A molecular design breeding method for salt-tolerant wheat

Abstract

The present invention relates to the field of biotechnology, and discloses a molecular design breeding method for salt-tolerant wheat, comprising the following steps: genotyping multiple wheat varieties with multiple SNP sites for controlling grain traits and ear traits of wheat under salt stress, selecting paired wheat varieties with complementary typing results of salt-tolerant grain traits and ear traits SNP sites as candidate hybrid parents; selecting a pair of candidate hybrid parents with good salt tolerance and complementary advantages as two hybrid parents by analyzing the salt-tolerant phenotype; genotyping the SNP sites with complementary typing results of the two parents for hybrid offspring single plants, using the typing results to assist in screening salt-tolerant offspring single plants, and combining the salt-tolerant phenotype to select salt-tolerant wheat varieties. The present invention scans the genotypes of breeding materials, purposefully screens the hybridization of varieties with complementary genotypes, and through precise marker selection, enables the excellent allele variation to aggregate in the offspring materials, and cultivates a designed salt-tolerant wheat new variety (line).

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a molecular design breeding method for salt-tolerant wheat. Background Art

[0002] The area of ​​saline-alkali land in the world is nearly 1 billion hectares, accounting for about 7.6% of the land area. Efficient use of salinized soil is of great significance to improving the agricultural production capacity of cultivated land. Common wheat originated in the Fertile Crescent and is one of the main cultivated crops on salinized soil. How to cultivate salt-tolerant wheat varieties suitable for planting in salinized medium and low-yield fields is an effective way to utilize the abundant saline-alkali land resources and increase wheat production.

[0003] Wheat yield is determined by the number of spikes, the number of spikelets and the grain weight per unit area, and there is a certain degree of restriction and coordination between the three factors. This research group used the (Zhongmai 175 / Xiaoyan 60) RIL population and a natural population consisting of 191 wheat varieties (lines) as experimental materials, and carried out linkage analysis and association analysis of wheat adult stage under different salt stress levels in coastal saline-alkali soils in my country to locate salt tolerance-related genetic loci (Hu et al. 2021; Luo et al. 2021). Thousand-grain weight is the yield trait with the most significant genetic improvement in modern breeding. It is mainly controlled by additive effect genes and has a high heritability. In the salt tolerance positioning study of the RIL population, compared with the low-salt (salt content 0.18%) environment, the correlation coefficient between wheat thousand-grain weight and yield increased significantly in the high-salt (salt content 0.3%) environment, indicating that breeding varieties with high thousand-grain weight is of great value for wheat breeding on moderately saline-alkali land. Through linkage analysis, a total of 5 QTLs related to thousand-grain weight under salt stress were located on chromosomes 2A, 2D, 3D, 4B and 6A. Among them, QTkw-2D (126.5-137.5cM) was detected in all 6 salt stress environments, which can explain an average of 8.67% of the phenotypic variation rate, and QTkw-4B (26.5-34.5cM) was detected in 5 salt stress environments, which can explain the largest phenotypic variation rate (22.40%), and its LOD value is as high as 20.15. Through association analysis, a total of 31 SNP markers significantly associated with thousand-grain weight under salt stress were detected, of which 25 markers constituted QTkw-4A, located in the 14.68-16.28Mb interval on chromosome 4A, which can explain 18.60-35.57% of the phenotypic variation rate.

[0004] In addition to the three elements of yield, wheat ear traits have a direct or indirect impact on wheat yield. In wheat breeding, ear traits such as ear length and spikelet number are often selected and studied as an important indicator in wheat breeding because of their easy early observation characteristics. GWAS analysis detected that the loci significantly associated with wheat ear length under salt stress were mainly located on wheat chromosomes 1B, 3B, 5B and 7A. Among them, the loci QSl-1B (674.96-675.13Mb) and QSl-3B (62.13-62.14Mb, ​​Jia Ji Zeng, personal communication; 63.40Mb, Chinese Spring Genome IWGSC RefSeq v1.0) include 11 and 5 SNP markers, respectively, which can explain 16.38-23.12% and 12.18-12.82% of the phenotypic variation rate. The composite locus Q-5B1 (574.06-578.35Mb) and the locus QS1-5B2 (583.48-584.89Mb) were significantly associated with relative spike length, explaining 12.94-14.29% and 13.03-15.28% of the phenotypic variation, respectively. The composite locus Q-7A, located in the 22.75-22.88Mb segment on chromosome 7A, carried 12 SNP markers significantly associated with spike length under salt stress, explaining 20.20-28.47% of the phenotypic variation. Through linkage analysis, a total of 11 QTLs controlling spikelet number under salt stress were located, among which QSps-6A (53.5-62.5cM) was detected in all salt stress environments, with the largest phenotypic explanation rate (9.10%) and LOD value (9.95). QSps-4B (27.5-34.5 cM) was also a stable QTL, explaining 7.48% of the phenotypic variation, while QSps-1D (152.5-155.0 cM) and QSps-2D (124.5-129.5 cM) explained 6.00% and 4.47% of the phenotypic variation, respectively.

[0005] Crop breeding has gone through the stages of traditional hybrid breeding, genetic engineering breeding and molecular marker-assisted breeding. At present, conventional breeding based on phenotypic selection is the basis of wheat breeding. As an effective supplement, marker-assisted breeding based on genotypic selection can shorten the breeding cycle and improve selection efficiency. With the advancement of high-throughput sequencing technology, wheat high-density SNP chips have emerged, which are used for automated analysis of single nucleotide polymorphisms at the genome level, realizing rapid scanning of wheat germplasm genotypes, and providing important support for gene positioning and molecular marker-assisted selection. Competitive allele-specific PCR (Kompetitive Allele Specific PCR, KASP) technology can achieve SNP typing based on the specific matching of primer terminal bases, and has become one of the mainstream methods of SNP analysis in the world. Using SNP chips to carry out linkage analysis or association analysis, after obtaining SNP markers linked to the target trait, using KASP markers to detect superior allele variations in other materials has become an economical, fast and efficient SNP marker-assisted selection breeding method.

[0006] References

[0007] (1)Hu, P., Zheng, Q., Luo, QL, Teng, W., Li, HW, Li, B., and Li, ZS (2021). Genome-wide association study of yield and related traits in common wheat under salt-stress conditions, BMC Plant Biology 21,27.

[0008] (2) Luo, QL, Zheng, Q., Hu, P., Liu, LQ, Yang, GT, Li, HW, Li, B., and Li, ZS (2021). Mapping QTL for agronomic traits under two levels of salt stress in a new constructed RIL wheat population. Theoretical and Applied Genetics 134, 171-189. Summary of the invention

[0009] A technical problem to be solved by the present invention is how to use molecular markers to assist in accurate salt-tolerant wheat design breeding.

[0010] In order to solve the above technical problems, the present invention provides a molecular design breeding method for salt-tolerant wheat, comprising the following steps: performing genotyping on multiple wheat varieties using multiple SNP sites for controlling grain traits and ear traits of wheat under salt stress, selecting paired wheat varieties whose typing results of salt-tolerant grain traits SNP sites and salt-tolerant ear traits SNP sites are complementary to each other as candidate hybrid parents; selecting a pair of candidate hybrid parents with good salt tolerance and complementary advantages as two hybrid parents by analyzing the salt-tolerant phenotype; performing genotyping on hybrid offspring single plants at SNP sites whose typing results of the two parents are complementary, using the typing results to assist in screening salt-tolerant offspring single plants, and breeding salt-tolerant wheat varieties in combination with the salt-tolerant phenotype.

[0011] A salt-tolerant wheat molecular design breeding method of the present invention specifically comprises the following steps:

[0012] 1) Genotyping multiple wheat varieties with N SNP loci for salt-tolerant grain traits and M SNP loci for salt-tolerant ear traits, selecting pairs of wheat varieties with complementary typing results of n SNP loci for salt-tolerant grain traits and m SNP loci for salt-tolerant ear traits from the multiple wheat varieties as candidate hybrid parents, analyzing the salt-tolerant phenotype, determining a pair from the candidate hybrid parents as two hybrid parents, and obtaining offspring individual plants;

[0013] 2) performing genotyping of the n SNP loci of salt-tolerant grain traits and the m SNP loci of salt-tolerant ear traits on the offspring individual plants, using the genotyping results to assist in screening salt-tolerant offspring individual plants, and breeding salt-tolerant wheat varieties in combination with the salt-tolerant phenotype;

[0014] Wherein, N≥n≥2, M≥m≥2, N, n, M, m are all positive integers. Preferably, N≥n≥3, M≥m≥3 or M≥m≥4. In one embodiment of the present invention, N=4, M=7, n=3, m=4.

[0015] In the above method, the grain trait may be thousand-grain weight, and the ear trait may be ear length and / or spikelet number.

[0016] In the above method, the candidate hybrid parents are preferably selected from wheat varieties that carry N salt-tolerant grain trait SNP loci and M salt-tolerant ear trait SNP loci and contain as many excellent genotypes as possible.

[0017] In the above method, the typing results are used to assist in screening salt-tolerant offspring plants, and plants containing more excellent allelic variations and better salt-tolerant phenotypes are selected.

[0018] In the above method, the genotyping results are used to assist in screening salt-tolerant offspring plants, specifically, in B xThe F2 generation selected plants that carried a salt-tolerant grain trait SNP locus as a homozygous superior genotype, b salt-tolerant grain trait SNP locus as a heterozygous genotype containing superior alleles, c salt-tolerant ear trait SNP locus as a homozygous superior genotype, d ear trait SNP locus as a heterozygous genotype containing superior alleles, and self-pollinated plants with salt-tolerant phenotypes to obtain B. x F3 generation; in B x The F3 generation performs genotyping on the b salt-tolerant grain trait SNP loci and the d salt-tolerant ear trait SNP loci; a, b, c, d, and x are all natural numbers, and 0 <a+b≤n,0<c+d≤m。

[0019] In one embodiment of the present invention, a=1, b=1; c=2, d=1.

[0020] The x represents the number of backcrossing generations after hybridization. When x=0, it means no backcrossing after hybridization; when x=1, it means one backcrossing generation after hybridization, and so on.

[0021] In the above method, the salt-tolerant phenotype is a phenotype of medium plant height, more ears, longer ears, more spikelets, more grains per ear, more grains per plant, heavier grain weight per plant, and heavier thousand-grain weight after planting in salt land.

[0022] In the above method, the N SNP sites of salt-tolerant grain traits are sites associated with wheat thousand-grain weight under salt stress, and can be selected from AX-109277923, AX-111571185, AX-111821007 and AX-110361956:

[0023] The AX-109277923 is a SNP site on chromosome 2D that controls wheat thousand-grain weight under salt stress, and is used to detect the QTL site QTkw-2D (126.5-137.5 cM). Its nucleotide type is C or G, which is the 36th nucleotide of SEQ ID No.1; the nucleotide type of the excellent gene variation of the SNP site is C, and the homozygous excellent genotype is CC; the CC is the homozygous genotype in which the 36th nucleotide of SEQ ID No.1 in the wheat genome is C;

[0024] The AX-111571185 is a SNP site on chromosome 3D that controls wheat thousand-grain weight under salt stress, and is used to detect the QTL site QTkw-3D (102.5-120.5 cM). Its nucleotide type is C or T, which is the 36th nucleotide of SEQ ID No.5; the nucleotide type of the superior gene variation of the SNP site is T, and the homozygous superior genotype is TT; the TT is the homozygous genotype in which the 36th nucleotide of SEQ ID No.5 in the wheat genome is T;

[0025] The AX-111821007 is a SNP site on chromosome 4A that controls wheat thousand-grain weight under salt stress, and is used to detect the QTL site QTkw-4A (14.68-16.28Mb), and its nucleotide type is C or T, which is the 36th nucleotide of SEQ ID No.9; the nucleotide type of the excellent gene variation of the SNP site is C, and the homozygous excellent genotype is CC; the CC is the homozygous genotype of the 36th nucleotide of SEQ ID No.9 in the wheat genome is C;

[0026] The AX-110361956 is a SNP site on chromosome 4B that controls the thousand-grain weight of wheat under salt stress, and is used to detect the QTL site QTkw-4B (26.5-34.5cM). Its nucleotide type is C or G, which is the 36th nucleotide of SEQ ID No.13; the nucleotide type of the excellent gene variation of the SNP site is C, and the homozygous excellent genotype is CC; the CC is the homozygous genotype in which the 36th nucleotide of SEQ ID No.13 in the wheat genome is C.

[0027] In the above method, the M SNP sites of salt-tolerant spike traits are SNP sites related to wheat spike length or spikelet number under salt stress, which can be selected from AX-109972158, AX-109460108, AX-111016259, AX-111578827, AX-109996563, AX-111675975 and AX-111449179:

[0028] The AX-109972158 is a SNP site on chromosome 1B that controls wheat spike length under salt stress, and is used to detect the QTL site QSl-1B (674.96-675.13Mb), and its nucleotide type is A or G, which is the 36th nucleotide of SEQ ID No.17; the nucleotide type of the superior allele variation of the SNP site is A, and the homozygous superior genotype is AA; the AA is the homozygous genotype in which the 36th nucleotide of SEQ ID No.17 in the wheat genome is A;

[0029] The AX-109460108 is a SNP site on chromosome 1D that controls the number of wheat spikelets under salt stress, and is used to detect the QTL site QSps-1D (152.5-155.0 cM). The nucleotide type is A or G, which is the 36th nucleotide of SEQ ID No.21; the nucleotide type of the superior allele variation of the SNP site is G, and the homozygous superior genotype is GG; the GG is the homozygous genotype in which the 36th nucleotide of SEQ ID No.21 in the wheat genome is G;

[0030] The AX-111016259 is a SNP site on chromosome 2D that controls the number of wheat spikelets under salt stress, and is used to detect the QTL site QSps-2D (124.5-129.5 cM). The nucleotide type is A or C, which is the 36th nucleotide of SEQ ID No. 25; the nucleotide type of the superior allele variation of the SNP site is A, and the homozygous superior genotype is AA; the AA is the homozygous genotype in which the 36th nucleotide of SEQ ID No. 25 in the wheat genome is A;

[0031] The AX-111578827 is a SNP site on chromosome 3B that controls wheat spike length under salt stress, and is used to detect the QTL site QS1-3B (62.13-62.14Mb, ​​Jia Ji Zeng, personal communication; 63.40Mb, Chinese Spring genome IWGSCRefSeq v1.0), whose nucleotide type is A or G, and is the 36th nucleotide of SEQ ID No.29; the nucleotide type of the superior allele of the SNP site is A, and the homozygous superior genotype is AA; the AA is the homozygous genotype of the 36th nucleotide of SEQ ID No.29 in the wheat genome as A;

[0032] The AX-109996563 is a SNP site on chromosome 5B that controls the number of wheat spikelets under salt stress, and is used to detect the QTL site QSps-5B (90.5-92.5 cM). The nucleotide type is G or T, which is the 36th nucleotide of SEQ ID No.33; the nucleotide type of the superior allele variation of the SNP site is G, and the homozygous superior genotype is GG; the GG is the homozygous genotype in which the 36th nucleotide of SEQ ID No.33 in the wheat genome is G;

[0033] The AX-111675975 is a SNP site on chromosome 5B that controls wheat spike length under salt stress, and is used to detect the QTL site QS1-5B2 (583.48-584.89Mb), and its nucleotide type is A or G, which is the 36th nucleotide of SEQ ID No.37; the nucleotide type of the superior allele variation of the SNP site is A, and the homozygous superior genotype is AA; the AA is the homozygous genotype in which the 36th nucleotide of SEQ ID No.37 in the wheat genome is A;

[0034] The AX-111449179 is a SNP site on chromosome 7A that controls wheat spike length under salt stress, and is used to detect the QTL site Q-7A (22.75-22.88Mb). Its nucleotide type is C or T, which is the 36th nucleotide of SEQ ID No.41; the nucleotide type of the superior allele variation of the SNP site is C, and the homozygous superior genotype is CC; the CC is the homozygous genotype in which the 36th nucleotide of SEQ ID No.41 in the wheat genome is C.

[0035] In the above method, a primer set is used for genotyping, and the primer set consists of at least two of the 11 combinations of primer set 1 to primer set 11.

[0036] The primer set for genotyping the AX-109277923 is primer set 1, and the primer set 1 is 1-1 or 1-2:

[0037] 1-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 48 of SEQ ID No. 2, a single-stranded DNA having a nucleotide sequence containing positions 22 to 48 of SEQ ID No. 3, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 4;

[0038] 1-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 2, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 3, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 4;

[0039] The primer set for genotyping the AX-111571185 is primer set 2, and the primer set 2 is 2-1 or 2-2:

[0040] 2-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 48 of SEQ ID No. 6, a single-stranded DNA having a nucleotide sequence containing positions 22 to 49 of SEQ ID No. 7, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 8;

[0041] 2-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.6, a single-stranded DNA having a nucleotide sequence of SEQ ID No.7, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.8;

[0042] The primer set for genotyping the AX-111821007 is primer set 3, and the primer set 3 is 3-1 or 3-2:

[0043] 3-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 41 of SEQ ID No. 10, a single-stranded DNA having a nucleotide sequence containing positions 22 to 41 of SEQ ID No. 11, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 12;

[0044] 3-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 10, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 11, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 12;

[0045] The primer set for genotyping the AX-110361956 is primer set 4, and the primer set 4 is 4-1 or 4-2:

[0046] 4-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 53 of SEQ ID No. 14, a single-stranded DNA having a nucleotide sequence containing positions 22 to 53 of SEQ ID No. 15, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 16;

[0047] 4-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 14, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 15, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 16;

[0048] The primer set for genotyping the AX-109972158 is primer set 5, and the primer set 5 is 5-1 or 5-2:

[0049] 5-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 47 of SEQ ID No. 18, a single-stranded DNA having a nucleotide sequence containing positions 22 to 46 of SEQ ID No. 19, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 20;

[0050] 5-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.18, a single-stranded DNA having a nucleotide sequence of SEQ ID No.19, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.20;

[0051] The primer set for genotyping the AX-109460108 is primer set 6, and the primer set 6 is 6-1 or 6-2:

[0052] 6-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 41 of SEQ ID No. 22, a single-stranded DNA having a nucleotide sequence containing positions 22 to 41 of SEQ ID No. 23, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 24;

[0053] 6-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 22, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 23, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 24;

[0054] The primer set for genotyping the AX-111016259 is primer set 7, and the primer set 7 is 7-1 or 7-2:

[0055] 7-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 45 of SEQ ID No. 26, a single-stranded DNA having a nucleotide sequence containing positions 22 to 45 of SEQ ID No. 27, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 28;

[0056] 7-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 26, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 27, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 28;

[0057] The primer set for genotyping the AX-111578827 is primer set 8, and the primer set 8 is 8-1 or 8-2:

[0058] 8-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 46 of SEQ ID No. 30, a single-stranded DNA having a nucleotide sequence containing positions 22 to 46 of SEQ ID No. 31, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 32;

[0059] 8-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 30, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 31, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 32;

[0060] The primer set for genotyping the AX-109996563 is primer set 9, and the primer set 9 is 9-1 or 9-2:

[0061] 9-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 43 of SEQ ID No. 34, a single-stranded DNA having a nucleotide sequence containing positions 22 to 43 of SEQ ID No. 35, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 36;

[0062] 9-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.34, a single-stranded DNA having a nucleotide sequence of SEQ ID No.35, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.36;

[0063] The primer set for genotyping the AX-111675975 is primer set 10, and the primer set 10 is 10-1 or 10-2:

[0064] 10-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 44 of SEQ ID No. 38, a single-stranded DNA having a nucleotide sequence containing positions 22 to 44 of SEQ ID No. 39, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 40;

[0065] 10-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.38, a single-stranded DNA having a nucleotide sequence of SEQ ID No.39, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.40;

[0066] The primer set for genotyping the AX-111449179 is primer set 11, and the primer set 11 is 11-1 or 11-2:

[0067] 11-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 49 of SEQ ID No. 42, a single-stranded DNA having a nucleotide sequence containing positions 22 to 51 of SEQ ID No. 43, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 44;

[0068] 11-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.42, a single-stranded DNA having a nucleotide sequence of SEQ ID No.43, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.44.

[0069] In the above method, the single-stranded DNA in the primer set can be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be attached to a nucleic acid. The marker includes but is not limited to dyes; radioactive labels such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The label can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moiety (positive or negative) or, alternatively, can be charge neutral. The label can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the label is detectable. For example, the primer set used for Kasp-7923 may be a primer set consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.2, a single-stranded DNA having a nucleotide sequence of SEQ ID No.3, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.4, wherein SEQ ID No.2 in the sequence list consists of 48 nucleotides, nucleotides at positions 1-21 are a FAM linker sequence (as a marker), and nucleotides at positions 22-48 are specific sequences; SEQ ID No.3 in the sequence list consists of 48 nucleotides, nucleotides at positions 1-21 are a VIC linker sequence (as a marker), and nucleotides at positions 22-48 are specific sequences.

[0070] The present invention also provides application of SNP sites of salt-tolerant grain traits in wheat breeding; the SNP sites of salt-tolerant grain traits are selected from two or more of the above-mentioned AX-109277923, AX-111571185, AX-111821007 and AX-110361956.

[0071] The present invention also provides application of SNP sites of salt-tolerant ear traits in wheat breeding; the SNP sites of salt-tolerant ear traits are selected from two or more of AX-109972158, AX-109460108, AX-111016259, AX-111578827, AX-109996563, AX-111675975 and AX-111449179.

[0072] The present invention also provides application of the primer set in wheat breeding.

[0073] The purpose of wheat breeding described in this article is to breed salt-tolerant wheat.

[0074] The present invention also provides products for the above method, which are reagents and / or instruments required for determining the polymorphism or genotype of the SNP site, and are selected from DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high performance liquid chromatography and SNP chips. Among them, the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein DNA binding reaction, and a chip based on fluorescent molecule DNA binding reaction.

[0075] In the above methods and products, the product may be a reagent or a kit or a system, and the system may include a combination product of a reagent or a kit, an instrument and an analysis software, such as a product consisting of PCR primers, PARMS master mix reagents, an ELISA reader and an online software SNP decoder (http: / / www.snpway.com / snpdecoder01 / ), and a combination product consisting of PCR primers, PARMS master mix reagents, an online software SNP decoder and a fluorescence quantitative PCR instrument. The product may include the above composition for detecting the genotype of the locus in the wheat genome.

[0076] The present invention discloses a molecular design breeding method for salt-tolerant wheat, designs KASP markers according to the located QTL loci controlling salt-tolerant traits of wheat in the adult stage, scans the genotypes of breeding materials, purposefully screens the variety combination hybridization combination with complementary genotypes, and achieves the aggregation of excellent allelic variation through marker tracking and systematic breeding to screen out new salt-tolerant wheat varieties (lines). An embodiment of the present invention uses KASP markers to accurately select parent materials, BC2F2 plants, and BC2F3 plants under salt stress, thereby achieving directional design breeding of new salt-tolerant wheat lines and breeding an excellent salt-tolerant new wheat line, Xiaoyan 156. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 The typing results of the marker Kasp-0108 in Example 1 of the present invention in the (Zhongmai 175 / Xiaoyan 60) RIL line and wheat varieties are shown.

[0078] Figure 2 The results are shown for typing the marker Kasp-6259 in Example 1 of the present invention in the (Zhongmai 175 / Xiaoyan 60) RIL line and wheat varieties.

[0079] Figure 3 The results are shown for typing the marker Kasp-6563 in Example 1 of the present invention in the (Zhongmai 175 / Xiaoyan 60) RIL line and wheat varieties.

[0080] Figure 4 These are phenotype photos of Shiyou 17 (left) and Xiaoyan 60 (right) in the adult stage in Example 2 of the present invention.

[0081] Figure 5 The statistical results of the properties of Shiyou 17 and Xiaoyan 60 in Example 2 of the present invention when planted in 0.18% field salt land are shown in the figure. In the figure, SY17 is Shiyou 17 and XY60 is Xiaoyan 60. The statistical analysis adopts a two-tailed t-test, where ** represents a significant analysis result of P<0.01, and *** represents a significant analysis result of P<0.001.

[0082] Figure 6 These are the typing results of the marker Kasp-1007 in Example 2 of the present invention in BC2F2 individual plants and some wheat varieties.

[0083] Figure 7 These are the typing results of the marker Kasp-1956 in Example 2 of the present invention in BC2F2 individual plants and some wheat varieties.

[0084] Figure 8 These are the typing results of the marker Kasp-7923 in Example 2 of the present invention in BC2F2 individual plants and some wheat varieties.

[0085] Fig. 9 These are the typing results of the marker Kasp-2158 in Example 2 of the present invention in BC2F2 individual plants and some wheat varieties.

[0086] Fig.10 These are the typing results of the marker Kasp-0108 in Example 2 of the present invention in BC2F2 individual plants and some wheat varieties.

[0087] Fig.11 These are the typing results of the marker Kasp-6259 in Example 2 of the present invention in BC2F2 individual plants and some wheat varieties.

[0088] Fig.12 These are the typing results of the marker Kasp-6563 in Example 2 of the present invention in BC2F2 individual plants and some wheat varieties.

[0089] Fig.13 These are the typing results of the marker Kasp-7923 in Example 2 of the present invention in BC2F3 individual plants and some wheat varieties.

[0090] Fig.14 These are the typing results of the marker Kasp-6259 in Example 2 of the present invention in BC2F3 individual plants and some wheat varieties. DETAILED DESCRIPTION

[0091] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0092] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0093] Example 1 Development of KASP markers

[0094] Wheat salt tolerance is a complex trait controlled by multiple genes, involving the accumulation and antagonism of inter-gene functions. KASP markers were designed based on the QTL loci that control salt tolerance-related traits in the adult stage of wheat located by the applicant, and the genotypes of breeding materials were scanned. The genotypes of complementary varieties were purposefully screened for hybrid combinations. Through marker tracking and systematic breeding, the aggregation of excellent alleles was achieved, and new salt-tolerant wheat varieties (lines) were screened. To this end, when scanning the genotypes of spikelet traits, three KASP markers were first developed to detect and control spikelet number variation under salt stress:

[0095] 1. KASP markers related to wheat grain traits under salt stress

[0096] The published SNP markers AX-109277923, AX-111571185, AX-111821007 and AX-110361956 related to wheat grain traits under salt stress were used to determine the merits and demerits of wheat salt tolerance thousand-grain weight QTL loci QTkw-2D (126.5-137.5 cM), QTkw-3D (102.5-120.5 cM), QTkw-4A (14.68-16.28 cM) and QTkw-4B (26.5-34.5 cM), respectively, as follows:

[0097] 1.1 Marking KASP-7923

[0098] Common wheat has a SNP marker at the physical position 569361770 of chromosome 2D (chip marker AX-109277923, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the locus QTkw-2D (126.5-137.5 cM) that controls wheat thousand-grain weight under salt stress, located at the 36th nucleotide of SEQ ID No.1 (5'-ACTCTAGGAAAAAATACAAAATTAAAGATGAATGASTGTAACAAAAAGGGACTACTTGCACAATTTTGGTA-3'), and its nucleotide type is C or G, represented by the letter S. One of its alleles is CC (i.e., homozygous type of nucleotide C at nucleotide 36 of SEQ ID No.1); the other allele is GG (i.e., homozygous type of nucleotide G at nucleotide 36 of SEQ ID No.1); and the heterozygous genotype is CG (i.e., heterozygous type of nucleotide C and G at nucleotide 36 of SEQ ID No.1). According to the applicant's previous positioning results, the CC genotype is an excellent variation for controlling wheat thousand-grain weight under salt stress, and QTkw-2D increases wheat thousand-grain weight by an average of 1.365 g. A set of KASP primers was designed for the two alleles of the target SNP marker AX-109277923, named Kasp-7923.

[0099] Table 1 Kasp-7923 primer sequences for detection site QTkw-2D (marker AX-109277923)

[0100]

[0101] The single-stranded DNA molecules shown in SEQ ID No. 2 and SEQ ID No. 4 amplify the fragment of SEQ ID No. 1 whose nucleotide at position 36 is C, and the fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0102] The single-stranded DNA molecules shown in SEQ ID No. 3 and SEQ ID No. 4 amplify the fragment of SEQ ID No. 1 in which the nucleotide at position 36 is G, and the fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescent quantitative PCR instrument.

[0103] 1.2 Marking Kasp-1185

[0104] Common wheat has a SNP marker at the physical position 520827884 of chromosome 3D (chip marker AX-111571185, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the site QTkw-3D (102.5-120.5 cM) of wheat thousand-grain weight under salt stress, located at the 36th nucleotide of SEQ ID No.5 (5'-GTTGCCCTGGGTTATTTTCGGTTTACAAGTTTTCCYGCGCACTAATTTTGAATCCTTGTGGTTGTTGAAGG-3'), and its nucleotide type is C or T, represented by the letter Y. One of its alleles is CC (i.e., homozygous type of nucleotide C at nucleotide 36 of SEQ ID No.5); the other allele is TT (i.e., homozygous type of nucleotide T at nucleotide 36 of SEQ ID No.5); and the heterozygous genotype is CT (i.e., heterozygous type of nucleotide C and T at nucleotide 36 of SEQ ID No.5). According to the applicant's previous positioning results, the TT genotype is an excellent variation for controlling the thousand-grain weight of wheat under salt stress, and QTkw-3D increases the thousand-grain weight by an average of 0.861 g. A set of KASP primers was designed for the two alleles of the target SNP marker AX-111571185, named Kasp-1185.

[0105] Table 2 Kasp-1185 primer sequences for detecting allelic variation at site QTkw-3D (marker AX-111571185)

[0106]

[0107] One strand of the amplified single-stranded DNA molecules shown in SEQ ID No.6 and SEQ ID No.8 is a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No.5 is C, and the fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0108] The single-stranded DNA molecules shown in SEQ ID No.7 and SEQ ID No.8 above amplify a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No.5 is T. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0109] 1.3 Marking Kasp-1007

[0110] Common wheat has a SNP marker at the physical position 14681814 of chromosome 4A (chip marker AX-111821007, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the site QTkw-4A (14.68-16.28 Mb) of wheat thousand-grain weight under salt stress, located at the 36th nucleotide of SEQ ID No.9 (5'-GAGGGGGCGTCCCACCCCCGTCGTTTGTCGAGAGTYAGCCTCCTCCAGCGGCATCGGGAAATTTGGTGGCT-3'), and its nucleotide type is C or T, represented by the letter Y. One of its alleles is CC (i.e., homozygous type with nucleotide C at position 36 of SEQ ID No.9); the other allele is TT (i.e., homozygous type with nucleotide T at position 36 of SEQ ID No.9); and the heterozygous genotype is CT (i.e., heterozygous type with nucleotide C and T at position 36 of SEQ ID No.9). According to the applicant's previous positioning results, the CC genotype is an excellent variation for controlling the thousand-grain weight of wheat under salt stress. QTkw-4A increases the thousand-grain weight by 4.06-5.32 g under low-salt conditions and 2.49-5.95 g under high-salt conditions on average. A set of KASP primers was designed for the two alleles of the target SNP marker AX-111821007, named Kasp-1007.

[0111] Table 3 Kasp-1007 primer sequences for detection site QTkw-4A (labeled AX-111821007)

[0112]

[0113] The amplified single-stranded DNA molecules shown in SEQ ID No. 10 and SEQ ID No. 12 are double-stranded DNA fragments in which the nucleotide at position 36 of SEQ ID No. 9 is C. The fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0114] The single-stranded DNA molecules shown in SEQ ID No. 11 and SEQ ID No. 12 amplify one strand of a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No. 9 is T. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0115] 1.4 Marking Kasp-1956

[0116] Common wheat has a SNP marker at the physical position 75741547 of chromosome 4B (chip marker AX-110361956, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the site QTkw-4B (26.5-34.5 cM) of wheat thousand-grain weight under salt stress, located at the 36th nucleotide of SEQ ID No.13 (5'-CCCACAACTACTTTCTTGCAGAAGTTTATAAAGTTSAACACAACATACTCTAGCAAGTAGAAACCGCAAAC-3'), and its nucleotide type is C or G, represented by the letter S. One of its alleles is CC (i.e., the homozygous type of nucleotide C at the 36th nucleotide of SEQ ID No.13); the other allele is GG (i.e., the homozygous type of nucleotide G at the 36th nucleotide of SEQ ID No.13); and the heterozygous genotype is CG (i.e., the heterozygous type of nucleotide C and G at the 36th nucleotide of SEQ ID No.13). According to the applicant's previous positioning results, the CC genotype is an excellent variation for controlling the thousand-grain weight of wheat under salt stress, and QTkw-4B increases the thousand-grain weight by an average of 2.314 g. A set of KASP primers was designed for the two alleles of the target SNP marker AX-110361956, named Kasp-1956.

[0117] Table 4 Kasp-1956 primer sequence for detection site QTkw-4B (marker AX-110361956)

[0118]

[0119]

[0120] The amplified single-stranded DNA molecules shown in SEQ ID No. 14 and SEQ ID No. 16 are double-stranded DNA fragments in which the nucleotide at position 36 of SEQ ID No. 13 is C. The fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0121] The single-stranded DNA molecules shown in SEQ ID No. 15 and SEQ ID No. 16 amplify one strand of a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No. 13 is G. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0122] 2. Development of KASP markers related to wheat spikelet number under salt stress

[0123] The SNP markers AX-109460108, AX-111016259 and AX-109996563, which were discovered to control wheat spikelet-related traits under salt stress, were converted into corresponding KASP markers and used to determine the merits and demerits of wheat salt-tolerant spikelet number QTL loci QSps-1D (152.5-155.0 cM), QSps-2D (124.5-129.5 cM) and QSps-5B (90.5-92.5 cM), respectively, as follows:

[0124] 2.1 Development of Marker Kasp-0108

[0125] Common wheat has a SNP marker at the physical position 490023985 of chromosome 1D (chip marker AX-109460108, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the site QSps-1D (152.5-155.0 cM) of wheat spikelet number under salt stress, located at the 36th nucleotide of SEQ ID No. 21 (5'-CTTCCTGCCGGCTACGGGTAGCATATCGTAGGCCCRTCTGTGTATAGAACCTTTTTTTTGGGCTTTGCGGG-3'), and its nucleotide type is A or G, represented by the letter R. One of its alleles is AA (i.e., homozygous type with nucleotide A at position 36 of SEQ ID No.21); the other allele is GG (i.e., homozygous type with nucleotide G at position 36 of SEQ ID No.21); and the heterozygous genotype is AG (i.e., heterozygous type with nucleotide A and G at position 36 of SEQ ID No.21). According to the applicant's previous positioning results, the GG genotype is an excellent variation for controlling the number of wheat spikelets, and QSps-1D increases the number of spikelets by 0.190 on average. A set of KASP primers was designed for the two alleles of the target SNP marker AX-109460108, named Kasp-0108.

[0126] Table 5 Kasp-0108 primer sequences for detection site QSps-1D (labeled AX-109460108)

[0127]

[0128] The amplified single-stranded DNA molecules shown in SEQ ID No. 22 and SEQ ID No. 24 are double-stranded DNA fragments in which the nucleotide at position 36 of SEQ ID No. 21 is A. The fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0129] The single-stranded DNA molecules shown in SEQ ID No. 23 and SEQ ID No. 24 amplify one strand of a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No. 21 is G. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0130] In order to verify the specificity and stability of the designed Kasp-0108 marker, 23 (Zhongmai 175 / Xiaoyan 60) RIL lines and wheat varieties Xiaoyan 60 and Shiyou 17 were selected for genotyping.

[0131] After amplification, the results were genotyped using StepOne software v2.3. Figure 1 As shown, the upper left of the figure shows the line with the genotype of SNP marker AX-109460108 as AA, the lower right shows the line with the genotype of SNP marker AX-109460108 as GG; the middle shows the line with the genotype of SNP marker AX-109460108 as AG, indicating that the marker can distinguish different genotypes of SNP marker AX-109460108. After testing, the typing results of KASP marker (Table 6) were consistent with the genotype results obtained by wheat 55K SNP chip (Luo et al. 2021), proving that the Kasp-0108 marker can achieve successful typing of SNP marker AX-109460108.

[0132] Table 6 Typing results of Kasp-0108 in (Zhongmai 175 / Xiaoyan 60) RIL lines and wheat varieties

[0133] Accession Allele1ΔRn Allele2ΔRn Chip typing KASP classification consistency ZX-1 0.196692571 2.628757715 AA AA Consistency ZX-3 0.814508379 -0.09837711 GG GG Consistency ZX-5 0.848710537 -0.24769261 GG GG Consistency ZX-12 0.817227662 -0.10029413 GG GG Consistency ZX-14 0.099947914 2.872364283 AA AA Consistency ZX-17 0.138873234 2.778931141 AA AA Consistency ZX-18 0.198443487 2.641737223 AA AA Consistency ZX-20 0.827156961 -0.26271158 GG GG Consistency ZX-21 0.187699914 2.813362837 AA AA Consistency ZX-22 0.870307684 -0.23836283 GG GG Consistency ZX-23 0.621789634 0.522078931 AG AG Consistency ZX-24 0.853668571 -0.26353484 GG GG Consistency ZX-25 0.820284605 -0.18288524 GG GG Consistency ZX-26 0.854203582 -0.23558989 GG GG Consistency ZX-28 0.076273702 2.810963869 AA AA Consistency ZX-31 0.199683115 2.64865613 AA AA Consistency ZX-32 0.174009413 2.744140625 AA AA Consistency ZX-33 0.830859303 -0.12927628 GG GG Consistency ZX-37 0.087064132 2.956903696 AA AA Consistency ZX-38 0.897901416 -0.39214447 GG GG Consistency ZX-47 0.167262152 2.81370163 AA AA Consistency ZX-48 0.850132644 -0.28371662 GG GG Consistency ZX-49 0.861219525 -0.29374105 GG GG Consistency Shiyou17 0.875644863 -0.21176603 GG GG Consistency Xiaoyan 60 0.072069637 3.149352789 AA AA Consistency

[0134] 2.2 Development of Marker Kasp-6259

[0135] Common wheat has a SNP marker at the physical position 571001972 of chromosome 2D (chip marker AX-111016259, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the wheat spikelet number locus QSps-2D (124.5-129.5cM) under salt stress, located at the 36th nucleotide of SEQ ID No.25 (5'-TGACGAGATTTTTGATCCATTCAAGAGGCTAGATGMTCTGTTCAGAAACACCGCGACGTGCTTGCAAGCTT-3'), and its nucleotide type is A or C, represented by the letter M. One of its alleles is AA (i.e., homozygous type with nucleotide A at position 36 of SEQ ID No. 25); the other allele is CC (i.e., homozygous type with nucleotide C at position 36 of SEQ ID No. 25); and the heterozygous genotype is AC (i.e., heterozygous type with nucleotide A and C at position 36 of SEQ ID No. 25). According to the applicant's previous positioning results, the AA genotype is an excellent variation for controlling the number of wheat spikelets, and QSps-2D increases the number of spikelets by 0.196 on average. A set of KASP primers was designed for the two alleles of the target SNP marker AX-111016259, named Kasp-6259.

[0136] Table 7 Kasp-6259 primer sequences for detection site QSps-2D (labeled AX-111016259)

[0137]

[0138] The single-stranded DNA molecules shown in SEQ ID No. 26 and SEQ ID No. 28 above amplify a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No. 25 is A, and the fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0139] The single-stranded DNA molecules shown in SEQ ID No. 27 and SEQ ID No. 28 above amplify a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No. 25 is C. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0140] In order to verify the specificity and stability of the designed Kasp-6259 marker, 23 (Zhongmai 175 / Xiaoyan 60) RIL lines and wheat varieties Xiaoyan 60 and Shiyou 17 were selected for genotyping.

[0141] After amplification, the results were genotyped using StepOne software v2.3. Figure 2 As shown, the upper left of the figure shows the line with the genotype of SNP marker AX-111016259 being AA, and the lower right shows the line with the genotype of SNP marker AX-111016259 being CC; indicating that this marker can distinguish different genotypes of SNP marker AX-111016259. After testing, the KASP marker typing results (Table 8) were consistent with the genotype results obtained by the wheat 55K SNP chip (Luo et al. 2021), proving that the Kasp-6259 marker can achieve successful typing of the SNP marker AX-111016259.

[0142] Table 8 Typing results of Kasp-6259 in (Zhongmai 175 / Xiaoyan 60) RIL lines and wheat varieties

[0143]

[0144]

[0145] 2.3 Development of Marker Kasp-6563

[0146] Common wheat has a SNP marker at the physical position 572164762 of chromosome 5B (chip marker AX-109996563, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the site QSps-5B (90.5-92.5cM) of wheat spikelet number under salt stress, located at the 36th nucleotide of SEQ ID No.33 (5'-ATAGGTAGTAGGGGATGGACCTCCACGGTGTCGTAKCATGATTTTAGGGCGATAGGTAGCATCGTTCGTTG-3'), and its nucleotide type is G or T, represented by the letter K. One of its alleles is GG (i.e., homozygous type with nucleotide G at position 36 of SEQ ID No.33); the other allele is TT (i.e., homozygous type with nucleotide T at position 36 of SEQ ID No.33); and the heterozygous genotype is GT (i.e., heterozygous type with nucleotide G and T at position 36 of SEQ ID No.33). According to the applicant's previous positioning results, the GG genotype is an excellent variation for controlling the number of wheat spikelets, and QSps-5B increases the number of spikelets by 0.182 on average. A set of KASP primers was designed for the two alleles of the target SNP marker AX-109996563, named Kasp-6563.

[0147] Table 9 Kasp-6563 primer sequence for detection site QSps-5B (labeled AX-109996563)

[0148]

[0149] The single-stranded DNA molecules shown in SEQ ID No.34 and SEQ ID No.36 amplify the fragment of SEQ ID No.33 in which the nucleotide at position 36 is T, and the fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescent quantitative PCR instrument;

[0150] The single-stranded DNA molecules shown in SEQ ID No. 35 and SEQ ID No. 36 amplify the fragment of SEQ ID No. 33 in which the nucleotide at position 36 is G, and the fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescent quantitative PCR instrument.

[0151] In order to verify the specificity and stability of the designed Kasp-6563 marker, 23 (Zhongmai 175 / Xiaoyan 60) RIL lines and wheat varieties Xiaoyan 60 and Shiyou 17 were selected for genotyping.

[0152] After amplification, the results were genotyped using StepOne software v2.3. Figure 3 As shown, the upper left of the figure shows a line with the genotype of SNP marker AX-109996563 as TT, and the lower right shows a line with the genotype of SNP marker AX-109996563 as GG; indicating that this marker can distinguish different genotypes of SNP marker AX-109996563. After testing, the KASP marker typing results (Table 10) are consistent with the genotype results obtained by the wheat 55K SNP chip (Luo et al. 2021), proving that the Kasp-6563 marker can achieve successful typing of the SNP marker AX-109996563.

[0153] Table 10 Typing results of Kasp-6563 in (Zhongmai 175 / Xiaoyan 60) RIL lines and wheat varieties

[0154]

[0155]

[0156] 3. KASP markers associated with wheat spike length under salt stress

[0157] 3.1 Marking KASP-2158

[0158] Common wheat has a SNP marker at the physical position 675705691 of chromosome 1B (chip marker AX-109972158, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the locus QSl-1B (674.96-675.13 Mb) of wheat spike length under salt stress, located at the 36th nucleotide of SEQ ID No.17 (5'-GTTAGAGGTGAGCACCTTTACAACAAAAGCTGAAGRACCGAACTGATATGTGTTGACCGATATTCTTGTTT-3'), and its nucleotide type is A or G, represented by the letter R. One of its alleles is AA (i.e., the homozygous type of the nucleotide at position 36 of SEQ ID No.17 is A); the other allele is GG (i.e., the homozygous type of the nucleotide at position 36 of SEQ ID No.17 is G); the heterozygous genotype is AG (i.e., the heterozygous type of the nucleotide at position 36 of SEQ ID No.17 is A and G). According to the applicant's previous positioning results, the AA genotype is an excellent variation for controlling wheat spike length under salt stress. The excellent haplotype of the site QSl-1B can increase the spike length by 0.67-0.72 cm under low-salt conditions and by 0.60-0.78 cm under high-salt conditions. A set of KASP primers was designed for the two alleles of the target SNP marker AX-109972158, named Kasp-2158.

[0159] Table 11 Kasp-2158 primer sequences for detection site QS1-1B (labeled AX-109972158)

[0160]

[0161] The amplified single-stranded DNA molecules shown in SEQ ID No. 18 and SEQ ID No. 20 are double-stranded DNA fragments in which the nucleotide at position 36 of SEQ ID No. 17 is A. The fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0162] The single-stranded DNA molecules shown in SEQ ID No. 19 and SEQ ID No. 20 above amplify a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No. 17 is G. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0163] 3.2 Marking KASP-8827

[0164] There is a SNP marker in common wheat at the physical position 63402397 of chromosome 3B (chip marker AX-111578827, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the locus QSl-3B (62.13-62.14 Mb, Jia Ji Zeng, personal communication; 63.40 Mb, Chinese Spring genome IWGSC RefSeq v1.0) of wheat spike length under salt stress, located at the 36th nucleotide of SEQ ID No. 29 (5′-CATTCAACACTGGTTAGAATTCGATTCAATTCCTCRTAAGTTGGATTCTCCAAATTGTTCGTCCAAGTGTA-3′), and the nucleotide type is A or G, represented by the letter R. One of its alleles is AA (i.e., the homozygous type of the nucleotide at position 36 of SEQ ID No. 29 is A); the other allele is GG (i.e., the homozygous type of the nucleotide at position 36 of SEQ ID No. 29 is G); the heterozygous genotype is AG (i.e., the heterozygous type of the nucleotide at position 36 of SEQ ID No. 29 is A and G). According to the applicant's previous positioning results, the AA genotype is an excellent variation for controlling wheat spike length under salt stress. The excellent haplotype of the site QS1-3B can increase the spike length by 0.54 cm under low salt conditions and by 0.76 cm under high salt conditions. A set of KASP primers was designed for the two alleles of the target SNP marker AX-111578827, named Kasp-8827.

[0165] Table 12 Kasp-8827 primer sequence for detection site QS1-3B (labeled AX-111578827)

[0166]

[0167] The single-stranded DNA molecules shown in SEQ ID No. 30 and SEQ ID No. 32 are amplified with one strand being a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No. 29 is A, and the fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0168] The single-stranded DNA molecules shown in SEQ ID No.31 and SEQ ID No.32 above amplify a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No.29 is G. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0169] 3.3 Marking KASP-5975

[0170] Common wheat has a SNP marker at the physical position 584109393 of chromosome 5B (chip marker AX-111675975, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the locus QSl-5B2 (583.48-584.89 Mb) of wheat spike length under salt stress, located at the 36th nucleotide of SEQ ID No.37 (5'-TAGTCGCATGGGAACACGTGTGCAATCTTAAGGAGRAAGGTGGGCTTGGCGTTCGGGCGCTTGCAATCCAG-3'), and its nucleotide type is A or G, represented by the letter R. One of its alleles is AA (i.e., homozygous type with nucleotide A at position 36 of SEQ ID No.37); the other allele is GG (i.e., homozygous type with nucleotide G at position 36 of SEQ ID No.37); and the heterozygous genotype is AG (i.e., heterozygous type with nucleotide A and G at position 36 of SEQ ID No.37). According to the applicant's previous positioning results, the AA genotype is an excellent variation for controlling wheat spike length under salt stress. The excellent haplotype of site QS1-5B2 can increase spike length by 2.31 cm under high salt conditions. A set of KASP primers was designed for the two alleles of the target SNP marker AX-111675975, named Kasp-5975.

[0171] Table 13 Kasp-5975 primer sequence for detection site QS1-5B2 (labeled AX-111675975)

[0172]

[0173] The single-stranded DNA molecules shown in SEQ ID No.38 and SEQ ID No.40 above amplify a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No.37 is A, and the fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0174] The single-stranded DNA molecules shown in SEQ ID No.39 and SEQ ID No.40 above amplify a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No.37 is G. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0175] 3.4 Development of Marker Kasp-9179

[0176] Common wheat has a SNP marker at the physical position 22783939 of chromosome 7A (chip marker AX-111449179, reference wheat variety Chinese Spring genome IWGSC RefSeq v1.0), which is used to detect the locus Q-7A (22.75-22.88Mb) of wheat spike length under salt stress, located at the 36th nucleotide of SEQ ID No.41 (5'-ATCAGCATTTAGTATATCCGAACCTGGAAAGTAAGYCGCTTTTAGAATTTGAGCGCTTAACGATTCAGGGT-3'), and its nucleotide type is C or T, represented by the letter Y. One of its alleles is CC (i.e., homozygous type of nucleotide C at nucleotide 36 of SEQ ID No.41); the other allele is TT (i.e., homozygous type of nucleotide T at nucleotide 36 of SEQ ID No.41); and the heterozygous genotype is CT (i.e., heterozygous type of nucleotide C and T at nucleotide 36 of SEQ ID No.41). According to the applicant's previous positioning results, the CC genotype is an excellent variation for controlling wheat ear length under salt stress. The excellent haplotype of locus Q-7A can increase the ear length by 0.62-0.90 cm under low salt conditions and 0.73 cm under high salt conditions. A set of KASP primers was designed for the two alleles of the target SNP marker AX-111449179, named Kasp-9179.

[0177] Table 14 Kasp-9179 primer sequences for detection site Q-7A (labeled AX-111449179)

[0178]

[0179] The amplified single-stranded DNA molecules shown in SEQ ID No. 42 and SEQ ID No. 44 are double-stranded DNA fragments in which the nucleotide at position 36 of SEQ ID No. 41 is C. The fluorescent signal of the fluorescent group bound to the FAM sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument;

[0180] The single-stranded DNA molecules shown in SEQ ID No.43 and SEQ ID No.44 amplify one strand of a double-stranded DNA fragment in which the nucleotide at position 36 of SEQ ID No.41 is T. The fluorescent signal of the fluorescent group bound to the VIC sequence can be read by an ELISA reader or a fluorescence quantitative PCR instrument.

[0181] Example 2 Breeding of salt-tolerant wheat

[0182] The design breeding of salt-tolerant wheat is a process of purposefully selecting and aggregating excellent salt-tolerant sites. Through genotype scanning, hybrid parents are systematically combined and hybrid offspring individuals are selected to avoid the environmental impact caused by the large spatial heterogeneity of saline-alkali land. However, since wheat is an allohexaploid with a large genome and complex coupling and coordination mechanisms between salt-tolerant genes with the same function, the cultivation of salt-tolerant wheat varieties cannot rely entirely on the genotype of the breeding material, but must be combined with systematic breeding based on phenotypic observation. The specific steps for salt-tolerant wheat breeding in this embodiment are as follows:

[0183] 1. Determination of hybrid parents

[0184] The salt tolerance-related KASP markers were scanned and the hybrid parents were determined by the SNP marker complementation principle and phenotypic selection. The specific method for determining the hybrid parents is as follows:

[0185] The genotyping of multiple wheat varieties was performed using N SNP markers for salt-tolerant grain traits and M SNP markers for salt-tolerant ear traits. According to the principle of complementarity, pairs of wheat varieties containing as many excellent genotypes as possible and with complementary typing results were selected as candidate hybrid parents. The salt-tolerant phenotypes of each pair of candidate hybrid parents were analyzed, and a pair of candidate hybrid parents with good salt tolerance were selected as two hybrid parents. Among them, N and M are both positive integers.

[0186] In this example, the typing of SNP sites was performed using KASP markers.

[0187] In this example, four KASP markers Kasp-7923, Kasp-1185, Kasp-1007, and Kasp-1956 related to wheat thousand-grain weight under salt stress and four KASP markers Kasp-2158, Kasp-8827, Kasp-5975, and Kasp-9179 related to wheat spikelet length were reported, as well as three KASP markers Kasp-0108, Kasp-6259, and Kasp-6563 related to wheat spikelet number developed in Example 1 to scan multiple wheat varieties. The method is as follows:

[0188] 1.1 Select candidate hybrid parents with complementary KASP marker typing results

[0189] The modified SLS method was used to extract DNA from wheat leaves. The integrity of the DNA samples was tested using agarose gel, and the DNA concentration and quality were tested using NanoDrop. The total amount of DNA was required to be greater than 1 μg, the DNA was intact, and there was no RNA contamination. The DNA that met the requirements was used as a DNA template for backup.

[0190] Use the microfluidic chip to detect the SNP typing results, configure the PCR amplification reaction system, use a pipette to inject the configured reaction system from the chip inlet into the chip, and seal the inlet and outlet. The PCR reaction system is shown in Table 15:

[0191] Table 15 Microfluidic PCR reaction system

[0192] 20-50ng / μL DNA template 0.15μL PrimerMix 0.01μL KASPMasterMix 0.25μL <![CDATA[ddH2O]]> 0.09μL Total volume 0.50μL

[0193] Place the chip in a centrifuge at 4000 rpm and centrifuge for 1 minute. Place it in a chip heat sealer and heat seal it for 1 second. Place it in a flat PCR instrument for amplification reaction. The PCR amplification program is shown in Table 16:

[0194] Table 16 Microfluidic PCR amplification program

[0195]

[0196] Genotypes were scanned using KASP markers. The upstream and downstream of the SNP marker probe sequence were extended to 120 bp, and then compared with the Chinese spring wheat genome (IWGSC RefSeq v1.0) to determine that the sequence existed as a single copy in the reference genome. The sequence was submitted to the KASP marker development program (https: / / galaxy.triticeaetoolbox.org / ) and primers were designed.

[0197] The PCR reaction system was 10 μL, and the specific volumes of each component are shown in Table 17.

[0198] Table 17 PCR reaction system

[0199] Components Volume (μL) 2×KASPmastermix 5 Primer mix 0.14 Template DNA (50ng / μL) 1 ddH2O 3.86

[0200] Place the prepared reaction solution into the ABI StepOnePlus instrument and operate according to the relevant operating instructions (https: / / biosearch-cdn.azureedge.net / assetsv6 / running-KASP-on-ABI-StepOne-and-StepOnePlus.pdf). The PCR amplification program is shown in Table 18.

[0201] Table 18 PCR amplification program

[0202]

[0203] If the fluorescence signal is low and the clusters are scattered, more cycles are needed. The procedure is as follows:

[0204]

[0205] One of the candidate hybrid parents was obtained to be composed of Xiaoyan 60 and Shiyou 17, and their typing results are shown in Table 19 and Table 20:

[0206] Table 19 Typing results of KASP markers related to thousand-grain weight under salt stress in candidate parents

[0207] KASPmarker SNPID Traits Chr. Position(bp) S.N.Palleles Favorableallele XY60 SY17 Kasp-7923 AX-109277923 TKW 2D 569361770 C / G C CC GG Kasp-1185 AX-111571185 TKW 3D 520827884 C / T T TT TT Kasp-1007 AX-111821007 TKW 4A 14681814 C / T C CC TT Kasp-1956 AX-110361956 TKW 4B 75741547 C / G C GG CC

[0208] Note: XY60 is Xiaoyan 60, SY17 is Shiyou 17.

[0209] Table 19 shows that: when amplified by primer Kasp-1185, Shiyou 17 and Xiaoyan 60 are both TT genotypes, indicating that they are both excellent genotypes, and the corresponding site QTkw-3D (102.5-120.5cM) does not need further breeding. When amplified by primer Kasp-7923, Shiyou 17 is GG genotype, and Xiaoyan 60 is CC genotype, indicating that the SNP site AX-109277923 of Xiaoyan 60 is an excellent type. When amplified by primer Kasp-1007, Shiyou 17 is TT genotype, and Xiaoyan 60 is CC genotype, indicating that the SNP site AX-111821007 of Xiaoyan 60 is an excellent type. Amplified with primer Kasp-1956, Shiyou 17 was of CC genotype, and Xiaoyan 60 was of GG genotype, indicating that the SNP site AX-110361956 of Shiyou 17 was of excellent type.

[0210] That is, Xiaoyan 60 and Shiyou 17 have complementary typing results of 3 (n) SNP loci related to wheat thousand-grain weight under salt stress, among which Xiaoyan 60 was detected as an excellent genotype by SNP markers AX-111821007 (can be typed by Kasp-1007) and AX-109277923 (can be typed by Kasp-7923), indicating that it carries excellent variations of QTL loci QTkw-4A (14.68-16.28Mb) and QTkw-2D (126.5-137.5cM); Shiyou 17 was detected as an excellent genotype by SNP marker AX-110361956 (can be typed by Kasp-1956), indicating that it carries excellent variations of QTL loci QTkw-4B (26.5-34.5cM).

[0211] Table 20 Typing of KASP markers related to spike length or spikelet number under salt stress in candidate parents

[0212]

[0213] Note: XY60 is Xiaoyan 60, SY17 is Shiyou 17.

[0214] Table 20 shows that the SNP markers AX-111578827, AX-111675975, and AX-111449179 are consistent in parental typing, indicating that the QTL loci QS1-3B (62.13-62.14Mb, ​​Jia Ji Zeng, personal communication; 63.40Mb, Chinese Spring Genome IWGSC RefSeq v1.0), QS1-5B2 (583.48-584.89Mb), and Q-7A (22.75-22.88Mb) of the hybrid parents have the same variation type and cannot be used for the next breeding selection. Amplified with primer Kasp-2158, Shiyou 17 is AA genotype, and Xiaoyan 60 is GG genotype, indicating that the SNP locus AX-109972158 of Shiyou 17 belongs to the excellent type. Amplified with primer Kasp-0108, Shiyou 17 was GG genotype, Xiaoyan 60 was AA genotype, and SNP site AX-109460108 of Shiyou 17 was of excellent type. Amplified with primer Kasp-6259, Shiyou 17 was CC genotype, Xiaoyan 60 was AA genotype, indicating that SNP site AX-111016259 of Xiaoyan 60 was of excellent type. Amplified with primer Kasp-6563, Shiyou 17 was TT genotype, Xiaoyan 60 was GG genotype, indicating that SNP site AX-109996563 of Xiaoyan 60 was of excellent type.

[0215] That is, Xiaoyan 60 and Shiyou 17 have 4 (m) SNP marker typing results related to wheat ear traits under salt stress that are complementary, among which the SNP sites AX-111016259 (can be typed with Kasp-6259) and AX-109996563 (can be typed with Kasp-6563) of Xiaoyan 60 are excellent genotypes, indicating that they carry the QTL sites QSps-2D (124.5-129.5cM) and QSps- 5B (90.5-92.5cM) is an excellent variation; the SNP sites AX-109972158 (which can be typed as Kasp-2158) and AX-109460108 (which can be typed as Kasp-0108) of Shiyou 17 are excellent genotypes, indicating that they carry the excellent variations of QTL sites QSl-1B (674.96-675.13Mb) and QSps-1D (152.5-155.0cM).

[0216] The above experimental results showed that Xiaoyan 60 and Shiyou 17 both carried superior alleles of the SNP locus AX-111571185 that controls thousand-grain weight under salt stress and the SNP loci AX-111675975 and AX-111449179 for ear length. In addition, Xiaoyan 60 carries the superior alleles of the SNP sites AX-109277923 and AX-111821007 that control thousand-grain weight under salt stress, while Shiyou 17 carries the superior allele of AX-110361956; Xiaoyan 60 carries the superior alleles of the SNP sites AX-111016259 and AX-109996563 that control the number of spikelets under salt stress, while Shiyou 17 carries the superior alleles of the SNP sites AX-109972158 and AX-109460108 that control the number of spikelets under salt stress. The superior sites of the two varieties are complementary. Therefore, Shiyou 17 and Xiaoyan 60 are selected as parents for hybridization, which can complement each other and achieve the aggregation of superior alleles, and Shiyou 17 and Xiaoyan 60 are selected as a pair of candidate hybrid parents.

[0217] 1.2 Analysis of salt tolerance phenotypes of candidate hybrid parents to determine hybrid parents

[0218] Analysis of candidate hybrid parents Shiyou 17 and Xiaoyan 60.

[0219] The wheat variety Xiaoyan 60 is a salt-tolerant and drought-resistant wheat variety bred by the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences. It is currently being demonstrated and promoted in Heilonggang area of ​​Hebei Province. This variety is a semi-winter mid-maturing variety. The seedlings are semi-creeping, the leaves are light green, and the tillering ability is strong. The plant type is semi-compact. The awns are long, the number of grains per ear is 26.9, and the maturity is good. The shell is white, the grains are hard, the grains are full, and the thousand-grain weight is 44.5 grams. According to the determination of the Hebei Provincial Crop Variety Quality Testing Center in 2016, the crude protein (dry basis) is 14.74%, the wet gluten (14% wet basis) is 35.1%, the formation time is 3.2 minutes, the stability time is 3.0 minutes, and the bulk density is 797 grams / liter. It passed the Hebei Provincial Approval in 2016 (Hebei Shenmai No. 2016030), and passed the National Approval in 2022 (National Shenmai No. 20220069).

[0220] Wheat variety Shiyou 17 is a high-quality wheat variety selected and bred by Shijiazhuang Academy of Agricultural Sciences. This variety is semi-winter. The seedlings are semi-creeping, with green leaves and strong tillering ability. The mature plants are compact. They have long awns, white shells, white grains, and are hard and full. The number of grains per ear is 32.3, and the thousand-grain weight is 38.5 grams. According to the test of Hebei Province Crop Variety Quality Testing Center, in 2006, the crude protein was 13.97%, the wet gluten was 30.2%, the water absorption rate was 60.7%, and the stable time was 15.7 minutes; in 2007, the protein was 13.65%, the wet gluten was 30.5%, the water absorption rate was 59.4%, and the stable time was 19.5 minutes. It passed the approval of Hebei Province in 2007, and the approval number is: Ji Shen Mai 2007007.

[0221] Phenotypic photos of Shiyou 17 (left) and Xiaoyan 60 (right) at the adult stage are shown in Figure 4 Through the description of variety characteristics, there are great differences in grain quality, number of grains per ear and 1000-grain weight between Xiaoyan 60 and Shiyou 17. In order to determine the performance of the two varieties in the field environment of saline land, a field experiment was designed at the Nanpi Ecological Agriculture Experimental Station of the Chinese Academy of Sciences (116°40'E, 38°00'N, 11m above sea level).

[0222] The soil salt content is 0.18% (m / m). Row spacing is 20 cm, plant spacing is 10 cm, 50 plants / row, and the plot area is 6 m 2 . The plot test was set up with 3 replicates. After seedlings emerged, there was no irrigation during the growth period, routine field management, and no serious pests and diseases and lodging during the growth period. Harvest individual plants at maturity, and harvest individual plants with consistent growth in the middle of each replicate row. Inspect plant height (cm), number of ears, ear length (cm), number of spikelets, number of ears, number of grains per plant, plant grain weight (g), and thousand-grain weight (g). In each replicate, 10-15 plants of each variety were inspected and the average value was taken. Among them, thousand-grain weight = (grain weight per plant / number of grains per plant) × 1000.

[0223] According to data statistics, when planted in 0.18% field salt land, there were significant differences in ear length, number of spikelets, number of grains per ear and thousand-grain weight between Xiaoyan 60 and Shiyou 17 ( Figure 5 ). The average spike length of Shiyou 17 is 1.28cm longer than that of Xiaoyan 60, the number of spikelets increases by 1.5, and the number of grains per spike increases by 6.3. The thousand-grain weight of Xiaoyan 60 is 5.23g higher than that of Shiyou 17. In summary, the two varieties have distinct characteristics and complementary traits under salt stress. In addition, Shiyou 17 is a high-quality wheat. When combined with Xiaoyan 60, it is expected to cultivate a new salt-tolerant, drought-resistant, and high-quality wheat line. Therefore, it was determined that Shiyou 17 and Xiaoyan 60 would be used as hybrid parents for the next step of breeding.

[0224] 2. Precise breeding of salt-tolerant offspring plants assisted by molecular markers

[0225] The salt-tolerant genetic loci of the offspring plants were identified and screened using KASP markers with different genotyping results from the two hybrid parents, and salt-tolerant phenotypes were combined to select salt-tolerant wheat varieties.

[0226] The details are as follows:

[0227] The hybrid combination was formed with Shiyou 17 as the female parent and Xiaoyan 60 as the male parent, and then backcrossed with Shiyou 17 twice to obtain BC2F1, which was then screened in the salt field (salt content was 0.18%). BC2F2 seeds were obtained by selfing BC2F1.

[0228] 2.1 Precision breeding of salt-tolerant BC2F2 plants

[0229] The KASP markers for salt-tolerant grain and ear traits that were genotyped differently in Shiyou 17 and Xiaoyan 60 were used to amplify and genotype BC2F2 plants and some wheat varieties (lines) to screen plants carrying superior alleles and having salt-tolerant phenotypes.

[0230] 2.1.1 Screening of superior alleles for grain trait loci in BC2F2 individual plants

[0231] The KASP markers Kasp-1007, Kasp-7923 and Kasp-1956, which were related to different grain traits in Shiyou 17 and Xiaoyan 60, were used to scan 15 BC2F2 plants. The results are shown in Table 21: The amplification results of Kasp-1007 were all TT, indicating that the SNP loci were all unfavorable genotypes ( Figure 6 ), none of them carried the excellent variation of the site QTkw-4A (14.68-16.28Mb); the amplification results of Kasp-1956 were all CC, indicating that the SNP sites were all excellent genotypes ( Figure 7 ), all carried the superior variation of the locus QTkw-4B (26.5-34.5 cM); the amplification results of Kasp-7923 showed separation, indicating that the genotypes of this locus were different in different individuals. The locus QTkw-2D (126.5-137.5 cM) of the BC2F2 population was not homozygous. Individuals 16NB4-7 and 16NB4-48 were homozygous excellent genotypes CC, and 4 other individuals including 16NB4-18 carried heterozygous genotypes CG. The remaining 9 individuals including 16NB4-15 were homozygous poor genotypes GG ( Figure 8 ).

[0232] Table 21 Genotypes of 1000-grain weight of BC2F2 plants

[0233]

[0234] 2.1.2 Screening of superior alleles for ear trait loci in BC2F2 individual plants

[0235] Fifteen BC2F2 plants were scanned using KASP markers Kasp-2158 for controlling spike length and KASP markers Kasp-0108, Kasp-6259 and Kasp-6563 for controlling spikelet number, which were genotyped differently in Shiyou 17 and Xiaoyan 60. The results are shown in Table 22: The amplification results of Kasp-2158 were all AA, indicating that its SNP locus was an excellent genotype ( Fig. 9 ), all carried the excellent variation of QTL site QSl-1B (674.96-675.13Mb); the amplification results of Kasp-0108 were all GG, indicating that its SNP site was an excellent genotype ( Fig.10 ), all carried the superior variation of site QSps-1D (152.5-155.0cM); the amplification results of Kasp-6259 showed separation, indicating that the genotypes of this site were different in different individuals, and the site QSps-2D (124.5-129.5cM) of the BC2F2 population was still not homozygous; 16NB4-7 and 16NB4-48 were homozygous superior genotypes AA, and 3 other individuals including 16NB4-18 carried heterozygous genotypes AC, and the remaining 9 individuals including 16NB4-15 were homozygous inferior genotypes CC ( Fig.11 ). The amplification results of Kasp-6563 were all TT, indicating that the SNP loci were all unfavorable genotypes and did not carry the superior variation of the locus QSps-5B (90.5-92.5cM) ( Fig.12 ).

[0236] Table 22 Genotypes of ear trait loci of BC2F2 individual plants

[0237]

[0238]

[0239] 2.1.3 Phenotypic investigation of BC2F2 individual plants

[0240] A total of 250 BC2F2 plants were planted in a salt field with a soil salt content of 0.18%. 17 of them were harvested to examine their phenotypes such as plant height, number of ears, ear length, number of spikelets, number of grains per ear, number of grains per plant, plant grain weight, and 1000-grain weight (Table 23). Among the grain-related phenotypes, the plant grain weight was 20.68-30.26g, the plant grain number was 433-740, and the 1000-grain weight was 40.89-49.30g. Among the ear-related phenotypes, the ear length was 8.0-10.0cm, the number of spikelets was 20-23, and the number of grains per ear was 50-59. Among the other phenotypes, the plant height was 71.0-80.8cm and the number of ears was 5-9. With plant height ≤75cm, ear length ≥8.5cm, and 1000-grain weight ≥45.00g as the selection criteria, a total of 7 individual plants were obtained, including 16NB-7, 16NB-18, 16NB-48, 16NB-51, 16NB-98, 16NB-106 and 16NB-188.

[0241] Table 23 BC2F2 single plant agronomic traits survey table

[0242] serial number Plant height (cm) Number of ears Ear length (cm) number of spikelets Number of grains per ear Plant weight (g) Plant grain number Thousand-grain weight (g) 16NB-7 75.0 6 9.5 22 58 21.92 485 45.20 16NB-15 76.0 9 9.0 21 57 23.39 509 45.96 16NB-18 73.0 6 10.0 22 59 24.47 515 47.52 16NB-48 74.5 5 9.0 21 50 24.12 516 46.74 16NB-51 73.0 5 9.0 21 55 21.79 499 43.67 16NB-98 74.7 8 9.0 21 57 20.88 451 46.30 16NB-106 71.0 7 8.5 20 56 21.35 433 49.30 16NB-128 75.3 5 9.2 21 55 20.68 455 45.45 16NB-135 72.4 9 8.2 21 57 21.42 477 44.91 16NB-188 75.0 8 8.8 21 54 25.47 563 45.25 16NB-189 73.0 9 8.0 21 55 22.24 504 44.13 16NB-190 80.8 8 9.5 21 55 30.26 740 40.89 16NB-213 77.5 8 8.0 21 55 21.84 467 46.77 16NB-223 79.5 7 8.7 21 56 22.51 489 46.03 16NB-224 77.0 7 9.0 21 56 22.04 489 45.06 16NB-230 76.8 7 9.5 23 58 22.11 475 46.56 16NB-235 77.7 8 9.4 22 57 26.38 580 45.48

[0243] Under salt stress, 7 plants, including 16NB-7, 16NB-18, 16NB-48, 16NB-51, 16NB-98, 16NB-106 and 16NB-188, all carried superior alleles controlling grain and ear traits from their parents. For loci with different genotypes of their parents, 16NB-7 and 16NB-48 aggregated two homozygous superior loci for grain-related and three ear-related traits; 16NB-18 and 16NB-106 aggregated one homozygous superior loci for grain-related and two ear-related traits, and carried one heterozygous locus for grain-related and one ear-related trait; 16NB-51, 16NB-98 and 16NB-188 aggregated one homozygous superior locus for grain-related and two ear-related traits. Based on the results of superior loci analysis and phenotypic identification, 100 full seeds from each of the above 7 plants were selected as BC2F3 seeds and planted in the saline fields of Nanpi Eco-Agriculture Experimental Station of the Chinese Academy of Sciences.

[0244] 2.2 Precision breeding of salt-tolerant BC2F3 plants

[0245] 2.2.1 Screening of superior variants for loci related to grain and ear traits in BC2F3

[0246] A total of 7 BC2F3 lines were planted in saline fields with a soil salt content of 0.18%. Based on the field performance, 26 plants from 6 of the lines were selected for subsequent genotype analysis.

[0247] According to the amplification results of the parental varieties Shiyou 17 and Xiaoyan 60 and BC2F2 plants, the grain-related SNP sites AX-111821007, AX-110361956 and the ear-related SNP sites AX-109972158, AX-109460108, AX-109996563 were all homozygous genotypes, while the grain-related marker AX-109277923 and the ear-related marker AX-111016259 were still not homozygous. Therefore, the markers Kasp-7923 and Kasp-6259 were selected to amplify the 26 BC2F3 plants of the selected 6 strains, and the results are shown in Table 24.

[0248] Table 24 Genotyping of grain weight and ear trait loci of BC2F3 individual plants

[0249]

[0250] The results of Kasp-7923 amplification showed that all the individuals from the lineages 17N284 and 17N287 carried the homozygous superior genotype CC; the individuals from the lineages 17N286 and 17N290 showed genotype separation, among which 17N286-9 and 17N286-37 carried the homozygous superior genotype CC, 17N290-4, 17N290-9 and 17N290-10 carried the heterozygous genotype CG, and the remaining individuals carried the inferior genotype GG; all the individuals from the lineage 17N288 and 17N293-1 carried the inferior genotype GG ( Fig.13 ). Kasp-6259 amplification results showed that all the individuals from group 17N284 carried the homozygous superior genotype AA; all the individuals from group 17N288 and 17N293-1 carried the inferior genotype CC; the individuals from groups 17N286, 17N287, and 17N290 showed genotype separation, among which 17N286-9, 17N286-37, 17N287-2 , 17N287-4, 17N287-6, 17N287-8, 17N287-131, 17N290-4, 17N290-9 and 17N290-10 carry the homozygous superior genotype AA, 17N287-1 carries the heterozygous genotype AC, and 17N286-1, 17N286-6, 17N286-8 and 17N290-1 carry the inferior genotype CC ( Fig.14 ). Considering that the genotype of the previous generation 16NB-48 of population 17N287 was AA when primer Kasp-6259 was used to amplify the genotype, the AC genotype of the single plant 17N287-1 may be derived from outcrossing.

[0251] 2.2.2 Phenotypic investigation of BC2F3 individual plants

[0252] 25 individual plants of the BC2F3 line planted in a salt field with a soil salt content of 0.18% were harvested and their phenotypes such as plant height, number of ears, ear length, number of spikelets, number of grains per ear, number of grains per plant, grain weight per plant, and 1000-grain weight were investigated (Table 25). Among the grain-related phenotypes, the grain weight per plant was 8.77-25.15 g, the number of grains per plant was 189-558, and the 1000-grain weight was 39.03-50.03 g. Among the ear-related phenotypes, the ear length was 7.5-8.6 cm, the number of spikelets was 14-19, and the number of grains per ear was 36-55. Among the other phenotypes, the plant height was 41.5-69.0 cm and the number of ears was 6-14. With grain weight ≥15.0 g per plant as the selection criterion, 11 individual plants were selected, including 17N286-9, 17N286-37, 17N287-2, 17N287-4, 17N287-8, 17N287-131, 17N288-8, 17N288-10, 17N290-1, 17N290-4 and 17N290-9.

[0253] Table 25 BC2F3 single plant agronomic traits survey table

[0254]

[0255] Under salt stress, 11 plants, including 17N286-9, 17N286-37, 17N287-2, 17N287-4, 17N287-8, 17N287-131, 17N288-8, 17N288-10, 17N290-1, 17N290-4, and 17N290-9, all carried the homozygous QTkw-3D (SNP site AX-1115 71185), QTkw-4B (SNP site AX-110361956), QSl-1B (SNP site AX-109972158), QSl-5B2 (SNP site AX-111675975), Q-7A (SNP site AX-111449179) and QSps-1D (SNP site AX-109460108) excellent genotypes. In addition, all the individuals from population 17N284 and the individuals 17N286-9, 17N286-37, 17N287-2, 17N287-4, 17N287-8, and 17N287-131 carried the homozygous superior loci of QTkw-2D (SNP site AX-109277923) and QSps-2D (SNP site AX-111016259); The loci QTkw-2D (SNP loci AX-109277923) and QSps-2D (SNP loci AX-111016259) of 90-9 are heterozygous; the loci QTkw-2D (SNP loci AX-109277923) and QSps-2D (SNP loci AX-111016259) of 17N288-8, 17N288-10 and 17N290-1 are homozygous unfavorable genotypes. 300 full seeds of each of the above 11 individual plants, i.e., BC2F4 generation seeds, were selected and planted in the salt field of the Nanpi Ecological Agriculture Experimental Station of the Chinese Academy of Sciences.

[0256] 2.3 Investigation of agronomic traits of BC2F4 strain and naming of Xiaoyan 156

[0257] Eleven BC2F4 generation materials planted in saline fields were observed, and nine new lines with consistent field phenotypes were selected. Six individual plants with consistent growth were selected from each line. The mean values ​​and standard deviations of traits such as plant height, number of ears, ear length, number of spikelets, number of grains per ear, number of grains per plant, grain weight per plant, and 1000-grain weight were investigated. SPSS software (IBM) was used for significance analysis. All remaining individual plants were threshed, and the total yield of the plot was calculated. The results are shown in Table 26.

[0258] Table 26 BC2F4 generation agronomic traits survey table

[0259]

[0260] The average plant height of the 9 lines was 68.1-76.2cm, the number of ears was 7-9, the ear length was 8.3-8.9cm, the number of spikelets was 19-21, the number of grains per ear was 46-54, the plant weight was 13.88-16.72g, the number of grains per plant was 304-358, and the thousand-grain weight was 43.81-49.44g. Comparison of the grain and ear traits of each line with the parents showed that the average thousand-grain weight of all lines was higher than that of the parent Shiyou 17, the average ear length and number of grains per ear of all lines were greater than that of the parent Xiaoyan 60, and the average number of spikelets was greater than or equal to Xiaoyan 60, indicating that effective selection of grain and ear traits under salt stress conditions was achieved in the new lines. The yields of the 9 plots were 2833-3298g, among which the yields of 18N210 and 18N218 were higher than those of the control Shiyou 17 and Xiaoyan 60, which were 3298g and 3248g respectively.

[0261] The 18N210 line with the highest yield was named Xiaoyan 156, with an average plant height of 74.2 cm, 9 ears, 8.3 cm ear length, 19 spikelets, 51 grains per ear, 16.72 g plant weight, 352 grains per plant, and 47.61 g thousand-grain weight. The T test of agronomic traits of Xiaoyan 156 and its parents showed that the thousand-grain weight of Xiaoyan 156 was significantly higher than that of Shiyou 17, the ear length was significantly longer than that of Xiaoyan 60, and the number of grains per ear was significantly more than that of Xiaoyan 60, indicating that Xiaoyan 156 successfully combined the advantages of high thousand-grain weight of its parent Xiaoyan 60 and long ears and more grains per ear of Shiyou 17, achieving the breeding goal.

[0262] 3 Analysis of superior variation of the new designed salt-tolerant wheat line Xiaoyan 156

[0263] Using KASP marker amplification, Xiaoyan 156 carried three excellent loci related to grains, QTkw-2D (SNP locus AX-109277923), QTkw-3D (SNP locus AX-111571185) and QTkw-4B (SNP locus AX-110361956), of which the excellent locus QTkw-2D (SNP locus AX-109277923) from Xiaoyan 60 and the excellent locus QTkw-4B (SNP locus AX-110361956) from Shiyou 17 were successfully aggregated in Xiaoyan 156. For the excellent genotype CC of the locus QTkw-4B (SNP locus AX-110361956) carried by the recurrent parent Shiyou 17, all the loci in the BC2F2 plants were homozygous excellent genotypes. For the other parent, Xiaoyan 60, which carries the excellent genotype CC at the site QTkw-2D (SNP site AX-109277923), only 2 of the 15 BC2F2 plants carried the homozygous excellent genotype. The superior allele type of this site in Xiaoyan 156 was obtained by precisely selecting the plant 16NB-48 carrying the homozygous excellent genotype in the BC2F2 generation, which was inherited by the BC2F3 plant 17PD287-2. Xiaoyan 156 also carries five excellent loci related to ear traits, namely QSl-1B (SNP locus AX-109972158), QSps-1D (SNP locus AX-109460108), QSps-2D (SNP locus AX-111016259), QSl-5B2 (SNP locus AX-111675975) and Q-7A (SNP locus AX-111449179), among which the excellent loci QSl-1B and QSps-1D derived from Shiyou 17 and the excellent locus QSps-2D derived from Xiaoyan 60 were successfully aggregated in Xiaoyan 156 (Table 27). For the excellent genotypes AA and GG of the loci QSl-1B and QSps-1D carried by the recurrent parent Shiyou 17, these two loci in the BC2F2 individual plants were homozygous excellent genotypes. For the other parent, Xiaoyan 60, which carries the excellent genotype AA at the QSps-2D locus, only 2 of the 15 BC2F2 plants carried the homozygous excellent genotype. The excellent allele type of this locus in Xiaoyan 156 was also due to the accurate retention of the single plant 16NB-48 carrying the homozygous excellent genotype in the BC2F2 generation, and then inherited by the BC2F3 single plant 17PD287-2.

[0264] When examining agronomic traits under salt stress, the BC2F2 generation focused on screening individual plants with moderate plant height, longer ear length and higher 1000-grain weight, the BC2F3 generation focused on screening individual plants with higher grain weight, and the BC2F4 generation focused on screening plant lines with higher plot yield.

[0265] In summary, with the help of KASP markers, the precise selection of parental materials, BC2F2 plants, and BC2F3 plants under salt stress, combined with the examination of wheat agronomic traits under combined salt stress, the directed design breeding of new salt-tolerant wheat lines was achieved.

[0266] Table 27 The excellent loci for wheat grain weight and ear traits under salt stress carried by Xiaoyan 156

[0267]

[0268] Note: XY60 is Xiaoyan 60, SY17 is Shiyou 17, XY56 is Xiaoyan 156.

[0269] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A molecular design breeding method for salt-tolerant wheat, characterized in that: The method comprises the following steps: genotyping multiple wheat varieties with multiple SNP loci for controlling grain traits and ear traits of wheat under salt stress, selecting paired wheat varieties with complementary typing results of salt-tolerant grain traits and ear traits SNP loci as candidate hybrid parents; selecting a pair of candidate hybrid parents with good salt tolerance and complementary advantages as two hybrid parents by analyzing the salt-tolerant phenotype; genotyping the hybrid offspring single plants at the SNP loci with complementary typing results of the two parents, using the typing results to assist in screening salt-tolerant offspring single plants, and breeding salt-tolerant wheat varieties in combination with the salt-tolerant phenotype; the method comprises the following steps: 1) performing genotyping on multiple wheat varieties using N salt-tolerant grain trait SNP loci and M salt-tolerant ear trait SNP loci, selecting pairs of wheat varieties with complementary typing results of n salt-tolerant grain trait SNP loci and m salt-tolerant ear trait SNP loci from the multiple wheat varieties as candidate hybrid parents, determining a pair from the candidate hybrid parents as two hybrid parents by analyzing the salt-tolerant phenotype, and obtaining offspring individual plants; 2) performing genotyping of the n SNP loci of salt-tolerant grain traits and the m SNP loci of salt-tolerant ear traits on the offspring individual plants, using the genotyping results to assist in screening salt-tolerant offspring individual plants, and breeding salt-tolerant wheat varieties in combination with the salt-tolerant phenotype; Wherein, N≥n≥2, M≥m≥2, N, n, M, m are all positive integers; The M SNP sites of salt-tolerant ear traits are selected from AX-109972158, AX-109460108, AX-111016259, AX-111578827, AX-109996563, AX-111675975 and AX-111449179: The AX-109972158 is a SNP site on chromosome 1B, and its nucleotide type is A or G, which is the 36th nucleotide of SEQ ID No. 17; the nucleotide type of the superior gene variation of the SNP site is A, and the homozygous superior genotype is AA; The AX-109460108 is a SNP site on chromosome 1D, and its nucleotide type is A or G, which is the 36th nucleotide of SEQ ID No. 21; the nucleotide type of the superior gene variation of the SNP site is G, and the homozygous superior genotype is GG; The AX-111016259 is a SNP site on chromosome 2D, and its nucleotide type is A or C, which is the 36th nucleotide of SEQ ID No. 25; the nucleotide type of the superior gene variation of the SNP site is A, and the homozygous superior genotype is AA; The AX-111578827 is a SNP site on chromosome 3B, and its nucleotide type is A or G, which is the 36th nucleotide of SEQ ID No. 29; the nucleotide type of the superior gene variation of the SNP site is A, and the homozygous superior genotype is AA; The AX-109996563 is a SNP site on chromosome 5B, and its nucleotide type is G or T, which is the 36th nucleotide of SEQ ID No. 33; the nucleotide type of the superior gene variation of the SNP site is G, and the homozygous superior genotype is GG; The AX-111675975 is a SNP site on chromosome 5B, the nucleotide type of which is A or G, and is the 36th nucleotide of SEQ ID No. 37; the nucleotide type of the superior gene variation of the SNP site is A, and the homozygous superior genotype is AA; The AX-111449179 is a SNP site on chromosome 7A, and its nucleotide type is C or T, which is the 36th nucleotide of SEQ ID No. 41; the nucleotide type of the excellent gene variation of the SNP site is C, and the homozygous excellent genotype is CC.

2. The method according to claim 1, characterized in that: The typing results are used to assist in screening salt-tolerant offspring plants, and plants with more excellent allelic variations and better salt-tolerant phenotypes are selected.

3. The method according to claim 2, characterized in that: The method of using the typing results to assist in screening the salt-tolerant offspring individual plants can be specifically: x The F2 generation selected plants that carried a salt-tolerant grain trait SNP locus as a homozygous superior genotype, b salt-tolerant grain trait SNP locus as a heterozygous genotype containing superior alleles, c salt-tolerant ear trait SNP locus as a homozygous superior genotype, d ear trait SNP locus as a heterozygous genotype containing superior alleles, and self-pollinated plants with salt-tolerant phenotypes to obtain B. x F3 generation; in B x The F3 generation performs genotyping on the b salt-tolerant grain trait SNP loci and the d salt-tolerant ear trait SNP loci; a, b, c, d, and x are all natural numbers, and 0 <a+b≤n,0<c+d≤m。 4. The method according to claim 1, characterized in that: The N SNP sites for salt-tolerant grain traits are selected from AX-109277923, AX-111571185, AX-111821007 and AX-110361956: The AX-109277923 is a SNP site on chromosome 2D, and its nucleotide type is C or G, which is the 36th nucleotide of SEQ ID No. 1; the nucleotide type of the superior gene variation of the SNP site is C, and the homozygous superior genotype is CC; The AX-111571185 is a SNP site on chromosome 3D, and its nucleotide type is C or T, which is the 36th nucleotide of SEQ ID No. 5; the nucleotide type of the superior gene variation of the SNP site is T, and the homozygous superior genotype is TT; The AX-111821007 is a SNP site on chromosome 4A, and its nucleotide type is C or T, which is the 36th nucleotide of SEQ ID No. 9; the nucleotide type of the superior gene variation of the SNP site is C, and the homozygous superior genotype is CC; The AX-110361956 is a SNP site on chromosome 4B, and its nucleotide type is C or G, which is the 36th nucleotide of SEQ ID No. 13; the nucleotide type of the excellent gene variation of the SNP site is C, and the homozygous excellent genotype is CC.

5. The method according to claim 4, characterized in that: Genotyping is performed using a primer set, wherein the primer set consists of at least two of the four combinations of primer set 1 to primer set 4, The primer set for genotyping the AX-109277923 is primer set 1, and the primer set 1 is 1-1 or 1-2: 1-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 48 of SEQ ID No. 2, a single-stranded DNA having a nucleotide sequence containing positions 22 to 48 of SEQ ID No. 3, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 4; 1-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 2, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 3, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 4; The primer set for genotyping the AX-111571185 is primer set 2, and the primer set 2 is 2-1 or 2-2: 2-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 48 of SEQ ID No. 6, a single-stranded DNA having a nucleotide sequence containing positions 22 to 49 of SEQ ID No. 7, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 8; 2-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.6, a single-stranded DNA having a nucleotide sequence of SEQ ID No.7, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.8; The primer set for genotyping the AX-111821007 is primer set 3, and the primer set 3 is 3-1 or 3-2: 3-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 41 of SEQ ID No. 10, a single-stranded DNA having a nucleotide sequence containing positions 22 to 41 of SEQ ID No. 11, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 12; 3-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 10, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 11, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 12; The primer set for genotyping the AX-110361956 is primer set 4, and the primer set 4 is 4-1 or 4-2: 4-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 53 of SEQ ID No. 14, a single-stranded DNA having a nucleotide sequence containing positions 22 to 53 of SEQ ID No. 15, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 16; 4-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.14, a single-stranded DNA having a nucleotide sequence of SEQ ID No.15, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.

16.

6. The method according to any one of claims 1 to 4, characterized in that: Genotyping is performed using a primer set, wherein the primer set consists of at least two of the seven combinations of primer set 5 to primer set 11, The primer set for genotyping the AX-109972158 is primer set 5, and the primer set 5 is 5-1 or 5-2: 5-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 47 of SEQ ID No. 18, a single-stranded DNA having a nucleotide sequence containing positions 22 to 46 of SEQ ID No. 19, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 20; 5-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.18, a single-stranded DNA having a nucleotide sequence of SEQ ID No.19, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.20; The primer set for genotyping the AX-109460108 is primer set 6, and the primer set 6 is 6-1 or 6-2: 6-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 41 of SEQ ID No. 22, a single-stranded DNA having a nucleotide sequence containing positions 22 to 41 of SEQ ID No. 23, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 24; 6-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 22, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 23, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 24; The primer set for genotyping the AX-111016259 is primer set 7, and the primer set 7 is 7-1 or 7-2: 7-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 45 of SEQ ID No. 26, a single-stranded DNA having a nucleotide sequence containing positions 22 to 45 of SEQ ID No. 27, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 28; 7-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 26, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 27, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 28; The primer set for genotyping the AX-111578827 is primer set 8, and the primer set 8 is 8-1 or 8-2: 8-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 46 of SEQ ID No. 30, a single-stranded DNA having a nucleotide sequence containing positions 22 to 46 of SEQ ID No. 31, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 32; 8-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No. 30, a single-stranded DNA having a nucleotide sequence of SEQ ID No. 31, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 32; The primer set for genotyping the AX-109996563 is primer set 9, and the primer set 9 is 9-1 or 9-2: 9-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 43 of SEQ ID No. 34, a single-stranded DNA having a nucleotide sequence containing positions 22 to 43 of SEQ ID No. 35, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 36; 9-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.34, a single-stranded DNA having a nucleotide sequence of SEQ ID No.35, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.36; The primer set for genotyping the AX-111675975 is primer set 10, and the primer set 10 is 10-1 or 10-2: 10-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 44 of SEQ ID No. 38, a single-stranded DNA having a nucleotide sequence containing positions 22 to 44 of SEQ ID No. 39, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 40; 10-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.38, a single-stranded DNA having a nucleotide sequence of SEQ ID No.39, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.40; The primer set for genotyping the AX-111449179 is primer set 11, and the primer set 11 is 11-1 or 11-2: 11-1. A composition consisting of a single-stranded DNA having a nucleotide sequence containing positions 22 to 49 of SEQ ID No. 42, a single-stranded DNA having a nucleotide sequence containing positions 22 to 51 of SEQ ID No. 43, and a single-stranded DNA having a nucleotide sequence of SEQ ID No. 44; 11-2. A composition consisting of a single-stranded DNA having a nucleotide sequence of SEQ ID No.42, a single-stranded DNA having a nucleotide sequence of SEQ ID No.43, and a single-stranded DNA having a nucleotide sequence of SEQ ID No.

44.

7. Application of SNP loci for salt-tolerant ear traits in wheat breeding; the SNP loci for salt-tolerant ear traits are selected from two or more of AX-109972158, AX-109460108, AX-111016259, AX-111578827, AX-109996563, AX-111675975 and AX-111449179 described in claim 1.

8. Use of the primer set described in claim 5 or 6 in wheat breeding.

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