Application of wheat TaOSBP-4B gene molecular markers in identifying spike length and yield traits

Abstract

This invention discloses the application of wheat TaOSBP-4B gene molecular markers in identifying spike length and yield traits. This invention belongs to the field of biotechnology, and particularly relates to the application of wheat TaOSBP-4B gene molecular markers in identifying spike length and yield traits. The substances of this invention for detecting SNP polymorphisms or genotypes in the wheat genome, or for detecting haplotypes, can be used to identify or assist in identifying wheat yield and wheat breeding. The SNPs are as follows: SNP1 is nucleotides 726-734 of sequence 1 in the sequence listing, containing 5'-CACCACCAC-3' or deleted; SNP2 is nucleotide 1128 of sequence 1, which is G or A; SNP3 is nucleotide 10620 of sequence 1, which is A or G; and SNP4 is nucleotide 10626 of sequence 1, which is A or G.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of wheat TaOSBP-4B gene molecular markers in identifying spike length and yield traits. Background Technology

[0002] Wheat is one of the world's most important food crops, and increasing wheat yield has become a key goal in wheat breeding. Traditional phenotypic selection-based breeding methods suffer from disadvantages such as long breeding cycles and low efficiency. Marker-assisted breeding can significantly shorten the breeding cycle and improve breeding efficiency. The number of ears per acre, the number of grains per ear, and the thousand-grain weight are the three key factors determining wheat yield. Ear length is an important ear trait with high heritability and a significant positive correlation with the number of grains per ear. Therefore, identifying genes controlling ear length and developing corresponding molecular markers can provide an accurate and effective approach for high-yield wheat breeding.

[0003] Oxidized sterol-binding proteins (OSBPs) are widely found in yeast, plants, and mammals, playing a crucial role in various cellular activities and regulatory networks related to abiotic stress responses, including signal transduction and lipid metabolism. They can significantly enhance the stress resistance of Arabidopsis thaliana, potato, and soybean. However, research on the wheat TaOSBP-4B gene has not been reported. Therefore, investigating the relationship between the wheat TaOSBP-4B gene and agronomic traits, and developing practical molecular markers, could provide effective marker resources for wheat genetic improvement and marker-assisted breeding. Summary of the Invention

[0004] The main problem this invention aims to solve is how to quickly and efficiently identify wheat ear length and yield traits.

[0005] To address the aforementioned problems, this invention provides a substance for detecting SNP polymorphisms or genotypes in the wheat genome, or for detecting haplotypes.

[0006] The application of the substance provided by this invention for detecting SNP polymorphisms or genotypes in the wheat genome, or for detecting haplotypes, in any of the following situations.

[0007] (1) To identify or assist in the identification of wheat yield;

[0008] (2) Screening or breeding high-yielding wheat individual plants, lines, strains or varieties;

[0009] (4) Wheat breeding;

[0010] (5) Prepare products for identifying or assisting in the identification of wheat yield;

[0011] (6) To prepare products for screening or breeding high-yielding wheat single plants, lines, strains or varieties;

[0012] (7) Prepare products for wheat breeding.

[0013] Based on the Chinese spring genome reference sequence, the wheat TaOSBP-4B gene (genome nucleotide sequence is Sequence 1 (SEQ ID No. 1)) has four SNPs with altered amino acids, located at positions 726-734 (containing nucleotide sequence 5'-CACCACCAC-3' / deleted nucleotide sequence 5'-CACCACCAC-3'), position 1128 (G / A), position 10620 (A / G), and position 10626 (A / G), respectively. Their corresponding physical locations in the Chinese spring reference genome sequence RefSeq v1.0 are 28950085-28950093, 28950487, 28959979, and 28959985. The molecular markers corresponding to these four SNP sites are named SNP-726 (SNP1), SNP-1128 (SNP2), SNP-10620 (SNP3), and SNP-10626 (SNP4), respectively. In sequence 1, r represents either a or g.

[0014] The SNPs mentioned are the four SNPs in the wheat genome named SNP1, SNP2, SNP3, and SNP4. SNP1 is nucleotide 726-734 of SEQ ID No.1 in the sequence listing, and its nucleotide type is either containing 5'-CACCACCAC-3' or lacking 5'-CACCACCAC-3'. SNP2 is nucleotide 1128 of SEQ ID No.1 in the sequence listing, and its nucleotide type is G or A. SNP3 is nucleotide 10620 of SEQ ID No.1 in the sequence listing, and its nucleotide type is A or G. SNP4 is nucleotide 10626 of SEQ ID No.1 in the sequence listing, and its nucleotide type is A or G.

[0015] The haplotype is a polymorphic combination of the four SNPs, namely SNP1, SNP2, SNP3, and SNP4, on a single chromosome of wheat.

[0016] This invention also provides a method for identifying or assisting in the identification of wheat yield, wherein the method may be method A or method B:

[0017] Method A is a method for identifying or assisting in identifying wheat yield, including detecting the genotype of the SNP mentioned above in the wheat to be tested, and identifying or assisting in identifying wheat yield based on the genotype of the wheat to be tested: the yield of wheat with the genotype AAAAGGGG is higher than or can be higher than that of wheat with the genotype AAGGAAAA and wheat with the genotype BBGGAAAA.

[0018] The AAAAGGGG is a combination of four SNP genotypes: SNP1 has a genotype of AA (homozygous for nucleotides 726-734 of SEQ ID No. 1 in the sequence listing, which is a deletion of 5'-CACCACCAC-3'), SNP2 has a genotype of AA (homozygous for nucleotide 1128 of SEQ ID No. 1 in the sequence listing, which is a deletion of A), SNP3 has a genotype of GG (homozygous for nucleotide 10620 of SEQ ID No. 1 in the sequence listing, which is a deletion of G), and SNP4 has a genotype of GG (homozygous for nucleotide 10626 of SEQ ID No. 1 in the sequence listing, which is a deletion of G).

[0019] The AAGGAAAA is a combination of four SNP genotypes: SNP1 has a genotype of AA (homozygous for nucleotides 726-734 of SEQ ID No. 1 in the sequence listing, which is a deletion of 5'-CACCACCAC-3'), SNP2 has a genotype of GG (homozygous for nucleotide 1128 of SEQ ID No. 1 in the sequence listing, which is a deletion of G), SNP3 has a genotype of AA (homozygous for nucleotide 10620 of SEQ ID No. 1 in the sequence listing, which is a deletion of A), and SNP4 has a genotype of AA (homozygous for nucleotide 10626 of SEQ ID No. 1 in the sequence listing, which is a deletion of A).

[0020] The BBGGAAAA genotype is a combination of four SNPs: SNP1 with genotype BB (homozygous for 5'-CACCACCAC-3' at nucleotides 726-734 of SEQ ID No. 1 in the sequence listing), SNP2 with genotype GG (homozygous for G at nucleotide 1128 of SEQ ID No. 1 in the sequence listing), SNP3 with genotype AA (homozygous for A at nucleotide 10620 of SEQ ID No. 1 in the sequence listing), and SNP4 with genotype AA (homozygous for A at nucleotide 10626 of SEQ ID No. 1 in the sequence listing).

[0021] Method B is a method for identifying or assisting in the identification of wheat yield, including detecting the haplotypes mentioned above in the wheat to be tested, and identifying or assisting in the identification of wheat yield based on the haplotypes of the wheat to be tested: the yield of homozygous genotype wheat corresponding to haplotype Hap1 is higher than or candidate higher than the yield of homozygous genotype wheat corresponding to haplotype Hap2 and the yield of homozygous genotype wheat corresponding to haplotype Hap3; the haplotype Hap1 is defined as SNP1 with a deletion of 5'-CACCACCAC-3' and the base of SNP2 is A and the S... The haplotype Hap2 is a haplotype in which SNP1 is missing 5'-CACCACCAC-3', SNP2 is a G, SNP3 is an A, and SNP4 is an A. The haplotype Hap3 is a haplotype in which SNP1 contains 5'-CACCACCAC-3', SNP2 is a G, SNP3 is an A, and SNP4 is an A.

[0022] In the above applications or methods, the wheat is a wheat inbred line.

[0023] This invention also provides the application of the above-mentioned identification method in wheat breeding.

[0024] The present invention also provides a method for wheat breeding, wherein the method comprises M1 and M2:

[0025] M1. The method includes detecting the genotype of the SNPs mentioned above in the wheat genome, selecting wheat with the genotype of the SNP AAAAGGGG as a parent for breeding, wherein the AAAAGGGG genotype is a homozygous type of SNP1 with a deletion of 5'-CACCACCAC-3', and SNP2 is a homozygous type of A, and SNP3 is a homozygous type of G, and SNP4 is a homozygous type of G, and the breeding objective of the method includes selecting high-yielding wheat;

[0026] M2. The method includes detecting the type of haplotype described above in the wheat genome, selecting wheat with haplotype Hap1 as the parent for breeding, wherein haplotype Hap1 is a haplotype in which SNP1 is missing 5'-CACCACCAC-3', SNP2 is A, SNP3 is G, and SNP4 is G.

[0027] The present invention also provides a product for detecting the polymorphism or genotype of the SNPs described above, the product containing the substances described above, and the product may be any one of them:

[0028] C1) Products that detect single nucleotide polymorphisms or genotypes related to wheat yield;

[0029] C2) Products used to identify or assist in identifying wheat yield;

[0030] C3) Products used in wheat breeding;

[0031] C4) Screening or breeding of high-yielding wheat single plants, lines, strains, or varieties.

[0032] In the above applications or products, the substance may be D1), D2), or D3):

[0033] D1) The substance is a primer composition for amplifying wheat genomic DNA fragments including SNP1, a primer composition for amplifying wheat genomic DNA fragments including SNP2, a primer composition for amplifying wheat genomic DNA fragments including SNP3, and a primer composition for amplifying wheat genomic DNA fragments including SNP4.

[0034] D2) The substance described is a PCR reagent containing the primer composition described in D1);

[0035] D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0036] In the above applications or products, 1) the primer composition for amplifying wheat genomic DNA fragments including SNP1 consists of primer F1-A and primer F1-B; 2) the primer composition for amplifying wheat genomic DNA fragments including SNP2 consists of primer F2-A and primer F2-B; 3) the primer composition for amplifying wheat genomic DNA fragments including SNP3 and SNP4 consists of primer F3-A and primer F3-B.

[0037] The primer F1-A is a single-stranded DNA molecule whose nucleotide sequence is sequence 22 in the sequence listing;

[0038] The primer F1-B is a single-stranded DNA molecule whose nucleotide sequence is sequence 23 in the sequence listing;

[0039] The primer F2-A is a single-stranded DNA molecule whose nucleotide sequence is sequence 4 in the sequence listing;

[0040] The primer F2-B is a single-stranded DNA molecule whose nucleotide sequence is sequence 5 in the sequence listing;

[0041] The primer F3-A is a single-stranded DNA molecule whose nucleotide sequence is sequence 20 in the sequence listing;

[0042] The primer F3-B is a single-stranded DNA molecule whose nucleotide sequence is sequence 21 in the sequence listing.

[0043] PCR amplification was performed using primer sets of molecular markers SNP1, SNP2, SNP3, and SNP4, yielding amplification products A, B, C, and D in sequence.

[0044] The PCR amplification reaction system is 25 μL, and the components include: 2 μL DNA, 8 μL ddHap-2O, 1 μL each of upper and lower primers, and 13 μL 2×Taq Master Mix.

[0045] The PCR amplification reaction conditions were as follows: 95℃ for 5 min, 95℃ for 30 s, 61℃ for 30 s, 72℃ for 15 s, 36 cycles, 72℃ for 10 min, and storage at 4℃.

[0046] The PCR results were determined as follows:

[0047] 1) If the PCR product A is 310 bp, the enzyme digestion products E are 663 bp and 134 bp, the enzyme digestion products F are 406 bp and 437 bp, and the enzyme digestion products G are 412 bp, 285 bp, and 146 bp, then the genotype of the sample to be tested is Hap-1. The specific genotype of Hap-1 is AAAAGGGG, wherein the genotype of SNP1 is AA (the 726th-734th nucleotides of SEQ ID No. 1 in the sequence listing are homozygous for the deletion of 5'-CACCACCAC-3'), the genotype of SNP2 is AA (the 1128th nucleotide of SEQ ID No. 1 in the sequence listing is homozygous for A), the genotype of SNP3 is GG (the 10620th nucleotide of SEQ ID No. 1 in the sequence listing is homozygous for G), and the genotype of SNP4 is GG (the 10626th nucleotide of SEQ ID No. 1 in the sequence listing is homozygous for G).

[0048] 2) If the PCR product A is 310 bp, the enzyme digestion product E is 797 bp, the enzyme digestion product F is 843 bp, and the enzyme digestion products G are 558 bp and 285 bp, then the genotype of the sample to be tested is Hap-2. The specific genotype of Hap-2 is AAGGAAAA, which is a combination of four SNPs: SNP1 is AA (homozygous with nucleotides 726-734 of SEQ ID No. 1 missing 5'-CACCACCAC-3'), SNP2 is GG (homozygous with nucleotide 1128 of SEQ ID No. 1), SNP3 is AA (homozygous with nucleotide 10620 of SEQ ID No. 1), and SNP4 is AA (homozygous with nucleotide 10626 of SEQ ID No. 1).

[0049] 3) If the PCR product A is 319 bp, the enzyme digestion product E is 797 bp, the enzyme digestion product F is 843 bp, and the enzyme digestion products G are 558 bp and 285 bp, then the genotype of the sample to be tested is Hap-3. The specific genotypes of Hap-3 are 5'-CACCACCAC-3', G, A, and A, where the genotype of SNP1 is BB (nucleotides 726-734 of SEQ ID No. 1 in the sequence listing are homozygous for 5'-CACCACCAC-3'), the genotype of SNP2 is G (nucleotide 1128 of SEQ ID No. 1 in the sequence listing is homozygous for G), the genotype of SNP3 is A (nucleotide 10620 of SEQ ID No. 1 in the sequence listing is homozygous for A), and the genotype of SNP4 is A (nucleotide 10626 of SEQ ID No. 1 in the sequence listing is homozygous for A), which is a combination of four SNP genotypes.

[0050] The present invention also provides a DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing.

[0051] The present invention also provides the use of the described DNA molecule in any of the following:

[0052] (1) To identify or assist in the identification of wheat yield;

[0053] (2) Screening or breeding high-yielding wheat individual plants, lines, strains or varieties;

[0054] (3) Wheat breeding;

[0055] (4) Prepare products for identifying or assisting in the identification of wheat yield;

[0056] (5) To prepare products for screening or breeding high-yielding wheat single plants, lines, strains or varieties;

[0057] (6) Prepare products for wheat breeding.

[0058] In this invention, the yield traits may specifically be spike length, number of spikelets, number of grains per spike, and thousand-grain weight.

[0059] This invention, through cloning the wheat TaOSBP-4B gene, discovered four SNPs with amino acid alterations, located at positions 726-734, 1128, 10620, and 10626 of sequence 1, respectively. These four SNPs have three haplotypes: haplotype Hap-1 (deleted 5'-CACCACCAC-3', A, G, G), haplotype Hap-2 (deleted 5'-CACCACCAC-3', G, A, A), and haplotype Hap-3 (5'-CACCACCAC-3', G, A, A). Association analysis and natural population validation confirmed that among the homozygous haplotypes of these three haplotypes, Hap-1 exhibited significantly higher spike length, spikelet number, grain number per spike, and thousand-grain weight than Hap-3. Hap-1 also showed significantly higher spike length than Hap-2. There were no significant differences in spikelet number, grain number per spike, and thousand-grain weight between Hap-1 and Hap-2, nor between Hap-2 and Hap-3. Experiments demonstrated that the wheat TaOSBP-4B gene molecular markers SNP-726 (SNP1), SNP-1128 (SNP2), SNP-10620 (SNP3), and SNP-10626 (SNP4) provided in this invention can rapidly screen for wheat spike length and yield-related traits, demonstrating significant application value in marker-assisted breeding and important implications for the genetic improvement of wheat varieties. Attached Figure Description

[0060] Figure 1 This is a schematic diagram of SNP sites in the TaOSBP-4B genome sequence. (a) is a schematic diagram of the TaOSBP-4B gene structure; (b) is a schematic diagram of the SNPs and their sites that cause amino acid changes in the TaOSBP-4B gene.

[0061] Figure 2 The images shown are molecular marker electrophoresis diagrams from Example 1. (a) is a polyacrylamide gel electrophoresis diagram of molecular marker SNP1; (b) is an agarose gel electrophoresis diagram of molecular marker SNP2; (c) is an agarose gel electrophoresis diagram of molecular marker SNP3; and (d) is an agarose gel electrophoresis diagram of molecular marker SNP4. The samples in lanes 1-6 are Xumai 24, Yangmai 158, Jimai 19, Jimai 23, Jimai 31, and Jimai 32, respectively.

[0062] Figure 3The statistical results of spike length and yield traits of the three haplotypes of the TaOSBP-4B gene in natural populations are as follows: (a) is the statistical results of spike length of the three haplotypes of the TaOSBP-4B gene; (b) is the statistical results of thousand-grain weight of the three haplotypes of the TaOSBP-4B gene; (c) is the statistical results of spikelet number per spike of the three haplotypes of the TaOSBP-4B gene; and (d) is the statistical results of grain number per spike of the three haplotypes of the TaOSBP-4B gene. Detailed Implementation

[0063] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0065] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0066] The wheat variety Wunong 988 in Example 1 below is an approved variety, which was approved by the National Crop Variety Approval Committee in 2021, with the variety approval number being Guoshenmai 20210153.

[0067] The wheat varieties in the natural wheat population in Example 2 below are from the National Germplasm Resource Bank. The public can obtain this biological material from the National Germplasm Resource Bank. This biological material is only used to repeat the experiments of this invention and cannot be used for other purposes.

[0068] The following examples used SPSS 26 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, P < 0.001 (***) indicates a highly significant difference, and P > 0.05 (ns) indicates no significant difference.

[0069] Example 1: Obtaining polymorphic sites (SNPs) and haplotypes of the TaOSBP-4B gene, a gene related to ear length and yield.

[0070] I. Obtaining the TaOSBP-4B gene polymorphism site

[0071] (1) Extract genomic DNA from wheat variety Wunong 988 (nationally approved wheat variety 20210153);

[0072] (2) Based on the Chinese spring genome reference sequence, specific primers for cloning TaOSBP-4B (genomic nucleotide sequence is sequence 1) were designed. The primer sequences are as follows:

[0073] SeqOSBP-4B-1F:5'-ACCCAGCCAGCAGTTCATTA-3' (Sequence 2)

[0074] SeqOSBP-4B-1R:5'-GCCAACCACCGATTTCGTTT-3' (sequence 3)

[0075] SeqOSBP-4B-2F:5'-GCATCTCGGGGGTCCCTCTA-3' (sequence 4)

[0076] SeqOSBP-4B-2R:5'-GCAAGGTCGGATAAATGTGA-3' (Sequence 5)

[0077] SeqOSBP-4B-3F:5'-CCTTGACACTCTTCCAACGC-3' (Sequence 6)

[0078] SeqOSBP-4B-3R:5'-ACCTATCCACTCCTCGTCCC-3' (Sequence 7)

[0079] SeqOSBP-4B-4F:5'-GGAGTCCTTGGTGTCCTTCC-3' (Sequence 8)

[0080] SeqOSBP-4B-4R:5'-GGAGTAGATGACACAGCGCA-3' (Sequence 9)

[0081] SeqOSBP-4B-5F:5'-GGTTATCGTTATCTCCTGCGT-3' (Sequence 10)

[0082] SeqOSBP-4B-5R:5'-CTTACTTTGTGCTAAGGCTAG-3' (Sequence 11)

[0083] SeqOSBP-4B-6F:5'-CCACTGTTCATCTCGCAAC-3' (Sequence 12)

[0084] SeqOSBP-4B-6R:5'-TATACCTGATGAGGATTGCG-3' (Sequence 13)

[0085] SeqOSBP-4B-7F:5'-AGTGAGTTTGTGTACCGTAA-3' (Sequence 14)

[0086] SeqOSBP-4B-7R:5'-ACATCACATTGAACACCCAC-3' (Sequence 15)

[0087] SeqOSBP-4B-8F:5'-AGCCAAATGTAGGACACTGC-3' (Sequence 16)

[0088] SeqOSBP-4B-8R:5'-CATTTACCAAAGAGCGTAGC-3 (Sequence 17)

[0089] SeqOSBP-4B-9F:5'-TCGAACTGTTGCTACGCTCT-3' (Sequence 18)

[0090] SeqOSBP-4B-9R:5'-TGCACAACAAGGCGTAGGTT-3' (Sequence 19)

[0091] SeqOSBP-4B-10F:5'-TGAGCATCACATTGGTGAAG-3' (Sequence 20)

[0092] SeqOSBP-4B-10R:5'-GAATAGACTTCAGGGGCAAA-3' (Sequence 21)

[0093] (3) Using the genomic DNA of wheat variety Wunong 988 as a template, PCR amplification was performed using primers SeqOSBP-4B-1F / R, SeqOSBP-4B-2F / R, SeqOSBP-4B-3F / R, SeqOSBP-4B-4F / R, SeqOSBP-4B-5F / R, SeqOSBP-4B-6F / R, SeqOSBP-4B-7F / R, SeqOSBP-4B-8F / R, SeqOSBP-4B-9F / R, and SeqOSBP-4B-10F / R. The PCR products were then sequenced and compared.

[0094] (4) The reaction system is 25 μL, and the components include: 2 μL DNA, 8 μL ddHap-2O, 1 μL each of upper and lower primers, and 13 μL 2×Taq Master Mix.

[0095] (5) The reaction conditions are: 95℃ for 5 min, 95℃ for 30 s, 61℃ for 30 s, 72℃ for 15 s, 36 cycles, 72℃ for 10 min, and stored at 4℃.

[0096] Sequence alignment revealed four amino acid-altering SNPs in the wheat TaOSBP-4B gene (genome nucleotide sequence is Sequence 1), located at positions 726-734 (containing nucleotide sequence 5'-CACCACCAC-3' / deleted nucleotide sequence 5'-CACCACCAC-3'), 1128 (G / A), 10620 (A / G), and 10626 (A / G), corresponding to physical positions 28950085-28950093, 28950487, 28959979, and 28959985 in the Chinese spring reference genome sequence RefSeq v1.0. The molecular markers corresponding to these four SNP sites are named SNP-726 (SNP1), SNP-1128 (SNP2), SNP-10620 (SNP3), and SNP-10626 (SNP4), respectively. In Sequence 1, r represents a or g.

[0097] II. Obtaining the TaOSBP-4B gene molecular marker and its application in haplotype identification

[0098] 1. Obtaining the TaOSBP-4B gene molecular marker

[0099] (1) Based on the SNP sites at positions 726-734, 1128, 10620, and 10626 of the TaOSBP-4B gene as described in the sequence alignment results, molecular markers SNP1, SNP2, SNP3, and SNP4 were designed, respectively. The marker primer sequences are as follows:

[0100] SNP1-F (abbreviated as F1-A): 5'-GGCGTGCTCTCCTACTACAA-3' (sequence 22)

[0101] SNP1-R (abbreviated as F1-B): 5'-AAGGTCATCGGCGAGGACTC-3' (sequence 23)

[0102] SeqOSBP-4B-2F (abbreviated as F2-A):5'-GCATCTCGGGGGTCCTCTA-3' (Sequence 4)

[0103] SeqOSBP-4B-2R (abbreviated as F2-B):5'-GCAAGGTCGGATAAATGTGA-3' (Sequence 5)

[0104] SeqOSBP-4B-10F (abbreviated as F3-A):5'-TGAGCATCACATTGGTGAAG-3' (Sequence 20)

[0105] SeqOSBP-4B-10R (abbreviated as F3-B):5'-GAATAGACTTCAGGGGCAAA-3' (Sequence 21)

[0106] The primer composition for amplifying wheat genomic DNA fragments including SNP1 consists of primers F1-A and F1-B; 2) the primer composition for amplifying wheat genomic DNA fragments including SNP2 consists of primers F2-A and F2-B; 3) the primer composition for amplifying wheat genomic DNA fragments including SNP3 and SNP4 consists of primers F3-A and F3-B.

[0107] (2) Using the genomic DNA of Xumai 24, Yangmai 158, Jimai 19, Jimai 23, Jimai 31 and Jimai 32 (from the National Germplasm Resource Bank) as templates, PCR amplification was performed using primers of molecular markers SNP1, SNP2, SNP3 and SNP4, and the amplification products A, B, C and D were obtained in sequence.

[0108] (3) The reaction system is 25 μL, and the components include: 2 μL DNA, 8 μL ddHap-2O, 1 μL each of upper and lower primers, and 13 μL 2×Taq Master Mix.

[0109] (4) The reaction conditions are: 95℃ for 5 min, 95℃ for 30 s, 61℃ for 30 s, 72℃ for 15 s, 36 cycles, 72℃ for 10 min, and stored at 4℃.

[0110] (5) The amplified product A was detected by polyacrylamide gel electrophoresis, and the results are as follows: Figure 2 As shown in (a); the amplified product B was digested with the restriction endonuclease BSRDI to obtain the digested product E, and the results are as follows. Figure 2 As shown in (b); the amplified product C was digested with the restriction endonuclease AluI to obtain the digested product F, and the results are as follows. Figure 2 As shown in (c); the amplified product D was digested with the restriction endonuclease MWOI to obtain the digested product G, as shown in the figure. Figure 2 As shown in (d).

[0111] The PCR results are interpreted as follows:

[0112] 1) If PCR product A is 310 bp, enzyme digestion product E is 663 bp and 134 bp, enzyme digestion product F is 406 bp and 437 bp, and enzyme digestion product G is 412 bp, 285 bp, and 146 bp, then the genotype of the sample to be tested is Hap-1. The specific genotype of Hap-1 is AAAAGGGG, which is a combination of four SNPs: SNP1 is AA (homozygous with nucleotides 726-734 of SEQ ID No. 1 missing 5'-CACCACCAC-3'), SNP2 is AA (homozygous with nucleotide 1128 of SEQ ID No. 1), SNP3 is GG (homozygous with nucleotide 10620 of SEQ ID No. 1), and SNP4 is GG (homozygous with nucleotide 10626 of SEQ ID No. 1).

[0113] 2) If PCR product A is 310 bp, enzyme digestion product E is 797 bp, enzyme digestion product F is 843 bp, and enzyme digestion products G are 558 bp and 285 bp, then the genotype of the sample to be tested is Hap-2. The specific genotype of Hap-2 is AAGGAAAA, which is a combination of four SNPs: SNP1 is AA (homozygous with nucleotides 726-734 of SEQ ID No. 1 missing 5'-CACCACCAC-3'), SNP2 is GG (homozygous with nucleotide 1128 of SEQ ID No. 1), SNP3 is AA (homozygous with nucleotide 10620 of SEQ ID No. 1), and SNP4 is AA (homozygous with nucleotide 10626 of SEQ ID No. 1).

[0114] 3) If PCR product A is 319 bp, enzyme digestion product E is 797 bp, enzyme digestion product F is 843 bp, and enzyme digestion products G are 558 bp and 285 bp, then the genotype of the sample to be tested is Hap-3. The specific genotypes of Hap-3 are 5'-CACCACCAC-3', G, A, and A, where the genotype of SNP1 is BB (nucleotides 726-734 of SEQ ID No. 1 in the sequence listing are homozygous for 5'-CACCACCAC-3'), the genotype of SNP2 is G (nucleotide 1128 of SEQ ID No. 1 in the sequence listing is homozygous for G), the genotype of SNP3 is A (nucleotide 10620 of SEQ ID No. 1 in the sequence listing is homozygous for A), and the genotype of SNP4 is A (nucleotide 10626 of SEQ ID No. 1 in the sequence listing is homozygous for A).

[0115] 2. Application of TaOSBP-4B gene molecular marker in haplotype identification

[0116] The genotype of the sample to be tested is determined based on the PCR product / enzyme digestion product in step 1, which is Hap-1, Hap-2, or Hap-3. The specific steps are as follows:

[0117] (1) Extract genomic DNA from wheat grains of the wheat samples to be tested;

[0118] (2) Using the genomic DNA of wheat grains to be tested as a template, PCR amplification was performed using primer pairs of molecular markers SNP1, SNP2, SNP3 and SNP4, and amplification products A, B, C and D were obtained in sequence.

[0119] (3) Amplified product A was detected by polyacrylamide gel electrophoresis; Amplified product B was digested with restriction endonuclease BSRDI to obtain digested product E; Amplified product C was digested with restriction endonuclease AluI to obtain digested product F; Amplified product D was digested with restriction endonuclease MWOI to obtain digested product G.

[0120] The genotype results of the samples to be tested were determined as follows:

[0121] 1) If PCR product A is 310 bp, enzyme digestion products E are 663 bp and 134 bp, enzyme digestion products F are 406 bp and 437 bp, and enzyme digestion products G are 412 bp, 285 bp, and 146 bp, then the genotype of the sample to be tested is Hap-1. The specific genotype of Hap-1 is AAAAGGGG, which is a combination of four SNPs: SNP1 is AA (homozygous with nucleotides 726-734 of SEQ ID No. 1 missing 5'-CACCACCAC-3'), SNP2 is AA (homozygous with nucleotide 1128 of SEQ ID No. 1), SNP3 is GG (homozygous with nucleotide 10620 of SEQ ID No. 1), and SNP4 is GG (homozygous with nucleotide 10626 of SEQ ID No. 1).

[0122] 2) If PCR product A is 310 bp, enzyme digestion product E is 797 bp, enzyme digestion product F is 843 bp, and enzyme digestion products G are 558 bp and 285 bp, then the genotype of the sample to be tested is Hap-2, and the specific genotype of Hap-2 is AAGGAAAA. This refers to the genotype combination of four SNPs: SNP1 (genotype AA, homozygous for nucleotides 726-734 of SEQ ID No. 1 in the sequence listing lacking 5'-CACCACCAC-3'), SNP2 (genotype GG, homozygous for nucleotide 1128 of SEQ ID No. 1 in the sequence listing), SNP3 (genotype AA, homozygous for nucleotide 10620 of SEQ ID No. 1 in the sequence listing), and SNP4 (genotype AA, homozygous for nucleotide 10626 of SEQ ID No. 1 in the sequence listing).

[0123] 3) If PCR product A is 319 bp, enzyme digestion product E is 797 bp, enzyme digestion product F is 843 bp, and enzyme digestion products G are 558 bp and 285 bp, then the genotype of the sample to be tested is Hap-3. The specific genotype of Hap-3 is BBGGAAAA, which is a combination of four SNPs: SNP1 is BB (nucleotides 726-734 of SEQ ID No. 1 in the sequence listing are homozygous for 5'-CACCACCAC-3'), SNP2 is GG (nucleotide 1128 of SEQ ID No. 1 in the sequence listing is homozygous for G), SNP3 is AA (nucleotide 10620 of SEQ ID No. 1 in the sequence listing is homozygous for A), and SNP4 is AA (nucleotide 10626 of SEQ ID No. 1 in the sequence listing is homozygous for A).

[0124] Example 2: U-test of haplotypes and wheat spike length and yield traits

[0125] Genotyping of the natural population (Table 1) was performed using the four molecular markers SNP-726 (SNP1), SNP-1128 (SNP2), SNP-10620 (SNP3), and SNP-10626 (SNP4) from Example 1. U-tests were then conducted on the genotypes and traits of spikelet length, spikelet number, number of grains per spike, and thousand-grain weight. The specific steps are as follows:

[0126] 1. Genotyping

[0127] A natural population of 333 local and bred wheat varieties (from the National Germplasm Bank) was used as the test samples. Genotyping was performed according to the method established in Example 1 to determine whether each test sample had the genotype of Hap-1, Hap-2, or Hap-3. The test results are shown in Table 1.

[0128] Table 1. Statistical results of haplotypes and wheat traits in natural populations

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] II. U-test of haplotypes and wheat spike length and yield traits

[0140] In 2022-2023, natural populations were planted at the teaching and experimental base of Anhui Agricultural University (Wupu Farm, Suixi County, Huaibei). During this period, spike length, spikelet number, grain number per spike, and thousand-grain weight of each wheat variety were investigated. The U-test was used to perform association analysis between the three haplotypes Hap-1, Hap-2, and Hap-3 described in Example 1 and spike length and yield traits. The statistical results of the three haplotypes and spike length and yield traits in the natural population are shown in Table 1 and... Figure 3 As shown.

[0141] The spike length, number of spikelets, number of grains per spike, and thousand-grain weight of Hap-1 were significantly higher than those of Hap-3. The spike length of Hap-1 was significantly higher than that of Hap-2. There were no significant differences in the number of spikelets, number of grains per spike, and thousand-grain weight between Hap-1 and Hap-2, and there were no significant differences in these parameters between Hap-2 and Hap-3. This indicates that the wheat TaOSBP-4B gene molecular markers SNP-726 (SNP1), SNP-1128 (SNP2), SNP-10620 (SNP3), and SNP-10626 (SNP4) in this invention can effectively distinguish the superior haplotype Hap-1 and play a role in breeding large-spike, high-yielding wheat varieties.

[0142] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Use of a reagent for detecting genotypes of a SNP combination or a reagent for detecting a haplotype in any one of the following, (1) identifying or assisting in identifying wheat yield; (2) screening or selecting a wheat single plant or strain or line or variety with high yield; (3) breeding wheat with high yield; (4) preparing a product for identifying or assisting in identifying wheat yield; (5) preparing a product for screening or selecting a wheat single plant or strain or line or variety with high yield; (6) preparing a product for breeding wheat with high yield; the SNP combination consists of four SNPs, SNP1, SNP2, SNP3 and SNP4, the SNP1 is nucleotide 726-734 of SEQ ID No. 1 in the sequence listing, the nucleotide species of which is containing 5'-CACCACCAC-3' or deleting 5'-CACCACCAC-3', the SNP2 is nucleotide 1128 of SEQ ID No. 1 in the sequence listing, the nucleotide species of which is G or A, the SNP3 is nucleotide 10620 of SEQ ID No. 1 in the sequence listing, the nucleotide species of which is A or G, and the SNP4 is nucleotide 10626 of SEQ ID No. 1 in the sequence listing, the nucleotide species of which is A or G; the haplotype is a combination of the four SNPs, SNP1, SNP2, SNP3 and SNP4 on a chromosome of wheat.

2. A method for identifying or assisting in identifying wheat yield, the method is method A or method B, the method A is a method for identifying or assisting in identifying wheat yield, comprising detecting genotypes of a SNP combination in the genome of a wheat to be tested as described in claim 1, identifying or assisting in identifying wheat yield according to the genotype of the wheat to be tested: the genotype of AAAAGGGG wheat has higher yield than the genotype of AAGGAAAA wheat and the genotype of BBGGAAAA wheat; wherein the genotype of AAAAGGGG is that the SNP1 is homozygous for deleting 5'-CACCACCAC-3', and the SNP2 is homozygous for A, and the SNP3 is homozygous for G, and the SNP4 is homozygous for G; the genotype of AAGGAAAA is that the SNP1 is homozygous for deleting 5'-CACCACCAC-3', and the SNP2 is homozygous for G, and the SNP3 is homozygous for A, and the SNP4 is homozygous for A; the genotype of BBGGAAAA is that the SNP1 is homozygous for containing 5'-CACCACCAC-3', and the SNP2 is homozygous for G, and the SNP3 is homozygous for A, and the SNP4 is homozygous for A. The method B is a method for identifying or assisting in identifying the yield of wheat, comprising detecting the haplotype as described in claim 1 in the genome of the wheat to be tested, and identifying or assisting in identifying the yield of wheat according to the haplotype of the wheat to be tested: the homozygous genotype of the wheat corresponding to the haplotype Hap1 has a higher yield than the homozygous genotype of the wheat corresponding to the haplotype Hap2 and the homozygous genotype of the wheat corresponding to the haplotype Hap3; the haplotype Hap1 is the haplotype that the SNP1 is a deletion of 5'-CACCACCAC-3', the base of the SNP2 is A, the base of the SNP3 is G, and the base of the SNP4 is G; the haplotype Hap2 is the haplotype that the SNP1 is a deletion of 5'-CACCACCAC-3', the base of the SNP2 is G, the base of the SNP3 is A, and the base of the SNP4 is A; and the haplotype Hap3 is the haplotype that the SNP1 contains 5'-CACCACCAC-3', the base of the SNP2 is G, the base of the SNP3 is A, and the base of the SNP4 is A.

3. The method of claim 2 is used in the breeding of high-yield wheat.

4. A method of breeding high yield wheat characterized by: The method is M1 and M2: M1, the method comprises detecting the genotype of the SNP combination as described in claim 1 in the genome of wheat, and selecting wheat with the genotype of the SNP combination AAAAGGGG as a parent for breeding, the genotype of AAAAGGGG is that the SNP1 is a homozygous deletion of 5'-CACCACCAC-3', the SNP2 is a homozygous A, the SNP3 is a homozygous G, and the SNP4 is a homozygous G, and the wheat is a wheat inbred line; M2, the method comprises detecting the type of the haplotype as described in claim 1 in the genome of wheat, and selecting wheat with the haplotype Hap1 as a parent for breeding, the haplotype Hap1 is the haplotype that the SNP1 is a deletion of 5'-CACCACCAC-3', the base of the SNP2 is A, the base of the SNP3 is G, and the base of the SNP4 is G.

5. A product for detecting the genotype of the SNP combination as described in claim 1, The product comprises a primer composition consisting of primer F1-A, primer F1-B, primer F2-A, primer F2-B, primer F3-A, and primer F3-B; The nucleotide sequence of the primer F1-A is shown in SEQ ID No. 22; The nucleotide sequence of the primer F1-B is shown in SEQ ID No. 23; The nucleotide sequence of the primer F2-A is shown in SEQ ID No. 4; The nucleotide sequence of the primer F2-B is shown in SEQ ID No. 5; The nucleotide sequence of the primer F3-A is shown in SEQ ID No. 20; The nucleotide sequence of the primer F3-B is shown in SEQ ID No. 21.

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