Wheat flour wet gluten content major site, SNP (Single Nucleotide Polymorphism) marker and application

Abstract

The invention provides a major site of the wet gluten content of wheat flour, an SNP marker and application, belongs to the technical field of wheat breeding, and mainly provides SNP molecular markers Marker 45889, Marker 45896, Marker 45348, Marker 45403, Marker 45397 and Marker 45431. The SNP site provided by the invention has important significance for detecting and breeding high-FWG varieties, strains and breeding materials of wheat flour, the molecular marker can be developed according to the SNP site, and the molecular marker can be used for detecting and breeding high-FWG varieties, strains and breeding materials of wheat flour. The wheat quality breeding efficiency is improved.

Description

[0001] This application is a divisional application with application number 202410541035.X, application date 2024.04.30, and invention name “Main effect loci and SNP markers of wet gluten content in wheat flour and their applications”. Technical Field

[0002] The invention belongs to the technical field of wheat breeding, and in particular relates to a major effect QTL site related to wheat flour wet gluten content (FWG) and application of a SNP molecular marker thereof. Background Art

[0003] Gluten is both a nutritional quality trait and a processing quality trait, and is a common indicator in the international trade of wheat. Generally speaking, wheat has a high protein content and a relatively high wet gluten content. Gluten is a substance that distinguishes wheat from other cereal crops, which also gives flour the characteristics of being able to be processed into a rich variety of foods. Gluten is an important indicator that determines the quality of wheat, involving two factors: gluten quantity and gluten quality, both of which have a great influence on the quality of flour and dough. A single factor cannot completely determine the quality of flour, and both must be considered at the same time. Previous studies have found that the main components that determine the quality of gluten are glutenin and alcohol-soluble protein, especially the ratio of the two is closely related to the formation of the network structure of gluten. The water absorption, elasticity, kneading resistance, viscosity and extensibility of gluten all affect the quality of dough and the final processing quality. Only when the gluten content is appropriate and the gluten quality is good, the gas generated during dough fermentation can be stored in the dough without escaping, so that the bread or steamed buns have a large volume and good internal texture quality; otherwise, it is poor. The national standard (GB / T17892-1999) stipulates that the wet gluten content of strong gluten wheat flour is >35.0%, and the wet gluten content of medium gluten wheat flour is ≥32.0%; the "Wheat Variety Quality Classification" (GB / T17320-2013) stipulates that the wet gluten content of strong gluten wheat is ≥30%, the wet gluten content of medium gluten wheat is 28%~30%, the wet gluten content of medium gluten wheat is 26%~28%, and the wet gluten content of weak gluten wheat is <26%. Therefore, the determination of wet gluten content is of great significance for the evaluation of the classification of wheat variety quality.

[0004] Previous researchers have conducted quantitative trait localization analysis on wet gluten content, gluten index and other traits that reflect gluten strength. Yu Haixia (2012) detected 40 significant associated markers related to wet gluten content and gluten index through association analysis, among which marker wPt-665169 was associated with both traits. Other studies have shown (Li Weihua et al., 2005) that there are three related loci on chromosomes 1DL, 2DS, and 3BL for the gluten index. McCartney et al. (2006) detected 6, 7, and 4 gluten-related genes on chromosomes 1B, 4D, and 7D, respectively. According to most previous studies, the loci of gluten strength involve a total of 19 chromosomes, with the largest number on chromosome 1B, followed by chromosomes 1D, 2D, 5D, 5B, and 2A.

[0005] Although some loci related to wheat FWG were located by predecessors, there are few major effect loci and SNP molecular markers that can be used to improve FWG in molecular marker-assisted breeding. Therefore, in the present invention, a RIL population was constructed using Nongda Nuowai No. 1 with different FWG as the female parent and Gaocheng 8901 as the male parent, and a high-density genetic map was constructed using SLAF-Seq technology to perform QTL positioning analysis on wheat FWG under three environments, in order to identify major effect QTL loci and their SNP markers and superior allele variation loci, so as to provide genetic resources and effective molecular markers for improving wheat FWG. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides major effect loci and SNP markers for wet gluten content in wheat flour and their applications.

[0007] The present invention uses Nongda Nuowai No. 1 with different FWG values ​​as the female parent and Gaocheng 8901 as the male parent, constructs a RIL population with different flour FWG values ​​as the material, and uses SLAF-Seq technology to construct a high-density genetic map containing 8,095 SNP markers, with a total map length of 2,554.10 cM and a linkage group length of 90.08 to 151.67 cM. Combined with FWG phenotypic data, QTL positioning analysis was performed under three environments.

[0008] Combined with the results of quantitative trait gene positioning analysis, and through excellent allele identification and gene locus phenotypic analysis, two major and tightly linked QTL loci QFwg-1D-1 and QFwg-1D-2 that control the FWG content of flour were identified. They are located on chromosome 1D and have six tightly linked SNP markers. This provides theoretical guidance and basis 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 controlling wheat flour FWG is located on chromosome 1D of wheat. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.1 or SEQ ID NO.2.

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

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

[0013] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45889 is as shown in SEQ ID NO.2, that is, the wheat flour FWG of the base type C at the SNP site is high.

[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, the wheat flour FWG of which the base type of the sequence at this position is C is high.

[0015] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45889 is as 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 as 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 base variation at the SNP site of 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 CC genotype of the SNP molecular marker is higher than that of individuals with TT genotype.

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

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

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

[0020] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45896 is as shown in SEQ ID NO.4, that is, the wheat flour FWG of the base type A at the SNP site is high.

[0021] 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, the wheat flour FWG of which the base type of the sequence at this position is A is high.

[0022] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45896 is as 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 as 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 base variation at the SNP site of wheat flour FWG, that is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is AA type, and the wheat flour FWG of individuals with AA genotype of the SNP molecular marker is higher than that of individuals with TT genotype.

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

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

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

[0027] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45348 is as shown in SEQ ID NO.6, that is, the wheat flour FWG of the base type A at the SNP site is high.

[0028] 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, the wheat flour FWG of which the base type of the sequence at this position is A is high.

[0029] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45348 is as 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 type; the nucleotide sequence of the SNP molecular marker Marker45348 is as 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 type; 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 base variation at the SNP site of wheat flour FWG, that is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is AA type, and the wheat flour FWG of individuals with AA genotype of the SNP molecular marker is higher than that of individuals with GG genotype.

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

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

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

[0034] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45403 is as shown in SEQ ID NO.8, that is, the wheat flour FWG of the SNP site base type T is high.

[0035] 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, the wheat flour FWG of which the base type of the sequence at this position is T is high.

[0036] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45403 is as 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 type; the nucleotide sequence of the SNP molecular marker Marker45403 is as 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 type; 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 base variation at the SNP site of 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 controlling wheat flour FWG is located on chromosome 1D of wheat. The nucleotide sequence of the molecular marker is shown in SEQ ID NO.9 or SEQ ID NO.10.

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

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

[0041] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45397 is as shown in SEQ ID NO.10, that is, the wheat flour FWG of the SNP site base type C is high.

[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, the wheat flour FWG of which the base type of the sequence at this position is C is high.

[0043] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45397 is as 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 as 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 base variation at the SNP site of 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 CC genotype of the SNP molecular marker is higher than that of individuals with GG genotype.

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

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

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

[0048] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45431 is as shown in SEQ ID NO.12, that is, the wheat flour FWG of the SNP site base type G is high.

[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, the wheat flour FWG of which the base type of the sequence at this position is G is high.

[0050] Preferably according to the present invention, the nucleotide sequence of the SNP molecular marker Marker45431 is as 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 as 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 base variation at the SNP site of 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 GG genotype of the SNP molecular marker is higher than that of individuals with the CC genotype.

[0052] A use of SNP molecular markers in breeding wheat varieties or strains with high FWG of wheat flour, wherein the SNP molecular markers include one or more of the above-mentioned SNP molecular markers Marker 45889, Marker 45896, Marker 45348, Marker 45403, Marker 45397, and Marker 45431.

[0053] Preferably, according to the present invention, in the use, wheat having one or more excellent bases among SNP sites Marker45889, Marker45896, Marker45348, Marker45403, Marker45397 and Marker45431 is selected;

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

[0055] Further preferably, in the use, wheat having four or more excellent bases among the SNP sites Marker45889, Marker45348, Marker45403, Marker45397, and Marker45431 is bred.

[0056] Preferably according to the present invention, the genotype of the selected SNP molecular marker Marker45889 is CC type, the genotype of Marker45896 is AA type, the genotype of Marker45348 is AA type, the genotype of Marker45403 is TT type, the genotype of Marker45397 is CC type, and the genotype of Marker45431 is GG type, or two or more of the following.

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

[0058] Preferably, according to the present invention, in the method, wheat having one or more excellent bases in SNP sites Marker45889, Marker45896, Marker45348, Marker45403, Marker45397, and Marker45431 is screened;

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

[0060] Further preferably, in the method, wheat having four or more excellent bases in SNP sites Marker45889, Marker45348, Marker45403, Marker45397, and Marker45431 is screened.

[0061] Preferably according to the present invention, the genotype of the screening SNP molecular marker Marker45889 is CC type, the genotype of Marker45896 is AA type, the genotype of Marker45348 is AA type, the genotype of Marker45403 is TT type, the genotype of Marker45397 is CC type, and the genotype of Marker45431 is GG type, or two or more of them are wheat.

[0062] SNP molecular marker Marker45889 is located on chromosome 1D of wheat. The nucleotide sequences of 300 bp before and after the molecular marker are shown in Table 8: Sequence information of SNP site for flour FWG trait; the base variation type of SNP molecular marker Marker45889 is shown in Table 7: Phenotypic effect of base variation of SNP site for wheat flour FWG trait, that is, wheat flour with T-type base of the SNP molecular marker at this location has low FWG, and the genotype of this type of wheat sample is TT type; wheat flour with C-type base of the SNP site has high FWG, and the genotype of this type of wheat sample is CC type, and the flour FWG value of CC genotype individuals of the SNP molecular marker is higher than that of TT genotype individuals;

[0063] The molecular marker includes SNP molecular marker Marker45896, which is located on chromosome 1D of wheat. The nucleotide sequences of 300 bp before and after the molecular marker are shown in Table 8: Sequence information of SNP site for flour FWG trait; the base variation type of SNP molecular marker Marker45896 is shown in Table 7: Phenotypic effect of base variation of SNP site for wheat flour FWG trait, that is, wheat flour with T-type base of the SNP molecular marker at this location has low FWG, and the genotype of this type of wheat sample is TT type; wheat flour with A-type base of the SNP site has high FWG, and the genotype of this type of wheat sample is AA type, and the flour FWG value of the AA genotype individual of the SNP molecular marker is higher than that of the TT genotype individual;

[0064] The molecular marker includes SNP molecular marker Marker45348, which is located on chromosome 1D of wheat. The nucleotide sequences of 300 bp before and after the molecular marker are shown in Table 8: Sequence information of SNP site for flour FWG trait; the base variation type of SNP molecular marker Marker45348 is shown in Table 7: Phenotypic effect of base variation of SNP site for wheat flour FWG trait, that is, wheat flour with G-type base of the SNP molecular marker at this location has low FWG, and the genotype of this type of wheat sample is GG type; wheat flour with A-type base of the SNP site has high FWG, and the genotype of this type of wheat sample is AA type, and the flour FWG value of the AA genotype individual of the SNP molecular marker is higher than that of the GG genotype individual;

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

[0066] The molecular marker includes SNP molecular marker Marker45397, which is located on chromosome 1D of wheat. The nucleotide sequences of 300 bp before and after the molecular marker are shown in Table 8: Sequence information of SNP site for flour FWG trait; the base variation type of SNP molecular marker Marker45397 is shown in Table 7: Phenotypic effect of base variation of SNP site for wheat flour FWG trait, that is, wheat flour with G-type base of the SNP molecular marker at this location has low FWG, and the genotype of this type of wheat sample is GG type; wheat flour with C-type base of the SNP site has high FWG, and the genotype of this type of wheat sample is CC type, and the flour FWG value of CC genotype individuals of the SNP molecular marker is higher than that of GG genotype individuals;

[0067] The molecular marker includes SNP molecular marker Marker45431, which is located on chromosome 1D of wheat. The nucleotide sequences of 300 bp before and after the molecular marker are shown in Table 8: Sequence information of SNP site for flour FWG trait; the base variation type of SNP molecular marker Marker45431 is shown in Table 7: Phenotypic effect of base variation of SNP site for wheat flour FWG trait, that is, wheat flour with C-type base of the SNP molecular marker at this location has low FWG, and the genotype of this type of wheat sample is CC type; wheat flour with G-type base of the SNP site has high FWG, and the genotype of this type of wheat sample is GG type. The flour FWG value of individuals with GG genotype of the SNP molecular marker is higher than that of individuals with CC genotype.

[0068] Beneficial effects of the present invention

[0069] The SNP loci provided by the present invention are of great significance for detecting and breeding wheat flour high FWG varieties, strains, and breeding materials, and molecular markers can be developed based on them to accelerate the efficiency of wheat quality breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the frequency distribution diagram of the average wet gluten content of the groups under three environments.

[0071] Figure 2 This is the genetic map result. DETAILED DESCRIPTION

[0072] 1. Materials and Methods

[0073] 1.1 Test materials

[0074] The experimental materials were Gaocheng 8901 (male parent) and Nongda Nuowai No. 1 (female parent) with different FWG contents, and a recombinant inbred line (RIL) population of 268 stable homozygous families was obtained by single-seed descent.

[0075] Nongda Glutinous Wheat No. 1 is a fully glutinous wheat variety bred by hybridizing "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 parents were planted in the experimental field of Shandong Agricultural University in Tai'an City, Shandong Province under the 2017-2018 environment (E1) and the 2018-2019 environment (E2), with a randomized block design; and were planted in the Wenyang experimental field in Tai'an City, Shandong Province under the 2019-2020 environment (E3), with a randomized block design; both were repeated 3 times; and the field management was the same as the local field management.

[0078] According to the local routine management methods, no serious pests and lodging occurred during the growth period. When mature, the crops were harvested by plot, dried and stored indoors according to standard methods.

[0079] 1.2 Test methods

[0080] (1) Preparation of wheat flour:

[0081] Wheat flour is tempered according to the standard, and the amount of water added is determined according to the original moisture content and hardness of the flour, so that the moisture content reaches 14%-15%, and then it is ground into flour using Buhler produced in Wuxi, China, with a flour yield of about 70%. The ground 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 is determined according to the method specified in GB / T 5506.2-2008. Gluten index = (total gluten weight - sieved gluten weight) / total gluten weight × 100%

[0084] (3) Construction of SLAF library and detection of SNP markers

[0085] The wheat genome was selected as the reference genome. After restriction prediction, the optimal endonuclease was finally determined to be HaeIII. The sequence with a length of 464-494bp was defined as the SLAF tag, and the SLAF tag data was predicted. The filtered sequencing data was analyzed for GC content and Q30, as well as the data volume. The Nipponbare rice was used as a control to evaluate the results of the control data, prove the correctness of the experimental process, and determine whether the restriction scheme is effective. (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:

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

[0087] According to the positioning results of sequencing reads on the reference genome, GATK performed local realignment, GATK variation detection, samtools variation detection, and took the intersection variation sites of the results of GATK and samtools to ensure the accuracy of the obtained SNPs, and obtained the final SNP site set, as shown in Table 1. Among them, the aaxbb type is suitable for the RIL population in this experiment, and the other markers are suitable for the hybrid population.

[0088] Table 1 SNP genotype coding rules

[0089]

[0090]

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

[0092] In order to ensure the quality of the genetic map, polymorphic SNP tags were filtered, and polymorphic markers with severe skewed separation (chi-square test P < 0.01) were filtered according to the standard. The SNP tags that were screened were filtered by calculating the MLOD value between the two tags of the filtered SNP data, setting the number of tags for the minimum and maximum groups, and arranging the tags according to the MLOD value of the preset MLOD value interval from small to large. The tags with the highest MLOD value between the tags will be divided into a linkage group, and the tags with MLOD values ​​lower than 5 with other SNP tags will be filtered out, and finally positioned as mapping markers (Marker). Taking the linkage group as the unit, HighMap software was used to analyze the linear arrangement of the markers in the linkage group, and then the genetic distance between the two adjacent markers was calculated to obtain the final genetic map. At the same time, the single source evaluation was carried out: the single source of each sample in all linkage groups was counted to find possible double exchange sites. In a linkage group, double exchange is usually controlled below 3%, and linkage relationship evaluation is also required. The closer the distance between markers, the lower the recombination rate. Linkage analysis was performed on the data from the RIL population using QTL IciMapping software and R / QTL mapping software. QTL naming was based on the method of McIntosh et al. (2005).

[0093] (5) Candidate gene prediction

[0094] The quantitative trait QTL loci with a high stable contribution rate to the composition of wheat FWG traits were selected as candidate regions for functional annotation. The wheat genome sequence on the public data platform of BioMed was used as the reference genome sequence. The located QTL locus region was aligned to the reference genome scaffold sequence using BLAST software, and the matching candidate interval was aligned with the published wheat template to obtain the corresponding coding gene information, such as genes. In addition, 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 overlap group, the IWGSC BLAST result was used to extend the sequence of each marker by 5000bp. Then, the extended sequence was used to search the database of the National Center for Biotechnology Information (NCBI) (http: / / www.ncbi.nlm.nih.gov) and Ensembl Plants (http: / /

[0095] plant.ensembl.org / Triticum_aestivum / Tools / Blast) was used to identify possible candidate genes and functions.

[0096] 2. Results

[0097] 2.1 Phenotypic analysis of FWG content in wheat flour of RIL population

[0098] In the three environments, the FWG content of wheat flour of Gaocheng 8901 was lower than that of Nongda Nuowai No. 1. The phenotypic variation of wheat flour FWG content of RIL population was large, and there was super-parent separation phenomenon. Under the three environments, the mean value of wheat flour FWG of the population was 34.03-34.82, the minimum value was 11.5, the maximum value was 44, and the coefficient of variation was 11.01%-11.77%, as shown in Table 2. The population separation was continuous and conformed to the normal distribution ( Figure 1 ), showing typical quantitative traits.

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

[0100]

[0101] Note: E1: 2017-2018 Taian, E2: 2018-2019 Taian, E3: 2019-2020 Taian

[0102] 2.2 Graph Information

[0103] After a systematic and rigorous screening, 13,658 SNP tags were finally obtained for mapping, and the linkage grouping is shown in Table 3. The MLOD values ​​of the 13,658 SNP tags were calculated between each tag, and the SNP tags with MLOD values ​​lower than 5 were filtered out. A total of 8,095 SNP tags were mapped, with a mapping rate of 59.27%.

[0104] Table 3Marker linkage grouping

[0105]

[0106]

[0107] Taking linkage groups as units, HighMap software was used to analyze the linear arrangement of markers within linkage groups and estimate the genetic distance between adjacent markers 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 Map information

[0109]

[0110] 2.3 QTL analysis of FWG content in wheat flour

[0111] Through QTL analysis of wheat flour FWG content (Table 5), four QTL loci related to flour wet gluten content were detected, namely 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, and the additive effects were -0.72, -0.78, 1.34 and 1.37, respectively, and the phenotypic contribution rates were 3.3%, 4.0%, 18.6% and 19.9%, respectively. Two QTLs were detected on chromosome 1D, and these two QTLs were detected in both E2 and E3 environments.

[0112] Table 5 QTL of wheat flour-related traits in RIL population

[0113]

[0114] As shown in Table 6, the main QTL loci controlling the stability of flour FWG content on chromosome 1D are QFwg-1D-1 and QFwg-1D-2, among which 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 the FWG content of flour.

[0115] Table 6 QTL and SNP markers for stable main effects of wheat flour whiteness traits in RIL population

[0116]

[0117] As shown in Table 7, the Marker45889 marker is located on the wheat chromosome 1D, and base C is an excellent variant base; the Marker45896 marker is located on the wheat chromosome 1D, and base A is an excellent variant base; the Marker45348 marker is located on the wheat chromosome 1D, and base A is an excellent variant base; the Marker45403 marker is located on the wheat chromosome 1D, and base T is an excellent variant base; the Marker45397 marker is located on the wheat chromosome 1D, and base C is an excellent variant base; the Marker45431 marker is located on the wheat chromosome 1D, and base G is an excellent variant base. Except for the Marker45896 marker, the effects of the SNP site base variation on the phenotypic effect of the other five markers are significantly different. The sequence information of the SNP sites of 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] As shown in Table 9, two common haplotype combinations that affect the FWG content of flour appeared in the sample materials, namely TAGAGC type and CTATCG type. Since the phenotypic effect of SNP Marker45896 base variation is basically the same, the variation of this marker base can be ignored in haplotype analysis. Overall, with the increase in the number of excellent bases, the wet gluten content gradually increases. Generally, the wet gluten content containing haplotype TGAGC is less than 30%, and at this time, the five SNP markers are all non-excellent variant base types. When the haplotype contains 1 to 3 excellent bases, the wet gluten content of flour is between 31%-33.73%; when it contains 4 excellent bases, the wet gluten content is between 33.98%-36%; when all 5 bases of the haplotype are excellent bases, the wet gluten content is generally greater than 36%. It can be seen that the excellent bases at SNP sites Marker45889, Marker45348, Marker45403, Marker45397, and Marker45431 can increase the FWG content of wheat, and the wheat varieties that have excellent variant bases at the five SNP sites have higher flour FWG content, 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 flour

[0127]

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

[0129] Through gene comparison, 11 genes were screened and predicted for the main QTL locus Qfwg1D-1 on chromosome 1D that controls the stability of FWG content in wheat flour (Table 10), and 14 genes were screened and predicted for the main QTL locus Qfwg1D-2 on chromosome 1D that controls the stability of FWG content in wheat flour (Table 11). The functions of TraesCS1D01G330200.1 and TraesCS1D01G330100.1 are related to α-amylase protease inhibitors and can inhibit α-amylase; the gene TraesCS1D01G310300.1 is related to the function of protease inhibitors and mainly inhibits the activity of proteases. The genes TraesCS1D01G328600.1 and TraesCS1D01G328000.1 control the photosynthesis of photosystem II, a photosynthetic protein in the thylakoid membrane, which in turn affects the light absorption rate, thereby affecting the synthesis of organic matter, and may eventually affect the wet gluten content of wheat flour.

[0130] Table 10Qfwg1D-1candidate gene predictions

[0131]

[0132] Table 11Qfwg1D-2candidate gene predictions

[0133]

[0134]

[0135] The SNP loci provided by the present invention are of great significance for detecting and breeding wheat flour high FWG varieties, strains, and breeding materials, and 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 controlling wheat flour FWG, which is located on chromosome 1D of wheat, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO.3 or SEQ ID NO.4; Preferably, the nucleotide sequence of the SNP molecular marker Marker45896 is as shown in SEQ ID NO.4, that is, the wheat flour FWG of the SNP site base type A is high; Preferably, the nucleotide sequence of the SNP molecular marker Marker45896 is as 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 as 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.

2. A SNP molecular marker Marker45348 associated with controlling wheat flour FWG, which is located on chromosome 1D of wheat, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO.5 or SEQ ID NO.6; Preferably, the nucleotide sequence of the SNP molecular marker Marker45348 is as shown in SEQ ID NO.6, that is, the wheat flour FWG of the SNP site base type A is high; Preferably, the nucleotide sequence of the SNP molecular marker Marker45348 is as 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 as 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.

3. A SNP molecular marker Marker45403 associated with controlling wheat flour FWG, which is located on chromosome 1D of wheat, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO.7 or SEQ ID NO.8; Preferably, the nucleotide sequence of the SNP molecular marker Marker45403 is as shown in SEQ ID NO.8, that is, the wheat flour FWG of the SNP site base type T is high; Preferably, the nucleotide sequence of the SNP molecular marker Marker45403 is as 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 as 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.

4. A SNP molecular marker Marker45397 associated with controlling wheat flour FWG, which is located on chromosome 1D of wheat, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO.9 or SEQ ID NO.10; Preferably, the nucleotide sequence of the SNP molecular marker Marker45397 is as shown in SEQ ID NO.10, that is, the wheat flour FWG of the SNP site base type C is high; Preferably, the nucleotide sequence of the SNP molecular marker Marker45397 is as 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 as 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.

5. A SNP molecular marker Marker45431 associated with controlling wheat flour FWG, which is located on chromosome 1D of wheat, and the nucleotide sequence of the molecular marker is shown in SEQ ID NO.11 or SEQ ID NO.12; Preferably, the nucleotide sequence of the SNP molecular marker Marker45431 is as shown in SEQ ID NO.12, that is, the wheat flour FWG of the SNP site base type G is high; Preferably, the nucleotide sequence of the SNP molecular marker Marker45431 is as 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 as 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.

6. A use of SNP molecular markers in breeding wheat varieties or strains with high FWG of wheat flour, wherein the SNP molecular markers include one or more of the SNP molecular markers Marker 45896, Marker 45348, Marker 45403, Marker 45397, and Marker 45431 according to any one of claims 1 to 5.

7. The use according to claim 6, characterized in that Breeding wheat with one or more excellent bases in SNP sites Marker45896, Marker45348, Marker45403, Marker45397, and Marker45431; Excellent bases at each SNP site: base A of SNP site Marker45896, base A of Marker45348, base T of Marker45403, base C of Marker45397, and base G of Marker45431; Preferably, in the use, wheat having more than 4 excellent bases in SNP sites Marker45348, Marker45403, Marker45397, and Marker45431 is selected; Preferably, the wheat selected has one or more of the following genotypes: AA type for SNP molecular marker Marker45896, AA type for Marker45348, TT type for Marker45403, CC type for Marker45397, and GG type for Marker45431.

8. A method for screening wheat flour with high FWG, using one or more of the SNP molecular markers Marker 45896, Marker 45348, Marker 45403, Marker 45397, and Marker 45431 described in any one of claims 1 to 5 for screening.

9. The method according to claim 8, characterized in that Screening wheat with one or more excellent bases among SNP sites Marker45896, Marker45348, Marker45403, Marker45397, and Marker45431; Excellent bases at each SNP site: base A of SNP site Marker45896, base A of Marker45348, base T of Marker45403, base C of Marker45397, and base G of Marker45431; Preferably, wheat having at least 4 excellent bases among SNP sites Marker45348, Marker45403, Marker45397, and Marker45431 is screened; Preferably, the wheat whose genotype of SNP molecular marker Marker45896 is AA type, the genotype of Marker45348 is AA type, the genotype of Marker45403 is TT type, the genotype of Marker45397 is CC type, and the genotype of Marker45431 is GG type, is screened.

Images