KASP molecular marker for detecting character genes of grain length and thousand grain weight of wheat grains and application of KASP molecular marker

Abstract

The invention discloses a KASP molecular marker for detecting wheat grain length and thousand grain weight character genes and application, and belongs to the technical field of biology. The primer group for detecting the SNP or the character of the KASP molecular marker is a primer group I and / or a primer group II, and specific sequences of the primer group I and the primer group II are given; meanwhile, the KASP molecular marker can be applied to the detection and identification of SNP allelic variation and haplotype of the wheat TaARF16-7D gene, and the auxiliary identification of the grain length and thousand grain weight characters of wheat grains or the auxiliary breeding of the molecular marker. The related primer group is developed on the basis of the grain length and thousand grain weight characters of the wheat grains, the SNP loci of the related characters and the excellent haplotypes of the SNP loci can be rapidly and conveniently detected, and the primer group has the advantages of being rapid in identification, high in accuracy and the like and has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a KASP molecular marker for detecting wheat grain length and thousand-grain weight trait genes and an application thereof. Background Art

[0002] wheat( Triticum aestivum L .) is one of the main grain crops, and its yield is mainly composed of three factors: grain weight, number of grains per ear, and number of ears per unit area. Among them, grain weight, as one of the core yield traits, has a high heritability (59-92%), and its genetic mechanism is mainly manifested as an additive effect between genes, which is a typical quantitative trait controlled by multiple genes. Studies have shown that grain weight is mainly determined by grain shape and grain filling rate, and grain shape can be further decomposed into grain shape trait indicators such as grain length, grain width and grain thickness. In production and breeding practice, thousand-grain weight has been widely used as a key indicator to measure grain size, and grain shape traits such as grain length, grain width and grain thickness have also been proven to be significantly positively correlated with yield. Therefore, in production, grain shape and thousand-grain weight complement each other and jointly determine and influence the formation of crop yield traits.

[0003] Traditional breeding methods have faced bottlenecks in improving yield, and the rapid development of molecular biology technology has provided a new breakthrough for the mining and utilization of wheat yield-related genes. Current research shows that through a variety of technical analysis methods such as functional genomics, transcriptomics, gene editing (such as CRISPR-Cas9) and bioinformatics, it is entirely possible to mine yield-related functional genes that control both grain shape and 1000-grain weight, and analyze their biological functions and regulatory networks, thereby providing efficient and accurate marker resources and gene resources for wheat molecular marker-assisted breeding (MAS), and accelerating the genetic improvement of high-yield and stable-yield wheat varieties.

[0004] In summary, mining and utilizing genes related to wheat grain shape and thousand-grain weight is not only a key way to increase wheat yield, but also an important strategy to address global food security challenges. Unfortunately, to date, less than 30 genes related to high wheat yield have been cloned and their functions have been analyzed. Therefore, there is still a lack of comprehensive understanding of the molecular mechanism of wheat yield trait formation and its regulatory network. The lack of important yield-related functional genes and unclear understanding of regulatory mechanisms have restricted the rapid development of the wheat industry. Therefore, it is urgently necessary to integrate and apply multi-omics technical analysis methods to mine new important yield functional genes that control multiple traits and clarify their biological effects and regulatory networks. This will not only provide important reference gene resources for molecular marker-assisted breeding of wheat in my country and accelerate the breeding process of wheat in my country, but also is expected to clarify the molecular mechanism of wheat yield trait formation and discover its regulatory network, thereby providing strong scientific and technological support for achieving high and stable wheat yields and sustainable agricultural production. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a KASP molecular marker for detecting wheat grain length and 1000-grain weight trait genes and its application, and a related primer set is developed based on wheat grain length and 1000-grain weight traits, which can quickly and conveniently detect SNP sites and excellent haplotypes of related traits, has the advantages of fast identification and high accuracy, and has good application prospects.

[0006] The technical solution of the present invention to solve the above technical problem is as follows: a KASP molecular marker for detecting wheat grain length and thousand-grain weight trait genes is provided, and the primer set for amplifying the KASP molecular marker is: Primer set 1, consisting of three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; and / or, Primer set 2 consists of three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

[0007] Furthermore, the genes for wheat grain length and thousand-grain weight are TaARF16-7D Gene.

[0008] The present invention provides a method for detecting wheat grain length and thousand-grain weight trait genes using the KASP molecular marker in wheat. TaARF16-7D Detection, identification and auxiliary identification of SNP allele variation and haplotype of genes for wheat grain length and thousand-grain weight traits or their application in molecular marker-assisted breeding.

[0009] The present invention provides a method for detecting wheat grain length and thousand-grain weight traits, comprising the following steps: (1) Extracting genomic DNA from the wheat material to be tested; (2) using the genomic DNA as a template and using the above-mentioned primer set 1 and / or primer set 2 to perform PCR amplification to obtain a PCR amplification product; (3) Clone and sequence the PCR amplified products and compare them; if the genotypes of the SNP1 site based on exon +2153 bp and the SNP2 site based on 3'UTR region +201 bp in the genomic DNA are AA and GG respectively, TaARF16-7D The genes were identified as SNP1 AA, SNP2 GG and haplotype HapⅠ -AGG, the trait candidate is long grain length, long ear length, high thousand-grain weight, but late flowering, late maturity, and tall stalk wheat; if the genotypes of the SNP1 site based on the exon +2153 bp and the SNP2 site based on the 3'UTR region +201 bp in the genomic DNA are GG and CC, respectively, TaARF16-7DThe genes in the tested wheat were identified as SNP1 GG and SNP2 CC and haplotype HapⅡ -GCT, the candidate wheat traits to be tested are short grain length, short ear length, low thousand-grain weight, but early flowering, early maturity, and short stem wheat.

[0010] The invention provides a kit for detecting wheat grain length and 1000-grain weight traits, comprising a primer set for amplifying KASP molecular marker genes for detecting wheat grain length and 1000-grain weight traits.

[0011] The present invention has the following beneficial effects: (1) This invention discovered a gene that regulates wheat grain length and thousand-grain weight (TKW) traits TaARF16- 7D . Through overexpression and knockout experiments, it was found that the transgenic plants had significant differences from wild-type wheat in two grain traits: thousand-grain weight and grain length; among them, the grains of the overexpression lines became smaller and TKW decreased, while the grains of the knockout lines became significantly longer and TKW increased. Compared with the wild-type receptor control, the average thousand-grain weight of the overexpression lines decreased by 4.67 g, while the thousand-grain weight of the knockout lines increased by 2.94 g, and the differences were significant; the average grain length of the wild-type control was 5.74 cm, which was 0.55 cm higher than the overexpression lines and 0.17 cm lower than the knockout mutants. These results indicate that the gene has a negative regulatory effect on wheat yield, and knockout is beneficial to improving the thousand-grain weight and grain length of wheat, and can be applied to wheat breeding improvement and germplasm innovation.

[0012] (2) Based on TaARF16-7D The KASP molecular marker was developed for the gene. If the genotypes of the SNP1 site at exon +2153 bp and the SNP2 site at 3'UTR region +201 bp in the genomic DNA are AA and GG, respectively, then TaARF16-7D The gene was identified as haplotype HapⅠ , the trait candidates are long grain length, long ear length, high thousand-grain weight, but late flowering, late maturity, and tall stalk wheat; if the genotypes of the SNP1 site based on the exon +2153 bp and the SNP2 site based on the 3'UTR region +201 bp in the genomic DNA are GG and CC respectively, then TaARF16-7D The gene was identified as haplotype HapⅡ The candidate wheat traits to be tested are short grain length, short ear length, low thousand-grain weight, but early flowering, early maturity, and short-stem wheat.

[0013] (3) The KASP molecular marker developed by the present invention not only improves the efficiency, accuracy and economy of genotyping, but also has significant advantages in terms of ease of operation and speed of obtaining results. Compared with previous technologies, KASP technology is more suitable for large-scale screening and marker-assisted selection breeding, which helps to accelerate the research progress of wheat grain development genes and the development of new varieties, and has important practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a map of the overexpression vector pWMB110; Figure 2 for TaARF16-7D T1 overexpression plant detection results; Figure 3 for TaARF16-7D T1 knockout plant test results; Figure 4 for TaARF16-7D Grain images of T2 overexpression and knockout plants; Figure 5 Schematic diagram of the KASP partial detection results of the SNP1 and SNP2 variant sites. DETAILED DESCRIPTION

[0015] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0016] Example 1: Cloning TaARF16-7D Gene A partial cDNA sequence of an interacting protein that interacts with genes related to regulating wheat thousand-grain weight was screened from the yeast two-hybrid cDNA library. The full-length cDNA and genomic sequence information of the candidate gene were first obtained using the sequence homology comparison method (NCBI website); based on its sequence information, specific genomic amplification primers TaARF-F1 and TaARF-R1 were designed, and the genomic DNA of Chinese spring wheat was extracted. The primer pair composed of TaARF-F1 and TaARF-R1 was used for the first round of PCR amplification. The first round of PCR amplification product was used as a template, and the primer pair composed of TaARF-cDNA-F1 and TaARF-cDNA-R1 was used for the second round of PCR amplification. The PCR amplification product was subjected to TA cloning and sequencing, and 15 positive clones were sequenced. Through comparison analysis, its genomic sequence was located in the chromosome interval of 114101652 bp to 114108455 bp of wheat homology group 7, and the gene was located on the positive strand, and it was named TaARF-7DThe cDNA nucleotide sequence is shown in SEQ ID NO.7, and the encoded protein is named TaARF-7D protein (the amino acid sequence is shown in SEQ ID NO.8).

[0017] The sequences of the amplification primers used are as follows: TaARF-F1: 5'-AGTCTGATGGATGCACTG-3' (SEQ ID NO. 9); TaARF-R1: 5'-GTTGCACTTCAACAGATG-3' (SEQ ID NO. 10); TaARF-cDNA-F1: 5'-ATGCCGCCGGTGGGCAGC-3' (SEQ ID NO. 11); TaARF-cDNA-R1: 5'-TTACTCCCATGGGTCATC-3' (SEQ ID NO. 12).

[0018] Example 2: Creation and Identification TaARF16-7D Transgenic plants Wheat KN199: wheat genetic transformation receptor material (reference: Richardson T, Thistleton J, Higgins TJ, Howitt C, Ayliffe M. Efficient Agrobacterium transformation ofelite wheat germplasm without selection. Plant Cell Tiss Organ Cult. 2014, 3(119): 647); Agrobacterium tumefaciens strain GV3101: a conventional strain in the laboratory of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (reference: Yadav S, Sharma P, Srivastava A, Desai P, Shrivastava N. Strainspecific Agrobacterium -mediated genetic transformation of Bacopa monnieri .Journal of Genetic Engineering and Biotechnology.2014,12(2):89-94).

[0019] (1) Construction of overexpression recombinant plasmid 1. Synthesis TaARF16-7D cDNA sequence of the gene; 2. Using the cDNA molecule synthesized in step 1 as a template, PCR amplification was performed using a primer pair consisting of ARF-TaA-F and ARF-TaA-R; wherein the primer sequences are as follows: ARF-TaA-F: 5'-CTCTAGAATGCCGCCGGTGGGCAGC-3' (SEQ ID NO. 13); ARF-TaA-R: 5'-CGAGCTCTTACTCCCATGGGTCATC-3' (SEQ ID NO. 14); 3. The product obtained in step 2 was cleaved with restriction endonuclease Xba Ⅰ and Sac Ⅰ Carry out double enzyme digestion and recover the digestion products; 4. Take the wheat overexpression vector pWMB110 (10800 bp, kanamycin resistance, vector map as shown Figure 1 as shown), using restriction endonucleases Xba Ⅰ and Sac Ⅰ Perform double enzyme digestion to recover the vector backbone; 5. Connect the enzyme digestion product of step 3 and the vector backbone of step 4 to obtain the recombinant plasmid of the overexpression plant.

[0020] (2) Construction of knockout recombinant plasmid 1. According to the characteristics of TaARF genome and cDNA sequence, a specific gene knockout guide RNA sequence (sgRNA) was designed to knock out the 9th exon cDNA sequence of the gene (the sgRNA target site was located 1118-1140bp after ATG of the cDNA sequence, and the nucleotide sequence was the same as that of sgRNA); sgRNA: 5'- CCA TGCCATGGCTTGGTGATGAC-3' (SEQ ID NO. 15, the underline is the PAM sequence); 2. TaARF16-7D The cDNA sequence of the gene was used as a template to amplify the TaARF-sgRNA fragment by PCR; wherein the nucleotide sequence of the amplification primer is as follows: ARF16F: 5'-GTCATCACCAGCCATGGCAgttttagagctagaaat-3' (SEQ ID NO. 16); ARF16R: 5'-TGCCATGGCTTGGTGATGACTGCTTCTTGGTGCCGCGCCTCC-3' (SEQ ID NO.17); 3. The TaARF-sgRNA fragment amplified in step 2 was digested with ASC and connected to the CRISPR / Cas9 expression vector pJIT163-2NLSCas9 to obtain the fusion expression vector pU6-TaARF-sgRNA (references for vector construction and transformation methods: Liu HX*, Si XM, Wang ZY, Cao LJ, Gao LF, Zhou XL, Wang WX, Wang K, Jiao CZ, Zhuang L, Liu YC, Hou J, Li T, Hao CY, Guo WL, Liu J, Zhang XY*. TaTPP- 7A Positive feedback regulates grain filling and wheat grain yield throughT6P-SnRK1 signaling pathway and sugar-ABA interaction. Plant BiotechnologyJournal.2023,21(6):1159-1175); 4. Use CeSg110F primer (5'-CCAGGCTTTACACTTTATGC-3', SEQ ID NO.18) to unidirectionally sequence the fusion expression vector. The fusion expression vector with the correct sequencing sequence is the knockout recombinant plasmid.

[0021] (3) Obtaining transgenic plants 1. The overexpression recombinant plasmid and the knockout recombinant plasmid prepared in step (1) and step (2) are respectively introduced into the Agrobacterium strain GV3101 to obtain overexpression and knockout recombinant Agrobacterium; 2. Take the recombinant Agrobacterium obtained in step 1, genetically transform the immature embryo callus of wheat KN199, and then culture it to obtain T0 regenerated plants and T1 seeds. Plant T1 positive plants in the second year, self-pollinate to obtain T2 grains, and test the grain traits such as thousand-grain weight and grain length of T2 grains. DNA needs to be extracted from transgenic plants in each generation, and PCR detection and sequencing are performed using the following methods to detect mutation knockout sites and obtain homozygous transgenic lines; The TaARF-OE-F1 / R1 primer pair was used for overexpression positive plant detection; the sequences of TaARF-OE-F1 and TaARF-OE-R1 are as follows: TaARF-OE-F1: 5'-TTTAGCCCTGCCTTCATAC-3' (SEQ ID NO. 19); TaARF-OE-R1: 5'-GTTGATTGACCAGCCAAC-3' (SEQ ID NO. 20); Depend on Figure 2 It can be seen that the amplified product of the overexpression positive plant is about 540 bp long.

[0022] The knockout mutant first used CeSg110F and TaU3F primers to amplify the DNA sequence of the transgenic plant; then the amplified fragment was sequenced using CeSg110F unidirectional primer; finally, the sequencing results were compared with the ARF target site for sequence mutation analysis to determine the existence, type and homozygosity of the mutant; wherein, the nucleotide sequence of CeSg110F is the same as above (SEQ ID NO.18); the nucleotide sequence of TaU3F is as follows: TaU3F: 5'-GAATTCATCCTCACGTTCAACACC-3' (SEQ ID NO. 21).

[0023] Depend on Figure 3 It can be seen that the cDNA sequence of exon 9 of the gene has been knocked out.

[0024] Depend on Figure 4 It can be seen that the transgenic plants have significant differences in the two grain traits of thousand-grain weight (TKW) and grain length compared with the wild-type wheat (KN199); among them, the overexpression line (OE- TaARF ) has smaller kernels and a lower TKW ( P <0.01, while the knockout strain ( KO - TaARF ) The grains became significantly longer ( P <0.05), TKW increased ( P <0.05). See Table 1 for the specific results.

[0025] Table 1 Phenotypic analysis of thousand-grain weight and grain traits of transgenic overexpression plants and knockout mutants

[0026] Note: One-way ANOVA was used. P <0.05,** P <0.01 As shown in Table 1, compared with the wild-type receptor control, the average thousand-grain weight of the overexpression strain decreased by 4.67 g, while the thousand-grain weight of the knockout strain increased by 2.94 g, and the differences were significant. The average grain length of the wild-type control was 5.74 cm, which was 0.55 cm higher than that of the overexpression strain and 0.17 cm lower than that of the knockout mutant ( P<0.05). These results indicate that this gene has a negative regulatory effect on wheat yield. Knockout of this gene is beneficial to improving wheat thousand-grain weight and increasing grain length, and can be applied to wheat breeding improvement and germplasm innovation.

[0027] Embodiment 3: TaARF16-7D Development and identification of KASP molecular markers for genes The full-length cDNA of the target gene was amplified from the transcriptome library of common hexaploid wheat (Chinese spring wheat) 10 days after anthesis. In the natural wheat population 262 Mini-Core Collection (262 MCC), the genotype information such as the differential SNP sites at this locus and the haplotype types formed by them were used to carry out phenotypic association analysis of the gene with important agronomic traits such as grain length, ear length and 1000-grain weight. The specific results are shown in Tables 2-8.

[0028] Table 2 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-1

[0029] Table 3 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-2

[0030] Table 4 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-3

[0031] Table 5 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-4

[0032] Table 6 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-5

[0033] Table 7 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-6

[0034] Table 8 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-7

[0035] Among them, in Tables 2 to 8, the haplotypes composed of SNP1 AA and SNP2 GG HapⅠ The genotype of the tested wheat material represented is AGG; the haplotype composed of SNP1 GG and SNP2 CC HapⅡ The genotype of the wheat material represented is GCT; further, HapⅠ The haplotype represents that the tested wheat material has long grain length, long ear length, high thousand-grain weight, but late flowering, late maturity, and tall stems; HapⅡ The haplotype represents that the tested wheat material has short grain length, short ear length, low thousand-grain weight, but early flowering, early maturity, and short stem traits.

[0036] From Tables 2 to 8, it can be seen that, regardless of whether it is a local variety or a selected variety, the allele variation of the SNP1 locus and the SNP2 locus can form two haplotypes in the natural population 262 MCC of wheat in my country. In local varieties, the representative long grain length, long ear length, high thousand-grain weight, but late flowering, late maturity, and tall stem traits have excellent haplotypes. HapⅠ The proportion of wheat varieties with short grain length, short ear length, low thousand-grain weight, but early flowering, early maturity, and short stem traits is only 14.38% in my country's 262 micro-core germplasms. HapⅡ The haplotype accounts for 85.62%; among the selected varieties, each major wheat region has a high demand for excellent haplotypes. HapⅠ The proportion of selected species increased to 29.52%, while HapⅡ It dropped to 70.48%, indicating that in the long-term artificial breeding process in my country, TaARF16-7D There are two important traits of the gene that have been strongly selected. Some wheat regions select grain length traits, while others select early flowering traits. Judging from the change trend of the two haplotypes from local varieties to bred varieties, Chinese breeders are currently inclined to select TaARF16-7D Genes for early flowering and early maturity traits.

[0037] Primers were designed according to the gene sequences of SNP1 and SNP2. The primer sequences are shown in Table 8.

[0038] Table 9 Primer sequences

[0039] Tissue sample detection: Extract genomic DNA from the tested wheat material; use the extracted genomic DNA as a template and use the primers in Table 9 for PCR amplification; the reaction procedure and reaction system are shown in Table 10; PCR amplification is run on the QuantStudio 7 instrument produced by ABI, and the genotyping results are automatically output. The results are shown in Figure 5 shown.

[0040] Table 10 Reaction procedure and reaction system

[0041] Each test material was planted in Luoyang (2002, 2005 and 2006) with conventional water and fertilizer management. The results of association analysis of local varieties in each genotype of the test wheat materials are shown in Table 11, and the results of association analysis of selected varieties are shown in Table 12. In Tables 11 and 12, 2002LY means Luoyang (2002); 2005LY means Luoyang (2005); 2006LY means Luoyang (2006); the data statistical software uses Tukey test, and the significant difference level is 0.05 or 0.01 (* P <0.05,** P <0.01).

[0042] Table 11 TaARF16-7D Association analysis of two haplotypes with grain TKW, grain shape traits, flowering period and maturity period in local varieties

[0043] As shown in Table 11, among the six agronomic traits of heading date (HD), maturity date (MD), plant height (PH), thousand kernel weight (TKW), grain length (GL) and spike length (SL), the two haplotypes showed statistically significant differences only in grain length. HapⅠ Compare HapⅡ The grain length of the rice increased by 0.04 cm (2002), 0.04 cm (2005) and 0.03 cm (2006) respectively, and the difference was extremely significant or significant in the three years of detection (* P <0.05,** P <0.01); HapⅠ Compare HapⅡ The thousand-grain weight of rice increased by 1.84 g (2002), 2.68 g (2005) and 1.55 g (2006) respectively, indicating that TaARF16-7D The gene is mainly closely related to the grain length and thousand-grain weight of wheat grains.

[0044] Table 12 TaARF16-7D Association analysis of two haplotypes with grain TKW, grain shape traits, plant flowering period and maturity period in selected varieties

[0045] As shown in Table 12, among the six agronomic traits of heading date (HD), maturity date (MD), plant height (PH), thousand kernel weight (TKW), grain length (GL) and spike length (SL), the two haplotypes showed statistically significant differences only in grain length. HapⅠ Compare HapⅡ The grain length of the rice increased by 0.04 cm (2002), 0.03 cm (2005) and 0.03 cm (2006) respectively, and the difference was extremely significant or significant in the three years of detection (* P <0.05,** P <0.01), HapⅠ Compare HapⅡ The thousand-grain weight of rice increased by 2.65 g (2002), 1.59 g (2005) and 3.04 g (2006) respectively, indicating that TaARF16-7D The gene is mainly closely related to the grain length trait of wheat grains.

[0046] In summary, SNP1, SNP2 and the two haplotypes formed by the two allele variant sites of SNP1 and SNP2 are significantly associated with the grain length and 1000-grain weight traits of wheat. The KASP molecular marker developed based on this can be used to detect the related traits in different wheat varieties. TaARF16-7D The alleles (SNPs) and / or haplotypes of the gene associated with the grain length and thousand-grain weight traits, and further, the superior alleles (SNP1 AA and SNP2 GG representing long grain length and high TKW traits) or haplotypes ( HapⅠ -AGG represents long grain length and high TKW traits) can be applied to molecular marker-assisted breeding. Through these KASP molecular markers, breeders can quickly screen out wheat germplasm with long grain length and high thousand-grain weight traits and apply them to genetic improvement of germplasm. Therefore, the present invention can accelerate the genetic improvement process of wheat varieties, improve breeding efficiency, and provide strong technical support for cultivating high-yield and high-quality wheat varieties that adapt to different ecological environments and cultivation requirements.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A KASP molecular marker for detecting wheat grain length and 1000-grain weight trait genes, characterized in that: The primer set used to amplify the KASP molecular marker is: Primer set 1, consisting of three primers whose nucleotide sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 respectively; and / or, Primer set 2 consists of three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively.

2. The KASP molecular marker for detecting wheat grain length and 1000-grain weight trait genes according to claim 1, characterized in that: The wheat grain length and thousand-grain weight trait genes are TaARF16-7D Gene.

3. The KASP molecular marker for detecting wheat grain length and 1000-grain weight trait genes according to any one of claims 1 to 2 is used in wheat TaARF16-7D Detection, identification and auxiliary identification of SNP allele variation and haplotype of genes for wheat grain length and thousand-grain weight traits or their application in molecular marker-assisted breeding.

4. A method for detecting wheat grain length and thousand-grain weight, characterized in that: The following steps are involved: (1) Extracting genomic DNA from the wheat material to be tested; (2) using genomic DNA as a template and using primer set 1 and / or primer set 2 in claim 1 to perform PCR amplification to obtain a PCR amplification product; (3) Clone and sequence the PCR amplified products and compare them; if the genotypes of the SNP1 site based on exon +2153 bp and the SNP2 site based on 3'UTR region +201 bp in the genomic DNA are AA and GG respectively, TaARF16-7D The genes were identified as SNP1 AA, SNP2 GG and haplotype HapⅠ -AGG, the trait candidate is long grain length, long ear length, high thousand-grain weight, but late flowering, late maturity, and tall stalk wheat; if the genotypes of the SNP1 site based on the exon +2153 bp and the SNP2 site based on the 3'UTR region +201 bp in the genomic DNA are GG and CC, respectively, TaARF16-7D The genes in the tested wheat were identified as SNP1 GG, SNP2 CC and haplotype HapⅡ -GCT, the candidate wheat traits to be tested are short grain length, short ear length, low thousand-grain weight, but early flowering, early maturity, and short stem wheat.

5. A kit for detecting wheat grain length and thousand-grain weight, characterized in that: The invention comprises a primer set for amplifying the KASP molecular marker for detecting wheat grain length and thousand-grain weight trait genes as described in any one of claims 1-2.

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