A KASP molecular marker for detecting wheat grain length and 1000-grain weight trait genes and its application

Abstract

The present invention discloses a KASP molecular marker for detecting wheat grain length and 1000-grain weight trait genes and its application, belonging to the field of biotechnology. The primer set for detecting SNP or traits by the KASP molecular marker is primer set 1 and / or primer set 2, and the specific sequences of primer set 1 and primer set 2 are given; at the same time, the KASP molecular marker can be applied to wheat <h2 style=";text-align:left;direction:ltr">TaARF16‑7D The present invention is aimed at detecting, identifying, and assisting in the identification of SNP alleles and haplotypes for wheat grain length and 1000-grain weight traits, or in molecular marker-assisted breeding. The present invention has developed a primer set based on wheat grain length and 1000-grain weight traits, which can quickly and conveniently detect SNP sites and their superior haplotypes for the relevant traits. The method has the advantages of rapid identification and high accuracy, and has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a KASP molecular marker for detecting wheat grain length and 1000-grain weight trait genes and its application. Background Art

[0002] wheat( Triticum aestivum L ( . ) is a major grain crop. Its yield is primarily determined by three factors: grain weight, number of grains per spike, and number of spikes per unit area. Grain weight, as a core yield trait, has a high heritability (59-92%), and its inheritance is primarily an additive effect between genes, making it a typical quantitative trait controlled by multiple genes. Research has shown that grain weight is primarily determined by grain shape and grain filling rate. Grain shape can be further broken down into grain morphological traits such as grain length, grain width, and grain thickness. In production and breeding practices, 1000-kernel weight has been widely used as a key indicator of grain size, and grain morphological traits such as grain length, grain width, and grain thickness have also been shown to be significantly positively correlated with yield. Therefore, in production, grain shape and 1000-kernel weight complement each other, jointly determining and influencing the formation of crop yield traits.

[0003] Traditional breeding methods have reached a bottleneck in increasing wheat yield, but the rapid development of molecular biology technologies has provided new breakthroughs for the discovery and utilization of wheat yield-related genes. Current research indicates that through a variety of analytical techniques, including functional genomics, transcriptomics, gene editing (such as CRISPR-Cas9), and bioinformatics, it is possible to identify yield-related functional genes that control both grain shape and 1000-grain weight, and to analyze their biological functions and regulatory networks. This will provide efficient and precise marker and gene resources for wheat marker-assisted breeding (MAS), accelerating the genetic improvement of high-yield and stable-yield wheat varieties.

[0004] In summary, identifying and utilizing genes associated with wheat grain shape and 1000-grain weight is not only a key approach to increasing wheat yield, but also a crucial strategy for addressing global food security challenges. Unfortunately, to date, fewer than 30 genes associated with high wheat yield have been cloned and functionally characterized. Consequently, a comprehensive understanding of the molecular mechanisms and regulatory networks underlying wheat yield traits remains elusive. The scarcity of key yield-related functional genes and the lack of a clear understanding of their regulatory mechanisms have hindered the rapid development of the wheat industry. Therefore, there is an urgent need to integrate and apply multi-omics analytical techniques to identify new key yield genes controlling multiple traits and elucidate their biological roles and regulatory networks. This will not only provide important reference gene resources for molecular marker-assisted wheat breeding in my country and accelerate wheat breeding efforts, but also offer the potential to elucidate the molecular mechanisms underlying yield traits and uncover their regulatory networks, 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 address the above-mentioned deficiencies in the prior art, the present invention aims to provide a KASP molecular marker and its application for detecting wheat grain length and 1000-grain weight trait genes. 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 their excellent haplotypes of related traits. It 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 problems is as follows: a KASP molecular marker for detecting wheat grain length and 1000-grain weight trait genes is provided, and the primer set for amplifying the KASP molecular marker is:

[0007] 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;

[0008] and / or,

[0009] 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.

[0010] Furthermore, the wheat grain length and 1000-grain weight trait genes are TaARF16-7D Gene.

[0011] The present invention provides a KASP molecular marker for detecting wheat grain length and thousand-grain weight trait genes in wheat TaARF16-7D Detection, identification, and auxiliary identification of SNP allele variation and haplotype of genes for wheat grain length and 1000-grain weight traits, or their application in molecular marker-assisted breeding.

[0012] The present invention provides a method for detecting wheat grain length and thousand-grain weight, comprising the following steps:

[0013] (1) Extracting genomic DNA from the wheat material to be tested;

[0014] (2) using the genomic DNA as a template, performing PCR amplification using the above-mentioned primer set 1 and / or primer set 2 to obtain a PCR amplification product;

[0015] (3) The PCR amplified products were cloned, sequenced, and compared; 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 were AA and GG, respectively, TaARF16-7D The gene was identified as SNP1 AA, SNP2 GG and haplotype in the wheat to be tested. HapⅠ-AGG, the trait candidate is long grain length, long ear length, high 1000-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 and SNP2 CC and haplotype HapⅡ -GCT, the wheat traits candidates to be tested are short grain length, short ear length, low thousand-grain weight, but early flowering, early maturity, and short stem wheat.

[0016] The invention provides a kit for detecting wheat grain length and 1000-grain weight traits, comprising a primer set for amplifying KASP molecular markers for detecting wheat grain length and 1000-grain weight trait genes.

[0017] The present invention has the following beneficial effects:

[0018] (1) This study discovered a gene that regulates wheat grain length and thousand-kernel 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 line became smaller and the TKW decreased, while the grains of the knockout line became significantly longer and the TKW increased. Compared with the wild-type receptor control, the average thousand-grain weight of the overexpression line decreased by 4.67 g, while the thousand-grain weight of the knockout line 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 average of the overexpression line and 0.17 cm lower than the average of the knockout mutant. 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 increasing grain length of wheat, and can be applied to wheat breeding improvement and germplasm innovation and utilization.

[0019] (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 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 HapⅡThe 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.

[0020] (3) The KASP molecular markers developed in this invention not only improve the efficiency, accuracy, and economy of genotyping, but also offer significant advantages in ease of operation and speed of result acquisition. Compared with previous technologies, KASP technology is more suitable for large-scale screening and marker-assisted selection breeding, helping to accelerate research progress on wheat grain development genes and the development of new varieties, and has important practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a map of the overexpression vector pWMB110;

[0022] Figure 2 for TaARF16-7D T1 overexpression plant detection results;

[0023] Figure 3 for TaARF16-7D T1 knockout plant test results;

[0024] Figure 4 for TaARF16-7D Grain images of T2 overexpression and knockout plants;

[0025] Figure 5 Schematic diagram of the KASP partial detection results of the SNP1 and SNP2 mutation sites. DETAILED DESCRIPTION

[0026] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0027] Example 1: Cloning TaARF16-7D Gene

[0028] A partial cDNA sequence of an interacting protein that interacts with genes regulating wheat thousand-grain weight was obtained 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 genomic DNA of Chinese spring wheat was extracted. The first round of PCR amplification was performed using the primer pair consisting of TaARF-F1 and TaARF-R1. The first round of PCR amplification product was then used as a template for the second round of PCR amplification using the primer pair consisting of TaARF-cDNA-F1 and TaARF-cDNA-R1. The PCR amplification product was subjected to TA cloning and sequencing, and 15 positive clones were sequenced. Through alignment analysis, its genomic sequence was located in the chromosomal 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-7D The cDNA nucleotide sequence is shown in SEQ ID NO.7, and the protein it encodes is named TaARF-7D protein (the amino acid sequence is shown in SEQ ID NO.8).

[0029] The sequences of the amplification primers used are as follows:

[0030] TaARF-F1: 5'-AGTCTGATGGATGCACTG-3' (SEQ ID NO. 9);

[0031] TaARF-R1: 5'-GTTGCACTTCAACAGATG-3' (SEQ ID NO. 10);

[0032] TaARF-cDNA-F1: 5'-ATGCCGCCGGTGGGCAGC-3' (SEQ ID NO. 11);

[0033] TaARF-cDNA-R1: 5'-TTACTCCCATGGGTCATC-3' (SEQ ID NO. 12).

[0034] Example 2: Creation and identification TaARF16-7D genetically modified plants

[0035] Wheat KN199: wheat genetic transformation recipient material (Reference: Richardson T, Thistleton J, Higgins TJ, Howitt C, Ayliffe M. Efficient Agrobacterium transformation of elite wheat germplasm without selection. Plant Cell Tiss Organ Cult. 2014, 3(119): 647);

[0036] Agrobacterium tumefaciens strain GV3101: a conventional laboratory strain of the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (references: 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).

[0037] (1) Construction of overexpression recombinant plasmid

[0038] 1. Synthesis TaARF16-7D cDNA sequence of the gene;

[0039] 2. Using the cDNA molecule synthesized in step 1 as a template, PCR amplification was performed using the primer pair consisting of ARF-TaA-F and ARF-TaA-R; wherein the primer sequences are as follows:

[0040] ARF-TaA-F: 5'-CTCTAGAATGCCGCCGGTGGGCAGC-3' (SEQ ID NO. 13);

[0041] ARF-TaA-R: 5'-CGAGCTCTTACTCCCATGGGTCATC-3' (SEQ ID NO. 14);

[0042] 3. The product obtained in step 2 was cleaved with restriction enzyme Xba Ⅰ and Sac Ⅰ Perform double enzyme digestion and recover the digestion products;

[0043] 4. Take the wheat overexpression vector pWMB110 (10800 bp, kanamycin resistance, vector map as shown Figure 1 as shown), using restriction enzymesXba Ⅰ and Sac Ⅰ Perform double enzyme digestion to recover the vector backbone;

[0044] 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.

[0045] (2) Construction of knockout recombinant plasmid

[0046] 1. Based on the characteristics of the TaARF genome and cDNA sequences, a specific gene knockout guide RNA sequence (sgRNA) was designed to knock out the cDNA sequence of exon 9 of the gene (the sgRNA target site is located 1118-1140 bp after the ATG of the cDNA sequence, and the nucleotide sequence is the same as that of the sgRNA);

[0047] sgRNA: 5'- CCA TGCCATGGCTTGGTGATGAC-3′ (SEQ ID NO. 15, the underlined sequence is the PAM sequence);

[0048] 2. TaARF16-7D The cDNA sequence of the gene was used as a template to amplify the TaARF-sgRNA fragment by PCR; the nucleotide sequences of the amplification primers are as follows:

[0049] ARF16F: 5'-GTCATCACCAGCCATGGCAgttttagagctagaaat-3' (SEQ ID NO. 16);

[0050] ARF16R: 5'-TGCCATGGCTTGGTGATGACTGCTTCTTGGTGCCGCGCCTCC-3'

[0051] (SEQ ID NO. 17);

[0052] 3. The TaARF-sgRNA fragment amplified in step 2 was digested with ASC and ligated into the CRISPR / Cas9 expression vector pJIT163-2NLSCas9 to generate 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- 7APositive feedback regulates grain filling and wheat grain yield throughT6P-SnRK1 signaling pathway and sugar-ABA interaction. Plant BiotechnologyJournal.2023,21(6):1159-1175);

[0053] 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.

[0054] (3) Obtaining transgenic plants

[0055] 1. The overexpression recombinant plasmid and knockout recombinant plasmid prepared in step (1) and step (2) are respectively introduced into Agrobacterium strain GV3101 to obtain overexpression and knockout recombinant Agrobacterium;

[0056] 2. Use the recombinant Agrobacterium obtained in step 1 to genetically transform the immature embryonic callus of wheat KN199, followed by culture to obtain T0 regenerated plants and T1 seeds. The following year, plant the T1-positive plants and self-pollinate to obtain T2 grains, which are then tested for grain traits such as thousand-grain weight and grain length. DNA is extracted from transgenic plants in each generation, and PCR and sequencing are performed using the following methods to detect the mutation knockout site and obtain homozygous transgenic lines.

[0057] The TaARF-OE-F1 / R1 primer pair was used to detect overexpression-positive plants. The sequences of TaARF-OE-F1 and TaARF-OE-R1 are shown below:

[0058] TaARF-OE-F1: 5'-TTTAGCCCTGCCTTCATAC-3' (SEQ ID NO. 19);

[0059] TaARF-OE-R1: 5'-GTTGATTGACCAGCCAAC-3' (SEQ ID NO. 20);

[0060] Depend on Figure 2 It can be seen that the amplified product of the overexpression positive plant is about 540 bp long.

[0061] Knockout mutants were first amplified using the CeSg110F and TaU3F primer pairs from transgenic plants. The amplified fragments were then sequenced using the CeSg110F unidirectional primer. Finally, the sequencing results were compared with the ARF target site for sequence mutation analysis to determine the presence, type, and homozygosity of the mutant. The nucleotide sequence of CeSg110F is the same as above (SEQ ID NO. 18); the nucleotide sequence of TaU3F is as follows:

[0062] TaU3F: 5'-GAATTCATCCTCACGTTCAACACC-3' (SEQ ID NO. 21).

[0063] Depend on Figure 3 It can be seen that the cDNA sequence of exon 9 of the gene has been targeted and knocked out.

[0064] Depend on Figure 4 It can be seen that the transgenic plants showed significant differences from the wild type wheat (KN199) in terms of two grain traits: thousand-grain weight (TKW) and grain length. TaARF )'s kernels became smaller and TKW decreased ( P <0.01, while the knockout lines ( KO - TaARF ) Grains become significantly longer ( P <0.05), TKW increased ( P <0.05). Specific results are shown in Table 1.

[0065] Table 1 Phenotypic analysis of 1000-grain weight and grain traits of transgenic overexpression plants and knockout mutants

[0066]

[0067] Note: One-way ANOVA was used. P <0.05,** P <0.01

[0068] As shown in Table 1, compared with the wild-type receptor control, the average thousand-grain weight of the overexpression strain decreased by 4.67g, while the thousand-grain weight of the knockout strain increased by 2.94g, and the differences were significant. The average grain length of the wild-type control was 5.74cm, which was 0.55cm higher than that of the overexpression strain and 0.17cm lower than that of the knockout mutant ( P <0.05). These results indicate that this gene negatively regulates wheat yield, and knocking it out is beneficial for increasing wheat thousand-grain weight and grain length, and can be applied to wheat breeding improvement and germplasm innovation.

[0069] Example 3: TaARF16-7D Development and identification of KASP molecular markers for genes

[0070] The full-length cDNA of the target gene was amplified from a transcriptome library of common hexaploid wheat (Chinese spring wheat) 10 days after anthesis. In the 262 Mini-Core Collection (262 MCC) of natural wheat populations, phenotypic association analyses of this gene with important agronomic traits such as grain length, spike length, and 1000-grain weight were conducted using genotypic information, including differential SNPs at this locus and the resulting haplotypes. The results are shown in Tables 2-8.

[0071] Table 2 TaARF-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-1

[0072]

[0073] Table 3 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-2

[0074]

[0075] Table 4 TaARF16-7D Genotyping and haplotype types of genes in the natural wheat population 262 MCC-3

[0076]

[0077] Table 5 TaARF16-7D Genotyping and haplotype types of genes in a natural wheat population 262 MCC-4

[0078]

[0079] Table 6 TaARF16-7D Genotyping and haplotype types of genes in a natural wheat population 262 MCC-5

[0080]

[0081] Table 7 TaARF16-7D Genotyping and haplotype types of genes in a natural wheat population 262 MCC-6

[0082]

[0083] Table 8 TaARF16-7D Genotyping and haplotype types of genes in a natural wheat population 262 MCC-7

[0084]

[0085] Among them, in Tables 2-8, the haplotype 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 the wheat material with 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 characteristics.

[0086] As shown in Tables 2-8, the allelic variation of SNP1 and SNP2 can form two haplotypes in the natural population 262 MCC of wheat in my country, regardless of whether it is a local variety or a selected variety. 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 wheat varieties with the characteristics of short grain length, short spike length, low thousand-grain weight, but early flowering, early maturity and short stem account for only 14.38% of the 262 micro-core germplasms in my country, while the wheat varieties with the characteristics of short grain length, short spike length, low thousand-grain weight, early flowering, early maturity and short stem HapⅡ The haplotype accounts for 85.62%; in the selected varieties, the major wheat regions have high demand for excellent haplotypes. HapⅠ The proportion of selective breeding 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 artificially selected. Some wheat regions select grain length traits, while others select early flowering traits. Judging from the changing trends of the two haplotypes from local varieties to developed varieties, Chinese breeders currently tend to select TaARF16-7D Genes for early flowering and early maturity traits.

[0087] Primers were designed based on the gene sequences of SNP1 and SNP2. The primer sequences are shown in Table 8.

[0088] Table 9 Primer sequences

[0089]

[0090] Tissue sample testing: Genomic DNA of the tested wheat material was extracted; PCR amplification was performed using the extracted genomic DNA as a template using the primers listed in Table 9; the reaction procedure and reaction system are shown in Table 10; PCR amplification was performed on a QuantStudio 7 instrument produced by ABI, and genotyping results were automatically output. Figure 5 shown.

[0091] Table 10 Reaction procedure and reaction system

[0092]

[0093] Each test material was planted in Luoyang (2002, 2005, and 2006) under conventional water and fertilizer management. The results of the association analysis of the local varieties of the test wheat materials of each genotype are shown in Table 11, and the results of the association analysis of the selected varieties are shown in Table 12. In Tables 11 and 12, 2002LY represents Luoyang (2002); 2005LY represents Luoyang (2005); and 2006LY represents Luoyang (2006). The data statistical software used Tukey's test, with a significant difference level of 0.05 or 0.01 (* P <0.05,** P <0.01).

[0094] Table 11 TaARF16-7D Association analysis of two haplotypes with grain TKW, grain shape traits, flowering period and maturity period in local varieties

[0095]

[0096] As shown in Table 11, among the six agronomic traits of local varieties, namely, 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. The differences were extremely significant or significant in all three years (* 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 closely related to the grain length and thousand-grain weight of wheat grains.

[0097] Table 12 TaARF16-7D Association analysis of two haplotypes with grain TKW, grain shape traits, plant flowering period and maturity period in selected varieties

[0098]

[0099] 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 differences were extremely significant or significant in all three years (* 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.

[0100] In summary, SNP1, SNP2 and the two haplotypes formed by the two allele variant sites of SNP1 and SNP2 are significantly associated with grain length and 1000-grain weight traits of wheat. The KASP molecular marker developed based on this can be used to detect the haplotypes in different wheat varieties. TaARF16-7D Alleles (SNPs) and / or haplotypes associated with grain length and 1000-grain weight traits of the gene, and further, 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. These KASP molecular markers enable breeders to quickly screen for wheat germplasm with long grain length and high 1000-grain weight traits and apply them to genetic improvement. Therefore, this invention can accelerate the genetic improvement of wheat varieties, improve breeding efficiency, and provide strong technical support for the cultivation of high-yield, high-quality wheat varieties adapted to different ecological environments and cultivation requirements.

[0101] 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A primer set for detecting KASP molecular marker genes for wheat grain length and 1000-grain weight traits in wheat TaARF16-7D Detection, identification, and auxiliary identification of SNP allele variation and haplotype of genes for wheat grain length and 1000-grain weight traits, or their application in molecular marker-assisted breeding; in, The primer set includes: 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, Primer set 2, consisting of three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively; described TaARF16-7D The nucleotide sequence of the cDNA of the gene is shown in SEQ ID NO.

7.

2. 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, TaARF16-7D The primer set of KASP molecular marker of the gene was used for PCR amplification to obtain PCR amplification products; (3) The PCR amplified products were cloned, sequenced, and compared; 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 were AA and GG, respectively, TaARF16-7D The gene was identified as SNP1 AA, SNP2 GG and haplotype in the wheat to be tested. HapⅠ -AGG, the trait candidate is long grain length, long ear length, high 1000-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 wheat traits candidates to be tested are short kernel length, short ear length, low thousand-kernel weight, but early flowering, early maturity, and short stem wheat; Wherein, the primer set includes: 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, Primer set 2, consisting of three primers whose nucleotide sequences are shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 respectively; described TaARF16-7D The nucleotide sequence of the cDNA of the gene is shown in SEQ ID NO.7.

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