A molecular marker for detecting protein content of wheat kernels and use thereof

Abstract

The application belongs to the technical field of molecular marker detection, and particularly relates to a molecular marker for detecting wheat kernel protein content and application thereof. TaGPC-6A a g.34 G>A site in the interior, such as the 123th nucleotide in SEQ ID NO:1, which is G or A in nucleotide type. The application further provides a method for identifying or assisting in identifying the wheat kernel protein content trait, which comprises detecting the genotype of the g.34 G>A site in the wheat to be detected, and the wheat to be detected with the genotype AA is high-protein wheat, and the kernel protein content of the high-protein wheat is higher than that of the wheat to be detected with the genotype GG. Accordingly, the application develops a dCAPS marker primer pair for efficiently screening the high and low wheat kernel protein content. The molecular marker provided by the application can be used for detecting the genotype of TaGPC-6A , and is applied to the wheat kernel protein content improvement breeding, and can greatly improve the breeding efficiency.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of molecular marker detection, and particularly relates to a molecular marker for detecting wheat grain protein content and application thereof. BACKGROUND

[0002] Wheat is one of the important food crops, which provides about 20% of the heat and protein required by human beings. Grain protein content is one of the important quality indicators of wheat, and its improvement will help to improve the nutritional quality and processing quality of wheat. Strengthening the basic research on the grain protein content of wheat will help to cultivate high-quality special-purpose wheat varieties suitable for making bread or biscuits and other foods. The grain protein content of wheat is affected by genetic factors and external environmental conditions. Because it is greatly affected by environmental effects, and a large amount of seed sample is required for determination, it cannot be effectively determined and screened in the early generations of breeding, resulting in low precision and efficiency in selecting protein content by using traditional breeding methods.

[0003] In recent years, the rapid development of wheat functional genomics and molecular markers provides an opportunity for genetic improvement of wheat grain protein content. Using molecular markers can effectively identify genetic loci related to wheat grain protein content at the genetic level, and using molecular marker assisted selection strategy for genetic improvement of wheat grain protein content can greatly improve the efficiency of breeding selection, thereby shortening the breeding period. SUMMARY

[0004] The present application provides a molecular marker for detecting the grain protein content of wheat, which can be used to identify or screen the grain protein content of wheat, and provides more options for wheat quality improvement.

[0005] In the first aspect of the present application, a product for detecting the g.34G>A site in the TaGPC-6A gene is provided, which comprises:

[0006] (1) a primer composition for detecting the genotype of the g.34G>A site in the TaGPC-6A gene, the g.34G>A site is a SNP site in the TaGPC-6A gene in the wheat genome, which is the 123rd nucleotide of SEQ ID NO: 1, and the nucleotide type is G or A; the primer composition comprises:

[0007] SNP34-F: 5'-AAGGCGTACTTGCGTGAGGTC-3';

[0008] SNP34-R: 5'-TGGCAAGCCTGGAGAAGATGG-3';

[0009] (2) a reagent or a kit comprising the primer composition described in (1). The reagent or the kit further comprises a restriction enzyme Sac II or a sequencing reagent.

[0010] In the second aspect of the present application, the above product is used in any of the following aspects:

[0011] (1) identifying the allelic genotype of the g.34G>A site in the TaGPC-6A gene of wheat;

[0012] (2) identifying or assisting in identifying the grain protein content trait of wheat;

[0013] (3) screening or assisting in screening wheat single plants or strains or lines or varieties with high grain protein content;

[0014] (4) preparing a product for identifying or assisting in identifying or comparing the grain protein content of wheat;

[0015] (5) wheat molecular marker assisted selection breeding.

[0016] In the third aspect of the present application, a method for identifying the allelic genotype of the g.34G>A site in the TaGPC-6A gene is provided, which specifically comprises the following operation steps: taking the genomic DNA of the wheat variety / line to be tested as a template, and performing PCR amplification on the primer composition in the above product, wherein the length of the amplification product is 1210bp;

[0017] Then, the PCR amplification product is subjected to enzyme digestion with a restriction enzyme Sac II, followed by agarose gel electrophoresis, and the band type is determined; the amplification product can be distinguished into two band types of 962bp and 248bp with the GG allelic genotype after enzyme digestion; the amplification product has only one band type of 1210bp with the AA allelic genotype after enzyme digestion; or the PCR amplification product is sequenced to obtain the allelic genotype of the g.34G>A site in the TaGPC-6A gene.

[0018] The application can identify or assist in identifying the grain protein content trait of the to-be-tested wheat according to the genotype of the to-be-tested wheat, and therefore in a fourth aspect, the application provides a method for identifying the grain protein content trait of wheat, comprising: obtaining the genotype of the to-be-tested wheat by using the method for identifying the allelic genotype of the g.34G>A site in the TaGPC-6A gene, and then identifying or assisting in identifying the grain protein content trait of the to-be-tested wheat according to the genotype, wherein the grain protein content of the to-be-tested wheat with the genotype AA of the g.34G>A site is higher than that of the to-be-tested wheat with the genotype GG of the g.34G>A site; the genotype GG indicates that the nucleotide type of the g.34G>A site in the wheat genome is homozygous G; and the genotype AA indicates that the nucleotide type of the g.34G>A site in the wheat genome is homozygous A.

[0019] In a fifth aspect, the application provides a method for breeding a new wheat variety / line, comprising the following steps:

[0020] (1) detecting the allelic genotype of the g.34G>A site in the TaGPC-6A gene in the candidate wheat breeding material by using the method described above;

[0021] (2) selecting the wheat breeding material with the AA allelic genotype as the paternal line / female line for subsequent breeding.

[0022] Compared with the prior art, the main beneficial technical effects of the application are as follows:

[0023] 1. The application utilizes the cloned wheat grain protein content regulating gene TaGPC-6A (SEQ ID NO: 1), and finds that the g.34G>A site in the gene is significantly associated with the grain protein content trait of wheat. On this basis, the application develops and converts the g.34G>A site into a dCAPS marker. Research proves that the dCAPS marker can be used to detect the genotype of TaGPC-6A, and the method is simple and accurate.

[0024] 2. The application can be applied to wheat molecular marker-assisted selection breeding, realize early screening of the grain protein content trait of wheat, and greatly improve the efficiency of selection breeding. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 FIG. 1 is a haplotype analysis diagram of the TaGPC-6A gene in the first embodiment of the application.

[0026] Figure 2 FIG. 2 is a comparison diagram of the grain protein content among the four haplotypes of the TaGPC-6A gene in the first embodiment of the application.

[0027] Figure 3Figure 2 shows the results of enzyme digestion of the amplification product of the SNP34-F and SNP34-R primer pair in Example Two of the present application. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0029] In the following examples, the instruments and equipment involved are all conventional instruments and equipment unless otherwise specified; the reagents, carriers and other test materials involved are all conventional commercial products unless otherwise specified; and the test methods and detection methods involved are all conventional methods unless otherwise specified. In the quantitative tests in the following examples, three repeated experiments are set up, and the average value is taken as the result. The primer synthesis and sequencing work are all completed by Shengong Bioengineering (Shanghai) Co., Ltd.

[0030] Wheat materials involved in the examples:

[0031] A natural population consisting of 451 wheat varieties / lines, which is used for haplotype analysis of the wheat grain protein content regulatory gene TaGPC-6A, and association analysis of the allelic genotype of the g.34G>A site in the TaGPC-6A gene and the grain protein content phenotype.

[0032] Discovery of SNPs closely linked to the wheat grain protein content trait in the wheat grain protein content regulatory gene TaGPC-6A in Example One

[0033] 1. Population planting

[0034] The 451 wheat varieties / lines used for haplotype analysis of the TaGPC-6A gene and determination of the grain protein content phenotype were planted in the Yuanyang scientific research base of Henan Agricultural University in the 2023-2024 growing season, using a completely randomized block design, with each planting plot being 2m long and 1.5m wide, and 12 rows being designed in each plot, with each variety being planted in two rows, single seed sowing, and the inter-row and intra-row plant spacings being 20cm and 10cm, respectively. The test field was managed according to the standard, and there was no occurrence of drought, lodging and diseases and pests during the entire growing period. After maturation, the grains were threshed, dried and stored uniformly.

[0035] 2. Determination of grain protein content

[0036] The grain protein content of 451 wheat varieties / lines was determined using a Perten DA7250 near-infrared analyzer (Perten, Sweden). To ensure the accuracy and reliability of the test results, 3 replicates were set for each sample, and each replicate was determined 3 times, and the average of 9 measurement values was taken as the final test value. The measurement results are shown in Table 1.

[0037] Table 1. The average grain protein content of 451 wheat varieties / lines and the genotype of g.34 G>A site

[0038]

[0039]

[0040]

[0041]

[0042]

[0043] 3. Haplotype analysis of wheat grain protein content regulating gene TaGPC-6A

[0044] The leaves of 451 wheat varieties / lines were sampled at the seedling stage, and the coding region of TaGPC-6A gene was amplified after extracting the genomic DNA, and then the amplification product was sequenced.

[0045] The specific steps are as follows:

[0046] ① DNA extraction: SLS method was used to extract the DNA of 451 wheat varieties / lines at the seedling stage.

[0047] The specific implementation steps are as follows:

[0048] (1) Take an appropriate amount of wheat leaves into a 2 mL centrifuge tube containing steel balls, freeze in liquid nitrogen, then grind with a grinder, add 800 μL SLS, shake for 20 min to make it fully mixed; the SLS contains 288 mM NaCl, 200 mM Tris-HCl, 25 mM EDTA, 0.5% SDS;

[0049] (2) Then add an equal volume of three-in-one solution (phenol: chloroform: isopropyl alcohol = 25:24:1);

[0050] (3) Shake for 10 min to make it fully mixed;

[0051] (4) Centrifuge at 12,000 rpm for 15 min, transfer the supernatant to a new 2 mL centrifuge tube, add an equal volume of pre-cooled isopropanol, and stand for 10 min for precipitation;

[0052] (5) 12,000 rpm centrifugation for 15 min, discard the supernatant, add 0.5 mL 75% (volume percent) ethanol aqueous solution, stand for 5 min.

[0053] (6) 12,000 centrifugation for 5 min, discard the supernatant, after vacuum drying, add 100 μL TE to dissolve the precipitate, and the wheat leaf genomic DNA is obtained.

[0054] 2. Amplification and sequence analysis of TaGPC-6A gene coding region

[0055] PCR primers were designed, TaGPC-6A-F and TaGPC-6A-R were designed at the positions of start codon ATG and stop codon TAG respectively, and the specific information is as follows:

[0056] TaGPC-6A-F: 5'-ATGGAGGTGGAGTCTAGC-3';

[0057] TaGPC-6A-R: 5'-CACATGCGCACCTTCCAC-3'.

[0058] Using the DNA of 451 wheat varieties / lines as templates, the primer pair composed of TaGPC-6A-F and TaGPC-6A-R was used for PCR amplification. The PCR amplification system is shown in Table 2, and the PCR amplification program is shown in Table 3.

[0059] Table 2. PCR amplification system of TaGPC-6A-F and TaGPC-6A-R primer pair for TaGPC-6A gene

[0060] Component Volume 2 x Phanta buffer 25 μL dNTP 1 μL Phanta Enzyme 1 μL Primer F 2 μL Primer R 2 μL DNA 2 μL ddH2O To 50 μL

[0061] Table 3. PCR amplification program of TaGPC-6A-F and TaGPC-6A-R primer pair for TaGPC-6A gene

[0062]

[0063] The PCR products were subjected to 1% agarose gel electrophoresis, and after detecting the amplification of the target band, they were sent to Shanghai Shengong for sequencing.

[0064] The sequencing results were analyzed, and the results are as follows: Figure 1As shown, in the 451 wheat varieties / lines detected, there are 4 SNP sites in the coding region of TaGPC-6A gene, which are located at the 19th, 21st, 34th and 1297th positions from the first start codon ATG in SEQ ID NO: 1, and the TaGPC-6A gene can be divided into four haplotypes according to the four SNP sites, which are named as Hap1, Hap2, Hap3 and Hap4 respectively. The four haplotypes of TaGPC-6A were combined with the grain protein content phenotype values for analysis, and the results are as follows Figure 2 As shown, the grain protein content of wheat varieties / lines with Hap3 type is significantly higher than that of the other three haplotypes. Further analysis found that the variation site g.34G>A located at the 34th position from the first start codon ATG in SEQ ID NO: 1 is a specific site for distinguishing Hap3 from the other three haplotypes, and this site is closely linked to the grain protein content.

[0065] Example Two Development of dCAPS marker of g.34G>A site in TaGPC-6A gene

[0066] The sequence of the position where the g.34G>A site in TaGPC-6A gene is located was analyzed, and it was found that the site is located on the recognition sequence of restriction enzyme Sac II. When the site is GG genotype, it can be recognized and cut by Sac II, while when the site is AA genotype, it cannot be recognized by Sac II. Based on this, the dCAPS marker was developed. First, specific primer pair SNP34-F and SNP34-R were designed according to the Chinese Spring reference genome sequence, which was used to amplify the DNA fragment containing the g.34G>A site. The length of the amplified fragment was 1210 bp, and the sequence of the amplified product was shown as SEQ ID NO: 2. The nucleotide sequences of the primer pair are as follows:

[0067] SNP34-F: 5'-AAGGCGTACTTGCGTGAGGTC-3';

[0068] SNP34-R: 5'-TGGCAAGCCTGGAGAAGATGG-3'.

[0069] DNA of some lines randomly selected from the 451 wheat varieties / lines was amplified using the above primer pair. The PCR amplification system is shown in Table 4, and the PCR amplification program is shown in Table 5.

[0070] Table 4. PCR amplification system of the primer pair composed of SNP34-F and SNP34-R for the DNA fragment containing g.34G>A

[0071] Component Volume 10 x Taq plus buffer 3 μL dNTP 1 μL Taq plus DNA Polymerase 1 μL Primer F 1 μL Primer R 1 μL DNA 2 μL ddH2O To 30 μL

[0072] Table 5. PCR amplification program for the DNA fragment containing g.34G>A using primer pairs composed of SNP34-F and SNP34-R.

[0073]

[0074]

[0075] Take 20 μL of the amplified product and digest it with the restriction endonuclease Sac II at 37 °C. The digestion reaction system is shown in Table 6.

[0076] Table 6. Enzyme digestion reaction system for SNP34-F and SNP34-R primer pair amplification products

[0077] Component Volume PCR product 20 μL 10 x buffer 3 μL Sac II 1 μL ddH2O To 30 μL

[0078] The enzyme digestion products were subjected to 1% agarose gel electrophoresis to determine whether the DNA fragments amplified using SNP34-F and SNP34-R primer pairs could be digested by the enzymes. The results are as follows: Figure 3 As shown, the DNA fragment of wheat lines with the GG genotype at the g.34G>A locus could be successfully cleaved into two fragments of length 962 bp and 248 bp, respectively, while the DNA fragment of wheat lines with the AA genotype at the g.34G>A locus could not be cleaved. This result indicates that the dCAPS marker can be used to determine the allele genotype at the g.34G>A locus.

[0079] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A product of detecting TaGPC-6A the g.34 G>A site within a gene, characterized by, The product comprises: (1) A primer composition and a restriction enzyme for detecting the genotype of a g.34 G>A site within a gene TaGPC-6A The primer composition and the restriction enzyme for detecting the genotype of a g.34 G>A site within a gene Sac II, the g.34 G>A site is a SNP site within a gene in a wheat genome, is the 123rd nucleotide of SEQ ID NO: 1, and the nucleotide species is G or A; the primer composition comprises: TaGPC-6A The primer composition and the restriction enzyme for detecting the genotype of a g.34 G>A site within a gene SNP34-F : 5'-AAGGCGTACTTGCGTGAGGTC-3'; SNP34-R : 5'-TGGCAAGCCTGGAGAAGATGG-3'; or (2) comprises the primer composition and a restriction endonuclease of (1) Sac reagents or kits of II.

2. Use of the product according to claim 1 in any one of the following, characterized in that, comprises: (1) identifying a wheat TaGPC-6A genotype of a g.34 G>A locus within the gene; (2) identifying or assisting in identifying the wheat grain protein content trait; (3) screening or assisting in screening wheat single plants or strains or lines or varieties with high wheat grain protein content; (4) preparing products for identifying or assisting in identifying or comparing the high or low wheat grain protein content of the test wheat; (5) wheat molecular marker assisted selection breeding; The grain protein content of the test wheat with the g.34 G>A site genotype AA is higher than that of the test wheat with the g.34 G>A site genotype GG.

3. A method of identifying TaGPC-6A the genotype of the g.34 G>A locus within a gene, characterized in that, comprises the following operation steps: Using the genomic DNA of the test wheat variety / line as the template, the primer composition in the product of claim 1 is used for PCR amplification; The restriction enzyme Sac II The PCR products were digested with restriction enzyme, and then agarose gel electrophoresis was performed, and the band types were determined; the amplified products could be distinguished into two band types of 962 bp and 248 bp with GG allele genotype; the amplified products had only one band type of 1210 bp with AA allele genotype; or using the genomic DNA of the test wheat variety / line as the template, the primer composition in the product of claim 1 is used for PCR amplification; The PCR amplification product was sequenced to obtain TaGPC-6A The g.34 G>A locus is a SNP locus in the wheat genome TaGPC-6A The SNP locus is nucleotide 123 of SEQ ID NO: 1, and the nucleotide species is G or A.

4. A method for identifying a wheat grain protein content trait, characterized in that, comprises: Using the method of claim 3 to obtain the genotype of the test wheat, and then identifying or assisting in identifying the grain protein content trait of the test wheat according to the genotype; The grain protein content of the test wheat with the g.34 G>A site genotype AA is higher than that of the test wheat with the g.34 G>A site genotype GG.

5. A method for selecting a new variety / line of high quality wheat, characterized by, comprises the following steps: (1) using the method of claim 3 to detect the allelic genotype of the g.34 G>A site in the candidate wheat breeding material TaGPC-6A gene (2) selecting wheat breeding materials with AA allelic genotype as the paternal / maternal parent for subsequent breeding.

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