Dcaps primer pairs for identifying or assisting in identifying wheat sds-sedimentation value and application

Abstract

The application discloses a dCAPS primer pair for identifying or assisting in identifying the SDS-sedimentation value of wheat and an application, wherein the primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 2. The dCAPS primer pair can be used for quickly and intuitively identifying the SDS-sedimentation value of wheat, is suitable for large-scale breeding screening, and shortens the breeding period of wheat quality.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of wheat breeding, and particularly relates to a dCAPS primer pair for identifying or assisting in identifying SDS-sedimentation value of wheat and application. BACKGROUND

[0002] Wheat is the most adaptable and widely planted grain crop in the world, and about 40% of the world's population relies on wheat as their main food, which provides 20% of the heat and protein needed by human beings. As the second largest grain crop in China, wheat can be processed into various flour products, and the processing quality of wheat is largely dependent on the content and type of seed storage protein in wheat. The glutenin and gliadin in wheat gluten protein endow wheat dough with unique rheological properties, thereby enabling the production of a series of food for human consumption.

[0003] SDS (sodium dodecyl sulfate) sedimentation value reflects the quantity and quality of wheat gluten protein, and the higher the content of wheat gluten protein and the better the quality, the higher the sedimentation value. SDS-sedimentation value has been used as one of the important indicators for wheat quality evaluation in wheat breeding work at home and abroad. By mining excellent haplotypes of SDS-sedimentation value regulating genes and developing specific molecular primers and applying them to molecular marker assisted breeding, the quality of breeding materials can be effectively improved. In the previous work of the laboratory, the wheat storage protein regulating gene TaMYB44 has been identified, which proves that TaMYB44 is significantly related to the SDS-sedimentation value of wheat. However, at present, molecular markers for TaMYB44 excellent haplotype have not been developed. SUMMARY

[0004] In view of the problems that the mining of excellent haplotypes of SDS-sedimentation value regulating genes is insufficient, specific molecular primers used in breeding are few, and molecular markers for excellent haplotypes of wheat SDS-sedimentation value related genes TaMYB44 have not been developed, the present application provides a dCAPS primer pair for identifying or assisting in identifying SDS-sedimentation value of wheat and application, which can quickly and intuitively identify the SDS-sedimentation value of wheat, is suitable for large-scale breeding screening, and improves the efficiency of molecular marker assisted selection for wheat quality breeding.

[0005] In order to achieve the above purpose, in one aspect, the present application provides a dCAPS primer pair for identifying or assisting in identifying SDS-sedimentation value of wheat, which comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO. 1 (5'-GGAACAAGTCATGGTGATGATC-3') and a reverse primer with a nucleotide sequence as shown in SEQ ID NO. 2 (5'-GTGGTGGTGTCTTTTCCTTTG-3').

[0006] The above primer of the present application can quickly and intuitively identify the SDS-sedimentation value of wheat, is suitable for large-scale breeding screening, and shortens the breeding cycle of wheat quality. TaMYB44 The present application can detect, screen and identify the high SDS-sedimentation value material of wheat, and improve the efficiency of molecular marker assisted selection of wheat quality breeding.

[0007] The second aspect of the present application provides the use of the above primer pair in identifying or assisting in identifying the SDS-sedimentation value of wheat.

[0008] The third aspect of the present application provides a method for identifying the SDS-sedimentation value of wheat, which comprises the following steps:

[0009] 1) performing PCR amplification reaction with the genomic DNA of the wheat to be tested as a template, using the above primer pair for PCR amplification, and the amplification product is a band with a size of 179 bp;

[0010] 2) performing enzyme digestion of the PCR product with the restriction endonuclease BclI, and then performing agarose gel electrophoresis, if the length of the fragment is 179 bp after electrophoresis, it proves that the amplification product is not cut by BclI, which indicates that the wheat to be tested contains Hap II haplotype and has low SDS-sedimentation value; if the length of the fragment is 161 bp after electrophoresis, it proves that the amplification product is cut by BclI, and the PCR product is smaller, which indicates that the wheat to be tested contains Hap I haplotype and has high SDS-sedimentation value.

[0011] Specifically, in step 1), the PCR amplification system (10 μL) is as follows: 20-100 ng / μL of the genomic DNA of the wheat to be tested 1 μL, 2X EasyTaq® PCR SuperMix 5 μL, 10 μmol / L of the forward primer and the reverse primer each 1 μL, and H2O is added to 10 μL.

[0012] The reaction conditions of PCR amplification are as follows: pre-denaturation at 94℃ for 5 min, followed by denaturation at 94℃ for 30 sec, annealing at 58℃ for 30 sec, extension at 72℃ for 20 sec, for 35 cycles; finally, extension at 72℃ for 5 min; and storage at 15℃.

[0013] In step 2), the enzyme digestion reaction system and the reaction conditions are as follows: 2X SE buffer containing BSA 1 μL, PCR product 2 μL, BclI restriction endonuclease 0.1 μL, H2O is added to 10 μL, incubation at 37°C for 1 hour. After enzyme digestion, the size of the product is 161 bp.

[0014] The electrophoretic separation of the enzyme digestion product in the agarose gel refers to: the amplified product is electrophoretically separated in a 2% agarose gel, the voltage is 120 V, and the electrophoretic time is 20 min, and then it is judged whether the enzyme digestion product band is 179 bp or 161 bp.

[0015] The fourth aspect of the present application provides a kit for identifying or assisting in identifying the SDS-sedimentation value of wheat.

[0016] Through the above technical solution, the present application achieves the following beneficial effects:

[0017] 1. The dCAPS molecular marker primer pair provided by the present application for identifying or assisting in identifying the SDS-sedimentation value of wheat can detect the wheat SDS-sedimentation value trait-related gene TaMYB44 Genotype, simple and fast, high visibility, high analysis throughput, and can be used for a large number of breeding material detection.

[0018] 2. The dCAPS molecular marker primer pair provided by the present application can quickly identify or assist in identifying the high and low of the SDS-sedimentation value of wheat, and the method is not affected by the environment, simple and efficient.

[0019] 3. The dCAPS molecular marker provided by the present application is used for TaMYB44 Haplotype identification of wheat, combined with wheat SDS-sedimentation value determination data, it is proved that the wheat variety containing Hap I haplotype has higher SDS-sedimentation value. It is proved that the molecular marker provided by the present application is used for molecular marker assisted breeding, aggregation of excellent haplotype, improvement of the SDS-sedimentation value of wheat breeding material, and acceleration of breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 For the sequence alignment results of two haplotypes in example 1 of the present application TaMYB44 The part with color difference is the position of SNP;

[0021] Figure 2 For the practicality test results of the dCAPS primer pair in example 2 of the present application in 15 wheat varieties in the middle and lower reaches of the Yangtze River, wherein the electrophoresis results represent the results of agarose gel electrophoresis after the wheat genomic DNA PCR amplification and the use of restriction endonuclease BclI for enzyme digestion of the PCR product.

[0022] Figure 3 For the correlation analysis of the varieties containing Hap I and Hap II haplotypes and their corresponding SDS-sedimentation value phenotypes in example 3 of the present application. DETAILED DESCRIPTION

[0023] The specific embodiments of the present application are described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and explanatory in nature and are not intended to limit the application.

[0024] The terms used in the present application, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. In the working examples, various processes and methods not described in detail are conventional methods known in the art.

[0025] The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially. The wheat materials used are all germplasm resources materials preserved by the Jiangsu Provincial Germplasm Repository (Crop) of Yangzhou University, which can be obtained and used by those skilled in the art and researchers.

[0026] In the following examples, two haplotypes Hap I and Hap II were obtained by using the published resequencing data of wheat, combined with specific PCR amplification and gene sequencing. TaMYB44 The difference SNPs were obtained by analyzing the sequences of the two haplotypes, and the dCAPS molecular markers were developed. The 239 varieties in the middle and lower reaches of the Yangtze River were genotyped using the molecular markers, combined with the SDS-sedimentation value phenotype determination data, and the specific sites of the excellent alleles were clarified. The developed dCAPS molecular markers can be used for high-throughput screening and auxiliary identification of wheat high SDS-sedimentation value. Specifically as follows:

[0027] Example 1 Development of dCAPS molecular markers for identifying wheat SDS-sedimentation value

[0028] Using the published wheat resequencing data in the wheat genome variation joint database, the sequences of the two haplotypes Hap I and Hap II were analyzed. TaMYB44 It was found that there were two haplotypes Hap I and Hap II. TaMYB44 After sequence alignment of the two haplotypes, a total of 5 SNP variation sites were found, which were located at the 448th, 493rd, 512th, 593rd and 672nd nucleotides of the coding region (see TaMYB44 ). The information results come from Hubs database, the website is http: / / plants.ensembl.org / Triticum_aestivum / Info / Index. Figure 1

[0029] ​Using the dCAPS primer design website http: / / helix.wustl.edu / dcaps / dcaps.html, dCAPS primers SEQ ID NO.1 and SEQ ID NO.2 were designed using the 493rd SNP site to distinguish between Hap I and Hap II. In this primer pair, the upstream primer SEQ ID NO.1 introduces a mutation of one T base, resulting in the formation of a BclI restriction site for TGATCA in the DNA amplification product containing the Hap I haplotype.

[0030] Example 2: Detection of SNP sites using dCAPS molecular marker primer pairs

[0031] DNA was extracted from 15 randomly selected wheat varieties from the middle and lower reaches of the Yangtze River. TaMYB44 The complete CDS region of the gene was amplified using the forward primer sequence 5'-AATGCAGACAACAACGCCT-3' (SEQ ID NO.3) and the reverse primer sequence 5'-CGGCCAGAAATCGACAG-3' (SEQ ID NO.4). The PCR products were sequenced and the gene sequences were analyzed, and these materials were divided into Hap I haplotypes and Hap II haplotypes (see Table 1). Simultaneously, the DNA of 15 samples was amplified using the dCAPS primers SEQ ID NO.1 and SEQ ID NO.2 designed in Example 1. The PCR products were then digested with the restriction endonuclease BclI, followed by agarose gel electrophoresis. If the fragment length after electrophoresis was 179 bp, it indicated that the amplified product was not cleaved by BclI, suggesting that the tested wheat contained the Hap II haplotype with a low SDS-precipitation value. If the fragment length after electrophoresis was 161 bp, it indicated that the amplified product was cleaved by BclI, and the PCR product became smaller, suggesting that the tested wheat contained the Hap I haplotype with a high SDS-precipitation value. The genotyping of the 15 samples was performed using the dCAPS primers SEQ ID NO.1 and SEQ ID NO.2, and the genotyping results were consistent with the sequencing results (see [link to sequencing results]). Figure 2 The above experiments demonstrate that the dCAPS molecular marker primers developed in Example 1 are accurate in detecting SNP sites.

[0032] The PCR amplification reaction system was as follows (10 μL): 1 μL of wheat genomic DNA to be tested (20-100 ng / μL), 5 μL of 2×EasyTaq® PCR SuperMix, 1 μL each of SEQ ID NO.1 and SEQ ID NO.2 (10 μmol / L), and H2O to bring the total volume to 10 μL.

[0033] The reaction conditions of PCR amplification are as follows: 5 min of pre-denaturation at 94 ℃, followed by 30 sec of denaturation at 94 ℃, 30 sec of annealing at 58 ℃, 20 sec of extension at 72 ℃, 35 cycles, and finally 5 min of extension at 72 ℃, and storage at 15 ℃.

[0034] The enzyme digestion reaction system and reaction conditions are as follows: 1 μL of 2X SE buffer containing BSA, 2 μL of PCR product, 0.1 μL of BclI restriction endonuclease, and H2O added to 10 μL, incubation at 37 °C for 1 h.

[0035] The electrophoretic separation of the enzyme digestion product in agarose gel refers to the electrophoretic separation of the amplification product in a 2% agarose gel, with a voltage of 120 V and an electrophoretic time of 20 min, to determine whether the enzyme digestion product band is 179 bp or 161 bp.

[0036] Table 1 15 wheat varieties in the middle and lower reaches of the Yangtze River TaMYB44 Haplotype

[0037]

[0038] Example 3 Application of dCAPS molecular marker primer pairs in identifying wheat SDS-sedimentation values

[0039] Using the verified dCAPS molecular markers obtained in Example 2, further genotyping was performed on other 224 wheat varieties in the middle and lower reaches of the Yangtze River in this example. The PCR reaction system, amplification procedure, enzyme digestion reaction system, electrophoresis method, and genotype determination method were consistent with the methods described in Example 2. The detection results are shown in Table 2, a total of 74 varieties containing Hap I haplotype and 150 varieties containing Hap II haplotype were obtained.

[0040] Of the 239 test materials in Table 1 and Table 2, 12 g of wheat was ground into whole wheat flour, and 2 g was weighed for micro-method SDS-sedimentation value determination in the experimental base of the Lower Reaches Institute in 2022. The determination method refers to the method disclosed by Axford et al. (1979) (Axford D, Mcdermott E, and Redman D (1979) Note on sodium dodecyl sulfate test of breadmaking quality; Comparison ith Pelshenke and Zeleny test. Cereal Chem 56:582-584.), with 3 technical repeats. The determination results were correlated with the haplotype typing results in Table 1 and Table 2, and the statistical analysis was performed using Graphpad Prism 5.02 software. The analysis results are as followsFigure 3 As shown in the table, the average SDS-sedimentation value of wheat carrying Hap I haplotype is 21.89 mL, and the average SDS-sedimentation value of wheat carrying Hap II haplotype is 19.65 mL. The t test analysis shows that t = 4.905, P < 0.0001, and the SDS-sedimentation value of wheat carrying Hap I haplotype is significantly higher than that of wheat carrying Hap II haplotype.

[0041] Table 2 224 wheat samples in the middle and lower reaches of the Yangtze River TaMYB44 Haplotype

[0042]

[0043] Sequence listing

[0044] SEQ ID NO. 1: 5'-GGAACAAGTCATGGTGATGATC-3';

[0045] SEQ ID NO. 2: 5'-GTGGTGGTGTCTTTTCCTTTG-3';

[0046] SEQ ID NO. 3: 5'-AATGCAGACAACAACGCCT-3';

[0047] SEQ ID NO. 4: 5'-CGGCCAGAAATCGACAG-3'.

[0048] The preferred embodiments of the present application are described in detail in combination with the embodiments above, but the present application is not limited to the specific details in the above-described embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0049] In addition, it should be noted that each specific technical feature described in the above-described specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present application will not further describe various possible combinations.

[0050] In addition, various different embodiments of the present application can also be combined in any appropriate manner, as long as it does not deviate from the idea of the present application, and it should also be considered as disclosed content of the present application.

Claims

1. Use of a dCAPS primer pair for discriminating or aiding in discriminating the SDS-sedimentation value in wheat, characterized in that, The primer pair comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.

2.

2. A method for the identification of the SDS-sedimentation value of wheat, characterized in that, The method comprises the following steps: 1) using the wheat genomic DNA to be tested as a template, performing PCR amplification with the primer pair in claim 1 to obtain a PCR product; 2) performing enzyme digestion on the PCR product with a restriction endonuclease BclI, and then performing agarose gel electrophoresis, if the length of the fragment after electrophoresis is 179 bp, it is proved that the amplification product is not cut by BclI, and then it is indicated that the wheat to be tested has a low SDS-sedimentation value; if the length of the fragment after electrophoresis is 161 bp, it is proved that the amplification product is cut by BclI, and then it is indicated that the wheat to be tested has a high SDS-sedimentation value.

3. The method of claim 2, wherein, In step 1), the length of the PCR amplification product is 179 bp.

4. The method of claim 2, wherein, In step 1), the PCR amplification system is as follows: 20-100 ng / μL of the wheat genomic DNA to be tested 1 μL, 2X EasyTaq PCR SuperMix 5 μL, 10 μmol / L of the forward primer and the reverse primer each 1 μL, and H2O is added to 10 μL. The reaction conditions for PCR amplification are as follows: 94℃ pre-denaturation for 5 min, followed by 94℃ denaturation for 30 sec, 58℃ annealing for 30 sec, 72℃ extension for 20 sec, 35 cycles; finally, 72℃ extension for 5 min; and storage at 15℃.

5. The method of claim 2, wherein, In step 2), the enzyme digestion reaction system and the reaction conditions are as follows: 2X SE buffer containing BSA 1 μL, the PCR product 2 μL, BclI restriction endonuclease 0.1 μL, H2O is added to 10 μL, and incubation at 37℃ for 1 hour.

6. The method of claim 2, wherein, In step 2), after enzyme digestion, the size of the product is 161 bp.

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