Wheat molecular marker ax-95660756 and its use in improving gluten aggregation properties

Abstract

The application discloses a molecular marker AX-95660756 related to the genetic correlation of the gluten aggregation characteristics of wheat, a KASP primer group related to the molecular marker and application of the KASP primer group, and a genetic marker with the name of AX-95660756 and located on the long arm of a 4D chromosome of wheat; a mutation of an A / G allele exists at the 36th base of the sequence; when the 36th base is AG, it is a genotype with strong gluten aggregation characteristics; and when the 36th base is GG, it is a genotype with weak gluten aggregation characteristics. The KASP primer group is used as a primer, a wheat genome to be tested is used as a template to perform PCR amplification on the KASP marker, and a wheat variety with strong gluten aggregation characteristics can be screened, so that the genetic method is used to change the gluten aggregation characteristics of the wheat, the gluten quality of the wheat is improved, and the wheat breeding method is improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular marker technology for wheat gluten aggregation characteristics, and relates to wheat molecular marker AX-95660756 and its application in improving gluten aggregation characteristics. Background Technology

[0002] With the continuous increase in population and the development of modern agricultural technology, high-yield and high-quality wheat has become an important goal in wheat breeding. Wheat quality is mainly reflected by the rheological properties of dough. Traditional methods for testing wheat rheological properties mainly utilize instruments such as farinographs and extensometers, but these methods are time-consuming and resource-intensive. To reduce experimental waste, a gluten peaker is used to directly measure whole wheat flour. The rapid stirring of the paddle in the cup causes the gluten to separate and then aggregate, allowing for the measurement of the gluten aggregation characteristics. Furthermore, this instrument requires a short measurement time (10 minutes), consumes minimal materials (10g), has high repeatability, and can rapidly test various wheat materials.

[0003] The quality of wheat dough is largely influenced by its gluten aggregation characteristics. Currently, there are numerous studies on the relationship between wheat gluten aggregation characteristics and quality, but there is little research on the genetic basis of wheat gluten aggregation characteristics. Existing research has indicated that wheat gluten aggregation characteristics are mainly determined by genetic factors. Therefore, genetic analysis of wheat gluten aggregation characteristics and understanding its molecular regulatory mechanisms are beneficial for optimizing wheat quality and early selection in high-quality wheat breeding. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, this invention utilizes previously constructed natural populations and combines them with 660K SNP microarray genotyping data to initially screen and obtain 224,706 effective SNP marker loci. Simultaneously, gluten aggregation characteristics of 207 wheat varieties from natural populations in three environments over three years were phenotyped using Glutopeak. A genome-wide association study (GWA) was conducted on gluten aggregation characteristics in wheat grains using a Q+K mixed linear model. The results showed that a significant marker AX-95660756 on the long arm of chromosome 4D was obtained from the aforementioned screened SNP marker loci.

[0005] In a first aspect, the present invention provides the application of wheat molecular marker AX-95660756 in improving the gluten aggregation characteristics of wheat. The wheat molecular marker AX-95660756 is located on the long arm of wheat chromosome 4D, and its 36th base is an A / G polymorphic site. The sequence of the wheat molecular marker AX-95660756 is: GATGTACGCCGGCTGCATGCAAACACAGTGATTCTRTGGGTGTTCACGTGTCTTTGGAGGCCACACATGCA (SEQ ID NO.1), where R represents the bases A / G. Based on the genotyping results: the homozygous genotype GG indicates weak gluten aggregation characteristics; the heterozygous genotype AG indicates strong gluten aggregation characteristics.

[0006] Secondly, the present invention also provides a substance for detecting the gluten aggregation characteristics of wheat, wherein the substance is a set of primers for detecting the genotype of the 36th position of the molecular marker AX-95660756 shown in SEQ ID No.1 on chromosome 4D in the wheat genome, or a detection reagent or kit containing the set of primers.

[0007] Furthermore, the primer set contains two upstream primers and one downstream primer; the upstream primers are designed based on the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 1 on chromosome 4D of the wheat genome and its upstream sequence, and the 3' end deoxyribonucleotide of one upstream primer is A, and the 3' end deoxyribonucleotide of the other upstream primer is G; the downstream primer is designed based on the downstream sequence of the 36th deoxyribonucleotide of the sequence shown in SEQ ID No. 1 on chromosome 4D of the wheat genome.

[0008] Furthermore, the upstream primers are AX-95660756-F1 and AX-95660756-F2, whose sequences are shown in SEQ ID No. 2 and SEQ ID No. 3, respectively, and the downstream primer is AX-95660756-R12, whose sequence is shown in SEQ ID No. 4.

[0009] Thirdly, the present invention provides for the use of the substance described in the second aspect of the present invention in any of the following:

[0010] (A) To identify or assist in the identification of wheat gluten aggregation characteristics;

[0011] (B) Compare the strength of the gluten aggregation properties of the wheat to be tested;

[0012] (C) Select or screen wheat plants, lines, strains or varieties with relatively strong gluten aggregation characteristics.

[0013] (D) Select or screen wheat plants, lines, strains or varieties with relatively weak gluten aggregation characteristics.

[0014] (E) Prepare products for identifying or assisting in the identification or comparison of the strength of the aggregation properties of wheat gluten to be tested;

[0015] (F) Prepare products for breeding or screening wheat single plants, lines, varieties or strains with relatively strong gluten aggregation characteristics;

[0016] (G) Prepare products for breeding or screening wheat plants, lines, strains or varieties with relatively weak gluten aggregation characteristics.

[0017] Fourthly, the present invention also provides any of the following methods:

[0018] Method A: A method for comparing the gluten aggregation characteristics of wheat to be tested, comprising the following steps (A1) or (A2):

[0019] (A1) Detect the genotype at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D in the wheat genome;

[0020] (A2) The strength of the gluten aggregation property of the wheat to be tested is determined as follows: if the deoxyribonucleotide at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D in the genome is a hybrid of A and G, then the gluten aggregation property of the wheat to be tested is the strongest.

[0021] Method B: A method for breeding or screening wheat plants, lines, strains, or varieties with relatively strong gluten aggregation characteristics, comprising the following steps:

[0022] (B1) Detect the genotype at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D in the wheat genome;

[0023] (B2) Select the test wheat with a heterozygote at the 36th deoxyribonucleic acid position being A and G as the parent for breeding, and select the heterozygote wheat with a heterozygote at the 36th deoxyribonucleic acid position being A and G in each generation of breeding, so as to finally obtain wheat single plants, lines, strains or varieties with relatively strong gluten aggregation characteristics.

[0024] Method C: A method for breeding or screening wheat plants, lines, strains, or varieties with relatively weak gluten aggregation characteristics, comprising the following steps:

[0025] (C1) Detect the genotype at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D in the wheat genome;

[0026] (C2) Select the test wheat that is homozygous for G at the 36th deoxyribonucleotide position as the parent for breeding, and select wheat that is homozygous for G at the 36th deoxyribonucleotide position in each generation of breeding, so as to obtain wheat single plants, lines, strains or varieties with relatively weak gluten aggregation characteristics.

[0027] Furthermore, the specific operation of (A1)(B1)(C1) is as follows:

[0028] The wheat genomic DNA to be tested was amplified by PCR using the aforementioned detection reagent or kit. The amplified product was scanned for fluorescence signals, and the scan data was analyzed. Then, the 36th deoxyribonucleotide on chromosome 4D of the wheat gene to be tested, as shown in SEQ ID No. 1, was determined as follows:

[0029] If the fluorescence signal data of the amplification product of the wheat to be tested is blue, then the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No. 1 on chromosome 4D of the wheat genome to be tested is a homozygous G;

[0030] If the fluorescence signal data of the amplification product of the wheat to be tested is green, then the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No. 1 on chromosome 4D of the wheat genome to be tested is a hybrid of A and G.

[0031] Furthermore, the PCR reaction system was designed as follows: 2×KASP Master Mix, 2.5 μl; Primer Mix, 0.7 μl; MgCl2, 0.04 μl; 100 ng / μl DNA, 1 μl; water, 0.76 μl.

[0032] Compared with existing technologies, this invention obtains a significant marker AX-95660756 on the long arm of chromosome 4D, which is associated with the gluten aggregation characteristics of wheat. This invention also designed and developed the KASP marker based on AX-95660756. Sequence analysis shows that the 36th base of this marker sequence is an A / G allele mutation. Genotyping analysis using the polymorphism at this site shows that when the base at this site is AG, the gluten aggregation characteristics in whole wheat flour are stronger. Effective screening of wheat with the AG genotype can lay a certain technical foundation for the breeding of new wheat varieties. Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below.

[0034] Figure 1Manhattan plot of BLUP values ​​for gluten aggregation characteristics in different environments on wheat chromosome SNP distribution: the red dashed line represents -log 10 The threshold line of P=4;

[0035] Figure 2 Genotyping diagram of KASP markers; where: HEX blue fluorescence (top left square) indicates the genotype GG; green fluorescence (top right triangle) indicates the heterozygous genotype AG; black fluorescence (bottom left square) is the blank control without template DNA;

[0036] Figure 3 Histogram of gluten aggregation characteristics for KASP marker genotypes in common wheat: Green boxes show gluten aggregation characteristics in wheat materials with genotype AG; blue boxes show gluten aggregation characteristics in wheat materials with genotype GG.

[0037] Figure 4 Box plot of gluten aggregation characteristics for KASP marker genotypes in common wheat: green indicates AG genotype with strong gluten aggregation characteristics; blue indicates GG genotype with weak gluten aggregation characteristics; *** indicates P<0.001. Detailed Implementation

[0038] The embodiments of the technical solution of the present invention will now be described in detail with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0039] It should be noted that, as a professional agricultural research institution, the applicant has long preserved relevant germplasm materials, and the relevant wheat varieties are all publicly available in the market or in existing germplasm banks.

[0040] In preliminary research, the inventors constructed natural populations based on different wheat varieties planted in two locations (Yuanyang (YY) and Shangqiu (SQ)) over three years. After sowing, management, and harvesting according to normal management practices, the inventors used 660K SNP chip genotyping data to preliminarily screen and identify effective SNPs in these natural populations, obtaining a total of 224,706 marker loci. The relevant marker loci in this application are within the scope of this screening.

[0041] Using Glutopeak, the inventors measured the gluten aggregation phenotype of 207 wheat varieties in a natural population. Based on these measurement results and the aforementioned SNP site screening results, the inventors used a Q+K mixed linear model to conduct a genome-wide association analysis on the gluten aggregation characteristics in wheat grains, in order to screen for potential superior loci and genes that are significantly associated with wheat gluten aggregation characteristics.

[0042] Example 1

[0043] During the screening process, the population structure of wheat varieties was calculated using STRUCTURE, and the population structure was divided using a Bayesian model. TASSEL analysis was then performed to analyze linkage disequilibrium within the population. The threshold was -log. 10 When P=4, SNP sites detected in all environments were considered significant sites. A summary of some research results is as follows: Figure 1 , Figure 2 As shown.

[0044] Analysis showed that a significantly associated marker, AX-95660756 (215185603 bp), was obtained through screening on the long arm of chromosome 4D, explaining 8.84%–15.35% of the phenotypic variation. A KASP marker was developed targeting this marker, which contains an A / G allele mutation at position 36. The specific nucleotide sequence is as follows:

[0045] GATGTACGCCGGCTGCATGCAAACACAGTGATTCTRTGGGTGTTCACGTGTCTTTGGAGGCCACACATGCA (SEQ ID NO.1) where R represents bases A and G; when the 36th base is AG, it is a genotype with strong gluten aggregation characteristics, and when the 36th base is GG, it is a genotype with weak gluten aggregation characteristics.

[0046] Example 2

[0047] Based on the nucleotide sequence of the SNP site AX-95660756 identified in Example 1, the inventors further designed the KASP marker. Combining this with genotyping analysis results from some common wheat varieties collected by the inventors, and considering the gluten aggregation characteristics of wheat materials with different genotypes, further functional verification and wheat variety screening were conducted, thus laying a foundation for wheat quality improvement. A brief description of the specific experimental process is as follows.

[0048] (I) Primer Design

[0049] Primers designed for this marker (AX-95660756) sequence are as follows:

[0050] AX-95660756-F1:

[0051] 5'-GAAGGTGACCAAGTTCATGCTGGCTGCATGCAAACACAGTGATTCTA-3' (SEQ ID NO. 2),

[0052] AX-95660756-F2:

[0053] 5'-GAAGGTCGGAGTCAACGGATTGGCTGCATGCAAACACAGTGATTCTG-3' (SEQ ID NO.3),

[0054] AX-95660756-R12:

[0055] 5'-ACATGCATGTGTGGCCTCCAAAGACA-3 (SEQ ID NO. 4).

[0056] (II) Genotyping Detection

[0057] First, the three primers, each with a concentration of 10 μmol / L, were mixed with water in a volume ratio of 12(AX-95660756-F1):12(AX-95660756-F2):30(AX-95660756-R12):46(water) to form the Primer Mix.

[0058] Then, the PCR reaction was performed, and the 5μl reaction system was designed as follows:

[0059] 2×KASP Master Mix, 2.5μl;

[0060] Primer Mix, 0.7 μl;

[0061] MgCl2, 0.04 μl;

[0062] DNA (100 ng / μl), 1 μl;

[0063] Water, 0.76 μl;

[0064] The reaction program was as follows: 95℃ for 15 min; 95℃ for 20 s, 65℃~55℃ for 1 min, 10 cycles (decreasing by 1℃ in each cycle); 95℃ for 20 s, 57℃ for 1 min, 30 cycles; 37℃ for 1 min; genotyping analysis was performed after the reaction.

[0065] Using the aforementioned KASP markers, reaction system, and reaction procedure, genotyping was performed on 207 common wheat materials collected by the inventors. The specific genotyping results, i.e., the gluten aggregation characteristics, are as follows: Figure 3 And as shown in Table 1 below.

[0066] Table 1. Genotyping and gluten aggregation characteristics of marker genes AX-95660756 in natural wheat populations.

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] The Glutopeak test yielded nine trait values: PMT (peak time), BEM (maximum torque), AM (torque in the first 15 seconds before BEM), PM (torque in the last 15 seconds after BEM), A1 (area from the start of the test to the first maximum value), A2 (area from the first maximum value to the first minimum value), A3 (area from the first minimum value to AM), A4 (area from AM to BEM), and A5 (area from BEM to PM). Previous experiments have demonstrated that the gluten aggregation characteristics measured by Glutopeak are significantly correlated with quality traits of wheat measured by other instruments. Glutopeak can rapidly evaluate wheat quality and can be used for early selection of breeding materials.

[0073] Further statistical classification of the results in Table 1 above is shown in Table 2 below.

[0074] Table 2 Comparative analysis of interfacial gluten aggregation characteristics of different genotypes in wheat material AX-95660756

[0075]

[0076] Combination Figure 3 It can be seen that the two allele types are distinct, which also shows that the marker can effectively separate allele types in different wheat varieties.

[0077] Analysis of the statistical results in Tables 1 and 2 shows that approximately 24.64% of the wheat materials have the AG genotype, while 71.98% contain the GG genotype. The PMT and A3 values ​​for wheat varieties with the AG genotype range from 28.17 to 82 and from 152.5 to 1210.67, respectively, while those for wheat varieties with the GG genotype range from 20.83 to 75.83 and from 34.83 to 1157.33, respectively.

[0078] Combination Figure 4Statistical results show that wheat materials with the AG genotype have strong gluten aggregation characteristics, while wheat materials with the GG genotype have weak gluten aggregation characteristics, and the gluten aggregation characteristics between the two genotypes have reached a significant difference level.

[0079] Based on the above results, it can be concluded that wheat varieties with the genotype AG have significantly stronger gluten aggregation characteristics than wheat varieties with the genotype GG. The genotype AG is an excellent allele that enhances wheat gluten aggregation characteristics and can be used to screen wheat varieties with strong gluten aggregation characteristics to improve wheat quality traits.

[0080] Unless otherwise specifically stated, the numerical values ​​set forth in these embodiments do not limit the scope of the invention. In all examples shown and described herein, any specific value should be interpreted as merely exemplary and not as a limitation, unless otherwise specified; therefore, other examples of exemplary embodiments may have different values.

Claims

1. The application of a reagent for detecting the wheat molecular marker AX-95660756 in the identification of wheat gluten aggregation characteristics, characterized in that, The wheat molecular marker AX-95660756 is located on the long arm of wheat chromosome 4D, with its 36th base being an A or G polymorphic site. The sequence of the wheat molecular marker AX-95660756 is: GATGTACGCCGGCTGCATGCAAACACAGTGATTCTRTGGGTGTTCACGTGTCTTTGGAGGCCACACATGCA, where R represents a base of A or G. Based on the genotyping results: the homozygous genotype GG indicates weak gluten aggregation characteristics; the heterozygous genotype AG indicates strong gluten aggregation characteristics.

2. The application of a set of primers for detecting the genotype at position 36 of the molecular marker AX-95660756 shown in SEQ ID No. 1 on chromosome 4D in the wheat genome in any of the following: (A) To identify or assist in the identification of wheat gluten aggregation characteristics; (B) Compare the strength of the gluten aggregation properties of the wheat to be tested; (C) Select or screen wheat plants, lines, strains or varieties with relatively strong gluten aggregation characteristics. (D) Select or screen wheat plants, lines, strains or varieties with relatively weak gluten aggregation characteristics. (E) Prepare products for identifying or assisting in the identification or comparison of the strength of the aggregation properties of wheat gluten to be tested; (F) Prepare products for breeding or screening wheat single plants, lines, varieties or strains with relatively strong gluten aggregation characteristics; (G) Prepare products for breeding or screening wheat plants, lines, varieties or strains with relatively weak gluten aggregation characteristics. The upstream primers of the primer set are AX-95660756-F1 and AX-95660756-F2, with sequences shown in SEQ ID No. 2 and SEQ ID No. 3, respectively. The downstream primer is AX-95660756-R12, with a sequence shown in SEQ ID No.

4. The wheat gluten aggregation characteristics are strongest when the deoxyribonucleotide at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D is a heterozygote of A and G; and weakest when the deoxyribonucleotide at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D is a homozygote of G.

3. Any of the following methods: Method A: A method for comparing the gluten aggregation characteristics of wheat samples, comprising the following steps: (A1) Detect the genotype at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D in the wheat genome; (A2) The strength of the gluten aggregation property of the wheat to be tested is determined as follows: if the deoxyribonucleotide at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D in the genome is a hybrid of A and G, then the gluten aggregation property of the wheat to be tested is the strongest. Method B: A method for breeding or screening wheat plants, lines, strains, or varieties with relatively strong gluten aggregation characteristics, comprising the following steps: (B1) Detect the genotype at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D in the wheat genome; (B2) Select the test wheat with a heterozygote at the 36th deoxyribonucleic acid position being A and G as the parent for breeding, and select the heterozygote wheat with a heterozygote at the 36th deoxyribonucleic acid position being A and G in each generation of breeding, so as to finally obtain wheat single plants, lines, strains or varieties with relatively strong gluten aggregation characteristics. Method C: A method for breeding or screening wheat plants, lines, strains, or varieties with relatively weak gluten aggregation characteristics, comprising the following steps: (C1) Detect the genotype at position 36 of the molecular marker shown in SEQ ID No. 1 on chromosome 4D in the wheat genome; (C2) Select the test wheat that is homozygous for G at the 36th deoxyribonucleotide position as the parent for breeding, and select wheat that is homozygous for G at the 36th deoxyribonucleotide position in each generation of breeding, so as to obtain wheat single plants, lines, strains or varieties with relatively weak gluten aggregation characteristics.

4. The method according to claim 3, characterized in that, The (A1) or (B1) or (C1) The specific operation is as follows: The wheat genomic DNA to be tested was amplified by PCR using the primer set described in claim 2. The amplified product was scanned for fluorescence signals, and the scan data was analyzed. Then, the 36th deoxyribonucleotide on chromosome 4D of the wheat gene to be tested, as shown in SEQ ID No. 1, was determined as follows: If the fluorescence signal data of the amplification product of the wheat to be tested is blue, then the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No. 1 on chromosome 4D of the wheat genome to be tested is a homozygous G; If the fluorescence signal data of the amplification product of the wheat to be tested is green, then the 36th deoxyribonucleotide of the molecular marker shown in SEQ ID No. 1 on chromosome 4D of the wheat genome to be tested is a hybrid of A and G.

5. The method according to claim 4, characterized in that, The PCR reaction system is designed as follows: 2×KASP Master Mix, 2.5µl; Primer Mix, 0.7µl; MgCl2, 0.04µl; 100ng / µl DNA, 1µl; Water, 0.76µl.

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