CN116814836A - Wheat grain weight and quality related gene TaPSK1-2A molecular marker and application - Google Patents
Wheat grain weight and quality related gene TaPSK1-2A molecular marker and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a molecular marker of a wheat grain weight and quality related gene TaPSK1-2A and application thereof. The molecular marker contains SNP loci related to the grain weight and quality of wheat, the SNP loci are positioned at 425870766bp of chromosome 2 of wheat, the polymorphism is C or T, and the genotype of 425870766bp of chromosome 2 is higher than that of wheat variety with genotype of CC. The molecular marker provided by the invention can be used for identifying whether the excellent allele of TaPSK1-2A exists in wheat varieties/strains or not, and can be applied to auxiliary selective breeding and other known grain weight and quality related gene polymerization breeding.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a molecular marker of a wheat grain weight and quality related gene TaPSK1-2A and application thereof.
Background
The yield of wheat is determined by the number of ears per unit area, the number of ears and thousand grain weight. Grain weight is one of the three elements of yield, and its genetic ability is more stable than yield. Thousand kernel weight is mainly controlled by the shapes such as kernel size, length and the like, while genotype is a decisive factor influencing the kernel weight of wheat, and has important influence on the kernel weight. The grain protein content is one of important wheat quality indexes, and influences the processing quality and the nutrition quality of wheat. Therefore, the development of functional molecular markers by exploiting excellent allelic variation of genes related to thousand kernel weight and protein content has an important role in high wheat yield and quality improvement.
PSK is a sulfonated pentapeptide Tyr (SO 3H) -Ile-Tyr (SO 3H) -Thr-Gln consisting of 5 amino acids, formed by cleavage modification of a precursor polypeptide of 80-120 amino acids in length, which belongs to a small gene family. The most pronounced physiological effect of PSK was found to be the stimulation of growth and proliferation of low density cell suspensions. In addition, exogenously applied PSK-alpha polypeptides or over-expressed PSK-alpha precursor genes can significantly increase the growth rate of Arabidopsis leaves, roots, and hypocotyls. 15 members of the small peptide family of PSK are contained in wheat, with only the specific expression of TaPSK1-2A in the endosperm. The protein encoded by TaPSK1-2A is one of the PSKs family members in wheat, but it is not known whether it relates to wheat grain weight and quality to date.
In view of this, the present invention has been made.
Disclosure of Invention
One of the purposes of the invention is to provide a SNP locus related to the grain weight and quality of wheat, wherein the SNP locus is positioned at 425870766bp of chromosome 2 of the wheat, and the polymorphism is C or T.
Further, the thousand grain weight, grain length and protein content of wheat with genotype TT at 425870766bp of chromosome 2 are higher than those of wheat variety with genotype CC.
The second object of the invention is to provide a molecular marker of the wheat grain weight and quality related gene TaPSK1-2A, wherein the molecular marker contains the SNP locus, the nucleotide sequence of the molecular marker is shown as 182-249bp of SEQ ID NO.1, and the degenerate base R of 201bp of the sequence is C or T.
The third object of the invention is to provide the molecular marked amplification primer, and the nucleotide sequence of the amplification primer is shown as SEQ ID NO. 2-4.
It is a fourth object of the present invention to provide the use of any one of the following for the SNP site, the molecular marker or the amplification primer:
1) Screening or identifying excellent wheat variety resources;
2) Cultivating wheat variety resources with excellent characters;
the superior traits are manifested by high thousand grain weight, grain length, and protein content.
The fifth object of the present invention is to provide a method for identifying wheat traits using said SNP sites, comprising the steps of:
detecting the genotype of the 425870766bp position of the No.2 chromosome in the genome of the wheat to be detected, wherein if the genotype of the 425870766bp position of the No.2 chromosome in the genome of the wheat to be detected is TT, the wheat is high in thousand kernel weight, grain length and protein content, and if the genotype of the 425870766bp position of the No.2 chromosome in the genome of the wheat to be detected is CC, the wheat is low in thousand kernel weight, grain length and protein content.
Further, the method for detecting the genotype of the genome of the wheat to be detected at 425870766bp of chromosome 2 comprises the following steps of 1) or 2):
1) Direct sequencing;
2) Designing a primer for amplifying a molecular marker containing the SNP locus, amplifying the wheat genome DNA to be detected by using the primer, and carrying out genotyping detection on an amplified product.
The sixth object of the present invention is to provide a method for breeding a wheat variety with excellent properties using said SNP locus, comprising the steps of:
firstly detecting the genotype of the 425870766bp part of the chromosome 2 of the genome of the wheat to be detected, and then selecting the wheat to be detected with the genotype TT as a parent for breeding;
the superior traits are manifested by high thousand grain weight, grain length, and protein content.
The invention has the following beneficial effects:
according to the invention, KASP molecular markers are developed for SNP loci of TaPSK1-2A promoter regions of sulfopeptide genes, and genotyping and phenotype association analysis are carried out on 220 parts of wheat germplasm resource materials in different ecological regions of China. The results show that KASP-TaPSK1-2A markers can divide different varieties of wheat into 2 haplotypes: haplotypes Hap-2A-1 and haplotypes Hap-2A-2. The genotype of the haplotype Hap-2A-1 is C/C, and the genotype of the haplotype Hap-2A-2 is T/T. And carrying out association analysis on materials with different genotypes by combining the phenotype data, and finding that the thousand grain weight, grain length and grain type of the wheat material with the T/T genotype are obviously larger than those of the wheat material with the C/C genotype. The genotype T/T is shown to be excellent allelic variation and has positive effect on wheat grain weight and grain type. In the breeding process, the aggregation of the favorable mutant alleles is consistent with the result of yield increase in the crop breeding process, so that the KASP-TaPSK1-2A molecular marker provided by the invention can efficiently detect and track the TaPSK-2A gene in wheat varieties/strains, and provides technical support for improving the high-yield breeding of wheat.
Drawings
FIG. 1 shows the position of the KASP marker on the TaPSK1-2A gene.
FIG. 2 shows the genotyping results of the KASP marker primer set on wheat germplasm resources in different wheat regions of China.
Wherein red dots in box A represent HEX type alleles C/C and blue dots in box B represent FAM type alleles T/T.
FIG. 3 is a comparison of grain weight (A), grain length (B) and protein content (C) of wheat germplasm resources of different genotypes of TaPSK 1-2A.
FIG. 4 shows the geographical distribution of the two genotypes of wheat germplasm resources of TaPSK1-2A in the main wheat planting area of China.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, 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 are commercially available unless otherwise indicated.
Example 1: obtaining of molecular marker of wheat grain weight and quality related gene TaPSK1-2A
The gene TaPSK1-2A related to the grain weight and quality of wheat of the embodiment codes for sulfopeptide (Phytosulfokine) and belongs to PSKs family members. Analysis was performed based on the wheat genome variation association database (http:// heat. Cau. Edu. Cn/wheatUnion /) 677 hexaploid wheat resequencing data, and it was found that the TaPSK1-2A gene had one SNP variation site 806bp from the promoter (FIG. 1) and was divided into two different haplotypes.
The SNP site related to the wheat grain weight and quality of this example is located at 425870766bp of the 2A chromosome. A KASP molecular marker is designed for the SNP locus, and is named as KASP-TaPSK1-2A, SEQ ID NO.1 is KASP-TaPSK1-2A (182-249 bp) and nearby sequence information, and degenerate base R of 201bp of the sequence shown in SEQ ID NO.1 is C or T.
Amplification primers were also designed for the KASP molecular markers, synthesized by the division of bioengineering (Shanghai) and the primer sequences were as follows:
KASP-TaPSK1-2A-F1(SEQ ID NO.2)：5′-GAAGGTCGGAGTCAACGGATTTCCCGTGTTTGACTCTCCAC-3′；
KASP-TaPSK1-2A-F2(SEQ ID NO.3)：5′-GAAGGTGACCAAGTTCATGCTTCCCGTGTTTGACTCTCCAT-3′；
common primer KASP-TaPSK1-2A-R (SEQ ID NO. 4): 5'-GCCGGTCCGTTTAGTTTTT-3'.
Example 2: application of KASP-TaPSK1-2A in identification of wheat genotype
1. Wheat genome DNA extraction
Collecting leaves of wheat seedlings in two leaves and one heart period, and extracting genome DNA of the wheat by using a CTAB method. DNA concentration and quality were measured using Nanodrop2000 and 1% agarose gel electrophoresis, with an A260/A280 ratio of about 1.8 indicating acceptable sample quality.
Kasp marker amplification and detection
A set of PCR reaction systems comprising two temperature steps was used: the DNA is denatured at a higher temperature, and then annealed and extended at a lower, same temperature. KASP-PCR amplification was performed on a fluorescence quantitative instrument (Quanskio) TM 7) And (3) performing the process. The PCR reaction system was 5. Mu.L, and contained KASP Master Mix (2X) 2. Mu.L, SNP Primer Mix (Primer Mix working solution) 1. Mu.L, DNA 1. Mu.L, ddH 2 0 was added to 5. Mu.L. The PCR amplification system is as follows: (1) 94 ℃ for 15min; (2) 94℃for 20s; decreasing the temperature by 0.6 ℃ every cycle at 61-55 ℃; 10 cycles were performed in total; (3) 94 ℃,20s; the work performed 37 cycles at 55℃for 45 s. After the PCR amplification cycle was completed, the fluorescence value was read by using an OMEGA SNP typing apparatus. In this method, SNP site detection uses the fluorophores FAM (485 nm for excitation light, 520nm for emission light) and VIC (535 nm for excitation light, 556nm for emission light) to distinguish between two isogenic sites. The passive reference dye ROX (excitation light 575nm, emission light 610 nm) was used to correct for the differences in signal from well to well due to reaction volume errors.
3. Data analysis
The data were analyzed using genotype reading software Kmaster Caller, in which the VIC and FAM data are plotted on the x-axis and y-axis, respectively. The VIC and FAM values of each reaction well are corrected by the specific Kong Can specific dye (ROX) value, and the data fluorescence values are subjected to standardized treatment to obtain the corresponding relative fluorescence values of the VIC and FAM of each PCR reaction well. And clustering samples according to the relative fluorescence values, and further determining genotypes according to the sample clusters and the fluorescence types.
Example 3: application of KASP-TaPSK1-2A in identification of wheat traits
The embodiment provides application of a molecular marker KASP-TaPSK1-2A in identifying thousand grain weight, grain length and protein content of wheat, which is specifically as follows:
1. 285 parts of wheat germplasm resource materials from different ecological areas in China in Table 1 are used, and the materials are planted in a Tougly county test station in Gansu province and a Zhuang Lang county and nan lake test station in Gansu province in 2020-2022. The three planting environments are respectively marked as follows: 2020TW (Tongwei, 2020), 2021TW (Tongwei, 2021) and 2022ZL (Zhuang Lang, 2022). The field test was performed on a random block, and 3 replicates were set. 3 rows of seeds are planted in each material, the row spacing is 20cm, the row length is 1m, and 60 seeds are planted in each row. After the seeds are mature, selecting 200 full seeds in each strain, checking the seeds, and performing three biological repetitions on each strain.
2. Seed weight, grain length, grain width and grain area were analyzed using a ten thousand depth SC-G automatic seed test analyzer, and specific data are shown in table 1.
3. The protein content of the natural population grains was determined using a near infrared grain analyzer.
4. Using the molecular markers provided in example 1, the significance between the wheat germplasm resource phenotypes of the two genotypes was analyzed using the method provided in example 2, and the typing results are shown in FIG. 2. The wheat grain weight, grain length and protein content carrying the different genotypes were analyzed for significant differences using the one-way anova method of Excel (2016) software, and specific data are shown in tables 1 and 3.
As a result, it was found that the thousand grain weight, grain length and grain type of the wheat material of the T/T genotype were significantly larger than those of the C/C genotype. The genotype T/T is shown to be excellent allelic variation and has positive effect on wheat grain weight and grain type.
Fig. 4 shows the distribution of wheat germ plasm materials of two genotypes in main wheat planting areas (Henan, hebei, shaanxi, shanxi, shandong, etc.) in China, with wheat materials of genotype TT occupying the main position. The geographical information of the specific wheat germplasm resources is shown in Table 2, and the information sources are 'first industry approval variety information inquiry' and Zhuang Qiaosheng 'Chinese wheat variety improvement and pedigree analysis'.
TABLE 1 seed data for wheat germ plasm resources in different ecoregions and genotypes thereof
Table 2 geographical distribution of wheat germ plasm resources
Example 4: application of KASP-TaPSK1-2A in cultivation of wheat variety with excellent properties
By adopting the molecular marker provided in the example 1 and adopting the method provided in the example 2, the genotype of the 425870766bp position of the chromosome 2 of the genome of the wheat to be detected is detected, then the wheat to be detected with the genotype TT is selected as a parent for breeding, and the wheat variety with high thousand grain weight, grain length and protein content is obtained through breeding.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (8)
1. A SNP locus related to wheat grain weight and quality, wherein the SNP locus is located at 425870766bp of chromosome 2 of wheat, and the polymorphism is C or T.
2. The SNP locus according to claim 1, wherein the thousand grain weight, grain length and protein content of wheat of genotype TT at 425870766bp of chromosome 2 are higher than those of wheat variety of genotype CC.
3. A molecular marker of a wheat grain weight and quality related gene TaPSK1-2A is characterized in that the molecular marker contains the SNP locus of claim 1, the nucleotide sequence of the molecular marker is shown as 182-249bp of SEQ ID NO.1, and the degenerate base R of 201bp of the sequence shown as SEQ ID NO.1 is C or T.
4. The molecular marker amplification primer of claim 3, wherein the nucleotide sequence of the amplification primer is shown in SEQ ID NO. 2-4.
5. Use of any one of the SNP sites as defined in claim 1, the molecular markers as defined in claim 3 or the amplification primers as defined in claim 4:
1) Screening or identifying excellent wheat variety resources;
2) Cultivating wheat variety resources with excellent characters;
the superior traits are manifested by high thousand grain weight, grain length, and protein content.
6. The method for identifying wheat traits by using the SNP locus according to claim 1, comprising the steps of:
detecting the genotype of the 425870766bp position of the No.2 chromosome in the genome of the wheat to be detected, wherein if the genotype of the 425870766bp position of the No.2 chromosome in the genome of the wheat to be detected is TT, the wheat is high in thousand kernel weight, grain length and protein content, and if the genotype of the 425870766bp position of the No.2 chromosome in the genome of the wheat to be detected is CC, the wheat is low in thousand kernel weight, grain length and protein content.
7. The method according to claim 6, wherein the method for detecting the genotype at 425870766bp of chromosome 2 in the genome of wheat under test comprises the following steps 1) or 2):
1) Direct sequencing;
2) Designing a primer for amplifying a molecular marker containing the SNP locus, amplifying the wheat genome DNA to be detected by using the primer, and carrying out genotyping detection on an amplified product.
8. The method for breeding a wheat variety with excellent properties by using the SNP locus according to claim 1, comprising the following steps:
firstly detecting the genotype of the 425870766bp part of the chromosome 2 of the genome of the wheat to be detected, and then selecting the wheat to be detected with the genotype TT as a parent for breeding;
the superior traits are manifested by high thousand grain weight, grain length, and protein content.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018113702A1 (en) * | 2016-12-21 | 2018-06-28 | Institute Of Crop Sciences, The Chinese Academy Of Agricultural Sciences | Plant grain trait-related protein, gene, promoter and snps and haplotypes |
| CN112176082A (en) * | 2019-07-04 | 2021-01-05 | 中国科学院植物研究所 | SNP molecular marker of wheat grain weight related gene and application thereof |
| CN114908188A (en) * | 2022-06-14 | 2022-08-16 | 河北省农林科学院粮油作物研究所 | Application of KASP molecular marker related to wheat grain weight and grain length and primer composition thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018113702A1 (en) * | 2016-12-21 | 2018-06-28 | Institute Of Crop Sciences, The Chinese Academy Of Agricultural Sciences | Plant grain trait-related protein, gene, promoter and snps and haplotypes |
| CN112176082A (en) * | 2019-07-04 | 2021-01-05 | 中国科学院植物研究所 | SNP molecular marker of wheat grain weight related gene and application thereof |
| CN114908188A (en) * | 2022-06-14 | 2022-08-16 | 河北省农林科学院粮油作物研究所 | Application of KASP molecular marker related to wheat grain weight and grain length and primer composition thereof |
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