat chromosome 2D, and has a physical position of 618.15Mb to 619.55Mb on a Chinese spring reference genome. The KASP primer set comprises a forward competitive primer KASP-2D_618149955-1 having a nucleotide sequence as shown in SEQ ID NO.1, a forward competitive primer KASP-2D_618149955-2 having a nucleotide sequence as shown in SEQ ID NO.2, and a reverse universal primer KASP-2D_618149955-3 having a nucleotide sequence as shown in SEQ ID NO.3. This KASP primer set provides a molecular marker for the coordinated screening of high-grain length and high-grain weight wheat, which has simple detection, high analysis throughput, and accurate and intuitive results. It is suitable for the efficient detection of excellent genotypes in large-scale genetic generations and breeding lines during the breeding process, meeting the breeding needs of early screening of target traits and evaluation of high-yield potential, improving the efficiency of coordinated improvement of wheat yield factors, and can also assist in the aggregation of excellent grain length / grain weight pleiotropic loci with other excellent traits, accelerating the selection and breeding of new excellent wheat varieties.

Description

Technical Field

[0001] The present invention relates to the field of wheat genetic breeding, and in particular to a KASP primer set for QTLs for co-enhancing wheat grain length and grain weight and an application thereof. Background Art

[0002] Wheat (Triticum aestivum L.) is one of the most important food crops in the world and an important source of energy and protein for people. Therefore, high-yield and super-high-yield breeding has always been one of the important goals of wheat breeding.

[0003] Grain weight (generally measured by thousand-grain weight), number of ears and number of grains per ear are three important factors in wheat yield. Their relationship with yield formation is relatively complex. The increase in the number of grains per ear is generally based on the reduction in the number of ears, while the increase in grain weight is relatively independent, with a heritability of 59%-80%. Therefore, in the process of wheat variety selection, while stabilizing the number of ears and the number of grains per ear, the increase in grain weight becomes the main driving force for further increasing wheat yield. Grain weight is a composite trait, which is jointly determined by grain size indicators such as grain length, grain width, and grain length-to-width ratio and related factors. Studies have shown that traits such as grain length and grain width are extremely significantly positively correlated with grain weight, and there is no negative correlation between the various components. Therefore, increasing wheat grain weight by synergistically improving the various components has always been the focus of breeders. However, grain weight, grain length, grain width and other grain size factors are quantitative traits controlled by multiple genes, with complex genetic basis and obvious influence of environmental conditions. The QTL / gene controlling grain weight is not completely consistent with the QTL / gene controlling independent factors such as grain length and grain width, and there may be complex genetic mechanisms and interaction effects. If a single grain size factor is screened, there may be a possibility that a single factor is improved but the comprehensive grain traits fail to increase synergistically. In fact, in the genetic study of target traits, there is a phenomenon of co-localization between traits, that is, a locus not only affects one trait, but may have pleiotropic effects in function. We call this type of QTL that affects two or more traits at the same time a pleiotropic QTL or a tightly linked QTL. The identification, evaluation and utilization of this type of QTL will have important practical application value for the synergistic improvement of wheat grain weight and one or more of its components.

[0004] Marker-assisted selection (MAS) is currently the most widely used and effective tool for assisting crop genetic improvement. This technology uses molecular markers that are closely linked or co-segregated with the QTL (Quantitative trait loci) or gene that controls the target trait to conduct targeted and whole-genome screening of individuals. It has the advantages of being fast, accurate, and not affected by environmental conditions. It has greatly overcome many difficulties in the identification of complex traits and the aggregation and utilization of functional genes in conventional breeding, and has greatly accelerated the breeding process. However, there are still the following defects in wheat: (1) Since wheat is an allohexaploid with a large genome and complex genetic basis, the complexity of yield trait inheritance and the limitations of QTL positioning have led to many restrictions on the application of MAS in improving quantitative traits such as grain weight and grain length, which are often controlled by multiple micro-effect genes; (2) Currently, most of the relevant QTLs and molecular markers are derived from conventional varieties or mutant materials, but the genetic background or comprehensive traits of their parents often have no obvious advantages, and their application value in breeding is not great; (3) Most of the commonly used molecular markers (such as AFLP, RFLP, SSR, etc.) have cumbersome detection steps, long time and high cost, which are difficult to meet the requirements of efficient screening of large-scale breeding offspring materials in the breeding process. Therefore, it has become an urgent need for wheat MAS breeding to use important resources with excellent genetic background and rich genetic basis to deeply explore major effects, especially pleiotropic favorable alleles / QTLs, and develop molecular breeding markers with strong practicality and high accuracy.

[0005] Most backbone parents have outstanding performance in one trait and no obvious disadvantages in other traits. They have low linkage drag and strong adaptability, and are valuable resources for wheat breeding. The wheat variety Yangmai 158 is a high-yield, high-quality, and disease-resistant wheat variety bred by the Lixiahe Regional Agricultural Science Research Institute in Jiangsu Province. It not only once dominated wheat production in the wheat region of the middle and lower reaches of the Yangtze River, but was also a backbone parent for wheat breeding. However, to date, there have been no reports on pleiotropic QTLs and molecular breeding markers related to its grain length and grain weight. Therefore, in-depth exploration of the important pleiotropic QTLs / genes that control yield factors such as grain weight and grain length in the wheat backbone parent Yangmai 158 will have important scientific research value and broad breeding application prospects for promoting the transfer and breeding utilization of genetic enhancement sites / genes to future generations and improving the synergistic improvement of wheat yield factors. Summary of the invention

[0006] In view of the above problems, the present invention provides a KASP primer set for wheat grain length and grain weight co-enhancing QTL and its application, which can simply, accurately and intuitively realize high-throughput identification and auxiliary selection of excellent genotype materials, and has high application value for high-yield molecular breeding of wheat.

[0007] The present invention is the first to detect a pleiotropic locus QgWT.L_2D that co-localizes with grain length and grain weight from Yangmai 158, a backbone parent of wheat in the middle and lower reaches of the Yangtze River. A KASP primer set is designed for the flanking SNP markers closely linked to the QTL locus QgWT.L_2D, and the primer set is applied to the simultaneous detection of wheat grain length and grain weight. The QTL locus QgWT.L_2D can significantly increase the grain length and grain weight of wheat, and the corresponding KASP marker amplification is sensitive and stable, the results are accurate and reliable, and the application is simple and fast, the detection throughput is high, the results are clear and intuitive, and the breeding practicability is strong.

[0008] Specifically, the technical solution provided by the present invention is as follows:

[0009] First, the present invention provides a set of KASP primer sets that are co-related to wheat grain length and grain weight, and the KASP primer set consists of a forward competitive primer KASP-2D_618149955-1 with a nucleotide sequence as shown in SEQ ID NO.1, a forward competitive primer KASP-2D_618149955-2 with a nucleotide sequence as shown in SEQ ID NO.2, and a reverse universal primer KASP-2D_618149955-3 with a nucleotide sequence as shown in SEQ ID NO.3.

[0010] The KASP primer set was developed based on the co-localized pleiotropic QTL locus QgWT.L_2D for wheat grain length and grain weight. QgWT.L_2D is located on chromosome 2D of wheat, corresponding to a physical position between 618.15Mb and 619.55Mb on the Chinese Spring reference genome IWGSC RefSeq v1.0. The SNP molecular markers on both sides are 2D_618149955 and 2D_619558727, respectively.

[0011] The 5' ends of the forward competitive primers KASP-2D_618149955-1 and KASP-2D_618149955-2 are each connected to different fluorescent labels, wherein the 5' end of KASP-2D_618149955-1 is connected to a FAM fluorescent probe label as shown in SEQ ID NO.4; and the 5' end of KASP-2D_618149955-2 is connected to a HEX fluorescent probe label as shown in SEQ ID NO.5.

[0012] This KASP primer set provides a molecular marker for the coordinated screening of high-grain length and high-grain weight wheat, which has simple detection, high analysis throughput, and accurate and intuitive results. It is suitable for the efficient detection of excellent genotypes in large-scale genetic generations and breeding lines during the breeding process, meeting the breeding needs of early screening of target traits and evaluation of high-yield potential, improving the efficiency of coordinated improvement of wheat yield factors, and can also assist in the aggregation of excellent grain length / grain weight pleiotropic loci with other excellent traits, accelerating the selection and breeding of new wheat varieties with outstanding yield and excellent comprehensive traits.

[0013] Secondly, the present invention also provides the use of the above KASP primer set in detecting wheat grain length and grain weight.

[0014] In addition, the application includes any one or more of the following:

[0015] (1) The KASP primer set is used to detect the wheat grain length and grain weight co-enhancing QTL QgWT.L_2D and the location of related genes;

[0016] (2) The KASP primer set is used to breed and create wheat varieties with high grain length and high grain weight;

[0017] (3) The KASP primer set is used to promote the aggregation breeding between the wheat grain length and grain weight co-enhancing QTL QgWT.L_2D and other excellent trait loci of wheat.

[0018] Specifically, the detection method comprises the following steps:

[0019] (1) using the genomic DNA of the wheat to be tested as a template, performing fluorescence quantitative PCR amplification using the above-mentioned KASP primers to obtain an amplification product;

[0020] (2) Using a fluorescent quantitative PCR instrument to scan the fluorescence signal of the amplified product, and perform typing and clustering of the wheat materials to be tested according to the fluorescence signal:

[0021] If the fluorescence signal of the amplified product appears blue in the obtained typing cluster diagram (the fluorescence signal value is aggregated close to the Y axis), it is considered that the wheat material to be tested contains the wheat grain length and grain weight co-enhancing site QgWT.L_2D, which is recorded as A genotype (Yangmai 158 genotype type); if the fluorescence signal of the amplified product appears red in the obtained typing cluster diagram (the fluorescence signal value is aggregated close to the X axis), it is considered that the wheat material to be tested does not contain the wheat grain length and grain weight co-enhancing site QgWT.L_2D, which is recorded as B genotype (Ningmai No. 9 genotype type); the grain length and grain weight of the A genotype wheat material are both higher than those of the B genotype wheat material. The above-mentioned wheat material to be tested is preferably wheat from the middle and lower reaches of the Yangtze River wheat region.

[0022] Specifically, in step (1), the amplification product is obtained by the following method:

[0023] The total volume of the PCR amplification reaction system was 10 μL, and its specific components included: 2.5 μL of genomic DNA with a concentration of 120 ng / μL, 5 μL of 2×KASP Master Mix, 0.14 μL of KASP Assay Mix, and 2.36 μL of ddH2O;

[0024] The preparation method of each 100 μL KASP Assay Mix is ​​as follows: 12 μL of the forward competitive primer KASP-2D_618149955-1 with a concentration of 100 μM, 12 μL of the forward competitive primer KASP-2D_618149955-2 with a concentration of 100 μM, 30 μL of the reverse universal primer KASP-2D_618149955-3 with a concentration of 100 μM, and 46 μL of ddH2O.

[0025] The PCR amplification reaction procedure was as follows: the first step, thermal activation at 94°C for 15 min; the second step, denaturation at 94°C for 20 s, 61-55°C for 60 s, 10 touch-down cycles, with the temperature decreasing by 0.6°C per cycle; the third step, denaturation at 94°C for 20 s, annealing at 55°C for 1 min, 28 cycles; finally, the amplified product was kept warm at 30°C.

[0026] Through the above technical solution, the present invention achieves the following beneficial effects:

[0027] 1. The present invention uses Yangmai 158, a backbone parent of wheat in the middle and lower reaches of the Yangtze River, and Ningmai 9, another backbone parent, to construct a high-generation recombinant selfing population. Through phenotypic collection and linkage analysis, a pleiotropic QTL site QgWT.L_2D originating from Yangmai 158 and increasing both wheat grain length and grain weight is finally located for the first time. QgWT.L_2D is located on chromosome 2D, and its physical position is 618.15Mb to 619.55Mb. This QTL can significantly increase both wheat grain length and grain weight at the same time, which can not only provide new functional sites and gene resources for high-yield wheat breeding, but also can be further combined with molecular marker-assisted selection to realize the transfer of genetic enhancement sites / genes in wheat backbone parents to offspring and breeding utilization, promote molecular improvement of grain traits, and has high utilization value for high-yield wheat breeding.

[0028] 2. Based on the SNPs closely linked to the QTL, the present invention has developed the KASP molecular marker and its application method, which can be used to accurately, efficiently and intuitively detect the co-synergistic site QgWT.L_2D of grain length and grain weight. The molecular marker can realize the accurate typing of dominant allele variation, and has the advantages of stable amplification, simple detection and intuitive results. It is very consistent with the needs of accurate detection of genotypes of large-scale genetic lines and auxiliary prediction of phenotypes in wheat breeding, and can be used for synchronous, high-throughput screening and evaluation of wheat grain length and grain weight traits.

[0029] 3. The molecular marker primer combination provided by the present invention can promote the application of the wheat grain length and grain weight co-enhancing QTL site QgWT.L_2D in high-yield wheat breeding, solve the problems of timeliness and accuracy of synergistic improvement of multiple traits, realize early identification and auxiliary selection of wheat with yield as breeding target, and improve breeding efficiency; it can also assist in the aggregation of grain length and grain weight co-enhancing sites with other excellent traits, and accelerate the selection of new wheat varieties with outstanding yield and coordinated comprehensive traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the genetic location of the QTL locus QgWT.L_2D for co-enhancing effect of wheat grain length and grain weight;

[0031] Figure 2 The diagram is a diagram of the typing effect of the KASP molecular marker combination co-related to wheat grain length and grain weight provided by the present invention and the genotype detection result of the verification material; wherein a is a diagram of the typing effect of the KASP molecular marker combination in the test sample, and b is a diagram of the detection result of the KASP molecular marker combination in the test verification wheat material; the coordinate axis value represents the intensity of the allele fluorescence signal, the black dot represents the negative control (NTC, i.e., ddH20), and the blue and red dots represent different genotypes respectively;

[0032] Figure 3 The figure is a comparison result of grain length (a) and grain weight (b) of two different genotypes detected by KASP molecular markers in the test wheat material in the embodiment of the present invention; wherein: A and B are two genotypes, GL represents grain length, and GW represents grain weight. DETAILED DESCRIPTION

[0033] The specific implementation of the present invention is described in detail below in conjunction with the examples. It should be understood that the specific implementation described herein is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0034] Unless otherwise specified, the reagents, equipment, etc. used in the following examples can be obtained from commercial sources.

[0035] The wheat materials used in the present invention are all germplasm resources preserved in the provincial crop germplasm resource bank of Jiangsu Province (crops, Yangzhou University bank), and can be obtained and used by technicians and researchers in this field.

[0036] Example 1 Obtaining QTL loci and marker intervals for co-enhancing wheat grain length and grain weight

[0037] (1) In this example, Yangmai 158, a main wheat parent in the middle and lower reaches of the Yangtze River, was used as the male parent and Ningmai No. 9 as the female parent to obtain the F1 generation through hybridization. The F1 generation was self-pollinated to obtain the F2 generation. A high-generation recombinant inbred line genetic population containing 282 strains was obtained through the single-seed descent method.

[0038] (2) The whole genome of a recombinant inbred population consisting of 282 lines constructed from Ningmai 9×Yangmai 158 was scanned using the Illumina 90k gene chip. IciMapping v4.1 software (http: / / www.isbreeding.net) was used to filter and remove redundancy. JoinMap v4.0 was used to construct and correct the linkage genetic map, which covers 21 chromosomes of wheat and has a total length of 3022 cM.

[0039] (3) The above-mentioned recombinant self-pollinated populations were planted in the experimental base of Yangzhou University (119.40°E, 32.34°N) and the Dantu experimental base in Zhenjiang, Jiangsu (119.31°E, 32.21°N) for two consecutive years in 2021-2022 and 2022-2023, respectively. Single-row sowing was carried out with a row length of 1.2 m and a row spacing of 0.3 m, with three replicates. The soil conditions in the experimental base were uniform. Before harvesting, the field management was completely consistent with the method disclosed by Ma Hongxiang et al. (2021) (Ma Hongxiang, Gu Kejun, Chen Huaigu (2021) "100 Questions on Key Practical Technologies in Wheat Industry", China Agricultural Press.). When the grains matured, 5 plants with uniform growth were randomly harvested from each line. After the grains were threshed and naturally dried, 1000 seeds were randomly selected from them for phenotypic investigation of grain-related traits. Grain length traits were collected using an SC-G automatic seed analyzer (Wanshen, China), and the average value of the test data was used for statistical analysis.

[0040] (4) Combined genotype and phenotype data, using QTL IciMapping v4.1 software to locate grain trait QTL (Meng L, Li H, Zhang L, et al. (2015) QTL IciMapping: Integrated software for genetic linkage map construction and quantitative trait locus mapping in biparental populations. Crop J 3: 269-283), using the complete interval mapping method (ICIM-ADD), with LOD value 2.5 as the threshold of significance. Finally, a stable pleiotropic QTL locus (hereinafter referred to as the co-enhancing QTL locus) derived from Yangmai 158 was located, which increased both wheat grain length and grain weight and was detected in different environments. The applicant named it QgWT.L_2D, and its genetic positioning results are shown in the following figure. Figure 1 QgWT.L_2D is located on wheat chromosome 2D, and the SNP molecular markers on both sides are 2D_618149955 and 2D_619558727, and their physical positions on the Chinese Spring reference genome IWGSC RefSeqv1.0 are between 618.15Mb and 619.55Mb.

[0041] Example 2 Development, screening and validation of KASP molecular markers

[0042] (1) In order to better utilize the grain length and grain weight co-enhancing locus QgWT.L_2D obtained by genetic mapping in Example 1 for breeding, based on the composition of closely linked marker SNPs within the interval, KASP markers suitable for high-throughput typing were further developed. Specifically:

[0043] As shown in Table 1, the SNPs are 2D_618149955 and 2D_619558727. In the first step, the sequence information of 150 bp upstream and downstream of each SNP was retrieved respectively, and the primers were used according to the base composition of the target SNP site. Premier5 (http: / / www.premierbiosoft.com / primerdesign / ) was used to design multiple sets of amplification primers. Each set of amplification primers included two forward primers with nucleotide sequence ends that specifically bound to the target SNP and one common reverse universal primer. In the second step, DNAMAN was used to evaluate the quality of the designed primers. At the same time, in order to ensure the primer amplification efficiency, sensitivity and resolution, the primers had to be manually screened again, and the following conditions must be met or met as much as possible: 1) There should not be 5 or more consecutive base sequence complementarity within a single primer; at the same time, 4 or more consecutive base sequences at the 3' end of the primer should not be complementary to other sequences within the primer; 2) There should not be 6 or more consecutive base complementarity between the forward primer and the reverse primer; 3) The Tm value of the primer should be controlled between 59℃ and 65℃; 4) The primer length should not be less than 19bp, and the amplification length should not be greater than 150bp.

[0044] Secondly, because wheat is an allohexaploid crop, the same fragment may have multiple copies in the whole genome. In order to avoid primer mismatch and nonspecific amplification, the primer sequence comparison analysis was further performed using the Ensembl Plants database to ensure that the primers can correctly identify the target site. In summary, the amplification primers that meet the above design principles and can specifically detect the target site were finally screened on the tightly linked marker 2D_618149955.

[0045] Finally, different fluorescent labels were added to the 5' ends of the screened primer sequences and converted into KASP primers (Yang Qingqing, Tang Jiaqi, Zhang Changquan, et al. (2022) Application and Prospect of KASP Labeling Technology in Major Crops. Biotechnology Bulletin 38(04):58-71). The KASP primer sequences involved are shown in Table 1. Each set of primers consists of 3 sequences, namely: forward competitive primer 1: FAM label sequence + amplification primer sequence; forward competitive primer 2: HEX label sequence + amplification primer sequence; reverse universal primer: amplification primer sequence; among which, the nucleotide sequences of the FAM label sequence and the HEX label sequence are as follows:

[0046] FAM tag sequence (SEQ ID NO. 4): 5'-GAAGGTGACCAAGTTCATGCT-3' (can bind to FAM fluorescent group);

[0047] HEX tag sequence (SEQ ID NO. 5): 5'-GAAGGTCGGAGTCAACGGATT-3' (can bind to HEX fluorescent group).

[0048] Table 1 KASP primer design

[0049]

[0050] Note: In the SNP composition (a / b), a represents the variation type of Yangmai 158, and b represents the variation type of Ningmai 9.

[0051] (2) In order to further test the accuracy and high resolution of the developed KASP molecular marker primer combination in identifying target SNPs and detecting co-enhancing sites of grain length and grain weight, and to prove the successful development of KASP markers, it is necessary to verify the typing effect of KASP markers. The specific method is as follows:

[0052] 1) Fifty accessions were randomly selected from the Ningmai 9×Yangmai 158 recombinant self-pollinated population, and young leaves were taken from the two parents. Genomic DNA was extracted from them using the CTAB method according to Stein et al. (2001) (Stein N, Herren G, and Keller B (2001) A new DNA extraction method for high-throughput marker analysis in a large-genome species such as Triticum aestivum. Plant Breed 120: 354-356), and then the DNA was uniformly diluted with sterile ultrapure water to a concentration of about 120 ng / μL.

[0053] 2) Fluorescence quantitative PCR amplification was performed using the KASP primer set obtained in Table 1. The reaction system, amplification procedure and genotyping method were as follows:

[0054] KASP reaction system: total volume is 10 μL, including sample DNA (120 ng / μL) 2.5 μL, 2×KASP MasterMix 5 μL, KASP Assay Mix 0.14 μL, and ddH2O 2.36 μL.

[0055] The preparation method of KASP Assay Mix is ​​as follows: every 100 μL of KASP Assay Mix contains 12 μL of each of two forward competitive primers with a concentration of 100 μM, 30 μL of a reverse universal primer with a concentration of 100 μM, and 46 μL of ddH2O.

[0056] KASP reaction procedure: the first step, 94℃ heat activation for 15min; the second step, 94℃ denaturation for 20s, 61-55℃ for 60s, 10 touch-down cycles, reducing 0.6℃ each cycle; the third step, 94℃ denaturation for 20s, 55℃ renaturation for 1min, 28 cycles; finally, the amplified product was kept warm at 30℃.

[0057] Fluorescence signal scanning and genotyping: The fluorescence quantitative PCR instrument Applied Biosystems ABIViia7 Real Time PCR System (Thermo Scientific, USA) was used to scan the fluorescence signal and perform genotyping clustering. Specifically, the samples with blue fluorescence signals in the genotyping cluster diagram (fluorescence signal values ​​aggregated close to the Y-axis) represent the binding of FAM fluorescent group, which is the Yangmai 158 genotype type; the samples with red fluorescence signals in the genotyping cluster diagram (fluorescence signal values ​​aggregated close to the X-axis) represent the binding of HEX fluorescent group, which is the Ningmai 9 genotype type; the samples aggregated close to the origin and displayed black are blank controls without added DNA.

[0058] like Figure 2 As shown in the typing results of a, the KASP marker developed by the present invention has high amplification efficiency, clear typing, and accurate detection of SNP sites, which is completely consistent with the gene chip detection results of the corresponding genotype parents and each strain, indicating that the KASP marker has been successfully developed. The applicant named the primer set KASP QgWT.L -2D_618149955, the nucleotide sequence of the KASP primer set includes three sequences, KASP-2D_618149955-1 (SEQ ID NO.1), KASP-2D_618149955-2 (SEQ ID NO.2) and KASP-2D_618149955-3 (SEQ ID NO.3), which can be applied to the molecular detection and breeding selection of the wheat grain length and grain weight co-enhancing site QgWT.L_2D, wherein the dominant allele variation is derived from Yangmai 158, and the non-dominant allele variation is derived from Ningmai 9.

[0059] Example 3 KASP molecular marker primer combination KASP QgWT.L -Application of 2D_618149955

[0060] In order to analyze the molecular marker KASP obtained in Example 2 QgWT.L-2D_618149955 was used for population typing and application verification, and a total of 300 wheat samples were randomly selected from different types of wheat populations and lines as verification materials, including: 1) 171 wheat varieties randomly selected from the main wheat cultivated in the middle and lower reaches of the Yangtze River that have passed variety approval (the materials were self-numbered as Va001-Va171, as shown in Table 2); 2) 129 wheat materials randomly selected from the wheat high-generation (F7-F8) breeding line identification test and variety comparison test obtained by the applicant using the varieties widely promoted in the middle and lower reaches of the Yangtze River (including Yangmai 20, Emai 16, Ningmai 33, Zhenmai 9, Yangfumai 9, Nongmai 161, Wanximai 0501, Emai 007, Yangmai 38, Ruihuamai 598, Baihumai 2, etc.) through hybridization and combination (they were self-named Line001-Line129, as shown in Table 2). The above-mentioned approved wheat variety materials and self-bred variety comparison test materials were planted in the Dantu test base in Zhenjiang, Jiangsu (119.31°E, 32.21°N) and the Liuhe test base in Nanjing, Jiangsu (118.68°E, 32.50°N) in 2022-2023, and the breeding line identification test materials were planted in the Dantu test base in Zhenjiang, Jiangsu (119.31°E, 32.21°N) in 2023-2024. The planting management methods and phenotypic investigation methods are consistent with those described in Example 1.

[0061] Using the KASP primer combination KASP obtained in Example 2 QgWT.L -2D_618149955, the 300 randomly selected wheat materials of different types (i.e., Va001-Va171, Line001-Line129 in Table 2) and the two parents of the recombinant selfing population described in Example 1 (Yangmai 158 and Ningmai 9) were extracted for genomic DNA and subjected to fluorescence quantitative PCR amplification and genotyping. DNA extraction, KASP reaction system, amplification procedure and genotyping method were consistent with the method described in Example 2. The results of fluorescence signal detection and genotyping clustering are shown in Figure 2. Figure 2 As shown in b.

[0062] Finally, the samples that appeared blue in the typing cluster diagram (the fluorescence signal values ​​were aggregated close to the Y-axis, the same as the fluorescence signal of Yangmai 158) were recorded as genotype A, and the samples that appeared red in the typing cluster diagram (the fluorescence signal values ​​were aggregated close to the X-axis, the same as the fluorescence signal of Ningmai No. 9) were recorded as genotype B (Table 2).

[0063] Table 2 Genotyping and phenotyping of 300 randomly selected wheat materials of different types

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] SPSS19.0 was used for statistical analysis. Comparing the phenotypic data of grain length and grain weight of genotypes A and B in Table 2, it was found that the grain length (6.52 mm) and grain weight (46.79 g) of wheat carrying genotype A were higher than those carrying genotype B (6.30 mm) and grain weight (43.93 g) (see Table 2). Figure 3 shown).

[0070] The independent sample t-test was further used to compare the phenotypic differences between wheat materials with high grain length and grain weight genotype (genotype A) and low grain length and grain weight genotype (genotype B). The results are shown in Table 3: The grain length and grain weight of wheat containing genotype A were significantly (p<0.01) higher than those of genotype B wheat.

[0071] Table 3 t test results

[0072]

[0073] Note: * indicates a difference at a significance level of 0.05, and ** indicates a difference at a significance level of 0.01

[0074] In summary, the present invention obtained a QTL locus QgWT.L_2D for co-enhancing grain length and grain weight from the main parent of wheat Yangmai 158 in the middle and lower reaches of the Yangtze River for the first time. The genotype (genotype A) carrying QgWT.L_2D can significantly increase wheat grain length and grain weight at the same time; the KASP molecular marker primer combination KASP closely linked to QgWT.L_2D developed thereby QgWT.L -2D_618149955 can complete the precise typing of dominant allele variation. The implementation method is simple and fast, the results are accurate and intuitive, and the analysis throughput is high. It is suitable for the efficient detection of excellent genotypes of large-scale genetic generations and breeding lines in the breeding process, and meets the breeding needs of early screening of target traits and evaluation of yield potential, thereby improving breeding efficiency. At the same time, it can also assist in the aggregation of the co-synergistic loci of grain length and grain weight with other excellent traits, and accelerate the selection of new wheat varieties with outstanding yield and excellent comprehensive traits.

[0075] The wheat tested in the above embodiments includes population materials derived from the backbone wheat varieties Yangmai 158 and Ningmai 9 in the middle and lower reaches of the Yangtze River as parents, wheat varieties that have passed the review in the middle and lower reaches of the Yangtze River wheat region, and wheat high-generation (F7-F8) line identification materials and variety comparison test materials bred by hybridization and combination using varieties in the middle and lower reaches of the Yangtze River wheat region, covering a wide range of types, so the identified grain length and grain weight co-enhancing QTL locus QgWT.L_2D and the developed KASP molecular marker KASP QgWT.L -2D_618149955 has a high application value in molecular design breeding of wheat, especially in the middle and lower reaches of the Yangtze River.

[0076] The preferred implementation modes of the present invention are described in detail above in combination with the embodiments; however, the present invention is not limited to the specific details in the above implementation modes. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical scheme of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0077] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0078] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. Application of a KASP primer set for QTLs for co-enhancing wheat grain length and grain weight, characterized in that: The co-synergistic QTL is named QgWT.L_2D, located on wheat chromosome 2D, and its physical position on the Chinese Spring reference genome is 618.15 Mb to 619.55 Mb; the KASP primer set includes a forward competitive primer KASP-2D_618149955-1 with a nucleotide sequence as shown in SEQ ID NO.1, a forward competitive primer KASP-2D_618149955-2 with a nucleotide sequence as shown in SEQ ID NO.2, and a reverse universal primer KASP-2D_618149955-3 with a nucleotide sequence as shown in SEQ ID NO.3; The application is any of the following: (1) Used to detect wheat grain length and weight traits; (2) Used for the selection and creation of wheat varieties with high grain length and high grain weight.

2. The use according to claim 1, characterized in that: The method for detecting wheat grain length and grain weight comprises the following steps: (1) Using the genomic DNA of the wheat to be tested as a template, the KASP primer set is used to perform fluorescence quantitative PCR amplification to obtain an amplification product, wherein the 5' end of KASP-2D_618149955-1 is connected to a FAM fluorescent probe label as shown in SEQ ID NO.4, and the 5' end of KASP-2D_618149955-2 is connected to a HEX fluorescent probe label as shown in SEQ ID NO.5; (2) The amplified product was subjected to fluorescence signal scanning and genotyping. If the fluorescence signal of the amplified product appeared blue in the typing cluster diagram, the wheat was recorded as genotype A; if the fluorescence signal of the amplified product appeared red in the typing cluster diagram, the wheat was recorded as genotype B. The grain length and grain weight of genotype A wheat were both higher than those of genotype B wheat.

3. The use according to claim 2, characterized in that: In step (1), the fluorescence quantitative PCR amplification system is 10 μL, comprising: 2.5 μL of wheat DNA template to be tested with a concentration of 120 ng / μL, 5 μL of 2×KASP Master Mix, 0.14 μL of KASP Assay Mix, and 2.36 μL of ddH2O; The preparation method of each 100 μL of KASP Assay Mix is ​​as follows: 12 μL of the forward competitive primer KASP-2D_618149955-1 with a concentration of 100 μM, 12 μL of the forward competitive primer KASP-2D_618149955-2 with a concentration of 100 μM, 30 μL of the reverse universal primer KASP-2D_618149955-3 with a concentration of 100 μM, and 46 μL of ddH2O; The PCR amplification procedure was as follows: the first step, thermal activation at 94°C for 15 min; the second step, denaturation at 94°C for 20 s, 61-55°C for 60 s, 10 touch-down cycles, with the temperature decreasing by 0.6°C per cycle; the third step, denaturation at 94°C for 20 s, annealing at 55°C for 1 min, 28 cycles; finally, the amplified product was kept at 30°C and the fluorescence signal was scanned.

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