Molecular markers and application of wheat grain weight and quality-related gene TaPSK1-2A

Abstract

The present invention belongs to the field of biotechnology, and in particular to molecular markers and applications of TaPSK1‑2A genes related to wheat grain weight and quality. The molecular marker contains a SNP site related to wheat grain weight and quality, and the SNP site is located at 425870766bp of chromosome 2 of wheat. The polymorphism is C or T. The thousand-grain weight, grain length and protein content of wheat with a genotype of TT at 425870766bp of chromosome 2 are higher than those of wheat varieties with a genotype of CC. The molecular markers provided by the present invention can be used to identify whether there are TaPSK1‑2A superior alleles in wheat varieties / lines, and can be used in assisted selection breeding and breeding with other known grain weight and quality-related genes.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a molecular marker of a gene TaPSK1-2A related to wheat grain weight and quality and an application thereof. Background Art

[0002] Wheat yield is determined by the number of ears per unit area, the number of grains per ear, and the thousand-grain weight. Grain weight is one of the three elements of yield, and its heritability is more stable than yield. The thousand-grain weight is mainly controlled by the grain size and length and other morphological factors, while the genotype is the decisive factor affecting the grain weight of wheat and has an important influence on the grain weight. Grain protein content is one of the important wheat quality indicators, which affects the processing quality and nutritional quality of wheat. Therefore, exploring the excellent allelic variation of genes related to thousand-grain weight and protein content and developing functional molecular markers are important for wheat high yield and quality improvement.

[0003] PSK is a sulfonated pentapeptide Tyr(SO 3H)-Ile-Tyr(SO3H)-Thr-Gln composed of 5 amino acids. It is formed by the cleavage modification of a precursor polypeptide of 80-120 amino acids in length. This precursor polypeptide belongs to a small gene family. Studies have found that the most significant physiological effect of PSK is that it can stimulate the growth and proliferation of low-density cell suspensions. In addition, exogenous application of PSK-α polypeptide or overexpression of PSK-α precursor gene can significantly increase the growth rate of Arabidopsis leaves, roots and hypocotyls. There are 15 members of the PSK small peptide family in wheat, among which TaPSK1-2A is highly expressed only in the endosperm. The protein encoded by TaPSK1-2A is one of the members of the PSKs family in wheat, but as of now, it is unknown whether it is related to wheat grain weight and quality.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a SNP site related to wheat grain weight and quality, wherein the SNP site is located at 425870766bp of wheat chromosome 2, and the polymorphism is C or T.

[0006] Furthermore, the thousand-grain weight, grain length and protein content of wheat with genotype TT at 425870766bp of chromosome 2 were higher than those of wheat varieties with genotype CC.

[0007] The second object of the present invention is to provide a molecular marker of wheat grain weight and quality related gene TaPSK1-2A, wherein the molecular marker contains the SNP site, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, 182-249bp, and the degenerate base R of the 201bp of the sequence is C or T.

[0008] The third object of the present invention is to provide amplification primers for the molecular markers, the nucleotide sequences of the amplification primers are shown in SEQ ID NO.2-4.

[0009] A fourth object of the present invention is to provide any of the following applications of the SNP site, the molecular marker or the amplification primer:

[0010] 1) Screening or identifying wheat variety resources with excellent traits;

[0011] 2) Cultivate wheat variety resources with excellent traits;

[0012] The excellent traits are high thousand-grain weight, grain length and protein content.

[0013] A fifth object of the present invention is to provide a method for identifying wheat traits using the SNP loci, comprising the following steps:

[0014] The genotype at 425870766bp of chromosome 2 in the wheat genome to be tested is detected. If the genotype at 425870766bp of chromosome 2 in the wheat genome to be tested is TT, it is wheat with high thousand-grain weight, grain length and protein content. If the genotype at 425870766bp of chromosome 2 in the wheat genome to be tested is CC, it is wheat with low thousand-grain weight, grain length and protein content.

[0015] Furthermore, the method for detecting the genotype at 425870766bp of chromosome 2 in the wheat genome to be tested includes the following 1) or 2):

[0016] 1) Direct sequencing;

[0017] 2) Designing primers for amplifying molecular markers containing the SNP site, using the primers to amplify the wheat genomic DNA to be tested, and performing genotyping detection on the amplified products.

[0018] The sixth object of the present invention is to provide a method for breeding wheat varieties with excellent traits using the SNP loci, comprising the following steps:

[0019] First, the genotype of the chromosome 2 at 425870766bp of the wheat genome to be tested is detected, and then the wheat to be tested with the genotype of TT is selected as the parent for breeding;

[0020] The excellent traits are high thousand-grain weight, grain length and protein content.

[0021] The present invention has the following beneficial effects:

[0022] The present invention develops a KASP molecular marker for the SNP site in the promoter region of the sulfopeptide gene TaPSK1-2A, and performs genotyping and phenotypic association analysis on 220 wheat germplasm resources in different ecological zones in my country. The results show that the KASP-TaPSK1-2A marker can divide different varieties of wheat into two haplotypes: haplotype Hap-2A-1 and haplotype Hap-2A-2. The genotype of haplotype Hap-2A-1 is C / C, and the genotype of haplotype Hap-2A-2 is T / T. Combined with the phenotypic data, an association analysis is performed on materials with different genotypes, and it is found that the thousand-grain weight, grain length and grain shape of the wheat material with the T / T genotype are significantly greater than those of the C / C genotype. It shows that the genotype T / T is an excellent allele variation, which has a positive effect on wheat grain weight and grain shape. During the breeding process, the aggregation of favorable mutant alleles is consistent with the result of increased yield in the crop breeding process, so the KASP-TaPSK1-2A molecular marker provided by the present invention can efficiently detect and track the TaPSK-2A gene in wheat varieties / lines, providing technical support for improving wheat high-yield breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the location of the KASP marker on the TaPSK1-2A gene.

[0024] Figure 2 The results of genotyping wheat germplasm resources from different wheat regions in my country using the KASP marker primer set.

[0025] Among them, the red dot in frame A represents the HEX type allele C / C, and the blue dot in frame B represents the FAM type allele T / T.

[0026] Figure 3 Comparison of grain weight (A), grain length (B) and protein content (C) of wheat germplasm resources with different genotypes of TaPSK1-2A. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0028] Example 1: Acquisition of molecular markers for wheat grain weight and quality-related gene TaPSK1-2A

[0029] The wheat grain weight and quality-related gene TaPSK1-2A in this example encodes phytosulfokine and belongs to the PSKs family. According to the analysis of 677 hexaploid wheat resequencing data from the Wheat Genome Variation Union Database (http: / / wheat.cau.edu.cn / WheatUnion / ), it was found that the TaPSK1-2A gene has a SNP mutation site (806 bp from the promoter) Figure 1 ), which can be divided into two different haplotypes.

[0030] The SNP site related to wheat grain weight and quality in this embodiment is located at 425870766bp on chromosome 2A. A KASP molecular marker was designed for this SNP site and named KASP-TaPSK1-2A. SEQ ID NO.1 is KASP-TaPSK1-2A (182-249bp) and its nearby sequence information. The degenerate base R of the 201bp of the sequence shown in SEQ ID NO.1 is C or T.

[0031] Amplification primers were also designed for the KASP molecular marker and synthesized by Sangon Biotech (Shanghai) Co., Ltd. The primer sequences are as follows:

[0032] KASP-TaPSK1-2A-F1 (SEQ ID NO.2): 5′-GAAGGTCGGAGTCAACGGATTTCCCGTGTTTGACTCTCCAC-3′;

[0033] KASP-TaPSK1-2A-F2 (SEQ ID NO.3): 5′-GAAGGTGACCAAGTTCATGCTTCCCGTGTTTGACTCTCCAT-3′;

[0034] Common primer KASP-TaPSK1-2A-R (SEQ ID NO. 4): 5′-GCCGGTCCGTTTAG TTTTT-3′.

[0035] Example 2: Application of KASP-TaPSK1-2A in Identifying Wheat Genotypes

[0036] 1. Wheat Genomic DNA Extraction

[0037] Leaves of wheat seedlings at the two-leaf and one-heart stage were collected, and wheat genomic DNA was extracted using the CTAB method. NanoDrop2000 and 1% agarose gel electrophoresis were used to detect DNA concentration and quality. An A260 / A280 ratio of about 1.8 indicated that the sample quality was qualified.

[0038] 2. KASP marker amplification and detection

[0039] A PCR reaction system with two temperature steps is used: DNA is denatured at a higher temperature, and then annealed and extended at a lower temperature. KASP-PCR amplification is performed on a fluorescence quantification instrument (QuanStudio TM 7). The PCR reaction system was 5 μL, including 2 μL KASP Master mix (2×), 1 μL SNP Primer Mix (primer mixed working solution), 1 μL DNA, and 5 μL ddH20. The PCR amplification system was: (1) 94°C, 15 min; (2) 94°C, 20 s; 61-55°C, decreasing by 0.6°C per cycle; 10 cycles in total; (3) 94°C, 20 s; 55°C, 45 s, 37 cycles in total. After the PCR amplification cycle, the fluorescence value was read using the OMEGA SNP typing instrument. In this method, SNP site detection uses the fluorophores FAM (excitation light 485 nm, emission light 520 nm) and VIC (excitation light 535 nm, emission light 556 nm) to distinguish two isogenic sites. The passive reference dye ROX (excitation light 575 nm, emission light 610 nm) is used to correct the signal difference between wells due to the reaction volume error.

[0040] 3. Data Analysis

[0041] The data were analyzed using the genotype reading software Kluster Caller, in which VIC and FAM data were plotted on the x-axis and y-axis, respectively. The VIC and FAM values ​​of each reaction well were corrected by the value of the reference dye (ROX) of that specific well, and the data fluorescence values ​​were standardized to obtain the relative fluorescence values ​​corresponding to VIC and FAM of each PCR reaction well. Based on the relative fluorescence values, the samples were clustered, and the genotypes were further determined based on the sample clusters and fluorescence types.

[0042] Example 3: Application of KASP-TaPSK1-2A in Identification of Wheat Traits

[0043] This example provides the application of molecular marker KASP-TaPSK1-2A in identifying wheat thousand-grain weight, grain length and protein content, as follows:

[0044] 1. 285 wheat germplasm resources from different ecological zones in my country were used in Table 1. The materials were planted in the Tongwei County Experimental Station in Gansu Province and the Nanhu Experimental Station in Zhuanglang County, Gansu Province in 2020-2022. The three planting environments were recorded as: 2020TW (Tongwei, 2020), 2021TW (Tongwei, 2021) and 2022ZL (Zhuanglang, 2022). The field experiment adopted a randomized block experiment with 3 repetitions. Each material was planted in 3 rows, with a row spacing of 20 cm, a row length of 1 m, and 60 seeds planted in each row. After the grains matured, 200 full seeds were selected from each strain for testing, and each strain was subjected to three biological replicates.

[0045] 2. The seed weight, length, width and area were analyzed using the Wanshen SC-G automatic seed analysis and thousand-grain weight meter. The specific data are shown in Table 1.

[0046] 3. Use near-infrared grain analyzer to determine the protein content of grains in natural populations.

[0047] 4. Using the molecular markers provided in Example 1 and the method provided in Example 2, the significance between the phenotypes of wheat germplasm resources of the two genotypes was analyzed. The typing results are as follows: Figure 2 The single-factor analysis of variance method using Excel (2016) software was used to analyze whether there were significant differences in grain weight, grain length and protein content among wheat with different genotypes. The specific data are shown in Tables 1 and Figure 3 .

[0048] The results showed that the thousand-grain weight, grain length and grain shape of the wheat materials with T / T genotype were significantly greater than those with C / C genotype, indicating that the genotype T / T was an excellent allele variation and had a positive effect on wheat grain weight and grain shape.

[0049] In the distribution of wheat germplasm materials of the two genotypes in the main wheat planting areas of my country (Henan, Hebei, Shaanxi, Shanxi, Shandong, etc.), wheat materials with genotype TT occupy a dominant position. The specific geographical information of wheat germplasm resources is shown in Table 2, and the information source is "The First Industry Approved Variety Information Query" and Zhuang Qiaosheng's "Chinese Wheat Variety Improvement and Pedigree Analysis".

[0050] Table 1 Grain data and genotypes of wheat germplasm resources in different ecological zones

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[0058] Table 2 Geographical release of wheat germplasm resources

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[0065]

[0066] Example 4: Application of KASP-TaPSK1-2A in breeding wheat varieties with superior traits

[0067] Using the molecular markers provided in Example 1 and the method provided in Example 2, the genotype at 425870766bp of chromosome 2 of the wheat genome to be tested is first detected, and then the wheat to be tested with a genotype of TT is selected as the parent for breeding, and a wheat variety with high thousand-grain weight, grain length and protein content is obtained through breeding.

[0068] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0069] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Amplification primers for the molecular marker TaPSK1-2A, a gene related to wheat grain weight and quality, characterized in that: The nucleotide sequence of the amplification primer is shown in SEQ ID NO.2-4, the nucleotide sequence of the molecular marker is shown in SEQ ID NO.1, bp 182-249, and the degenerate base R in the molecular marker is C or T.

2. The use of any one of the following amplification primers according to claim 1: 1) Screening or identifying wheat variety resources with excellent traits; 2) Cultivate wheat variety resources with excellent traits; The excellent traits are high thousand-grain weight, grain length and protein content.

3. A method for identifying wheat traits, characterized in that: The following steps are involved: The genomic DNA of the wheat to be tested is used as a template and amplified using the amplification primers described in claim 1. If the amplified product is as shown in 182-249 bp of SEQ ID NO.1 and the genotype at the degenerate base R is TT, it is wheat with high thousand-grain weight, grain length and protein content; if the amplified product is as shown in 182-249 bp of SEQ ID NO.1 and the genotype at the degenerate base R is CC, it is wheat with low thousand-grain weight, grain length and protein content.

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