A taMYB44-4D gene haplotype SNP molecular marker for identifying wheat kernel starch content and application thereof

Abstract

This invention discloses a haplotype SNP molecular marker for identifying wheat grain starch content in the TaMYB44-4D gene and its application. Specifically, the SNP is T or C at position 1557bp of the sequence in SEQ ID NO.1. Based on this SNP, primers for detecting this haplotype SNP molecular marker have also been developed, and a kit containing these primers has been prepared. By PCR and polyacrylamide gel electrophoresis, the SNP and genotype can be accurately identified, thereby distinguishing between superior haplotypes (high starch content) and non-superior haplotypes (low starch content) in wheat.

Description

Technical Field

[0001] This invention belongs to the field of wheat breeding technology and relates to a TaMYB44-4D gene haplotype SNP molecular marker for identifying wheat grain starch content and its application. Background Technology

[0002] Wheat (Triticum aestivum L.) is a globally important food crop, and its grain starch content directly affects its processing quality and nutritional value. Starch is not only a key factor influencing grain yield but also an important raw material for food production and industrial processing. Wheat grain starch trait is a complex quantitative trait, controlled by multiple quantitative trait loci (QTLs). Its low heritability and high selection difficulty make traditional breeding methods time-consuming and costly. Screening for high-starch-content wheat using traditional breeding methods is time-consuming, labor-intensive, and easily affected by environmental factors. Molecular marker-assisted selection (MAS) technology, by identifying molecular markers closely linked to the target trait, can accurately screen for target genotypes in early generations, significantly improving breeding efficiency.

[0003] In recent years, with the deepening of wheat genomics research, several key genes and their molecular markers related to starch synthesis have been identified. Using these markers, wheat materials carrying superior alleles can be rapidly screened at the molecular level. Combined with traditional breeding methods, this allows for the cultivation of new wheat varieties with high starch content and excellent processing quality. Therefore, applying marker-assisted selection technology to the breeding of high-starch-content wheat has significant theoretical and practical value. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a TaMYB44-4D gene haplotype SNP molecular marker for identifying the starch content of wheat grains and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] 1. A haplotype SNP molecular marker of the TaMYB44-4D gene for identifying starch content in wheat grains, namely SNP1, whose nucleotide sequence is shown in SEQ ID NO.1, and the position at 1557bp of the sequence is T or C.

[0007] As one of the preferred technical solutions, the genotype of SNP1 is T / T, corresponding to the HapⅠ haplotype, and the corresponding wheat is low starch content wheat; the genotype of SNP1 is C / C, corresponding to the HapⅡ haplotype, and the corresponding wheat is high starch content wheat.

[0008] 2. Primers used to detect the aforementioned haplotype SNP molecular markers, including two upstream primers and one downstream primer:

[0009] STARP-F1: 5'-TTATCCCGTGTGAAACTCCTTTG-3', as shown in SEQ ID NO.2;

[0010] STARP-F2: 5'-ACGACTCAATTTATCCCGTGTGAAACTCCCCTA-3', as shown in SEQ ID NO.3;

[0011] STARP-R: 5'-CTCGTCACGGTTCATGATCTATC-3', as shown in SEQ ID NO.4.

[0012] As one of the preferred technical solutions, the primer pair STARP-F1 / STARP-R composed of STARP-F1 and STARP-R amplifies the fragment with SNP site T in SEQ ID NO.1, corresponding to the Hap I haplotype; the primer pair STARP-F2 / STARP-R composed of STARP-F2 and STARP-R amplifies the fragment with SNP site C in SEQ ID NO.1, corresponding to the Hap II haplotype.

[0013] 3. Application of the aforementioned primers in the preparation of kits for detecting the aforementioned haplotype SNP molecular markers.

[0014] 4. A kit for detecting the aforementioned haplotype SNP molecular markers, containing the aforementioned primers.

[0015] 5. Application of the aforementioned haplotype SNP molecular markers, primers, or kits in the identification of starch content in wheat grains.

[0016] 6. Application of the aforementioned haplotype SNP molecular markers, primers, or kits in wheat breeding.

[0017] 7. A method for identifying the starch content of wheat grains, comprising detecting the aforementioned haplotype SNP molecular markers of the wheat to be tested, and identifying the starch content of wheat grains based on genotype.

[0018] As one of the preferred technical solutions, the genotype of SNP1 is T / T, corresponding to the HapⅠ haplotype, and the corresponding wheat is low starch content wheat; the genotype of SNP1 is C / C, corresponding to the HapⅡ haplotype, and the corresponding wheat is high starch content wheat.

[0019] 8. A wheat breeding method, wherein the genotype of the aforementioned haplotype SNP molecular markers in wheat samples is detected, and superior haplotype wheat samples with high starch content in grains are selected for breeding.

[0020] As one of the preferred technical solutions, the genotype of the SNP1 locus is selected as C / C, and the superior haplotype wheat sample corresponding to the HapⅡ haplotype is used for breeding.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention discloses a haplotype SNP molecular marker for identifying wheat grain starch content in the TaMYB44-4D gene and its application. Specifically, the SNP is T or C at position 1557bp of the sequence in SEQ ID NO.1. Based on this SNP, primers for detecting this haplotype SNP molecular marker have also been developed, and a kit containing these primers has been prepared. By PCR and polyacrylamide gel electrophoresis, the SNP and genotype can be accurately identified, thereby distinguishing between superior haplotypes (high starch content) and non-superior haplotypes (low starch content) in wheat.

[0023] The results of testing on existing wheat varieties show that the superior haplotypes detected using this molecular marker have significantly higher starch content than non-superior haplotypes. Therefore, this molecular marker can be used in the early stages of wheat breeding to rapidly screen wheat lines with higher starch content from different populations, improving breeding efficiency and accelerating the process of high-yield wheat breeding.

[0024] This invention has the following advantages:

[0025] 1. High amplification specificity and easy detection.

[0026] 2. The marker is a codominant marker, which can accurately identify heterozygous genotypes.

[0027] 3. High amplification abundance, which can be detected by polyacrylamide gel electrophoresis with high resolution. Attached Figure Description

[0028] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0029] Figure 1 This is a schematic diagram of the haplotype SNP molecular marker developed in this invention.

[0030] Figure 2The results of polyacrylamide gel electrophoresis for some natural wheat populations are shown. Note: During electrophoresis, PCR products amplified by STARP-F1, STARP-F2, and STARP-R were first spotted into the gel wells, and a marker was spotted before the first sample and after each of the 12 samples. The wheat varieties used, from left to right, are: 1. Jimai 20, 2. Jining 13, 3. Liaomai 19, 4. Lumai 14, 5. SN055849, 6. Shannong 22, 7. Shannong 23, 8. Shannong 2149, 9. Taishan 4606, 10. Yannong 19, 11. Yannong 21, 12. Yan 09135, 13. Jimai 19, 14. Jimai 21, 15. Jimai 22, 16. Jimai 23, 17. Jinan No. 8, 18. Jinan 13, 19. Jinan 16, 20. Jinan 17, 21. Jining No. 16, 22. Liangxing 66, 23. Liangxing 77, 24. Linmai No. 2.

[0031] Figure 3 Comparison of starch content among different haplotypes of TaMYB44-4Dpro in natural populations. The vertical axis represents starch content (%). ** indicates P < 0.01. Detailed Implementation

[0032] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] The following examples use GraphPad Prism 8.0 plotting and statistical software to process the data. The experimental results are expressed as mean ± standard deviation. Student's t-test is used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0035] Example:

[0036] Detection of superior haplotypes of the TaMYB44-4D gene

[0037] 1. Discovery and detection of superior haplotypes that regulate grain starch content

[0038] The differences between the superior haplotype (Hap II) and the non-superior haplotype (Hap I) of the TaMYB44 gene in wheat discovered in this invention are as follows: Figure 1As shown, this differential haplotype includes one SNP site, named SNP1. SNP1 corresponds to position 467226046 on chromosome 4D of the wheat Chinese Spring reference genome (IWGSC v1.1), and its nucleotide is T or C, corresponding to position 1557 of sequence 1 in the sequence listing. Y in SEQ ID NO.1 represents T or C.

[0039] Three primers were designed to detect different genotypes at the SNP1 site, including two upstream primers and one downstream primer.

[0040] The upstream primer with a "C" at the 3' end was named "STARP-F1": 5'-TTATCCCGTGTGAAACTCCTTTG-3', as shown in SEQ ID NO.2;

[0041] The upstream primer with a "T" at the 3' end is named "STARP-F2": 5'-ACGACTCAATTTATCCCGTGTGAAACTCCCCTA-3', as shown in SEQ ID NO.3;

[0042] The common downstream primer is named “STARP-R”: 5'-CTCGTCACGGTTCATGATCTATC-3', as shown in SEQ ID NO.4.

[0043] The primer pair consisting of SEQ ID NO.2 and SEQ ID NO.4 is the fragment with SNP site T in sequence 1 of the STARP-F1 / STARP-R amplification sequence listing (corresponding to haplotype I); the primer pair consisting of SEQ ID NO.3 and SEQ ID NO.4 is the fragment with SNP site C in sequence 1 of the STARP-F2 / STARP-R amplification sequence listing (corresponding to haplotype II).

[0044] The specific procedure for detecting superior haplotypes in the promoter region of the TaMYB44-4D gene using a designed primer set:

[0045] 1.1 Extraction of genomic DNA

[0046] Genomic DNA was extracted from wheat leaves using the CTAB method;

[0047] 1.2 PCR amplification

[0048] Using genomic DNA as a template, PCR amplification was performed using the primer set “STARP-F1 / STARP-F2 / STARP-R” to obtain PCR products.

[0049] PCR amplification system (10μL system): 2μL template DNA, 5μL 2×PCR Mix (Genestar), 0.5μL each of STARP-F1 and STARP-F2 (concentration 2μmol / L), 1μL STARP-R (concentration 2μmol / L), and make up to 10μL with double-distilled water.

[0050] PCR amplification program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s (from the second cycle onwards, the annealing temperature is decreased by 1.2℃ for each cycle), 72℃ extension for 30 s, 10 cycles; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 28 cycles; 72℃ extension for 10 min; store at 4℃.

[0051] 1.3 PCR product detection

[0052] The obtained PCR products were separated by electrophoresis on a 10.0% (v / v) non-denaturing polyacrylamide gel. The electrophoresis buffer was 1×TBE (Coollabo, Beijing), and the gel was kept at 180V for 4 hours. The gel was stained with 0.1% silver nitrate solution (0.3g silver nitrate in 300mL deionized water) for 20 min, then rinsed three times with deionized water. The gel was then developed with developing solution (0.3g Na2CO3, 10g NaOH, 750μL formaldehyde, diluted to 500mL) for about 10 min. After rinsing twice with water, the gel was laid flat on a gel lamp stand and photographed to obtain gel images.

[0053] Electrophoresis results showed that if the PCR product amplified using the primer set (STARP-F1 / STARP-F2 / STARP-R) contained only a 207 bp DNA fragment, the genotype of the wheat SNP1 locus to be tested was T / T (i.e., homozygous for the T locus in the wheat genome), corresponding to Hap I. The tested wheat did not contain the superior allele, meaning it did not have the superior haplotype with high starch content in its grains. If the PCR product contained only a 197 bp DNA fragment, the genotype of the wheat SNP1 locus to be tested was C / C (i.e., homozygous for the C locus in the wheat genome), corresponding to Hap II. The tested wheat contained the superior allele, meaning it had the superior haplotype with high starch content in its grains. If the PCR product contained both a 197 bp and a 207 bp DNA fragment, the genotype of the wheat SNP1 locus to be tested was T / C (i.e., heterozygous for both T and C loci in the wheat genome).

[0054] 2. Screening of natural wheat populations using the superior TaMYB44-4D allele.

[0055] The primer set (STARP-F1 / STARP-F2 / STARP-R) obtained in step 1 for detecting the superior allele (SNP1 locus genotype) of TaMYB44-4D was used to detect a natural population containing 489 bred varieties (preserved in this laboratory, publicly available from the applicant, and used only for replicating this invention). Related literature: Lou H, Zhang R, Liu Y, Guo D, Zhai S, Chen A, Zhang Y, Xie C, You M, Peng H, Liang R, Ni Z, Sun Q, Li

[0056] B.Genome-wide association study of six quality-related traits incommon wheat(Triticum aestivum L.) under two sowing conditions.TheorAppl.Genet.2021,134(1):399-418.

[0057] doi:10.1007 / s00122-020-03704-y.Epub 2020Nov 5), some strains and electrophoresis test results can be found in Figure 2 .

[0058] Figure 2 The lane numbers and their corresponding wheat varieties are as follows: 1. Jimai 20, 2. Jining 13, 3. Liaomai 19, 4. Lumai 14, 5. SN055849, 6. Shannong 22, 7. Shannong 23, 8. Shannong 2149, 9. Taishan 4606, 10. Yannong 19, 11. Yannong 21, 12. Yan 09135, 13. Jimai 19, 14. Jimai 21, 15. Jimai 22, 16. Jimai 23, 17. Jinan 8, 18. Jinan 13, 19. Jinan 16, 20. Jinan 17, 21. Jining 16, 22. Liangxing 66, 23. Liangxing 77, 24. Linmai 2. A comparative analysis was conducted based on the phenotypic analysis of grain starch content determination for each variety within the population. This population was planted in Linfen in 2019, in single-row plots with a row length of 1m and a row spacing of 0.2m, with 3 replicates. The starch content of the harvested seeds was determined using near-infrared spectroscopy (Perten Instruments, USA), with 3 technical replicates for each determination.

[0059] Analysis results as follows Figure 3As shown, the starch content of wheat materials with the HapⅠ haplotype (genotype T / T at the SNP1 locus, i.e., homozygous for T at the SNP1 locus in the wheat genome) in the natural population was lower than that of superior haplotype wheat materials with the HapⅡ haplotype (p<0.01), and the proportion of superior haplotypes in this population was relatively large (82.01%).

[0060] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. The application of primers for detecting haplotype SNP molecular markers in the preparation of a kit for identifying starch content in wheat grains, characterized in that, The primers consist of two upstream primers and one downstream primer: STARP-F1: 5'-TTATCCCGTGTGAAACTCCTTTG-3', as shown in SEQ ID NO.2; STARP-F2: 5'-ACGACTCAATTTATCCCGTGTGAAACTCCCCTA-3', as shown in SEQ ID NO.3; STARP-R: 5'-CTCGTCACGGTTCATGATCTATC-3', as shown in SEQ ID NO.4; The haplotype SNP molecular marker, namely SNP1, has the nucleotide sequence shown in SEQ ID NO.1, where the position at 1557bp is either T or C. The genotype at SNP1 is T / T, corresponding to the Hap I haplotype, and the corresponding wheat is low-starch wheat. The genotype at SNP1 is C / C, corresponding to the Hap II haplotype, and the corresponding wheat is high-starch wheat.

2. The application according to claim 1, characterized in that, The primer pair STARP-F1 / STARP-R, consisting of STARP-F1 and STARP-R, amplifies the fragment with SNP site T in SEQ ID NO.1, corresponding to haplotype I; the primer pair STARP-F2 / STARP-R, consisting of STARP-F2 and STARP-R, amplifies the fragment with SNP site C in SEQ ID NO.1, corresponding to haplotype II.

3. The application of primers or kits containing such primers in the identification of starch content in wheat grains, characterized in that, The primers consist of two upstream primers and one downstream primer: STARP-F1: 5'-TTATCCCGTGTGAAACTCCTTTG-3', as shown in SEQ ID NO.2; STARP-F2: 5'-ACGACTCAATTTATCCCGTGTGAAACTCCCCTA-3', as shown in SEQ ID NO.3; STARP-R: 5'-CTCGTCACGGTTCATGATCTATC-3', as shown in SEQ ID NO.

4.

4. The application of primers or kits containing such primers in the breeding of wheat varieties with high starch content, characterized in that, The primers consist of two upstream primers and one downstream primer: STARP-F1: 5'-TTATCCCGTGTGAAACTCCTTTG-3', as shown in SEQ ID NO.2; STARP-F2: 5'-ACGACTCAATTTATCCCGTGTGAAACTCCCCTA-3', as shown in SEQ ID NO.3; STARP-R: 5'-CTCGTCACGGTTCATGATCTATC-3', as shown in SEQ ID NO.

4.

5. A method for determining the starch content of wheat grains, characterized in that, The haplotype SNP molecular markers of the wheat to be tested were detected, and the starch content of wheat grains was identified based on the genotype. The haplotype SNP molecular marker, namely SNP1, has the nucleotide sequence shown in SEQ ID NO.1, where the position at 1557bp is either T or C. The genotype at SNP1 is T / T, corresponding to the Hap I haplotype, and the corresponding wheat is low-starch wheat. The genotype at SNP1 is C / C, corresponding to the Hap II haplotype, and the corresponding wheat is high-starch wheat.

6. A method for wheat breeding, characterized in that, Genotyping of haplotype SNP molecular markers in wheat samples was performed, and superior haplotype wheat samples with high starch content were selected for breeding. The haplotype SNP molecular marker, namely SNP1, has the nucleotide sequence shown in SEQ ID NO.1, where the position at 1557bp is either T or C. The genotype at SNP1 is T / T, corresponding to the Hap I haplotype, and the corresponding wheat is low-starch wheat. The genotype at SNP1 is C / C, corresponding to the Hap II haplotype, and the corresponding wheat is high-starch wheat.

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