Molecular marker related to grain width and thousand grain weight characters of sorghum and application of molecular marker

Abstract

The invention belongs to the technical field of biology, and particularly relates to a molecular marker related to grain width and thousand grain weight characters of sorghum and application of the molecular marker. According to the molecular marker provided by the invention, excellent haplotypes SbTGW3-hap5 and SbGS5-hap6 can be accurately and efficiently screened out, and both the excellent haplotypes SbTGW3-hap5 and SbGS5-hap6 are sorghum with large grain width and high thousand grain weight. The molecular marker provided by the invention is closely linked with the grain width and thousand seed weight characters of sorghum, the excellent haplotype sorghum germplasm material can be quickly, stably and efficiently identified at low cost based on the molecular marker provided by the invention, the grain width and thousand seed weight characters of sorghum can be identified, the accuracy rate is extremely high, and a foundation is laid for breeding of sorghum with large grain width and high thousand seed weight.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a molecular marker related to sorghum grain width and 1000-grain weight traits and an application thereof. Background Art

[0002] Sorghum has become a globally important crop due to its drought tolerance and adaptability to adverse conditions, but its average yield per unit area is significantly lower than that of other cereals such as corn and rice. Currently, sorghum germplasm breeding for grain size is still primarily based on macroselection. However, traditional breeding methods for sorghum yield traits have reached a bottleneck.

[0003] Functional markers are molecular markers that associate gene alleles with phenotypic traits based on candidate gene polymorphisms. The development of molecular markers associated with specific traits is the core method of marker-assisted breeding. Among them, KASP (competitive allele-specific PCR) technology is a high-throughput genotyping technology based on SNPs, which has played an important role in crop improvement and marker-assisted breeding research in recent years. It has accuracy, flexibility, rapid analysis and good visualization effects. The use of SNP molecular markers to discover and utilize excellent genetic resources, screen out germplasm materials carrying target genotypes, and is of great significance for the directed transfer of target traits, as well as the creation of new sorghum germplasm in sorghum breeding, and the improvement of grain width and 1000-grain weight. Summary of the Invention

[0004] The purpose of the present invention is to develop molecular markers related to sorghum grain width and 1000-grain weight traits, to identify sorghum grain width and 1000-grain weight traits quickly, stably, efficiently and at low cost, and to lay the foundation for the breeding of sorghum with large grain width and high 1000-grain weight.

[0005] The present invention provides a molecular marker related to sorghum grain width and 1000-grain weight traits, wherein the molecular marker comprises a first molecular marker combination and / or a second molecular marker combination;

[0006] The first molecular marker combination includes TGW3-SNP1 and TGW3-SNP2;

[0007] The second molecular marker combination includes GS5-SNP1 and GS5-SNP2;

[0008] The TGW3-SNP1 is located at 1529622bp of chromosome 1 of the sorghum genome, and the polymorphism is G / A; the TGW3-SNP2 is located at 1526093bp of chromosome 1 of the sorghum genome, and the polymorphism is T / G;

[0009] The GS5-SNP1 is located at 5404386bp of chromosome 9 in the sorghum genome, and the polymorphism is T / C; the GS5-SNP2 is located at 5403747bp of chromosome 9 in the sorghum genome, and the polymorphism is A / T.

[0010] Preferably, the TGW3-SNP1 is located at 196 bp of the nucleotide sequence shown in SEQ ID NO: 13, and the polymorphism is G / A; the TGW3-SNP2 is located at 196 bp of the nucleotide sequence shown in SEQ ID NO: 14, and the polymorphism is T / G;

[0011] The GS5-SNP1 is located at 181 bp of the nucleotide sequence shown in SEQ ID NO: 15, and the polymorphism is T / C; the GS5-SNP2 is located at 249 bp of the nucleotide sequence shown in SEQ ID NO: 16, and the polymorphism is A / T;

[0012] In SEQ ID NOs: 13 to 16, R, W, S, K, M and Y are degenerate bases, respectively.

[0013] The present invention also provides a primer combination for detecting the molecular markers described in the above technical solution, wherein the primer combination includes a first primer combination for detecting the first molecular marker combination of the above technical solution and / or a second primer combination for detecting the second molecular marker combination of the above technical solution;

[0014] The first primer combination includes a TGW3-SNP1 primer combination for detecting TGW3-SNP1 described in the above technical solution and a TGW3-SNP2 primer combination for detecting TGW3-SNP2 described in the above technical solution;

[0015] The TGW3-SNP1 primer combination includes TGW3-SNP1-F1 with a nucleotide sequence as shown in SEQ ID NO: 17, TGW3-SNP1-F2 with a nucleotide sequence as shown in SEQ ID NO: 18, and TGW3-SNP1-R with a nucleotide sequence as shown in SEQ ID NO: 3; the 5' ends of the TGW3-SNP1-F1 and the TGW3-SNP1-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the TGW3-SNP1-F1 and the TGW3-SNP1-F2 excite different fluorescence colors;

[0016] The TGW3-SNP2 primer combination includes TGW3-SNP2-F1 with a nucleotide sequence as shown in SEQ ID NO: 19, TGW3-SNP2-F2 with a nucleotide sequence as shown in SEQ ID NO: 20, and TGW3-SNP2-R with a nucleotide sequence as shown in SEQ ID NO: 6; the 5' ends of the TGW3-SNP2-F1 and the TGW3-SNP2-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the TGW3-SNP2-F1 and the TGW3-SNP2-F2 excite different fluorescence colors;

[0017] The second primer combination includes a GS5-SNP1 primer combination for detecting GS5-SNP1 described in the above technical solution and a GS5-SNP2 primer combination for detecting GS5-SNP2 described in the above technical solution;

[0018] The GS5-SNP1 primer combination includes GS5-SNP1-F1 with a nucleotide sequence as shown in SEQ ID NO: 21, GS5-SNP1-F2 with a nucleotide sequence as shown in SEQ ID NO: 22, and GS5-SNP1-R with a nucleotide sequence as shown in SEQ ID NO: 9; the 5' ends of the GS5-SNP1-F1 and the GS5-SNP1-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the GS5-SNP1-F1 and the GS5-SNP1-F2 excite different fluorescence colors; wherein, R in SEQ ID NO: 21 and SEQ ID NO: 22 is a degenerate base;

[0019] The GS5-SNP2 primer combination includes GS5-SNP2-F1 with a nucleotide sequence as shown in SEQ ID NO:23, GS5-SNP2-F2 with a nucleotide sequence as shown in SEQ ID NO:24, and GS5-SNP2-R with a nucleotide sequence as shown in SEQ ID NO:12; the 5' ends of the GS5-SNP2-F1 and the GS5-SNP2-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the GS5-SNP2-F1 and the GS5-SNP2-F2 excite different fluorescence colors.

[0020] Preferably, the fluorescent groups connected to TGW3-SNP1-F1, TGW3-SNP2-F1, GS5-SNP1-F1 and GS5-SNP2-F1 are respectively FAM.

[0021] The fluorescent groups connected to the TGW3-SNP1-F2, TGW3-SNP2-F2, GS5-SNP1-F2 and GS5-SNP2-F2 are HEX;

[0022] The present invention also provides a kit for detecting the molecular markers described in the above technical solution, comprising the primer combination described in the above technical solution.

[0023] The present invention also provides the use of a substance for detecting a molecular marker as described in the above technical solution, or a primer combination as described in the above technical solution, or a kit as described in the above technical solution in one or more of the following:

[0024] (1) Identify or assist in identifying sorghum with large kernel width and high 1000-kernel weight;

[0025] (2) screening or assisting in screening sorghum with large kernel width and high 1000-kernel weight;

[0026] (3) Identifying or assisting in identifying haplotype sorghum; the haplotype sorghum includes SbTGW3-hap5 haplotype sorghum and / or SbGS5-hap6 haplotype sorghum;

[0027] (4) Screening or auxiliary screening of haplotype sorghum; the haplotype sorghum includes SbTGW3-hap5 haplotype sorghum and / or SbGS5-hap6 haplotype sorghum.

[0028] The present invention also provides a method for identifying sorghum haplotypes and / or identifying sorghum grain width and 1000-grain weight traits, wherein the haplotype is a SbTGW3-hap5 haplotype and / or a SbGS5-hap6 haplotype, including the following I and / or II:

[0029] I: Using the gene DNA of the sorghum to be tested as a template, PCR amplification is performed using the TGW3-SNP1 primer combination and the TGW3-SNP2 primer combination in the primer combination described in the above technical solution, respectively, to obtain a TGW3-SNP1 amplification product and a TGW3-SNP2 amplification product; and the haplotype and / or grain width and 1000-grain weight traits of the sorghum to be tested are determined based on the TGW3-SNP1 amplification product and the TGW3-SNP2 amplification product:

[0030] The TGW3-SNP1 amplification product is DNA fragment 1 and the TGW3-SNP2 amplification product is DNA fragment 2, then the haplotype of the sorghum to be tested is SbTGW3-hap5, and the sorghum has a larger grain width and a higher 1000-grain weight; the genotype corresponding to the 1529622 bp position of chromosome 1 of the sorghum genome in the DNA fragment 1 is GG; the genotype corresponding to the 1526093 bp position of chromosome 1 of the sorghum genome in the DNA fragment 2 is GG;

[0031] II: Using the genomic DNA of the sorghum to be tested as a template, PCR amplification is performed using the GS5-SNP1 primer combination and the GS5-SNP2 primer combination described in the above technical solution to obtain a GS5-SNP1 amplification product and a GS5-SNP2 amplification product; the haplotype of the sorghum to be tested is determined based on the GS5-SNP1 amplification product and the GS5-SNP2 amplification product:

[0032] The GS5-SNP1 amplification product is DNA fragment 3 and the GS5-SNP2 amplification product is DNA fragment 4, then the haplotype of the sorghum to be tested is SbGS5-hap6, and it is a sorghum with a larger grain width and a higher 1000-grain weight; the genotype corresponding to the 5404386bp of chromosome 9 of the sorghum genome in the DNA fragment 3 is CC; the genotype corresponding to the 5403747bp of chromosome 9 of the sorghum genome in the DNA fragment 4 is AA.

[0033] The present invention also provides the application of the method described in the above technical solution in the breeding or auxiliary breeding of sorghum with large grain width and high 1000-grain weight.

[0034] The present invention also provides a method for cultivating sorghum with large grain width and high 1000-grain weight, comprising the following steps:

[0035] Identify the haplotype of sorghum according to the method described in the above technical solution;

[0036] Sorghum with the SbTGW3-hap5 haplotype and / or the SbGS5-hap6 haplotype was retained as a parent for breeding.

[0037] Beneficial effects:

[0038] The molecular markers provided by the present invention include a first molecular marker combination and / or a second molecular marker combination; the first molecular marker combination includes TGW3-SNP1 and TGW3-SNP2; the second molecular marker combination includes GS5-SNP1 and GS5-SNP2. The molecular markers provided by the present invention can accurately and efficiently screen out excellent genotypes SbTGW3-hap5 and SbGS5-hap6, and SbTGW3-hap5 and SbGS5-hap6 are both sorghums with large grain width and high thousand-grain weight. The molecular markers provided by the present invention are closely linked to the grain width and thousand-grain weight traits of sorghum. Based on the molecular markers provided by the present invention, high-quality haplotype sorghum can be identified quickly, stably, efficiently and at low cost, and the grain width and thousand-grain weight traits of sorghum can be identified, laying the foundation for the breeding of large grain width and high thousand-grain weight sorghum. The results of the embodiment show that the accuracy of typing using the first molecular marker combination provided by the present invention is 98.88%, and the accuracy of screening for excellent genotypes is 100%. The accuracy of typing using the second molecular marker combination provided by the present invention is 94.9%, and the accuracy of screening for excellent genotypes is 100%. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0040] Figure 1 The results of association analysis between sorghum yield traits and SbTGW3 gene genotypes;

[0041] Figure 2 The results of association analysis between sorghum yield traits and SbGS5 genotypes;

[0042] Figure 3 The results of association analysis between sorghum yield traits and genotypes of SbTGW3 and SbGS5 genes based on SNP loci; different lowercase letters represent significant differences;

[0043] Figure 4 is the KASP typing result based on TGW3-SNP1 of the present invention;

[0044] Figure 5 is the KASP typing result based on TGW3-SNP2 of the present invention;

[0045] Figure 6 is the KASP typing result based on GS5-SNP1 of the present invention;

[0046] Figure 7 is the KASP typing result based on GS5-SNP2 of the present invention;

[0047] Figures 4 to 7In the figure, red and blue represent materials corresponding to two different bases carrying the SNP site, green represents heterozygotes, and pink represents problems with the DNA sample. DETAILED DESCRIPTION

[0048] The present invention provides a molecular marker associated with sorghum grain width and 1000-grain weight traits, wherein the molecular marker comprises a first molecular marker combination and / or a second molecular marker combination; the first molecular marker combination comprises TGW3-SNP1 and TGW3-SNP2; the second molecular marker combination comprises GS5-SNP1 and GS5-SNP2;

[0049] The TGW3-SNP1 is located at 1529622bp of chromosome 1 of the sorghum genome, and the polymorphism is G / A; the TGW3-SNP2 is located at 1526093bp of chromosome 1 of the sorghum genome, and the polymorphism is T / G;

[0050] The GS5-SNP1 is located at 5404386bp of chromosome 9 in the sorghum genome, and the polymorphism is T / C; the GS5-SNP2 is located at 5403747bp of chromosome 9 in the sorghum genome, and the polymorphism is A / T.

[0051] As an embodiment, the reference genome of the sorghum described in the present invention is BTx623.

[0052] As an embodiment, the TGW3-SNP1 of the present invention is located at 196bp of the nucleotide sequence shown in SEQ ID NO:13, and the polymorphism is G / A; the TGW3-SNP2 is located at 196bp of the nucleotide sequence shown in SEQ ID NO:14, and the polymorphism is T / G; the GS5-SNP1 is located at 181bp of the nucleotide sequence shown in SEQ ID NO:15, and the polymorphism is T / C; the GS5-SNP2 is located at 249bp of the nucleotide sequence shown in SEQ ID NO:16, and the polymorphism is A / T; R, W, S, K, M and Y in SEQ ID NOs:13 to 16 are degenerate bases, respectively.

[0053] The nucleotide sequences of SEQ ID NOs: 13 to 16 are as follows:

[0054] SEQ ID NO: 13:

[0055] Among them, the bold part is the polymorphic site, and the polymorphism is G / A;

[0056] SEQ ID NO: 14:

[0057] Among them, the bold part is the polymorphic site, and the polymorphism is T / G;

[0058] SEQ ID NO: 15:

[0059] Among them, the bold part is the polymorphic site, and the polymorphism is T / C;

[0060] SEQ ID NO: 16:

[0061] Among them, the bold part is the polymorphic site, and the polymorphism is A / T;

[0062] The present invention also provides a primer combination for detecting the molecular markers described in the above technical solution, wherein the primer combination includes a first primer combination for detecting the first molecular marker combination of the above technical solution and / or a second primer combination for detecting the second molecular marker combination of the above technical solution;

[0063] The first primer combination includes a TGW3-SNP1 primer combination for detecting TGW3-SNP1 described in the above technical solution and a TGW3-SNP2 primer combination for detecting TGW3-SNP2 described in the above technical solution;

[0064] The TGW3-SNP1 primer combination includes TGW3-SNP1-F1 with a nucleotide sequence as shown in SEQ ID NO: 17, TGW3-SNP1-F2 with a nucleotide sequence as shown in SEQ ID NO: 18, and TGW3-SNP1-R with a nucleotide sequence as shown in SEQ ID NO: 3; the 5' ends of the TGW3-SNP1-F1 and the TGW3-SNP1-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the TGW3-SNP1-F1 and the TGW3-SNP1-F2 excite different fluorescence colors;

[0065] The TGW3-SNP2 primer combination includes TGW3-SNP2-F1 with a nucleotide sequence as shown in SEQ ID NO: 19, TGW3-SNP2-F2 with a nucleotide sequence as shown in SEQ ID NO: 20, and TGW3-SNP2-R with a nucleotide sequence as shown in SEQ ID NO: 6; the 5' ends of the TGW3-SNP2-F1 and the TGW3-SNP2-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the TGW3-SNP2-F1 and the TGW3-SNP2-F2 excite different fluorescence colors;

[0066] The second primer combination includes a GS5-SNP1 primer combination for detecting GS5-SNP1 described in the above technical solution and a GS5-SNP2 primer combination for detecting GS5-SNP2 described in the above technical solution;

[0067] The GS5-SNP1 primer combination includes GS5-SNP1-F1 with a nucleotide sequence as shown in SEQ ID NO: 21, GS5-SNP1-F2 with a nucleotide sequence as shown in SEQ ID NO: 22, and GS5-SNP1-R with a nucleotide sequence as shown in SEQ ID NO: 9; the 5' ends of the GS5-SNP1-F1 and the GS5-SNP1-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the GS5-SNP1-F1 and the GS5-SNP1-F2 excite different fluorescence colors; wherein, R in SEQ ID NO: 21 and SEQ ID NO: 22 is a degenerate base;

[0068] The GS5-SNP2 primer combination includes GS5-SNP2-F1 with a nucleotide sequence as shown in SEQ ID NO:23, GS5-SNP2-F2 with a nucleotide sequence as shown in SEQ ID NO:24, and GS5-SNP2-R with a nucleotide sequence as shown in SEQ ID NO:12; the 5' ends of the GS5-SNP2-F1 and the GS5-SNP2-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the GS5-SNP2-F1 and the GS5-SNP2-F2 excite different fluorescence colors.

[0069] The specific sequences of SEQ ID NO: 17 to SEQ ID NO: 24 of the present invention are as follows:

[0070] SEQ ID NO: 17: 5'-GTTTCCATAATTGCCTTGTTTTTCTG-3';

[0071] SEQ ID NO: 18: 5'-GGTTTCCATAATTGCCTTGTTTTTCTA-3';

[0072] SEQ ID NO: 19: 5'-CCAGTAACTCAGACTGAAACCTGGA-3';

[0073] SEQ ID NO:20: 5'-CAGTAACTCAGACTGAAACCTGGC-3';

[0074] SEQ ID NO:21:5'-GTTGCRCAAAAGCTTCTGGATATTT-3'; wherein R is a degenerate base;

[0075] SEQ ID NO:22:5'-GTTGCRCAAAAGCTTCTGGATATTC-3'; wherein R is a degenerate base;

[0076] SEQ ID NO: 23: 5'-CCAGGTGAGCTAGGCGAGAGA-3';

[0077] SEQ ID NO: 24: 5'-CCAGGTGAGCTAGGCGAGAGT-3';

[0078] As an embodiment, the fluorescent groups connected to TGW3-SNP1-F1, TGW3-SNP2-F1, GS5-SNP1-F1 and GS5-SNP2-F1 of the present invention are respectively FAM; the fluorescent groups connected to TGW3-SNP1-F2, TGW3-SNP2-F2, GS5-SNP1-F2 and GS5-SNP2-F2 are respectively HEX.

[0079] The present invention also provides a kit for detecting the molecular markers described in the above technical solution, comprising the primer combination described in the above technical solution.

[0080] This study resequenced sorghum germplasm from a natural population and compared it to the sorghum reference genome, BTx623. High-quality SNP markers were retained by removing markers with deletion rates greater than 20% and allele frequencies less than 5%. Using the resequencing data to extract single nucleotide polymorphism (SNP) information from the target gene region, the researchers found that the aforementioned molecular markers were tightly linked to sorghum grain width and 1000-grain weight. Haplotype analysis of the target genes based on the resequencing data also identified the superior genotypes SbTGW3-hap5 and SbGS5-hap6.

[0081] In view of the advantages of the molecular markers provided by the present invention, the use of the molecular marker substance or the primer combination described in the above technical solution or the kit described in the above technical solution in one or more of the following also falls within the scope of protection of the present invention: (1) identifying or assisting in the identification of sorghum with large grain width and high 1000-grain weight; (2) screening or assisting in the screening of sorghum with large grain width and high 1000-grain weight; (3) identifying or assisting in the identification of haplotype sorghum; the haplotype sorghum includes SbTGW3-hap5 haplotype sorghum and / or SbGS5-hap6 haplotype sorghum; (4) screening or assisting in the screening of haplotype sorghum; the haplotype sorghum includes SbTGW3-hap5 haplotype sorghum and / or SbGS5-hap6 haplotype sorghum.

[0082] The present invention also provides a method for identifying sorghum haplotypes and / or identifying sorghum grain width and 1000-grain weight traits, wherein the haplotype is a SbTGW3-hap5 haplotype and / or a SbGS5-hap6 haplotype, including the following I and / or II:

[0083] I: Using the gene DNA of the sorghum to be tested as a template, PCR amplification is performed using the TGW3-SNP1 primer combination and the TGW3-SNP2 primer combination in the primer combination described in the above technical solution, respectively, to obtain a TGW3-SNP1 amplification product and a TGW3-SNP2 amplification product; and the haplotype and / or grain width and 1000-grain weight traits of the sorghum to be tested are determined based on the TGW3-SNP1 amplification product and the TGW3-SNP2 amplification product:

[0084] The TGW3-SNP1 amplification product is DNA fragment 1 and the TGW3-SNP2 amplification product is DNA fragment 2, then the haplotype of the sorghum to be tested is SbTGW3-hap5, and the sorghum has a larger grain width and a higher 1000-grain weight; the genotype corresponding to the 1529622 bp position of chromosome 1 of the sorghum genome in the DNA fragment 1 is GG; the genotype corresponding to the 1526093 bp position of chromosome 1 of the sorghum genome in the DNA fragment 2 is GG;

[0085] II: Using the genomic DNA of the sorghum to be tested as a template, PCR amplification is performed using the GS5-SNP1 primer combination and the GS5-SNP2 primer combination described in the above technical solution to obtain a GS5-SNP1 amplification product and a GS5-SNP2 amplification product; the haplotype of the sorghum to be tested is determined based on the GS5-SNP1 amplification product and the GS5-SNP2 amplification product:

[0086] The GS5-SNP1 amplification product is DNA fragment 3 and the GS5-SNP2 amplification product is DNA fragment 4, then the haplotype of the sorghum to be tested is SbGS5-hap6, and it is a sorghum with a larger grain width and a higher 1000-grain weight; the genotype corresponding to the 5404386bp of chromosome 9 of the sorghum genome in the DNA fragment 3 is CC; the genotype corresponding to the 5403747bp of chromosome 9 of the sorghum genome in the DNA fragment 4 is AA.

[0087] The present invention has no strict requirements on the PCR amplification method, and conventional operations in the art can be used.

[0088] The present invention also provides the application of the method described in the above technical solution in the breeding or auxiliary breeding of sorghum with large grain width and high 1000-grain weight.

[0089] The present invention also provides a method for cultivating sorghum with large grain width and high 1000-grain weight, comprising the following steps:

[0090] Identify the haplotype of sorghum according to the method described in the above technical solution;

[0091] The sorghum with the SbTGW3-hap5 haplotype and / or the SbGS5-hap6 haplotype is retained as a parent for breeding. The method of the present invention can be used to optimize the yield traits of sorghum and cultivate sorghum with large grain width and high 1000-grain weight.

[0092] To further illustrate the present invention, a molecular marker related to sorghum grain width and 1000-grain weight traits and its application provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0093] Example 1

[0094] The inventors' team resequenced 340 sorghum germplasm resources from natural populations (Table 1) and compared them with the sorghum reference genome BTx623, obtaining a total of 8,151,131 SNP markers. By removing markers with a deletion rate greater than 20% and an allele frequency less than 5%, 3,084,386 high-quality SNP markers were retained. The resequencing data was used to extract single nucleotide polymorphism (SNP) information in the target gene (SbTGW3 gene and SbGS5 gene) region. Based on the resequencing data, different haplotypes of the two key genes were obtained. The different haplotypes were mapped to the sorghum yield trait phenotypes, and the results are shown below. Figures 1-2 and as shown in Table 2. Among them, the yield traits include grain length (GL), grain width (GW), grain area (GA), grain circumference (GC), grain length-to-width ratio (LWR) and thousand-grain weight (TGW).

[0095] Table 1 Information on 340 sorghum germplasms

[0096]

[0097]

[0098]

[0099] Table 2 Yield traits of sorghum of different haplotypes

[0100]

[0101] according to Figures 1-2 As can be seen from Table 2, there are five haplotypes of SbTGW3 gene in natural sorghum populations, among which haplotype 5 (SbTGW3-hap5, Figure 1 The SbGS5 gene has 6 haplotypes in the natural population of sorghum, among which haplotype 6 (SbGS5-hap6, Figure 2 Middle H6) was an excellent haplotype for grain width (GW) and thousand-grain weight (TGW).

[0102] Example 2

[0103] 1. Experimental materials: 340 sorghum germplasms in Example 1.

[0104] 2. Genomic DNA was extracted from 340 sorghum seedling leaves using the TIANGEN kit DNAquickPlant System. The concentration and purity of the extracted DNA were determined using a micro-spectrophotometer. 260 / OD 280 The ratio is between 1.7-2.1, and the sample DNA concentration is greater than 25 ng / μl.

[0105] 3. Obtaining SbTGW3 gene polymorphic sites and haplotypes

[0106] 3.1 Acquisition of SbTGW3 gene polymorphic sites

[0107] Resequencing data was used to extract single nucleotide polymorphism (SNP) information in the SbTGW3 gene region in the sample DNA. The results showed that there were two SNP sites in the SbTGW3 gene, named TGW3-SNP1 and TGW3-SNP2; among them, the genomic position of TGW3-SNP1 was 1529622bp on chromosome 1 of the sorghum genome (with G and A polymorphisms); the genomic position of TGW3-SNP2 was 1526093bp on chromosome 1 of the sorghum genome (with T and G polymorphisms).

[0108] 3.2 Acquisition of superior haplotypes of the SbTGW3 gene

[0109] Sequence analysis revealed that the two SNP sites of the SbTGW3 gene had three haplotypes in the natural sorghum population, which were named haplotype SbTGW3-GGTT, haplotype SbTGW3-GGGG and haplotype SbTGW3-AAGG respectively; among them, haplotype SbTGW3-GGTT was the SbTGW3-hap1 haplotype of the SbTGW3 gene in the natural sorghum population in Example 1 (i.e. Figure 1 H1), haplotype SbTGW3-GGGG is the haplotype SbTGW3-hap5 (i.e. Figure 1 H5), haplotype SbTGW3-AAGG is the haplotype SbTGW3-hap2, SbTGW3-hap3 and SbTGW3-hap4 of the SbTGW3 gene in the natural population of sorghum in Example 1 (i.e. Figure 1 The nucleotide sequences of each haplotype at positions 1529622 and 1526093 of chromosome 1 of the sorghum genome are shown in Table 3.

[0110] Table 3 Nucleotide of each SbTGW3 gene haplotype at each SNP site

[0111]

[0112] 4. Obtaining SbGS5 gene polymorphic sites and haplotypes

[0113] 4.1 Acquisition of polymorphic sites in the SbGS5 gene

[0114] Resequencing data was used to extract single nucleotide polymorphism (SNP) information in the SbGS5 gene region of the sample DNA. The results showed that there were two SNP sites in the SbGS5 gene, named GS5-SNP1 and GS5-SNP2. Among them, the genomic position of GS5-SNP1 was 5404386bp on chromosome 9 of the sorghum genome (with T and C polymorphisms); the genomic position of GS5-SNP2 was 5403747bp on chromosome 9 of the sorghum genome (with A and T polymorphisms).

[0115] 4.2 Acquisition of superior haplotypes of the SbGS5 gene

[0116] Sequence analysis revealed that the two SNP sites of the SbGS5 gene had three haplotypes in the natural sorghum population, which were named haplotype SbGS5-CCAA, haplotype SbGS5-CCTT and haplotype SbGS5-TTAA respectively; among them, haplotype SbGS5-CCAA was the haplotype SbGS5-hap6 (i.e. Figure 2 H6), haplotype SbGS5-CCTT is the SbGS5-hap2 haplotype of the SbGS5 gene in the natural population of sorghum in Example 1 (i.e. Figure 2 H2), haplotype SbGS5-TTAA is the haplotypes SbGS5-hap1, SbGS5-hap3, SbGS5-hap4 and SbGS5-hap5 of the SbGS5 gene in the natural population of sorghum in Example 1 (i.e. Figure 2 The nucleotide sequences of each haplotype at positions 5404386 and 5403747 on chromosome 9 of the sorghum genome are shown in Table 4.

[0117] Table 4 Nucleotide of each SbGS5 gene haplotype at each SNP site

[0118]

[0119] 5. According to the steps of Example 1, the association analysis of sorghum yield phenotype and genotype at the target gene SNP site was performed. The results are as follows: Figure 3 Among them, the yield traits are grain width (GW) and thousand-grain weight (TGW). Figure 3 As can be seen, the sorghum grain width and 1000-grain weight of the haplotype SbTGW3-GGGG were significantly higher than those of the other two haplotypes; the sorghum grain width and 1000-grain weight of the haplotype SbGS5-CCAA were significantly higher than those of the other two haplotypes. The two SNPs corresponding to the SbTGW3 gene were closely linked to sorghum grain width and 1000-grain weight, with GGGG being the superior genotype for grain width and 1000-grain weight (SbTGW3-hap5). The two SNPs corresponding to the SbGS5 gene were closely linked to sorghum grain width and 1000-grain weight, with CCAA being the superior genotype for grain width and 1000-grain weight (SbGS5-hap6).

[0120] Example 3

[0121] 1. Based on the conclusion of Example 2, the sequence of 150 bp upstream and downstream of the SNP site was obtained, and primers for the SNP site were designed and synthesized based on the basic principles and process of AQPTMSNP typing primer design. The specific primer information is shown in Table 5.

[0122] Table 5 KASP primers designed based on molecular marker sites

[0123]

[0124] Note: The bold sequences in Table 4 represent FAM fluorescent tag sequences, and the underlined sequences represent HEX fluorescent tag sequences.

[0125] 2. Select genomic DNA from 178 of the 340 sorghum accessions extracted in step 2 of Example 2 as templates and load them into 96-well PCR plates according to their number. PCR detection was performed according to the operating instructions of the OMEGAF Reader 96- and 384-well compatible OMEGAFSNP typing detector. The KASP genotyping system PCR amplification system consisted of: 2.5 μL KASP 2× Mix (HiGeno 2x Probe Mix A), 0.07 μL SNP Primer Mix, and 25-100 ng template. The volume was made up to 5 μL with sterile water. The SNP Primer Mix was a mixture of F1, F2, and R corresponding to the SNP locus.

[0126] The PCR amplification program of the KASP genotyping system was as follows: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 20 s, annealing / extension at 61-55°C for 45 s, percycle with a drop of 0.6°C; denaturation at 95°C for 20 s, annealing / extension at 55°C for 45 s; and storage at 25°C.

[0127] 3. After the PCR amplification cycle is completed, the fluorescence value is read using a fluorescence quantitative PCR instrument at 35°C and the results are analyzed using the LGCKlusterCaller software. In this software, HEX and FAM data are plotted on the x-axis and y-axis respectively. The results are as follows Figures 4 to 7 As shown in Tables 6 and 7.

[0128] Table 6 KASP typing results and resequencing results based on SNP sites

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Note: In Table 6, the KASP marker detection results marked as “-” indicate that there are problems with the material samples. The materials marked in bold are the resequencing results that are inconsistent with the KASP marker results, and the materials marked in the underlined part are heterozygous.

[0135] Table 7 Results of screening materials carrying excellent genotypes based on SNP sites

[0136]

[0137] according to Figures 4 to 7 As shown in Tables 6 and 7, the KASP marker detection results for 178 accessions were highly consistent with the sequencing results, with a 98.88% match for the SbTGW3 gene and a 94.94% match for the SbGS5 gene. Among the selected experimental accessions, seven accessions with the SbTGW3-hap5 genotype corresponded to the GGGG genotype, while five accessions with the SbGS5-hap6 genotype corresponded to the CCAA genotype. The marker results had a 100% selectivity rate for both genotypes, accurately identifying the target genotype.

[0138] Based on the above content, it can be seen that the molecular markers provided by the present invention are closely linked to the grain width and 1000-grain weight traits of sorghum, and can accurately and efficiently screen out excellent genotypes SbTGW3-hap5 and SbGS5-hap6, both of which are sorghums with large grain width and high 1000-grain weight, laying the foundation for the breeding of sorghums with large grain width and high 1000-grain weight and the creation of new sorghum germplasm.

[0139] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A molecular marker associated with grain width and 1000-grain weight traits of sorghum, characterized in that: The molecular markers include a first molecular marker combination and / or a second molecular marker combination; The first molecular marker combination includes TGW3-SNP1 and TGW3-SNP2; The second molecular marker combination includes GS5-SNP1 and GS5-SNP2; The TGW3-SNP1 is located at 1529622bp of chromosome 1 of the sorghum genome, and the polymorphism is G / A; the TGW3-SNP2 is located at 1526093bp of chromosome 1 of the sorghum genome, and the polymorphism is T / G; The GS5-SNP1 is located at 5404386bp of chromosome 9 in the sorghum genome, and the polymorphism is T / C; the GS5-SNP2 is located at 5403747bp of chromosome 9 in the sorghum genome, and the polymorphism is A / T.

2. The molecular marker according to claim 1, characterized in that The TGW3-SNP1 is located at 196 bp of the nucleotide sequence shown in SEQ ID NO: 13, and the polymorphism is G / A; the TGW3-SNP2 is located at 196 bp of the nucleotide sequence shown in SEQ ID NO: 14, and the polymorphism is T / G; The GS5-SNP1 is located at 181 bp of the nucleotide sequence shown in SEQ ID NO: 15, and the polymorphism is T / C; the GS5-SNP2 is located at 249 bp of the nucleotide sequence shown in SEQ ID NO: 16, and the polymorphism is A / T; In SEQ ID NOs: 13 to 16, R, W, S, K, M and Y are degenerate bases, respectively.

3. A primer combination for detecting the molecular marker according to claim 1 or 2, characterized in that: The primer combination includes a first primer combination for detecting the first molecular marker combination according to claim 1 or 2 and / or a second primer combination for detecting the second molecular marker combination according to claim 1 or 2; The first primer combination includes a TGW3-SNP1 primer combination for detecting the TGW3-SNP1 according to claim 1 or 2 and a TGW3-SNP2 primer combination for detecting the TGW3-SNP2 according to claim 1 or 2; The TGW3-SNP1 primer combination includes TGW3-SNP1-F1 with a nucleotide sequence as shown in SEQ ID NO: 17, TGW3-SNP1-F2 with a nucleotide sequence as shown in SEQ ID NO: 18, and TGW3-SNP1-R with a nucleotide sequence as shown in SEQ ID NO: 3; the 5' ends of the TGW3-SNP1-F1 and the TGW3-SNP1-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the TGW3-SNP1-F1 and the TGW3-SNP1-F2 excite different fluorescence colors; The TGW3-SNP2 primer combination includes TGW3-SNP2-F1 with a nucleotide sequence as shown in SEQ ID NO: 19, TGW3-SNP2-F2 with a nucleotide sequence as shown in SEQ ID NO: 20, and TGW3-SNP2-R with a nucleotide sequence as shown in SEQ ID NO: 6; the 5' ends of the TGW3-SNP2-F1 and the TGW3-SNP2-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the TGW3-SNP2-F1 and the TGW3-SNP2-F2 excite different fluorescence colors; The second primer combination includes a GS5-SNP1 primer combination for detecting the GS5-SNP1 according to claim 1 or 2 and a GS5-SNP2 primer combination for detecting the GS5-SNP2 according to claim 1 or 2; The GS5-SNP1 primer combination includes GS5-SNP1-F1 with a nucleotide sequence as shown in SEQ ID NO: 21, GS5-SNP1-F2 with a nucleotide sequence as shown in SEQ ID NO: 22, and GS5-SNP1-R with a nucleotide sequence as shown in SEQ ID NO: 9; the 5' ends of the GS5-SNP1-F1 and the GS5-SNP1-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the GS5-SNP1-F1 and the GS5-SNP1-F2 excite different fluorescence colors; wherein R in SEQ ID NO: 21 and SEQ ID NO: 22 is a degenerate base; The GS5-SNP2 primer combination includes GS5-SNP2-F1 with a nucleotide sequence as shown in SEQ ID NO:23, GS5-SNP2-F2 with a nucleotide sequence as shown in SEQ ID NO:24, and GS5-SNP2-R with a nucleotide sequence as shown in SEQ ID NO:12; the 5' ends of the GS5-SNP2-F1 and the GS5-SNP2-F2 are respectively connected to fluorescent groups, and the fluorescent groups connected to the GS5-SNP2-F1 and the GS5-SNP2-F2 excite different fluorescence colors.

4. The primer combination according to claim 3, characterized in that The fluorescent groups connected to TGW3-SNP1-F1, TGW3-SNP2-F1, GS5-SNP1-F1 and GS5-SNP2-F1 are FAM; The fluorescent groups connected to TGW3-SNP1-F2, TGW3-SNP2-F2, GS5-SNP1-F2 and GS5-SNP2-F2 are HEX respectively.

5. A kit for detecting the molecular marker according to claim 1 or 2, characterized in that: Comprising the primer combination according to claim 3 or 4.

6. Use of the molecular marker substance of claim 1 or 2, the primer combination of claim 3 or 4, or the kit of claim 5 for detecting one or more of the following: (1) Identify or assist in identifying sorghum with large kernel width and high 1000-kernel weight; (2) screening or assisting in screening sorghum with large kernel width and high 1000-kernel weight; (3) Identifying or assisting in identifying haplotype sorghum; the haplotype sorghum includes SbTGW3-hap5 haplotype sorghum and / or SbGS5-hap6 haplotype sorghum; (4) Screening or auxiliary screening of haplotype sorghum; the haplotype sorghum includes SbTGW3-hap5 haplotype sorghum and / or SbGS5-hap6 haplotype sorghum.

7. A method for identifying sorghum haplotypes and / or identifying sorghum grain width and 1000-grain weight traits, wherein the haplotype is a SbTGW3-hap5 haplotype and / or a SbGS5-hap6 haplotype, characterized in that: Includes the following I and / or II: I: Using the gene DNA of the sorghum to be tested as a template, PCR amplification is performed using the TGW3-SNP1 primer combination and the TGW3-SNP2 primer combination in the primer combination of claim 3 or 4, respectively, to obtain a TGW3-SNP1 amplification product and a TGW3-SNP2 amplification product; and the haplotype and / or grain width and 1000-grain weight traits of the sorghum to be tested are determined based on the TGW3-SNP1 amplification product and the TGW3-SNP2 amplification product: The TGW3-SNP1 amplification product is DNA fragment 1 and the TGW3-SNP2 amplification product is DNA fragment 2, then the haplotype of the sorghum to be tested is SbTGW3-hap5, and the sorghum has a larger grain width and a higher 1000-grain weight; the genotype corresponding to the 1529622 bp position of chromosome 1 of the sorghum genome in the DNA fragment 1 is GG; the genotype corresponding to the 1526093 bp position of chromosome 1 of the sorghum genome in the DNA fragment 2 is GG; II: Using the gene DNA of the sorghum to be tested as a template, PCR amplification is performed using the GS5-SNP1 primer combination and the GS5-SNP2 primer combination in the primer combination of claim 3 or 4, respectively, to obtain a GS5-SNP1 amplification product and a GS5-SNP2 amplification product; and the haplotype of the sorghum to be tested is determined based on the GS5-SNP1 amplification product and the GS5-SNP2 amplification product: The GS5-SNP1 amplification product is DNA fragment 3 and the GS5-SNP2 amplification product is DNA fragment 4, then the haplotype of the sorghum to be tested is SbGS5-hap6, and it is a sorghum with a larger grain width and a higher 1000-grain weight; the genotype corresponding to the 5404386bp of chromosome 9 of the sorghum genome in the DNA fragment 3 is CC; the genotype corresponding to the 5403747bp of chromosome 9 of the sorghum genome in the DNA fragment 4 is AA.

8. Use of the method according to claim 7 in breeding or auxiliary breeding of sorghum with large grain width and high 1000-grain weight.

9. A method for cultivating sorghum with large grain width and high 1000-grain weight, characterized in that: The steps include: Identifying the haplotype of sorghum according to the method of claim 7; Sorghum with the SbTGW3-hap5 haplotype and / or the SbGS5-hap6 haplotype was retained as a parent for breeding.

Images