Major loci and SNP markers for wheat kernel hardness and use thereof

Abstract

The application provides a wheat kernel hardness major locus and SNP marker and application, belongs to the wheat breeding technical field, and mainly provides SNP molecular markers Marker287880, Marker287947, Marker287946, Marker288023, Marker287945, Marker288063 and Marker288351; the SNP locus provided by the application has important significance for detecting and breeding wheat kernel high HI varieties, lines and breeding materials, and a molecular marker can be developed according to the SNP locus, so that the wheat quality breeding efficiency is accelerated.

Description

Technical Field

[0001] 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 wheat grain hardness (HI). Background Technology

[0002] Wheat can be classified into three types based on its hardness: soft wheat, mixed wheat, and hard wheat. Different hardness types of wheat have different uses. Hard wheat flour has a large particle size, strong water absorption capacity, and contains more broken starch, making it more suitable for bread and noodle making. Conversely, soft wheat flour has a smaller particle size, weaker water absorption capacity compared to hard wheat, and a lower content of broken starch, making it relatively more suitable for making cookies, cakes, etc. Grain hardness has a significant impact on wheat flour quality, flour color, protein content, and the quality of processed products. Previous studies have found that increasing the hardness index gradually increases the flour yield and protein content of wheat flour. Furthermore, increasing grain hardness has a more significant effect on improving bread quality than other flour products, possibly because hard wheat dough has higher elasticity and lower extensibility (Hu Wenjing, 2021). Other studies have shown that wheat grain hardness affects flour whiteness, and there is a significant negative correlation between grain hardness and flour whiteness. This is because the granular starch and protein in the endosperm of hard wheat are tightly bound, but the endosperm is easily separated from the bran. During milling, most of the grains break along the direction of the endosperm cell wall, resulting in large and uniform flour particles. However, the opposite is true for flour from soft wheat (Hu Ruibo, 2006).

[0003] Early studies showed that the heritability of grain hardness is controlled by one or two major genes, in addition to some modifying genes. In 1986, Greenwell and Schofield first discovered Frabilin protein from wheat washed starch using 1% SDS, leading to significant progress and breakthroughs in the molecular study of wheat grain hardness. Experiments confirmed that Frabilin protein is mainly composed of two proteins, named puroindolinea (Pina) and puroindoline b (Pinb) because they are rich in tryptophan-rich regions (An Jianghong, 2020). This protein is present in soft wheat but is partially or completely absent in hard wheat. Jolly et al. (1996) located the gene (Ha) encoding the protein on chromosome 5DS. Currently, most researchers agree that wheat grain hardness is controlled by the dominant gene Ha (Breseghello et al., 2005; Nelson et al., 2006). Some studies have also located a major gene locus controlling grain hardness on chromosome 1B, which can explain the higher rate of variation in the hardness phenotype compared to the QTL hardness phenotype on chromosome 5DS. Hu Wenjing et al. (2021) detected 20 SNPs significantly associated with wheat grain hardness using 171 wheat varieties. Of these, 14 SNPs (7 loci) were located on chromosomes 1A, 1B, 1D, 2A, 5A, and 7A. Four loci were significantly associated with wheat grain hardness under all conditions, located on chromosomes 1A, 1B, 2A, and 7A.

[0004] Although previous researchers have located some QTL sites related to grain hardness, there are very few effective markers for marker-assisted breeding. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes major sites and SNP markers for wheat grain hardness and their applications.

[0006] This invention uses Nongda Nuomai No. 1, which exhibits significant differences in grain hardness HI, as the female parent and Gaocheng 8901 as the male parent to construct a RIL population with differences in grain hardness. 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. Combined with grain HI phenotypic data, QTL mapping analysis was performed under three different environmental conditions.

[0007] Based on the QTL mapping results, and through superior allele identification and phenotypic analysis of the mapping site, a stable major QTL locus QHi-5A located on chromosome 5A controlling grain hardness (HI) and its seven closely linked SNP markers were identified. The major locus and its linked molecular markers provide a theoretical basis for the discovery of wheat grain hardness genes and the molecular improvement of wheat quality breeding.

[0008] The technical solution of the present invention is as follows:

[0009] A molecular marker for an SNP, Marker287880, associated with the control of HI in wheat grains, is located on chromosome 5A of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] The nucleotide sequence of this molecular marker is shown in Table 8: Seed HI SNP site sequence information.

[0011] The physical location of the SNP molecular marker Marker287880 is 415435115 on chromosome 5A, and the base is a C / T variation.

[0012] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker287880 is shown in SEQ ID NO.1, that is, wheat grains with a base type of C at this SNP site have a high HI.

[0013] The base variation type and phenotypic value of the SNP molecular marker Marker287880 are shown in Table 7: Phenotypic effect of SNP site base variation in grain HI, that is, wheat grains with a C base type at this position have a high HI.

[0014] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker287880 is shown in SEQ ID NO.1, 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 Marker287880 is shown in SEQ ID NO.2, that is, the base type of the SNP site is T, and the genotype of the SNP molecular marker is TT; the wheat grain HI of individuals with the SNP molecular marker TT genotype is lower than that of individuals with the CC genotype.

[0015] The base variation type and phenotypic value of the SNP molecular marker Marker287880 are shown in Table 7: Phenotypic effect of base variation at SNP site in wheat grain HI. 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 grain HI of individuals with the SNP molecular marker TT genotype is lower than that of individuals with the CC genotype.

[0016] A molecular marker for an SNP, Marker287947, associated with the control of HI in wheat grains, is located on chromosome 5A of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.3 or SEQ ID NO.4.

[0017] The nucleotide sequence of this molecular marker is shown in Table 8: Seed HI SNP site sequence information.

[0018] The physical location of the SNP molecular marker Marker287947 is 416481113 on chromosome 5A, and the base is a T / C variant.

[0019] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker287947 is shown in SEQ ID NO.3, that is, wheat grains with a base type of T at this SNP site have a high HI.

[0020] The base variation type and phenotypic value of the SNP molecular marker Marker287947 are shown in Table 7: Phenotypic effect of SNP site base variation in grain HI, that is, wheat grain HI is high when the base type of the sequence at this position is T.

[0021] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker287947 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 nucleotide sequence of the SNP molecular marker Marker287947 is shown in SEQ ID NO.4, that is, the base type of the SNP site is C, and the genotype of the SNP molecular marker is CC; the wheat grain HI of individuals with the SNP molecular marker CC genotype is lower than that of individuals with the TT genotype.

[0022] The base variation type and phenotypic value of the SNP molecular marker Marker287947 are shown in Table 7: Phenotypic effect of base variation at SNP site in wheat grain HI. 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 grain HI of individuals with the SNP molecular marker CC genotype is lower than that of individuals with the TT genotype.

[0023] A molecular marker for an SNP, Marker287946, associated with the control of HI in wheat grains, is located on chromosome 5A of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.5 or SEQ ID NO.6.

[0024] The nucleotide sequence of this molecular marker is shown in Table 8: Seed HI SNP site sequence information.

[0025] The physical location of the SNP molecular marker Marker287946 is 416480728 on chromosome 5A, and the base is a C / T variation.

[0026] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker287946 is shown in SEQ ID NO.5, that is, wheat grains with a base type of C at this SNP site have a high HI.

[0027] The base variation type and phenotypic value of the SNP molecular marker Marker287946 are shown in Table 7: Phenotypic effect of SNP site base variation in grain HI, that is, wheat grains with a C base type at this position have a high HI.

[0028] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker287946 is shown in SEQ ID NO.5, 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 Marker287946 is shown in SEQ ID NO.6, that is, the base type of the SNP site is T, and the genotype of the SNP molecular marker is TT; the wheat grain HI of individuals with the SNP molecular marker TT genotype is lower than that of individuals with the CC genotype.

[0029] The base variation type and phenotypic value of the SNP molecular marker Marker287946 are shown in Table 7: Phenotypic effect of base variation at SNP site in wheat grain HI. 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 grain HI of individuals with the SNP molecular marker TT genotype is lower than that of individuals with the CC genotype.

[0030] A molecular marker for an SNP, Marker288023, associated with the control of HI in wheat grains, is located on chromosome 5A of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.7 or SEQ ID NO.8.

[0031] The nucleotide sequence of this molecular marker is shown in Table 8: Seed HI SNP site sequence information.

[0032] The physical location of the SNP molecular marker Marker288023 is 417907856 on chromosome 5A, and the base is a C / T variation.

[0033] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker288023 is shown in SEQ ID NO.8, that is, wheat grains with a base type of T at this SNP site have a high HI.

[0034] The base variation type and phenotypic value of the SNP molecular marker Marker288023 are shown in Table 7: Phenotypic effect of SNP site base variation in grain HI, that is, wheat grains with a base type of T at this position have a high HI.

[0035] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker288023 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 Marker288023 is shown in SEQ ID NO.7, that is, the base type of the SNP site is C, and the genotype of the SNP molecular marker is CC; the wheat grain HI of individuals with the SNP molecular marker TT genotype is higher than that of individuals with the CC genotype.

[0036] The base variation type and phenotypic value of the SNP molecular marker Marker288023 are shown in Table 7: Phenotypic effect of base variation at SNP site in wheat grain HI. 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 grain HI of individuals with the SNP molecular marker TT genotype is higher than that of individuals with the CC genotype.

[0037] A molecular marker for an SNP, Marker287945, associated with the control of HI in wheat grains, is located on chromosome 5A of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.9 or SEQ ID NO.10.

[0038] The nucleotide sequence of this molecular marker is shown in Table 8: Seed HI SNP site sequence information.

[0039] The physical location of the SNP molecular marker Marker287945 is 416449401 on chromosome 5A, and the base is an A / G variation.

[0040] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker287945 is shown in SEQ ID NO.10, that is, wheat grains with a high HI level have a base type of G at this SNP site.

[0041] The base variation type and phenotypic value of the SNP molecular marker Marker287945 are shown in Table 7: Phenotypic effect of SNP site base variation in grain HI, that is, wheat grain HI with a base type of G at this position is high.

[0042] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker287945 is shown in SEQ ID NO.10, 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 Marker287945 is shown in SEQ ID NO.9, that is, the base type of the SNP site is A, and the genotype of the SNP molecular marker is AA; the wheat grain HI of individuals with the SNP molecular marker GG genotype is higher than that of individuals with the AA genotype.

[0043] The base variation type and phenotypic value of the SNP molecular marker Marker287945 are shown in Table 7: Phenotypic effect of base variation at SNP site in wheat grain HI. 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 grain HI of individuals with the SNP molecular marker GG genotype is higher than that of individuals with the AA genotype.

[0044] A molecular marker for an SNP, Marker288063, associated with the control of HI in wheat grains, is located on chromosome 5A of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.11 or SEQ ID NO.12.

[0045] The nucleotide sequence of this molecular marker is shown in Table 8: Seed HI SNP site sequence information.

[0046] The physical location of the SNP molecular marker Marker288063 is 418428459 on chromosome 5A, and the base is an A / C variation.

[0047] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker288063 is shown in SEQ ID NO.11, that is, wheat grains with a base type of A at this SNP site have a high HI.

[0048] The base variation type and phenotypic value of the SNP molecular marker Marker288063 are shown in Table 7: Phenotypic effect of SNP site base variation in grain HI, that is, wheat grains with a base type of A at this position have high HI.

[0049] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker288063 is shown in SEQ ID NO.11, 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 Marker288063 is shown in SEQ ID NO.12, that is, the base type of the SNP site is C, and the genotype of the SNP molecular marker is CC; the wheat grain HI of individuals with the SNP molecular marker CC genotype is lower than that of individuals with the AA genotype.

[0050] The base variation type and phenotypic value of the SNP molecular marker Marker288063 are shown in Table 7: Phenotypic effect of base variation at SNP site in wheat grain HI. 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 grain HI of individuals with the SNP molecular marker CC genotype is lower than that of individuals with the AA genotype.

[0051] A molecular marker for an SNP, Marker288351, associated with the control of HI in wheat grains, is located on chromosome 5A of wheat. The nucleotide sequence of this molecular marker is shown in SEQ ID NO.13 or SEQ ID NO.14.

[0052] The nucleotide sequence of this molecular marker is shown in Table 8: Seed HI SNP site sequence information.

[0053] The physical location of the SNP molecular marker Marker288351 is 422827157 on chromosome 5A, and the base is a G / A variant.

[0054] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker288351 is shown in SEQ ID NO.13, that is, wheat grains with a high HI level have a base type of G at this SNP site.

[0055] The base variation type and phenotypic value of the SNP molecular marker Marker288351 are shown in Table 7: Phenotypic effect of SNP site base variation in grain HI, that is, wheat grain HI with a base type of G at this position is high.

[0056] According to a preferred embodiment of the present invention, the nucleotide sequence of the SNP molecular marker Marker288351 is shown in SEQ ID NO.13, 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 Marker288351 is shown in SEQ ID NO.14, that is, the base type of the SNP site is A, and the genotype of the SNP molecular marker is AA; the wheat grain HI of individuals with the SNP molecular marker AA genotype is lower than that of individuals with the GG genotype.

[0057] The base variation type and phenotypic value of the SNP molecular marker Marker288351 are shown in Table 7: Phenotypic effect of base variation at SNP site in wheat grain HI. That is, after the base variation occurs, the genotype of the sample where the SNP molecular marker is located is AA, and the wheat grain HI of individuals with the SNP molecular marker AA genotype is lower than that of individuals with the GG genotype.

[0058] The use of SNP molecular markers in the breeding of wheat varieties or lines with high HI in wheat grains, wherein the SNP molecular markers include one or more of the above-mentioned SNP molecular markers Marker287880, Marker287947, Marker287946, Marker288023, Marker287945, Marker288063, and Marker288351.

[0059] According to a preferred embodiment of the present invention, the intended use involves selecting wheat species in which one or more of the following SNP sites—Marker287880, Marker287947, Marker287946, Marker288023, Marker287945, Marker288063, and Marker288351—are superior bases.

[0060] The dominant bases at each SNP site: C at SNP site Marker 287880, T at SNP site Marker 287947, C at SNP site Marker 287946, T at SNP site Marker 288023, G at SNP site Marker 287945, A at SNP site Marker 288063, and G at SNP site Marker 288351.

[0061] According to a preferred embodiment of the present invention, in the stated use, wheat varieties selected are those with one or more of the following genotypes: Marker287880 (CC type), Marker287947 (TT type), Marker287946 (CC type), Marker288023 (TT type), Marker287945 (GG type), Marker288063 (AA type), and Marker288351 (GG type).

[0062] A method for screening wheat grains with high HI levels involves using one or more of the above-mentioned SNP molecular markers, namely Marker287880, Marker287947, Marker287946, Marker288023, Marker287945, Marker288063, and Marker288351.

[0063] According to a preferred embodiment of the present invention, in the method, wheat with one or more of the following SNP sites (Marker287880, Marker287947, Marker287946, Marker288023, Marker287945, Marker288063, Marker288351) as superior bases is selected.

[0064] The dominant bases at each SNP site: C at SNP site Marker 287880, T at SNP site Marker 287947, C at SNP site Marker 287946, T at SNP site Marker 288023, G at SNP site Marker 287945, A at SNP site Marker 288063, and G at SNP site Marker 288351.

[0065] According to a preferred embodiment of the present invention, in the method, wheat samples are screened for one or more of the following genotypes: Marker287880 (CC type), Marker287947 (TT type), Marker287946 (CC type), Marker288023 (TT type), Marker287945 (GG type), Marker288063 (AA type), and Marker288351 (GG type).

[0066] The SNP molecular marker Marker 287880 is located on chromosome 5A of wheat. The nucleotide sequences 300 bp before and after this molecular marker are shown in Table 8: Sequence Information of SNP Sites for the HI Trait in Grains. The base variation types of the SNP molecular marker Marker 287880 are shown in Table 7: Phenotypic Effects of Base Variations at SNP Sites for the HI Trait in Wheat Grains. That is, wheat grains with a C-type base of the SNP molecular marker at this location have high HI, and the genotype of this type of wheat sample is CC. The wheat grain HI of individuals with the CC genotype of the SNP molecular marker is higher than that of individuals with the TT genotype.

[0067] The SNP molecular marker Marker 287947 is located on chromosome 5A of wheat. The nucleotide sequences 300 bp before and after this marker are shown in Table 8: SNP site sequence information for traits. The base variation types of SNP molecular marker Marker 287947 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the HI trait in wheat grains. That is, wheat grains with a T-type base at this SNP molecular marker have high HI, and the genotype of this type of wheat sample is TT. The wheat grain HI of individuals with the TT genotype of the SNP molecular marker is higher than that of individuals with the CC genotype.

[0068] The SNP molecular marker Marker 287946 is located on chromosome 5A of wheat. The nucleotide sequences 300 bp before and after this molecular marker are shown in Table 8: SNP site sequence information for traits. The base variation types of SNP molecular marker Marker 287946 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the HI trait in wheat grains. That is, wheat grains with a C-type base at this SNP molecular marker have high HI, and the genotype of this type of wheat sample is CC. The wheat grain HI of individuals with the CC genotype of the SNP molecular marker is higher than that of individuals with the TT genotype.

[0069] The SNP molecular marker Marker 288023 is located on chromosome 5A of wheat. The nucleotide sequences 300 bp before and after this molecular marker are shown in Table 8: SNP site sequence information for traits. The base variation types of the SNP molecular marker Marker 288023 are shown in Table 7: Phenotypic effects of base variation at the SNP site for the HI trait in wheat grains. That is, wheat grains with a T-type base at this SNP molecular marker have high HI, and the genotype of this type of wheat sample is TT. The wheat grain HI of individuals with the TT genotype of the SNP molecular marker is higher than that of individuals with the CC genotype.

[0070] The SNP molecular marker Marker 287945 is located on chromosome 5A of wheat. The nucleotide sequences 300 bp before and after this marker are shown in Table 8: SNP site sequence information for traits. The base variation types of SNP molecular marker Marker 287945 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the HI trait in wheat grains. That is, wheat grains with a G base at this SNP molecular marker have high HI, and the genotype of this type of wheat sample is GG. The wheat grain HI of individuals with the SNP molecular marker GG genotype is higher than that of individuals with the AA genotype.

[0071] The SNP molecular marker Marker 288063 is located on chromosome 5A of wheat. The nucleotide sequences 300 bp before and after this molecular marker are shown in Table 8: SNP site sequence information for traits. The base variation types of SNP molecular marker Marker 288063 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the HI trait in wheat grains. That is, wheat grains with the base type A of the SNP molecular marker at this location have high HI, and the genotype of this type of wheat sample is AA. The wheat grain HI of individuals with the AA genotype of the SNP molecular marker is higher than that of individuals with the CC genotype.

[0072] The SNP molecular marker Marker 288351 is located on chromosome 5A of wheat. The nucleotide sequences 300 bp before and after this molecular marker are shown in Table 8: SNP site sequence information for traits. The base variation types of the SNP molecular marker Marker 288351 are shown in Table 7: Phenotypic effects of base variation at SNP sites for the HI trait in wheat grains. That is, wheat grains with the base type G of the SNP molecular marker at this location have high HI, and the genotype of this type of wheat sample is GG. The wheat grain HI of individuals with the SNP molecular marker GG genotype is higher than that of individuals with the AA genotype.

[0073] Beneficial effects of the present invention

[0074] 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 HI in grains. Molecular markers can be developed based on them to accelerate the efficiency of wheat quality breeding. Attached Figure Description

[0075] Figure 1 This is a frequency distribution diagram of the average grain hardness content of the population under three environmental conditions.

[0076] Figure 2 This is a diagram of the genetic map results. Detailed Implementation

[0077] 1. Materials and Methods

[0078] 1.1 Test Materials

[0079] 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 different grain hardness and Nongda Nuomai No. 1 (female parent) through single-grain transmission.

[0080] The two parents showed significant differences in grain and flour quality traits:

[0081] Nongda Nuomai No. 1 is a fully glutinous wheat variety bred by crossing "Jiangsu Baihuomai" and "Guandong 107", with a relatively low grain HI value.

[0082] Gaocheng 8901 is a high-quality, strong-gluten wheat variety bred from "77546-2" and "Linzhang", with a relatively high grain HI value.

[0083] The RIL population was 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.

[0084] 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.

[0085] 1.2 Test Methods

[0086] (1) Grain hardness: Grain hardness was measured using a JYDB100X40 wheat hardness index tester, with three replicates.

[0087] (2) SLAF library construction and SNP marker detection

[0088] 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])

[0089] http: / / rapdb.dna.affrc.go.jp

[0090] 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.

[0091] Table 1 SNP genotype coding rules

[0092]

[0093] (3) Construction of linkage genetic maps

[0094] 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).

[0095] (4) Candidate gene prediction

[0096] QTL loci with stable and high contribution rates to grain hardness 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 to confirm possible candidate genes and functions in the National Center for Biotechnology Information (NCBI) database (http: / / www.ncbi.nlm.nih.gov) and Ensembl Plants (http: / / plant.ensembl.org / Triticum_aestivum / Tools / Blast).

[0097] 2. Results

[0098] 2.1 Phenotypic Analysis of Wheat Grains in the RIL Population

[0099] Under all three environmental conditions, Nongda Nuomai No. 1 had lower grain hardness than Gaocheng 8901. The grain hardness phenotype of the RIL population showed significant variation, with over-parental segregation observed in all cases. Under the three environmental conditions, the mean grain hardness of the population ranged from 60.79 to 62.79, with a minimum of 41.7 and a maximum of 74.4, and a coefficient of variation of 12.8% to 12.82% (see Table 2). The segregation within the population was continuous and conformed to a normal distribution. Figure 1 It exhibits typical quantitative traits.

[0100] Table 2 Phenotypic analysis of hardness index traits in wheat grains of the RIL population.

[0101]

[0102]

[0103] Note: E1: 2017-2018 Tai'an, E2: 2018-2019 Tai'an, E3: 2019-2020 Tai'an

[0104] 2.2 Atlas Information

[0105] 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%.

[0106] Table 3. Statistical Table of Marker Linkage Grouping Projects

[0107]

[0108] 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).

[0109] Table 4. Basic Map Information

[0110]

[0111]

[0112] 2.3 QTL Analysis of HI in Wheat Grains

[0113] QTL analysis of wheat grain HI (Table 5) revealed one QTL locus, QHi-5A, located on chromosome 5A, which controls wheat grain HI. This locus was detected in all three environments, with an additive effect of 1.2 and a phenotypic contribution of 11.4%, making it a stable major additive effect gene.

[0114] Table 5. QTL loci of HI traits in wheatgrain of RIL population

[0115]

[0116] As shown in Table 6, the major QTL locus QHi-5A on chromosome 5A that controls grain HI stability has seven SNP loci, namely Marker287880, Marker287947, Marker287946, Marker288023, Marker287945, Marker288063 and Marker288351, and their variation information is related to grain HI.

[0117] Table 6. QTL and SNP markers for grain HI traits of wheat in RIL population

[0118]

[0119] As shown in Table 7, Marker 287880 is located on wheat chromosome 5A, with base C being a superior variant; Marker 287947 is located on wheat chromosome 5A, with base T being a superior variant; Marker 287946 is located on wheat chromosome 5A, with base C being a superior variant; Marker 288023 is located on wheat chromosome 5A, with base T being a superior variant; Marker 287945 is located on wheat chromosome 5A, with base G being a superior variant; Marker 288063 is located on wheat chromosome 5A, with base A being a superior variant; and Marker 288351 is located on wheat chromosome 5A, with base G being a superior variant. The SNP site sequence information for each marker is shown in Table 8.

[0120] Table 7. Phenotypic effect of SNP base variation of wheat grain HI traits

[0121]

[0122] Table 8

[0123]

[0124]

[0125]

[0126] Table 9 shows that the most superior haplotype among the seven SNP markers affecting the grain HI value is CCTGAG, while the least superior haplotype is TCTCACA. The grain HI value gradually increases with the increase in the number of superior alleles at the SNP marker sites. When all seven SNP markers are simultaneously the less superior haplotype TCTCACA, the grain HI value is <50. The grain HI value gradually increases with the increase in the number of superior SNP bases. When all seven SNP markers are simultaneously superior, i.e., the haplotype is CCTGAG, the grain HI value is significantly increased, reaching as high as 72.4. Therefore, the superior haplotype combination CCTGAG results in a higher grain HI value, while the less superior haplotype combination TCTCACA results in a lower grain HI value. Furthermore, the number of superior variant sites has a cumulative effect on the grain HI value. Therefore, these seven SNP sites play a crucial role in the level of wheat grain HI.

[0127] Table 9. Haplotypes of high and low HI samples associated with grain HI sites.

[0128]

[0129]

[0130] 2.4 Prediction of candidate genes for major loci of wheat grain HI value

[0131] Gene alignment revealed nine genes (Table 10) associated with the major QTL locus Qhi5A on chromosome 5A, which controls the stability of the HI value in wheat grains. Among them, the gene TraesCS5A01G208200.1 is related to RNA polymerase II, primarily responsible for transcribing over 20,000 coding genes in the genome, producing messenger RNA, which is then translated into protein by ribosomes. The gene TraesCS5A01G205800.2 is related to the ubiquitin system, a crucial system for the selective degradation of intracellular proteins. The gene TraesCS5A01G207500.2 controls diacylglycerol acyltransferase, the final step in triacylglycerol synthesis and the only rate-limiting enzyme, which is closely related to lipid metabolism and lipid deposition in tissues. The gene TraesCS5A01G208900.2 controls aspartic acid peptiderase, a cytoplasmic protein involved in general intracellular protein metabolism. The gene TraesCS5A01G207000.1 controls the transcription initiation factor TFIID, enhancing or inhibiting transcription by binding to specific transcription factors on a core promoter element. The functions of these genes are related to protein synthesis and may have some influence on wheat grain hardness.

[0132] Table 10 Qhi 5A 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 HI in grains. Molecular markers can be developed based on them to accelerate the efficiency of wheat quality breeding.

Claims

1. The use of SNP molecular markers in the breeding of wheat varieties or lines with high grain hardness, wherein the SNP molecular marker is SNP molecular marker Marker287880; Alternatively, the SNP molecular markers, in addition to the SNP molecular marker Marker287880, may include one or more of Marker287947, Marker287946, Marker288023, Marker287945, Marker288063, and Marker288351. The SNP molecular marker Marker287880 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2. The SNP molecular marker Marker287947 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.3 or SEQ ID NO.

4. The SNP molecular marker Marker287946 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.5 or SEQ ID NO.

6. The SNP molecular marker Marker288023 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.7 or SEQ ID NO.

8. The SNP molecular marker Marker287945 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.9 or SEQ ID NO.

10. The SNP molecular marker Marker 288063 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO. 11 or SEQ ID NO.

12. The SNP molecular marker Marker288351 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.13 or SEQ ID NO.

14. Selecting wheat varieties with superior bases at the SNP site Marker287880; Alternatively, in addition to selecting wheat with superior bases at the SNP site Marker287880, wheat with one or more of the following superior bases: Marker287947, Marker287946, Marker288023, Marker287945, Marker288063, and Marker288351 may also be selected. The dominant bases at each SNP site: C at SNP site Marker 287880, T at SNP site Marker 287947, C at SNP site Marker 287946, T at SNP site Marker 288023, G at SNP site Marker 287945, A at SNP site Marker 288063, and G at SNP site Marker 288351.

2. The use as described in claim 1, characterized in that, The genotype of SNP molecular marker Marker 287880 was selected to be CC type; Alternatively, in addition to selecting wheat with the SNP molecular marker Marker287880 having the genotype CC, one or more of the following can be selected: Marker287947 having the genotype TT, Marker287946 having the genotype CC, Marker288023 having the genotype TT, Marker287945 having the genotype GG, Marker288063 having the genotype AA, and Marker288351 having the genotype GG.

3. A method for screening wheat grains with high hardness, using the SNP molecular marker Marker287880 for screening; Alternatively, in addition to using the SNP molecular marker Marker287880, one or more of Marker287947, Marker287946, Marker288023, Marker287945, Marker288063, and Marker288351 can be used for screening. The SNP molecular marker Marker287880 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.1 or SEQ ID NO.

2. The SNP molecular marker Marker287947 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.3 or SEQ ID NO.

4. The SNP molecular marker Marker287946 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.5 or SEQ ID NO.

6. The SNP molecular marker Marker288023 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.7 or SEQ ID NO.

8. The SNP molecular marker Marker287945 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.9 or SEQ ID NO.

10. The SNP molecular marker Marker 288063 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO. 11 or SEQ ID NO.

12. The SNP molecular marker Marker288351 is located on chromosome 5A of wheat, and its nucleotide sequence is shown in SEQ ID NO.13 or SEQ ID NO.

14. Screening wheat for superior bases at SNP site Marker 287880; Alternatively, in addition to screening wheat with superior bases at SNP site Marker287880, we can also screen wheat with one or more of Marker287947, Marker287946, Marker288023, Marker287945, Marker288063, and Marker288351 as superior bases. The dominant bases at each SNP site: C at SNP site Marker 287880, T at SNP site Marker 287947, C at SNP site Marker 287946, T at SNP site Marker 288023, G at SNP site Marker 287945, A at SNP site Marker 288063, and G at SNP site Marker 288351.

4. The method as described in claim 3, characterized in that, The genotype of SNP molecular marker Marker 287880 was determined to be CC. Alternatively, in addition to screening for SNP molecular marker Marker287880 with the genotype CC, wheat with the genotypes TT (Marker287947), CC (Marker287946), TT (Marker288023), GG (Marker287945), AA (Marker288063), and GG (Marker288351) can be screened for, or one or more of these genotypes.

Images