Wheat flour wet gluten content major locus and SNP marker and application

Abstract

The application provides a wheat flour wet gluten content main effect site and SNP marker and application, and belongs to the wheat breeding technical field, and mainly provides SNP molecular markers Marker45889, Marker45896, Marker45348, Marker45403, Marker45397 and Marker45431, and the SNP site provided by the application has important significance for detecting and breeding wheat flour high FWG varieties, lines and breeding materials, and molecular markers can be developed according to the SNP site, so that the wheat quality breeding efficiency is accelerated.

Description

[0001] This application is a divisional application of application number 202410541035.X, application date 2024.04.30, entitled "Major effective sites and SNP markers of wet gluten content in wheat flour and their application". Technical Field

[0002] This invention belongs to the field of wheat breeding technology, specifically involving the application of major QTL sites and their SNP molecular markers related to wet gluten content (FWG) in wheat flour. Background Technology

[0003] Gluten is both a nutritional and processing quality trait, and a commonly used indicator in international wheat trade. Generally, wheat with high protein content also has a relatively high wet gluten content. Gluten is a unique substance that distinguishes wheat from other cereal crops, thus giving flour the ability to be processed into a wide variety of foods. Gluten is an important indicator of wheat quality, involving two factors: gluten quantity and gluten quality, both of which have a significant impact on flour and dough quality. No single factor can completely determine flour quality; both must be considered simultaneously. Previous research has found that the main components determining gluten quality are glutenin and gliadin, especially their ratio, which is closely related to the formation of the gluten network structure. The water absorption, elasticity, kneadability, viscosity, and extensibility of gluten all affect the quality of dough and the final processed quality. Only when the gluten content is appropriate and the gluten quality is good can the gas produced during dough fermentation be retained within the dough, resulting in larger bread or steamed buns with better internal texture; conversely, poor gluten content leads to poorer texture. The national standard (GB / T17892-1999) stipulates that the wet gluten content of equal-strength wheat flour is >35.0%, and that of equal-strength wheat is ≥32.0%. The "Classification of Wheat Varieties" (GB / T17320-2013) specifies that strong-gluten wheat has a wet gluten content ≥30%, medium-strength wheat has a wet gluten content of 28%–30%, medium-gluten wheat has a wet gluten content of 26%–28%, and weak-gluten wheat has a wet gluten content <26%. Therefore, the determination of wet gluten content is of great significance for evaluating the classification of wheat varieties.

[0004] Previous studies have conducted quantitative trait mapping analyses on traits reflecting gluten strength, such as wet gluten content and gluten index. Yu Haixia (2012) detected 40 significant association markers related to wet gluten content and gluten index using association analysis, with marker wPt-665169 associated with both traits. Other studies (Li Weihua et al., 2005) have shown three associated loci on chromosomes 1DL, 2DS, and 3BL. McCartney et al. (2006) detected 6, 7, and 4 gluten-related genes on chromosomes 1B, 4D, and 7D, respectively. Based on previous research, a total of 19 chromosomes are involved in gluten strength loci, with the largest number on chromosome 1B, followed by chromosomes 1D, 2D, 5D, 5B, and 2A.

[0005] Although previous researchers have located some loci associated with wheat FWG, there are very few major loci and SNP molecular markers that can be used for marker-assisted breeding to improve FWG. Therefore, this invention uses a RIL population constructed with Nongda Nuomai 1 (a wheat variety with FWG differences) as the female parent and Gaocheng 8901 as the male parent as the material. Combined with a high-density genetic map constructed using SLAF-Seq technology, QTL mapping analysis of wheat FWG was performed in three environments to identify major QTL loci, their SNP markers, and superior allelic variation loci, so as to provide genetic resources and effective molecular markers for improving wheat FWG. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides major active sites and SNP markers for wet gluten content in wheat flour, and their applications.

[0007] This invention uses Nongda Nuomai No. 1 (with differential FWG values) as the female parent and Gaocheng 8901 as the male parent to construct a RIL population with differential FWG values ​​in wheat flour. Using SLAF-Seq technology, a high-density genetic map containing 8,095 SNP markers was constructed, with a total map length of 2,554.10 cM and linkage group lengths ranging from 90.08 to 151.67 cM. Based on the FWG phenotypic data, QTL mapping analysis was performed under three different environments.

[0008] Based on the results of quantitative trait gene mapping analysis, and through superior allele identification and gene locus phenotypic analysis, two major and closely linked QTL loci, QFwg-1D-1 and QFwg-1D-2, controlling the FWG content of wheat flour were identified. These loci are located on chromosome 1D and contain six closely linked SNP markers. This provides theoretical guidance and a foundation for the discovery of wheat flour FWG genes and molecular marker-assisted quality selection breeding.

[0009] The technical solution of the present invention is as follows:

[0010] A SNP molecular marker, Marker45889, associated with the control of wheat flour FWG, is located on the 1D chromosome of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0011] The nucleotide sequence of this molecular marker is shown in Table 8: SNP site sequence information of flour FWG.

[0012] The physical location of the SNP molecular marker Marker45889 is 419880366 on chromosome 1D, and the base is a T / C variant.

[0013] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45889 is shown in SEQ ID NO.2, that is, wheat flour with high FWG is C at this SNP site.

[0014] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in flour FWG, that is, wheat flour FWG with a base type of C at this position is high.

[0015] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45889 is shown in SEQ ID NO.2, that is, the base type of the SNP site is C, and the genotype of the SNP molecular marker is CC; the nucleotide sequence of the SNP molecular marker Marker45889 is shown in SEQ ID NO.1, that is, the base type of the SNP site is T, and the genotype of the SNP molecular marker is TT; the wheat flour FWG of individuals with the SNP molecular marker CC genotype is higher than that of individuals with the TT genotype.

[0016] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in wheat flour FWG. That is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is CC type, and the wheat flour FWG of individuals with the CC genotype of the SNP molecular marker is higher than that of individuals with the TT genotype.

[0017] A SNP molecular marker, Marker45896, associated with the control of wheat flour FWG, is located on the 1D chromosome of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0018] The nucleotide sequence of this molecular marker is shown in Table 8: SNP site sequence information of flour FWG.

[0019] The physical location of the SNP molecular marker Marker45896 is 420997301 on chromosome 1D, and the base is a T / A variant.

[0020] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45896 is shown in SEQ ID NO.4, that is, wheat flour with a base type of A at this SNP site has a high FWG.

[0021] The base variation type and phenotypic value of the SNP molecular markers are shown in Table 7: Phenotypic effect of SNP site base variation in flour FWG, that is, wheat flour FWG with base type A at this position is high.

[0022] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45896 is shown in SEQ ID NO.4, that is, the base type of the SNP site is A, and the genotype of the SNP molecular marker is AA; the nucleotide sequence of the SNP molecular marker Marker45896 is shown in SEQ ID NO.3, that is, the base type of the SNP site is T, and the genotype of the SNP molecular marker is TT; the wheat flour FWG of individuals with the SNP molecular marker AA genotype is higher than that of individuals with the TT genotype.

[0023] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in wheat flour FWG. That is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is AA, and the wheat flour FWG of individuals with the AA genotype of the SNP molecular marker is higher than that of individuals with the TT genotype.

[0024] A SNP molecular marker, Marker45348, associated with the control of wheat flour FWG, is located on the 1D chromosome of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.5 or SEQ ID NO.6.

[0025] The nucleotide sequence of this molecular marker is shown in Table 8: SNP site sequence information of flour FWG.

[0026] The physical location of the SNP molecular marker Marker45348 is chromosome 1D at 405902894, and the base is a G / A variant.

[0027] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45348 is as shown in SEQ ID NO.6, that is, wheat flour with a base type of A at this SNP site has a high FWG.

[0028] The base variation type and phenotypic value of the SNP molecular markers are shown in Table 7: Phenotypic effect of SNP site base variation in flour FWG, that is, wheat flour FWG with base type A at this position is high.

[0029] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45348 is shown in SEQ ID NO.6, that is, the base type of the SNP site is A, and the genotype of the SNP molecular marker is AA; the nucleotide sequence of the SNP molecular marker Marker45348 is shown in SEQ ID NO.5, that is, the base type of the SNP site is G, and the genotype of the SNP molecular marker is GG; the wheat flour FWG of individuals with the SNP molecular marker AA genotype is higher than that of individuals with the GG genotype.

[0030] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in wheat flour FWG. That is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is AA, and the wheat flour FWG of individuals with the SNP molecular marker AA genotype is higher than that of individuals with the GG genotype.

[0031] A SNP molecular marker, Marker45403, associated with the control of wheat flour FWG, is located on the 1D chromosome of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0032] The nucleotide sequence of this molecular marker is shown in Table 8: SNP site sequence information of flour FWG.

[0033] The physical location of the SNP molecular marker Marker45403 is 408888749 on chromosome 1D, and the base is an A / T variation.

[0034] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45403 is shown in SEQ ID NO.8, that is, wheat flour with high FWG is characterized by a base type of T at this SNP site.

[0035] The base variation type and phenotypic value of the SNP molecular markers are shown in Table 7: Phenotypic effect of SNP site base variation in flour FWG, that is, wheat flour FWG with a base type of T at this position is high.

[0036] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45403 is shown in SEQ ID NO.8, that is, the base type of the SNP site is T, and the genotype of the SNP molecular marker is TT; the nucleotide sequence of the SNP molecular marker Marker45403 is shown in SEQ ID NO.7, that is, the base type of the SNP site is A, and the genotype of the SNP molecular marker is AA; the wheat flour FWG of individuals with the SNP molecular marker TT genotype is higher than that of individuals with the AA genotype.

[0037] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in wheat flour FWG. That is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is TT type, and the wheat flour FWG of individuals with the SNP molecular marker TT genotype is higher than that of individuals with the AA genotype.

[0038] A SNP molecular marker, Marker45397, associated with the control of wheat flour FWG, is located on chromosome 1D of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.9 or SEQ ID NO.10.

[0039] The nucleotide sequence of this molecular marker is shown in Table 8: SNP site sequence information of flour FWG.

[0040] The physical location of the SNP molecular marker Marker45397 is 408741172 on chromosome 1D, and the base is a G / C variant.

[0041] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45397 is shown in SEQ ID NO.10, that is, wheat flour with a base type of C at this SNP site has a high FWG.

[0042] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in flour FWG, that is, wheat flour FWG with a base type of C at this position is high.

[0043] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45397 is shown in SEQ ID NO.10, that is, the base type of the SNP site is C, and the genotype of the SNP molecular marker is CC; the nucleotide sequence of the SNP molecular marker Marker45397 is shown in SEQ ID NO.9, that is, the base type of the SNP site is G, and the genotype of the SNP molecular marker is GG; the wheat flour FWG of individuals with the SNP molecular marker CC genotype is higher than that of individuals with the GG genotype.

[0044] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in wheat flour FWG. That is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is CC type, and the wheat flour FWG of individuals with the CC genotype of the SNP molecular marker is higher than that of individuals with the GG genotype.

[0045] A SNP molecular marker, Marker45431, associated with the control of wheat flour FWG, is located on the 1D chromosome of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.11 or SEQ ID NO.12.

[0046] The nucleotide sequence of this molecular marker is shown in Table 8: SNP site sequence information of flour FWG.

[0047] The physical location of the SNP molecular marker Marker45431 is 409391287 on chromosome 1D, and the base is a C / G variation.

[0048] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45431 is shown in SEQ ID NO.12, that is, wheat flour with high FWG is characterized by a base type of G at this SNP site.

[0049] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in flour FWG, that is, wheat flour FWG with a base type of G at this position is high.

[0050] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker45431 is shown in SEQ ID NO.12, that is, the base type of the SNP site is G, and the genotype of the SNP molecular marker is GG; the nucleotide sequence of the SNP molecular marker Marker45431 is shown in SEQ ID NO.11, that is, the base type of the SNP site is C, and the genotype of the SNP molecular marker is CC; the wheat flour FWG of individuals with the SNP molecular marker GG genotype is higher than that of individuals with the CC genotype.

[0051] The base variation type and phenotypic value of the SNP molecular marker are shown in Table 7: Phenotypic effect of SNP site base variation in wheat flour FWG. That is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is GG type, and the wheat flour FWG of individuals with the SNP molecular marker GG genotype is higher than that of individuals with the CC genotype.

[0052] The use of SNP molecular markers in the breeding of wheat varieties or lines with high FWG in wheat flour, wherein the SNP molecular markers include one or more of the above-mentioned SNP molecular markers Marker45889, Marker45896, Marker45348, Marker45403, Marker45397, and Marker45431.

[0053] According to a preferred embodiment of the present invention, the intended use involves selecting wheat varieties in which one or more of the SNP sites Marker45889, Marker45896, Marker45348, Marker45403, Marker45397, and Marker45431 are superior bases.

[0054] The dominant bases at each SNP site: C at Marker 45889, A at Marker 45896, A at Marker 45348, T at Marker 45403, C at Marker 45397, and G at Marker 45431.

[0055] More preferably, in the intended use, wheat with four or more superior bases among the SNP sites Marker45889, Marker45348, Marker45403, Marker45397, and Marker45431 are selected.

[0056] According to a preferred embodiment of the present invention, wheat is bred with one or more of the following genotypes: Marker45889 (CC type), Marker45896 (AA type), Marker45348 (AA type), Marker45403 (TT type), Marker45397 (CC type), and Marker45431 (GG type).

[0057] A method for screening wheat flour with high FWG content involves using one or more of the above-mentioned SNP molecular markers, including Marker45889, Marker45896, Marker45348, Marker45403, Marker45397, and Marker45431.

[0058] According to a preferred embodiment of the present invention, the method involves screening wheat samples from one or more superior bases among the SNP sites Marker45889, Marker45896, Marker45348, Marker45403, Marker45397, and Marker45431.

[0059] The dominant bases at each SNP site: C at Marker 45889, A at Marker 45896, A at Marker 45348, T at Marker 45403, C at Marker 45397, and G at Marker 45431.

[0060] More preferably, in the method, wheat with more than 4 superior bases in the SNP sites Marker45889, Marker45348, Marker45403, Marker45397, and Marker45431 is screened.

[0061] According to a preferred embodiment of the present invention, wheat samples selected from the following genotypes are selected: Marker 45889 (CC type), Marker 45896 (AA type), Marker 45348 (AA type), Marker 45403 (TT type), Marker 45397 (CC type), and Marker 45431 (GG type), or two or more of these genotypes.

[0062] The SNP molecular marker Marker 45889 is located on chromosome 1D of wheat. The nucleotide sequences 300 bp before and after this molecular marker are shown in Table 8: SNP site sequence information for the FWG trait in wheat flour. The base variation types of the SNP molecular marker Marker 45889 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the FWG trait in wheat flour. That is, wheat flour with a T-type base at this SNP molecular marker has low FWG, and the genotype of this type of wheat sample is TT. Wheat flour with a C-type base at this SNP site has high FWG, and the genotype of this type of wheat sample is CC. The flour FWG value of individuals with the CC genotype of the SNP molecular marker is higher than that of individuals with the TT genotype.

[0063] The molecular markers include the SNP marker Marker45896, located on wheat chromosome 1D. The nucleotide sequences of the 300 bp before and after this marker are shown in Table 8: SNP site sequence information for the flour FWG trait. The base variation types of the SNP marker Marker45896 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the wheat flour FWG trait. That is, wheat flour with a T-type base at this SNP site has low FWG, and the genotype of this type of wheat sample is TT. Wheat flour with an A-type base at this SNP site has high FWG, and the genotype of this type of wheat sample is AA. The flour FWG value of individuals with the AA genotype of the SNP marker is higher than that of individuals with the TT genotype.

[0064] The molecular markers include the SNP marker Marker45348, located on wheat chromosome 1D. The nucleotide sequences 300 bp before and after this marker are shown in Table 8: SNP site sequence information for the flour FWG trait. The base variation types of the SNP marker Marker45348 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the wheat flour FWG trait. That is, wheat flour with a base of type G at this SNP site has low FWG, and the genotype of this type of wheat sample is GG. Wheat flour with a base of type A at this SNP site has high FWG, and the genotype of this type of wheat sample is AA. The flour FWG value of individuals with the AA genotype of the SNP marker is higher than that of individuals with the GG genotype.

[0065] The molecular marker includes SNP marker Marker45403, located on wheat chromosome 1D. The nucleotide sequences 300 bp before and after this marker are shown in Table 8: SNP site sequence information for flour FWG trait. The base variation types of SNP marker Marker45403 are shown in Table 7: Phenotypic effects of base variation at SNP sites for wheat flour FWG trait. That is, wheat flour with base type A at this SNP site has low FWG, and the genotype of this type of wheat sample is AA; wheat flour with base type T at this SNP site has high FWG, and the genotype of this type of wheat sample is TT. The flour FWG value of individuals with the TT genotype of the SNP marker is higher than that of individuals with the AA genotype.

[0066] The molecular markers include SNP marker Marker45397, located on wheat chromosome 1D. The nucleotide sequences 300 bp before and after this marker are shown in Table 8: SNP site sequence information for the FWG trait in wheat flour. The base variation types of SNP marker Marker45397 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the FWG trait in wheat flour. That is, wheat flour with a base of type G at this SNP site has low FWG, and the genotype of this type of wheat sample is GG. Wheat flour with a base of type C at this SNP site has high FWG, and the genotype of this type of wheat sample is CC. The flour FWG value of individuals with the CC genotype of the SNP marker is higher than that of individuals with the GG genotype.

[0067] The molecular markers include SNP marker Marker45431, located on wheat chromosome 1D. The nucleotide sequences 300 bp before and after this marker are shown in Table 8: SNP site sequence information for flour FWG trait. The base variation types of SNP marker Marker45431 are shown in Table 7: Phenotypic effects of base variation at SNP sites for wheat flour FWG trait. That is, wheat flour with a C base at this SNP site has low FWG, and the genotype of this type of wheat sample is CC. Wheat flour with a G base at this SNP site has high FWG, and the genotype of this type of wheat sample is GG. The flour FWG value of individuals with the GG genotype of the SNP marker is higher than that of individuals with the CC genotype.

[0068] Beneficial effects of the present invention

[0069] The SNP sites provided by this invention are of great significance for the detection and selection of wheat varieties, lines and breeding materials with high FWG in wheat flour. Molecular markers can be developed based on them to accelerate the efficiency of wheat quality breeding. Attached Figure Description

[0070] Figure 1 The frequency distribution of average wet gluten content in the population under three different environments is shown in the figure.

[0071] Figure 2 This is a diagram of the genetic map results. Detailed Implementation

[0072] 1. Materials and Methods

[0073] 1.1 Test Materials

[0074] The material used in this experiment was a recombinant inbred line (RIL) population of 268 stable homozygous families obtained by crossing Gaocheng 8901 (male parent) with FWG content differences with Nongda Nuomai 1 (female parent) through single-seed transmission.

[0075] Nongda Nuomai No. 1 is a fully glutinous wheat variety bred by crossing "Jiangsu Baihuomai" and "Guandong 107".

[0076] Gaocheng 8901 is a high-quality, strong-gluten wheat variety bred from "77546-2" and "Linzhang".

[0077] The RIL population and its parents were planted in the experimental field of Shandong Agricultural University in Tai'an City, Shandong Province in the 2017-2018 environment (E1) and 2018-2019 environment (E2) using a randomized block design; and in the 2019-2020 environment (E3) using the Wenyang experimental field in Tai'an City, Shandong Province using a randomized block design; all were replicated three times; field management was the same as local field management.

[0078] Grown according to local standard management methods, no serious pests, diseases, or lodging occurred during the growing period. Harvested by plot at maturity, dried, and then stored indoors using standard methods.

[0079] 1.2 Test Methods

[0080] (1) Preparation of wheat flour:

[0081] According to standards, wheat flour is moistened by adding water according to the original moisture content and hardness of the flour, so that the moisture content reaches 14%-15%. Then it is milled using Buhler milling equipment produced in Wuxi, China, with a flour yield of about 70%. The milled flour is stored in a cold storage at 4℃ for later use.

[0082] (2) Determination of FWG content in wheat flour:

[0083] The wet gluten content of wheat was determined according to the method specified in GB / T 5506.2-2008. The gluten index was calculated as: (Total gluten weight - sieved gluten weight) / Total gluten weight × 100%.

[0084] (3) SLAF library construction and SNP marker detection

[0085] The wheat genome was selected as the reference genome. After enzyme digestion prediction, the optimal restriction enzyme was determined to be HaeIII. Sequences with digestion fragment lengths between 464-494 bp were defined as SLAF tags. The predicted SLAF tag data was then analyzed. GC content analysis and Q30 analysis were performed on the filtered sequencing data, and the data volume was evaluated. Nipponbare rice was used as a control to evaluate and monitor the results of this control data, verifying the correctness of the experimental procedure and determining the effectiveness of the enzyme digestion scheme. (Note: Wheat reference genome download address: http: / / www.wheatgenome.org / News / Latest-news / IWGSC-Reference-Sequence-v1.0-browser-now-available-at-URGI; Nipponbare rice genome download address: [link missing])

[0086] http: / / rapdb.dna.affrc.go.jp

[0087] Based on the sequencing reads' localization results on the reference genome, GATK was used for local realignment, GATK variant detection, and samtools variant detection. Intersection of the GATK and samtools results was also performed to ensure the accuracy of the obtained SNPs, resulting in the final SNP set, as shown in Table 1. The aaxbb type was suitable for the RIL population in this experiment, while the other markers were suitable for the hybrid population.

[0088] Table 1 SNP genotype coding rules

[0089]

[0090]

[0091] (4) Construction of high-density genetic maps and QTL mapping

[0092] To ensure the quality of the genetic map, polymorphic SNP tags were filtered. Severely segregated polymorphic markers (chi-square test P < 0.01) were filtered according to standards. The selected SNP tags were then analyzed by calculating the MLOD value between two tags, setting minimum and maximum population numbers, and sorting tags by MLOD value from smallest to largest according to a pre-defined MLOD value range. Tags with the highest MLOD values ​​were grouped into the same linkage group. Tags with MLOD values ​​below 5 with other SNP tags were filtered out. These were then designated as mapping markers. Using linkage groups as units, HighMap software was used to analyze the linear arrangement of markers within each group, and the genetic distance between adjacent markers was calculated to obtain the final genetic map. Simultaneously, monomeric origin assessment was performed: the monomeric origin of each sample in all linkage groups was statistically analyzed to identify potential double crossover sites. Double crossovers within a linkage group are typically controlled to below 3%. Linkage assessment is also conducted; the closer the markers are, the lower the recombination rate. Linkage analysis was performed on the RIL population data using QTL IciMapping software and R / QTL localization software. The QTL naming followed the method of McIntosh et al. (2005).

[0093] (5) Candidate gene prediction

[0094] QTL loci with stable and high contribution rates in wheat FWG trait composition were selected as candidate regions for functional annotation. The wheat genome sequence from the BioMed public database platform was used as the reference genome sequence. The located QTL locus regions were aligned to the reference genome scaffold sequence using BLAST software. Matching candidate regions were compared with published wheat templates to obtain corresponding coding gene information, such as genes. Furthermore, a BLAST (Basic Local Alignment Search Tool) search was performed on the International Wheat Genome Sequencing Consortium database (IWGSC; http: / / www.wheatgenome.org / ). When the SNP marker sequence was 100% identical to any wheat contig, the IWGSC BLAST results were used to extend the sequence of each marker by 5000 bp. Then, the extended sequences were used in the National Center for Biotechnology Information (NCBI) database (http: / / www.ncbi.nlm.nih.gov) and Ensembl Plants (http: / / ...

[0095] The `plant.ensembl.org / Triticum_aestivum / Tools / Blast` function is used to identify potential candidate genes and their functions.

[0096] 2. Results

[0097] 2.1 Phenotypic Analysis of FWG Content in Wheat Flour from the RIL Population

[0098] Under all three environmental conditions, the FWG content in wheat flour from the Gaocheng 8901 population was lower than that of the Nongda Nuomai 1 population. The RIL population exhibited significant phenotypic variation in FWG content in wheat flour, showing overpopulation segregation. Under the three environmental conditions, the mean FWG content in wheat flour ranged from 34.03 to 34.82, with a minimum of 11.5 and a maximum of 44, and a coefficient of variation ranging from 11.01% to 11.77% (see Table 2). The segregation within the population was continuous and conformed to a normal distribution. Figure 1 It exhibits typical quantitative traits.

[0099] Table 2. Phenotypic analysis of wheat flour FWG traits in RIL population

[0100]

[0101] Note: E1: 2017-2018 Tai'an, E2: 2018-2019 Tai'an, E3: 2019-2020 Tai'an

[0102] 2.2 Atlas Information

[0103] After rigorous screening, 13,658 SNP tags were finally obtained that could be used for mapping, and the linkage clustering is shown in Table 3. The 13,658 selected SNP tags were then processed by calculating the MLOD values ​​between each pair of tags. SNP tags with MLOD values ​​lower than 5 were filtered out, resulting in 8,095 tags being mapped, representing a mapping rate of 59.27%.

[0104] Table 3. Statistical Table of Marker Linkage Grouping Projects

[0105]

[0106]

[0107] Using linkage groups as units, HighMap software was used to analyze and obtain the linear arrangement of markers within each linkage group, and the genetic distance between adjacent markers was estimated, as follows: Figure 2 As shown, the final genetic map with a total map distance of 2,554.10 cM was obtained (Table 4).

[0108] Table 4. Basic Map Information

[0109]

[0110] 2.3 QTL Analysis of FWG Content in Wheat Flour

[0111] QTL analysis of wheat flour FWG content (Table 5) revealed four QTL loci associated with wet gluten content: QFwg-4A, QFwg-5D, QFwg-1D-1, and QFwg-1D-2. Their LOD values ​​were 3.0, 3.0, 7.2, and 8.2, respectively; their additive effects were -0.72, -0.78, 1.34, and 1.37, respectively; and their phenotypic contribution rates were 3.3%, 4.0%, 18.6%, and 19.9%, respectively. Two QTLs were detected on chromosome 1D, and both QTLs were detected in both E2 and E3 environments.

[0112] Table 5. QTLs of wheat flour-related traits in the RIL population

[0113]

[0114] Table 6 shows that the major QTL loci controlling the stability of flour FWG content on chromosome 1D are QFwg-1D-1 and QFwg-1D-2. Among them, QFwg-1D-1 has two SNP loci, Marker45889 and Marker45896; QFwg-1D-2 has four SNP loci, Marker45348, Marker45403, Marker45397 and Marker45431, and their variation information is related to flour FWG content.

[0115] Table 6. QTLs and SNPs for stable main effects of wheat flour whiteness traits in the RIL population.

[0116]

[0117] As shown in Table 7, Marker 45889 is located on wheat chromosome 1D, with base C being a superior variant; Marker 45896 is located on wheat chromosome 1D, with base A being a superior variant; Marker 45348 is located on wheat chromosome 1D, with base A being a superior variant; Marker 45403 is located on wheat chromosome 1D, with base T being a superior variant; Marker 45397 is located on wheat chromosome 1D, with base C being a superior variant; and Marker 45431 is located on wheat chromosome 1D, with base G being a superior variant. Except for Marker 45896, the SNP site variations of the other five markers showed significant differences in their phenotypic effects. The SNP site sequence information for each marker is shown in Table 8.

[0118] Table 7. Phenotypic effect of base variation at SNP site of flour FWG content

[0119]

[0120]

[0121] Table 8. SNP site sequence information of flour FWG content

[0122]

[0123]

[0124]

[0125] Table 9 shows that two common haplotype combinations affecting flour FWG content appeared in the sample materials: TAGAGC and CTATCG. Since the phenotypic effect of SNP Marker 45896 base variation is basically consistent, the variation of this marker base can be disregarded in haplotype analysis. Overall, the wet gluten content gradually increases with the increase in the number of superior bases. Generally, the wet gluten content of haplotype TGAGC is less than 30%, at which point all five SNP markers are non-superior variant base types. When the haplotype contains 1 to 3 superior bases, the wet gluten content of the flour is between 31% and 33.73%; when it contains 4 superior bases, the wet gluten content is between 33.98% and 36%; when all 5 bases of the haplotype are superior bases, the wet gluten content is generally greater than 36%. Therefore, it can be seen that the superior bases at SNP sites Marker45889, Marker45348, Marker45403, Marker45397, and Marker45431 can increase the FWG content of wheat. Furthermore, wheat varieties that simultaneously possess superior variant bases at all five SNP sites have higher FWG content in their flour, which plays an important role in the screening of high-FWG flour materials.

[0126] Table 9. Haplotypes of flour wet gluten content with different base variation in wheat

[0127]

[0128] 2.4 Prediction of candidate genes for major loci of FWG content in wheat flour

[0129] Gene comparison revealed 11 genes (Table 10) at the major QTL site Qfwg1D-1 on chromosome 1D that control the stability of FWG content in wheat flour, and 14 genes (Table 11) at the major QTL site Qfwg1D-2 on chromosome 1D. Among these genes, TraesCS1D01G330200.1 and TraesCS1D01G330100.1 are associated with α-amylase protease inhibitors and can inhibit α-amylase; TraesCS1D01G310300.1 is associated with protease inhibitors and mainly inhibits protease activity. TraesCS1D01G328600.1 and TraesCS1D01G328000.1 control a photosynthetic protein in photosystem II (thylakoid membrane) during photosynthesis, thus affecting the light absorption rate and consequently the synthesis of organic matter, potentially influencing the wet gluten content of wheat flour.

[0130] Table 10 Qfwg1D-1 candidate gene predictions

[0131]

[0132] Table 11 Qfwg1D-2 candidate gene predictions

[0133]

[0134]

[0135] The SNP sites provided by this invention are of great significance for the detection and selection of wheat varieties, lines and breeding materials with high FWG in wheat flour. Molecular markers can be developed based on them to accelerate the efficiency of wheat quality breeding.

Claims

1. A SNP molecular marker, Marker45896, associated with the control of FWG in wheat flour, characterized in that, It is located on chromosome 1D of wheat, and the nucleotide sequence of this molecular marker is shown in SEQ ID NO.3 or SEQ ID NO.

4.

2. The SNP molecular marker Marker 45896 as described in claim 1, characterized in that, The nucleotide sequence of the SNP molecular marker Marker45896 is shown in SEQ ID NO.4, that is, wheat flour FWG with an SNP site of Marker45896 having a base type of A.

3. The SNP molecular marker Marker 45896 as described in claim 1, characterized in that, The nucleotide sequence of the SNP molecular marker Marker45896 is shown in SEQ ID NO.4, that is, the base type of the SNP site of Marker45896 is A, and the genotype of the SNP molecular marker is AA; the nucleotide sequence of the SNP molecular marker Marker45896 is shown in SEQ ID NO.3, that is, the base type of the SNP site is T, and the genotype of the SNP molecular marker is TT; the wheat flour FWG of individuals with the SNP molecular marker AA genotype is higher than that of individuals with the SNP molecular marker TT genotype.

4. The use of SNP molecular markers in the breeding of wheat varieties or lines with high FWG in wheat flour, wherein the SNP molecular marker is the SNP molecular marker Marker45896 as described in any one of claims 1-3; Alternatively, the SNP molecular marker may include, in addition to the SNP molecular marker Marker45896 as described in any one of claims 1-3, one or more of Marker45348, Marker45403, Marker45397, and Marker45431. The SNP molecular marker Marker45896 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.3 or SEQ ID NO.

4. The SNP molecular marker Marker45348 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.5 or SEQ ID NO.

6. The SNP molecular marker Marker45403 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.7 or SEQ ID NO.

8. The SNP molecular marker Marker45397 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.9 or SEQ ID NO.

10. The SNP molecular marker Marker45431 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.11 or SEQ ID NO.

12.

5. The use as described in claim 4, characterized in that, Selecting wheat varieties with superior bases at the SNP site Marker45896; We also selected wheat varieties with one or more superior bases from Marker45348, Marker45403, Marker45397, and Marker45431. The dominant bases at each SNP site: base A at SNP site Marker 45896, base A at SNP site Marker 45348, base T at SNP site Marker 45403, base C at SNP site Marker 45397, and base G at SNP site Marker 45431.

6. The use as described in claim 5, characterized in that, In the aforementioned application, wheat with four superior bases in SNP sites Marker45348, Marker45403, Marker45397, and Marker45431 is selected.

7. The use as described in claim 5, characterized in that, Wheat varieties were selected with one or more of the following SNP molecular markers: Marker45896 (genotype AA), Marker45348 (genotype AA), Marker45403 (genotype TT), Marker45397 (genotype CC), and Marker45431 (genotype GG).

8. A method for screening wheat flour with high FWG (Ferrous Gluten) content, characterized in that, Wheat with superior bases of SNP site Marker45896 was screened using the SNP molecular marker Marker45896 as described in any one of claims 1-3. Alternatively, in addition to using the SNP molecular marker Marker45896 as described in any one of claims 1-3, one or more of Marker45348, Marker45403, Marker45397, and Marker45431 may be used for screening to select wheat varieties for which one or more of Marker45348, Marker45403, Marker45397, and Marker45431 are superior bases; The dominant bases at each SNP site: base A at SNP site Marker 45896, base A at SNP site Marker 45348, base T at SNP site Marker 45403, base C at SNP site Marker 45397, and base G at SNP site Marker 45431. The SNP molecular marker Marker45896 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.3 or SEQ ID NO.

4. The SNP molecular marker Marker45348 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.5 or SEQ ID NO.

6. The SNP molecular marker Marker45403 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.7 or SEQ ID NO.

8. The SNP molecular marker Marker45397 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.9 or SEQ ID NO.

10. The SNP molecular marker Marker45431 is located on chromosome 1D of wheat, and its nucleotide sequence is shown in SEQ ID NO.11 or SEQ ID NO.

12.

9. The method as described in claim 8, characterized in that, Wheat samples were screened for four superior bases among the SNP sites Marker45348, Marker45403, Marker45397, and Marker45431.

10. The method as described in claim 8, characterized in that, Wheat samples were screened for one or more of the following SNP molecular markers: Marker45896 (genotype AA), Marker45348 (genotype AA), Marker45403 (genotype TT), Marker45397 (genotype CC), and Marker45431 (genotype GG).

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