SNP molecular marker combination related to gossypium hirsutum resistance to verticillium wilt and application thereof

Abstract

The present application provides a SNP molecular marker related to the resistance to Verticillium wilt of Gossypium hirsutum, and particularly relates to 10 SNP molecular markers, and provides specific SNP sites and polymorphisms. The present application provides two sets of primer groups based on the 10 SNP molecular markers: one set (10 pairs) of sequencing primer groups for amplifying the DNA fragments where the SNPs are located, which can be combined with barcodes to obtain the resistance / susceptibility genotype information of multiple samples in a high-throughput manner; and another set (10 pairs) of typing primer groups for ordinary PCR, which can distinguish the resistance / susceptibility genotype through electrophoresis band type. The combination of the 10 SNP markers related to the resistance to Verticillium wilt can be used for early prediction and screening of the resistance to Verticillium wilt of Gossypium hirsutum, and can assist in improving the disease resistance of cotton varieties.

Description

Technical Field

[0001] This invention belongs to the technical field of bio-agriculture, specifically relating to SNP molecular markers related to resistance to Verticillium wilt in upland cotton and their applications. Background Technology

[0002] Existing research indicates that cotton resistance to Verticillium wilt is a complex quantitative trait, controlled by multiple genes and easily influenced by the environment. In recent years, researchers have located over 200 QTLs for Verticillium wilt resistance in upland cotton using linkage analysis across different genetic populations. However, to date, there have been no reports of cloning major resistance QTLs or resistance genes. Most of the identified genes involved in Verticillium wilt resistance are located downstream of the resistance pathway and have weak effects. Studies of single genes are insufficient to fully understand the resistance mechanism, and marker-assisted breeding based on single loci suffers from low selection efficiency and unsatisfactory results. Therefore, in the era of modern molecular breeding, for the complex trait of cotton Verticillium wilt significantly influenced by the environment, identifying loci with broad-spectrum resistance and strong effects in complex field environments requires multi-year, multi-location trait surveys of genetic populations to identify a batch of stable, key genetic loci. In multiple environments, the stable emergence of multiple loci represents a combination of pressures from different regions, temperature and humidity conditions, soil environments, and various dominant microbial strains. By utilizing this set of loci, a set of molecular marker combinations can be developed. By aggregating disease-resistant alleles, genomic selection breeding can be carried out to comprehensively improve the disease resistance of cotton varieties, accelerate the innovation of cotton germplasm resistant to Verticillium wilt, and improve breeding effectiveness. Summary of the Invention

[0003] The purpose of this invention is to provide an SNP molecular marker related to resistance to Verticillium wilt in upland cotton, wherein the SNP molecular marker combination includes 10 SNP molecular markers from Lsnp1 to Lsnp10;

[0004] The nucleotide sequence of the SNP molecular marker of Lsnp1 is shown in SEQ ID NO.1, and the polymorphism site is located at position 66 of the sequence shown in SEQ ID NO.1, with a polymorphism of A / G.

[0005] The nucleotide sequence of the SNP molecular marker of Lsnp2 is shown in SEQ ID NO.2, and the polymorphism site is located at position 144 of the sequence shown in SEQ ID NO.2, with a polymorphism of C / T.

[0006] The nucleotide sequence of the SNP molecular marker of Lsnp3 is shown in SEQ ID NO.3, and the polymorphism site is located at position 67 of the sequence shown in SEQ ID NO.3, with a polymorphism of T / A;

[0007] The nucleotide sequence of the SNP molecular marker of Lsnp4 is shown in SEQ ID NO.4, and the polymorphism site is located at position 127 of the sequence shown in SEQ ID NO.4, with a polymorphism of C / T.

[0008] The nucleotide sequence of the SNP molecular marker of Lsnp5 is shown in SEQ ID NO.5, and the polymorphism site is located at position 126 of the sequence shown in SEQ ID NO.5, with a polymorphism of T / C.

[0009] The nucleotide sequence of the SNP molecular marker of Lsnp6 is shown in SEQ ID NO.6, and the polymorphic site is located at position 125 of the sequence shown in SEQ ID NO.6, with a polymorphism of T / A.

[0010] The nucleotide sequence of the SNP molecular marker of Lsnp7 is shown in SEQ ID NO.7, and the polymorphic site is located at position 112 of the sequence shown in SEQ D NO.7, with a polymorphism of T / A.

[0011] The nucleotide sequence of the SNP molecular marker of Lsnp8 is shown in SEQ ID NO.8, and the polymorphic site is located at position 112 of the sequence shown in SEQ ID NO.8, with a polymorphism of T / C.

[0012] The nucleotide sequence of the SNP molecular marker of Lsnp9 is shown in SEQ ID NO.9, and the polymorphism site is located at position 93 of the sequence shown in SEQ ID NO.9, with a polymorphism of C / T.

[0013] The nucleotide sequence of the SNP molecular marker of Lsnp10 is shown in SEQ ID NO.10, and the polymorphic site is located at position 90 of the sequence shown in SEQ ID NO.10, with a polymorphism of T / C.

[0014] Furthermore, the disease-resistant dominant alleles of the SNP molecular markers Lsnp1-Lsnp10 are, in order, AA, CC, TT, CC, TT, TT, TT, TT, CC, TT.

[0015] This invention also provides a method for detecting Verticillium wilt resistance in upland cotton: the disease resistance of upland cotton is determined by detecting the genotypes of 10 SNP molecular markers;

[0016] The physical location information of the 10 SNP molecular markers is as follows:

[0017] qVWR.A01.1 Lsnp1 A01 111929609 A / G qVWR.A01.2 Lsnp2 A01 117983536 C / T qVWR.A07.2 Lsnp3 A07 90971603 T / A qVWR.A10.1 Lsnp4 A10 108893925 C / T qVWR.A11.2 Lsnp5 A11 119799615 T / C qVWR.A13.1 Lsnp6 A13 105434827 T / A qVWR.D01.1 Lsnp7 D01 1696495 T / A qVWR.D07.1 Lsnp8 D07 13371656 T / C qVWR.D08.2 Lsnp9 D08 61445110 C / T qVWR.D10.1 Lsnp10 D10 22542365 T / C

[0018] The physical location information of SNP molecular markers was determined based on the Gossypium hirsutum(AD1)'TM-1'genome ZJU-improved_v2.1_a1 version of the upland cotton standard line Texas Marker-1.

[0019] Furthermore, the criterion for judging the resistance of upland cotton to Verticillium wilt is: the more disease-resistant genotypes there are, the stronger the resistance.

[0020] The present invention also provides primer sequences for amplifying the 10 SNP molecular markers Lsnp1-Lsnp10, as shown in SEQ ID NO.11-30.

[0021] This invention also provides the application of the above-mentioned SNP molecular markers or primer sets in the early prediction, screening and breeding of resistance to Verticillium wilt in upland cotton.

[0022] The present invention also provides a primer set for detecting resistance to Verticillium wilt in upland cotton. The primer set is used to amplify nucleotide fragments near the above-mentioned 10 SNP molecular markers. The primer sequences corresponding to the 10 SNP molecular markers Lsnp1-Lsnp10 are shown in SEQ ID NO.31-50 in sequence.

[0023] Specifically, a positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.31-32 indicates a susceptible genotype, and the annealing temperature is 54-57℃;

[0024] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.33-34 indicates a susceptible genotype, and the annealing temperature is 50-55℃;

[0025] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.35-36 indicates a disease-resistant genotype, and the annealing temperature is 65-67℃;

[0026] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.37-38 indicates a disease-resistant genotype, and the annealing temperature is 50-52℃;

[0027] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.39-40 indicates a disease-resistant genotype, and the annealing temperature is 50-55℃;

[0028] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.41-42 indicates a susceptible genotype, and the annealing temperature is 50-55℃;

[0029] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.43-44 indicates a disease-resistant genotype, and the annealing temperature is 50-53.5℃;

[0030] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.45-46 indicates a disease-resistant genotype, and the annealing temperature is 55-57℃;

[0031] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.47-48 indicates a susceptible genotype, and the annealing temperature is 58-60℃;

[0032] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.49-50 indicates a disease-resistant genotype, and the annealing temperature is 55-58℃.

[0033] This application also provides the application of the above primer set in the early prediction, screening and breeding of Verticillium wilt resistance in upland cotton.

[0034] This application also provides a kit for detecting resistance to Verticillium wilt in upland cotton, the kit comprising one of the primer sets described above.

[0035] The beneficial effects of this invention are:

[0036] (1) This invention investigated the resistance to Verticillium wilt by planting 290 natural cotton populations in three natural disease nurseries in Xinjiang over three years, obtaining a total of 20 phenotypic values. Association analysis of these 20 phenotypic values ​​with high-density SNP markers covering the entire genome identified 10 stable QTLs that could be repeatedly detected in three or more analyses. The most significant SNP site among these 10 stable QTLs was designated as the Lead-SNP. The correlation between different haplotypes of these 10 Lead-SNPs and the disease index differences reached a highly significant level. Figure 2 The number of disease-resistant genotypes carried at 10 Lsnp sites in cotton materials is positively correlated with disease resistance. Figure 3 Prior to this invention, no invention had provided a set of key loci for the complex quantitative genetics of cotton resistance to Verticillium wilt, which is beneficial for comprehensively and effectively improving the disease resistance of cotton varieties.

[0037] (2) Based on the 10 Lsnp, this invention provides two sets of primers for application: one set (10 pairs) of genotyping primers for ordinary PCR and one set (10 pairs) of sequencing primers for amplifying the DNA fragment containing the SNP. These can be used to detect genotypic information at the 10 Lsnp in single and multiple samples, respectively. The marker set and primer set provided by this invention can be used for early prediction and assessment of cotton Verticillium wilt resistance, and can assist in the breeding of cotton varieties resistant to Verticillium wilt. Furthermore, this application method yields results at the DNA level; the detection does not require consideration of the cotton's growth stage or tissue type, nor does it require inoculation or disease resistance identification experiments. It is not limited by pathogens, temperature, humidity, or other environmental factors, and can accurately and quickly obtain genotypic information from single and multiple samples, which is beneficial for accelerating the innovation of cotton germplasm resistant to Verticillium wilt and improving breeding efficiency. Attached Figure Description

[0038] Figure 1 Heatmap of multi-year, multi-location phenotypic correlation analysis of resistance to Verticillium wilt in 290 natural upland cotton populations.

[0039] Figure 2 Box plot of disease resistance haplotype analysis of 10 Lsnp.

[0040] Figure 3 Box plot of the number of disease-resistant genotypes carried by natural population materials and disease resistance, with the vertical axis representing the disease index and the horizontal axis representing the number of disease-resistant genotypes among 10 Lsnp carriers.

[0041] Figure 4 This indicates that the number of disease-resistant genotypes carried at 10 Lsnp sites in cotton materials is positively correlated with disease resistance.

[0042] Figure 5 A schematic diagram showing the simultaneous detection of genotypes at 5 Lsnp locations in 272 samples using a sequencing primer set designed based on 10 Lsnp.

[0043] Figure 6 :according to Figure 5 A schematic diagram showing the results of genotyping 272 individual plants at Lsnp4, 5, 7, 8, and 10 using peak shapes.

[0044] Figure 7 Schematic diagram of electrophoresis amplification of specific DNA fragments between resistant materials using a genotyping primer set designed based on 10 Lsnp. Detailed Implementation

[0045] This invention investigated resistance to Verticillium wilt by planting 290 natural cotton populations in three natural disease nurseries in Xinjiang over three years, obtaining 20 phenotypic values. Association analysis of these 20 phenotypic values ​​with high-density SNP markers covering the entire genome identified 10 SNP markers associated with Verticillium wilt resistance in cotton. The 290 upland cotton cultivars used in this invention were selected from upland cotton cultivars whose genomes had been resequencing by the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (see Wang et al. Asymmetric subgenome selection and cis-regulatory divergenceduring cotton domestication. Nat Genet, 2017, 49:579-587).

[0046] This invention provides 10 SNP molecular markers, Lsnp1-Lsnp10, associated with resistance to Verticillium wilt in upland cotton:

[0047] The nucleotide sequence of the SNP molecular marker of Lsnp1 is shown in SEQ ID NO.1. The polymorphic site is located at position 66 of the sequence shown in SEQ ID NO.1, and the polymorphism is A / G.

[0048] The nucleotide sequence of the SNP molecular marker of Lsnp2 is shown in SEQ ID NO.2. The polymorphism site is located at position 144 of the sequence shown in SEQ ID NO.2, and the polymorphism is C / T.

[0049] The nucleotide sequence of the SNP molecular marker of Lsnp3 is shown in SEQ ID NO.3. The polymorphism site is located at position 67 of the sequence shown in SEQ ID NO.3, and the polymorphism is T / A.

[0050] The nucleotide sequence of the SNP molecular marker of Lsnp4 is shown in SEQ ID NO.4. The polymorphic site is located at position 127 of the sequence shown in SEQ ID NO.4, and the polymorphism is C / T.

[0051] The nucleotide sequence of the SNP molecular marker Lsnp5 is shown in SEQ ID NO.5. The polymorphic site is located at position 126 of the sequence shown in SEQ ID NO.5, and the polymorphism is T / C.

[0052] The nucleotide sequence of the SNP molecular marker Lsnp6 is shown in SEQ ID NO.6. The polymorphic site is located at position 125 of the sequence shown in SEQ ID NO.6, and the polymorphism is T / A.

[0053] The nucleotide sequence of the SNP molecular marker Lsnp7 is shown in SEQ ID NO.7, and the polymorphism site is located at position 112 of the sequence shown in SEQ D NO.7, with a polymorphism of T / A.

[0054] The nucleotide sequence of the SNP molecular marker Lsnp8 is shown in SEQ ID NO.8. The polymorphic site is located at position 112 of the sequence shown in SEQ ID NO.8, and the polymorphism is T / C.

[0055] The nucleotide sequence of the SNP molecular marker of Lsnp9 is shown in SEQ ID NO.9. The polymorphism site is located at position 93 of the sequence shown in SEQ ID NO.9, and the polymorphism is C / T.

[0056] The nucleotide sequence of the SNP molecular marker Lsnp10 is shown in SEQ ID NO.10. The polymorphic site is located at position 90 of the sequence shown in SEQ ID NO.10, and the polymorphism is T / C.

[0057] Furthermore, the disease resistance dominant alleles of the SNP molecular markers Lsnp1-Lsnp10 are AA, CC, TT, CC, TT, TT, TT, TT, CC, TT in sequence.

[0058] The present invention also provides a method for detecting resistance to Verticillium wilt in upland cotton, which determines the disease resistance of upland cotton by detecting the genotypes of SNP molecular markers Lsnp1-Lsnp10;

[0059] The physical location information of the 10 SNP molecular markers is as follows:

[0060] qVWR.A01.1 Lsnp1 A01 111929609 A / G qVWR.A01.2 Lsnp2 A01 117983536 C / T qVWR.A07.2 Lsnp3 A07 90971603 T / A qVWR.A10.1 Lsnp4 A10 108893925 C / T qVWR.A11.2 Lsnp5 A11 119799615 T / C qVWR.A13.1 Lsnp6 A13 105434827 T / A qVWR.D01.1 Lsnp7 D01 1696495 T / A qVWR.D07.1 Lsnp8 D07 13371656 T / C qVWR.D08.2 Lsnp9 D08 61445110 C / T qVWR.D10.1 Lsnp10 D10 22542365 T / C

[0061] The physical location information of the above SNP molecular markers was determined based on the Gossypium hirsutum(AD1)'TM-1'genome ZJU-improved_v2.1_a1 version of the Texas Marker-1 upland cotton standard line genome.

[0062] Furthermore, the criterion for judging the resistance of upland cotton to Verticillium wilt is: the more disease-resistant genotypes there are, the stronger the resistance.

[0063] The present invention also provides a primer set for amplifying the nucleotide sequences of the above 10 SNP molecular markers, the primer sequences being shown in SEQ ID NO.11-30 in sequence.

[0064] Preferably, the primer set serves as a sequencing primer set, and when combined with barcodes, it can obtain high-throughput anti / susceptible genotype information from multiple samples.

[0065] Preferably, the method for detecting Verticillium wilt resistance in upland cotton is as follows: rapid acquisition of resistance / susceptibility genotype information from multiple samples is achieved through barcode and next-generation sequencing. The specific steps are as follows:

[0066] (1) Extract genomic DNA from the sample to be tested;

[0067] (2) Design barcodes and adapters according to the needs of multiple samples. Add adapters to the series of primers shown in SEQ ID NO. 11-30 as needed and synthesize them. Perform PCR reactions on multiple samples and mix and purify the products.

[0068] (3) Perform second-generation sequencing, extract information from multiple samples based on barcode, and analyze the genotypes of each cotton sample at 10 Lsnp sites to analyze the resistance to Verticillium wilt.

[0069] This invention also provides the application of the above 10 SNP molecular markers in the early prediction, screening and breeding of Verticillium wilt resistance in upland cotton.

[0070] Furthermore, the present invention also provides a primer set for detecting resistance to Verticillium wilt in upland cotton. The primer set is used to amplify nucleotide fragments near the above-mentioned SNP molecular markers. The primer sequences corresponding to the 10 SNP molecular markers Lsnp1-Lsnp10 are shown in SEQ ID NO.31-50 in sequence.

[0071] Preferably, the primer set, as a PCR typing primer set, can specifically amplify nucleotide fragments near the above 10 SNP molecular markers in disease-resistant or disease-susceptible materials.

[0072] Specifically, a positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.31-32 indicates a susceptible genotype, and the annealing temperature is 54-57℃;

[0073] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.33-34 indicates a susceptible genotype, and the annealing temperature is 50-55℃;

[0074] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.35-36 indicates a disease-resistant genotype, and the annealing temperature is 65-67℃;

[0075] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.37-38 indicates a disease-resistant genotype, and the annealing temperature is 50-52℃;

[0076] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.39-40 indicates a disease-resistant genotype, and the annealing temperature is 50-55℃;

[0077] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.41-42 indicates a susceptible genotype, and the annealing temperature is 50-55℃;

[0078] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.43-44 indicates a disease-resistant genotype, and the annealing temperature is 50-53.5℃;

[0079] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.45-46 indicates a disease-resistant genotype, and the annealing temperature is 55-57℃;

[0080] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.47-48 indicates a susceptible genotype, and the annealing temperature is 58-60℃;

[0081] A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.49-50 indicates a disease-resistant genotype, and the annealing temperature is 55-58℃.

[0082] The application of the above primer set in the early prediction, screening and breeding of resistance to Verticillium wilt in upland cotton.

[0083] The present invention also provides a kit for detecting resistance to Verticillium wilt in upland cotton, the kit comprising one of the primer pair combinations described above.

[0084] In this invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art. The technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0085] Example 1

[0086] Acquisition of SNP markers associated with resistance to Verticillium wilt in upland cotton:

[0087] (1) Survey of planting and disease resistance phenotypes of natural cotton populations:

[0088] The 290 upland cotton cultivars used in this invention were selected from upland cotton cultivars whose genomes had been resequencing by the State Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University (see Wang et al. Asymmetric subgenome selection and cis-regulatory divergence during cotton domestication. Nat Genet, 2017, 49: 579-587). From 2018 to 2020, this natural population was planted in three Verticillium wilt natural nurseries in Manas, Korla, and Kuqa, Xinjiang. Disease index surveys were conducted after the onset of disease in July and August. Disease index data were collected for 1, 2, and 2 years in the three locations. Except for Kuqa in 2020, where there was only one replicate, all other locations had two replicates. Analysis of the manually counted disease index showed a normal distribution, consistent with the genetic characteristics of quantitative traits. The Pearson coefficient (R²) was used to analyze the disease index frequency distribution. 2 Correlation analysis of multiple environmental disease indicators showed that R at the same location 2 (0.33-0.67) higher than the R of the same year. 2 (0.13-0.63), indicating higher stability at the same location, such as Figure 1 As shown: The figure is a heatmap of correlation analysis of the Verticillium wilt resistance phenotypes of 290 natural upland cotton populations at three locations over three years. The values ​​are Pearson correlation coefficients calculated pairwise, and the heatmap thermodynamic values ​​range from -1 to 1, with positive and negative values ​​representing positive and negative correlations, respectively. The phenotypes include 9 sets of original phenotype values, numbered in the format of year, location, DI, and repeatability (18M_DI_1 represents the first repeating disease index in Manas in 2018). The results indicate a higher correlation between different years at the same location. After BLUE processing with location as a fixed factor, 7 sets of processed phenotype values ​​were obtained, numbered in the format of year, location, DI, and BLUE (18_19K_DI_BLUE represents the disease index after BLUE processing of four phenotypes from Korla in 2018 and 2019).

[0089] By using the best linear unbiased estimates (BLUE) for fixed effects to address the issues of location and material duplication, the R-value for the same location was improved. 2 (0.47-0.66), and in addition, all phenotypic data from three years and three locations were processed to obtain a comprehensive phenotypic value, with correlation coefficients ranging from 0.61 to 0.85 with each phenotypic. Thus, 9 sets of original phenotypic values ​​were obtained from the three years and three locations, 7 sets of processed phenotypic values ​​were obtained by BLUE processing, and based on practical experience, in order to reduce the error caused by uneven pathogen content in natural disease nurseries, duplicate data were merged according to location to obtain 5 sets of merged phenotypic values, for a total of 20 sets of phenotypic values.

[0090] (2) Genome-wide association analysis of cotton resistance to Verticillium wilt:

[0091] Based on the 290 upland cotton cultivars whose genomes have been resequencing by the State Key Laboratory of Crop Genetic Improvement at Huazhong Agricultural University, resequencing data were downloaded for population SNP detection. Using BWA (0.7.17), the sequencing data were aligned to the genome of the upland cotton standard line Texas Marker-1, specifically the genome of Gossypium hirsutum (AD1)'TM-1'genome ZJU-improved_v2.1_a1. After deduplication and back-alignment using GATK, gvcf files were generated for each sample using GATKGenotypeGVCFs. GATK CombineGVCF was used to merge the gvcf files from all samples and generate a total vcf file. SNPs were filtered using VCFtools based on DP (depth, sequencing depth > 5), missing rate (missing > 50), and minimum allele frequency (MAF > 0.05). The final retained vcf files were used for association analysis using GEMMA. The 20 phenotypic values ​​described in (1) were subjected to GWAS analysis, and a total of 10 stable QTLs that could be repeatedly detected in three or more analyses were located. The most significant SNP site among the 10 stable QTLs was denoted as Lead-SNP and named Lsnp1 to 10. Sequence information:

[0092] The nucleotide sequence of the SNP molecular marker of Lsnp1 is shown in SEQ ID NO.1. The polymorphic site is located at position 66 of the sequence shown in SEQ ID NO.1, and the polymorphism is A / G.

[0093] The nucleotide sequence of the SNP molecular marker of Lsnp2 is shown in SEQ ID NO.2. The polymorphism site is located at position 144 of the sequence shown in SEQ ID NO.2, and the polymorphism is C / T.

[0094] The nucleotide sequence of the SNP molecular marker of Lsnp3 is shown in SEQ ID NO.3. The polymorphic site is located at position 67 of the sequence shown in SEQ ID NO.3, and the polymorphism is T / A.

[0095] The nucleotide sequence of the SNP molecular marker of Lsnp4 is shown in SEQ ID NO.4. The polymorphic site is located at position 127 of the sequence shown in SEQ ID NO.4, and the polymorphism is C / T.

[0096] The nucleotide sequence of the SNP molecular marker Lsnp5 is shown in SEQ ID NO.5. The polymorphic site is located at position 126 of the sequence shown in SEQ ID NO.5, and the polymorphism is T / C.

[0097] The nucleotide sequence of the SNP molecular marker Lsnp6 is shown in SEQ ID NO.6. The polymorphic site is located at position 125 of the sequence shown in SEQ ID NO.6, and the polymorphism is T / A.

[0098] The nucleotide sequence of the SNP molecular marker Lsnp7 is shown in SEQ ID NO.7, and the polymorphism site is located at position 112 of the sequence shown in SEQ D NO.7, with a polymorphism of T / A.

[0099] The nucleotide sequence of the SNP molecular marker Lsnp8 is shown in SEQ ID NO.8. The polymorphic site is located at position 112 of the sequence shown in SEQ ID NO.8, and the polymorphism is T / C.

[0100] The nucleotide sequence of the SNP molecular marker of Lsnp9 is shown in SEQ ID NO.9. The polymorphism site is located at position 93 of the sequence shown in SEQ ID NO.9, and the polymorphism is C / T.

[0101] The nucleotide sequence of the SNP molecular marker Lsnp10 is shown in SEQ ID NO.10. The polymorphic site is located at position 90 of the sequence shown in SEQ ID NO.10, and the polymorphism is T / C.

[0102] (3) Correlation analysis of 10 Lsnp and cotton resistance to Verticillium wilt:

[0103] Analysis of the correlation between genotype and disease index showed that the correlation between different haplotypes of Lead-SNPs in the 10 QTLs and the differences in disease index was highly significant. For example... Figure 2 As shown: The 10 box plots represent the classification and statistical analysis of the variation of Lead-SNPs and the disease index in 10 stable QTLs. The horizontal axis represents the base type of the Lead-SNP, and the vertical axis represents the disease index. The disease-resistant dominant alleles of Lsnp1 to Lsnp10 are AA, CC, TT, CC, TT, TT, TT, TT, CC, TT, respectively.

[0104] Analysis of the relationship between genotypes at 10 Lsnp positions and disease resistance in cotton materials from 290 natural populations revealed that the more disease-resistant genotypes present at 10 Lsnp positions, the lower the disease index and the stronger the disease resistance of the cotton material. Figure 3As shown in Figure A, the 290 natural populations of upland cotton carried a minimum of 1 disease-resistant genotype and a maximum of 10 disease-resistant genotypes, indicating that the number of disease-resistant genotypes carried at the 10 Lsnp sites in cotton materials is positively correlated with disease resistance.

[0105] Example 2

[0106] The physical locations of 10 Lsnp were determined based on the Gossypium hirsutum(AD1)'TM-1' genome ZJU-improved_v2.1_a1 version of the upland cotton standard line Texas Marker-1.

[0107] The physical location information of the 10 SNP molecular markers is as follows:

[0108] qVWR.A01.1 Lsnp1 A01 111929609 A / G qVWR.A01.2 Lsnp2 A01 117983536 C / T qVWR.A07.2 Lsnp3 A07 90971603 T / A qVWR.A10.1 Lsnp4 A10 108893925 C / T qVWR.A11.2 Lsnp5 A11 119799615 T / C qVWR.A13.1 Lsnp6 A13 105434827 T / A qVWR.D01.1 Lsnp7 D01 1696495 T / A qVWR.D07.1 Lsnp8 D07 13371656 T / C qVWR.D08.2 Lsnp9 D08 61445110 C / T qVWR.D10.1 Lsnp10 D10 22542365 T / C

[0109] Example 3

[0110] To further objectively evaluate the accuracy of the 10 Verticillium wilt resistance SNP markers in Example 1, a natural population of 419 materials from other teams used in the association analysis of Verticillium wilt resistance in upland cotton was analyzed (see: Ma et al. High-quality genome assembly and resequencing of modern cotton cultivars provide resources for crop improvement. Nat Genet, 2021, 53: 1385-1391). The same analysis was performed on the 10 Lsnp markers, revealing that 9 SNPs other than Lsnp2 were detectable in the population of 419 cotton materials. All SNPs at Lsnp2 were resistance genotypes. The more resistance genotypes present at the 10 Lsnp markers in the population, the lower the disease index and the stronger the resistance of the cotton material. Figure 3 As shown in Figure B, the 419 natural populations of upland cotton carried a minimum of 2 disease-resistant genotypes and a maximum of 9 disease-resistant genotypes, indicating that the number of disease-resistant genotypes carried at 10 Lsnp sites in cotton materials is positively correlated with disease resistance.

[0111] The above results indicate that the 10 Lsnp genotypes detected through multi-year, multi-location disease resistance identification and association analysis are closely related to the Verticillium wilt resistance trait in cotton. The number of disease-resistant genotypes carried by cotton materials at these 10 Lsnp genotypes is positively correlated with disease resistance, and this conclusion can be verified in different natural populations. Analysis of the resistance / susceptibility genotype frequencies of these 10 Lsnp genotypes in Chinese varieties using publicly available resequencing data from 2033 Chinese varieties revealed that the major alleles of all 10 Lsnp genotypes are TM-1, and Lsnp1, Lsnp4, and Lsnp9 are predominantly susceptible alleles in Chinese varieties, indicating significant potential for future Verticillium wilt resistance breeding.

[0112] like Figure 4 As shown: A total of 2033 cotton varieties were used in the statistics. The vertical axis represents the number of varieties, and the horizontal axis represents 10 Lsnp loci. 0 / 0 and 1 / 1 represent the same base type as the upland cotton standard line Texas Marker-1 and the variant base type, respectively. The circled type represents the disease-resistant allele; Lsnp1, Lsnp4, and Lsnp9 are rare disease-resistant gene loci.

[0113] The genome resequencing data of the above 2033 Chinese varieties were obtained from the following five research papers:

[0114] He et al. The genomic basis of geographic differentiation and fiber improvement in cultivated cotton. Nat Genet, 2021,53:916-924;

[0115] Li et al. Cotton pan-genome retrieves the lost sequences and genes during domestication and selection. Genome Biol, 2021a, 22:119;

[0116] Li et al. Genomic analyzes reveal the genetic basis of early maturity and identification of loci and candidate genes in upland cotton (Gossypiumhirsutum L.). Plant Biotechnol J, 2021b, 19:109-123;

[0117] Li et al.Combined GWAS and eQTL analysis uncovers a geneticregulatory network orchestrating the initiation of secondary cell wall development in cotton.New Phytol,2020,226:1738-1752;

[0118] Ma et al.Resequencing a core collection ofupland cotton identifiesgenomic variation and lociinfluencing fiberquality and yield.Nat Genet,2018,50:803-813.

[0119] Example 4

[0120] (1) Sequencing primer set designed based on 10 Lsnp:

[0121] First, 500 bp DNA sequences were extracted from the front and back of 10 Lsnp sites for primer design to ensure specific amplification of the Lsnp DNA sequence. After primer optimization, 10 sequencing primer pairs were finally obtained, with primer sequences shown in SEQ ID NO.11-30.

[0122] Since this application aims to provide a high-throughput detection method for multiple samples, a method using barcodes to label multiple samples combined with next-generation sequencing was developed. The core ideas for barcodes and sequencing in this application are derived from the invention patent: A method, kit, and application for genotyping using high-throughput sequencing (202010523088.0), which discloses a method for genotyping using high-throughput sequencing. According to the patent, based on the 10 primer pairs specifically amplifying the Lsnp DNA sequence, an 18bp adapter sequence 5'ATAGCGACGCGTTTCAAC3' is uniformly added to the 5' end of the reverse primers of the G-Lsnp series primers. Subsequent construction of barcode fragments and amplification of SNP fragments are carried out according to the requirements of this patent (202010523088.0).

[0123] (2) Genotyping of 272 individuals from the F2 population was performed using sequencing primers.

[0124] Previously, an F2 population was constructed between the disease-resistant variety Zhongzhimian 2 and the susceptible variety Xinluzao 36. Sequencing revealed variations at five Lsnp positions (Lsnp4, Lsnp5, Lsnp7, Lsnp8, and Lsnp10) in both parents. Therefore, the genotyping primer set was validated using 272 individual plants from this F2 population. F2 individual plants were planted and DNA was extracted. The SNP fragments were amplified using the method described in (1) above. Then, according to the patent (202010523088.0), overlap extension, product mixing, product purification, and sequencing were performed. After the sequencing data was returned, the data was processed by the server, the barcode was split, and the genotype was extracted.

[0125] like Figure 5 The image shows the genotyping density of the disease-resistant allele reads at Lsnp4, 5, 7, 8, and 10 for 272 samples. The horizontal axis represents the percentage of disease-resistant allele reads, and the vertical axis represents the density value. The three peaks, from left to right, represent materials with homozygous susceptible alleles, heterozygous alleles, and homozygous resistant alleles, respectively. The 272 individual plants were genotyped based on the percentage of disease-resistant allele reads for each material.

[0126] like Figure 6 As shown: According to Figure 5 The peak shape was used to genotype 272 individual plants at Lsnp4, 5, 7, 8, and 10. SS represents homozygous susceptible allele, RR represents homozygous resistant allele, and SR represents heterozygous genotype. The results showed that among the 272 individual plants, 42, 15, 64, 59, and 62 were homozygous resistant at Lsnp4, Lsnp5, Lsnp7, Lsnp8, and Lsnp10, respectively, and 152, 212, 68, 101, and 80 were homozygous susceptible genotypes, respectively.

[0127] The results showed that among the 272 individual plants, 42, 15, 64, 59, and 62 were homozygous for disease resistance at the five Lsnp sites (Lsnp4, Lsnp5, Lsnp7, Lsnp8, and Lsnp10), respectively, and 152, 212, 68, 101, and 80 were homozygous for disease susceptibility, respectively. 22 plants were homozygous at all five Lsnp sites, resulting in 13 different genotype combinations. This demonstrates that the developed 10 pairs of sequencing primers can accurately and rapidly detect genotypes in multiple samples.

[0128] Example 5

[0129] (1) Design of a genotyping primer set based on 10 Lsnp:

[0130] Since all 10 Lsnp variants are single nucleotide variants, designing annealing temperature-sensitive primers based on SNPs would have limitations such as strict PCR reaction conditions and unstable results. Therefore, it was prioritized to design genotyping primer sets using Indels linked to Lsnp. First, using the vcf file obtained in Example 1 above, PLINK was used to calculate Indels highly linked to Lsnp (R2>0.9). Then, 400bp DNA sequences before and after the target variant were extracted for primer design. After optimizing the primers and PCR reaction conditions, 10 pairs of genotyping primer sets were finally obtained, and the primer sequences are shown in SEQ ID NO.31-50 as follows:

[0131]

[0132]

[0133] (2) Ten pairs of genotyping primers were used for PCR reaction and banding detection with antibody / susceptible materials.

[0134] The resistant cotton variety 2 and the susceptible variety 36 were selected, and genomic DNA was extracted using the CTAB method. Primers were synthesized according to the 10 primer pairs in claim 3, and the PCR program was set according to the annealing temperature specified in (1), with all cycles set to 34×. Gel electrophoresis imaging was then performed.

[0135] like Figure 7 As shown: Markers 1-10 represent genotyping primer pairs designed based on 10 Lsnp molecules. R represents resistant material, and S represents susceptible material. The annealing temperature range is indicated above the R row in the 10 sets of graphs. Different lanes represent electrophoresis results within the set annealing temperature range. M-Lsnp1, 2, 6, and 9 can specifically amplify bands in susceptible material at the set annealing temperature, but cannot amplify bands in resistant material; M-Lsnp3, 4, 5, 7, 8, and 10 can specifically amplify bands in resistant material at the set annealing temperature, but cannot amplify bands in susceptible material.

[0136] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A group of SNP molecular markers associated with resistance to Verticillium wilt in upland cotton, characterized in that, The SNP molecular marker combination includes 10 SNP molecular markers, Lsnp1-Lsnp10; The nucleotide sequence of the SNP molecular marker of Lsnp1 is shown in SEQ ID NO.1, and the polymorphism site is located at position 66 of the sequence shown in SEQ ID NO.1, with a polymorphism of A / G. The nucleotide sequence of the SNP molecular marker of Lsnp2 is shown in SEQ ID NO.2, and the polymorphism site is located at position 144 of the sequence shown in SEQ ID NO.2, with a polymorphism of C / T. The nucleotide sequence of the SNP molecular marker of Lsnp3 is shown in SEQ ID NO.3, and the polymorphism site is located at position 67 of the sequence shown in SEQ ID NO.3, with a polymorphism of T / A. The nucleotide sequence of the SNP molecular marker of Lsnp4 is shown in SEQ ID NO.4, and the polymorphism site is located at position 127 of the sequence shown in SEQ ID NO.4, with a polymorphism of C / T. The nucleotide sequence of the SNP molecular marker of Lsnp5 is shown in SEQ ID NO.5, and the polymorphic site is located at position 126 of the sequence shown in SEQ ID NO.5, with a polymorphism of T / C. The nucleotide sequence of the SNP molecular marker of Lsnp6 is shown in SEQ ID NO.6, and the polymorphic site is located at position 125 of the sequence shown in SEQ ID NO.6, with a polymorphism of T / A. The nucleotide sequence of the SNP molecular marker of Lsnp7 is shown in SEQ ID NO.7, and the polymorphic site is located at position 112 of the sequence shown in SEQ ID NO.7, with a polymorphism of T / A. The nucleotide sequence of the SNP molecular marker of Lsnp8 is shown in SEQ ID NO.8, and the polymorphic site is located at position 112 of the sequence shown in SEQ ID NO.8, with a polymorphism of T / C. The nucleotide sequence of the SNP molecular marker of Lsnp9 is shown in SEQ ID NO.9, and the polymorphism site is located at position 93 of the sequence shown in SEQ ID NO.9, with a polymorphism of C / T. The nucleotide sequence of the SNP molecular marker of Lsnp10 is shown in SEQ ID NO.10, and the polymorphic site is located at position 90 of the sequence shown in SEQ ID NO.10, with a polymorphism of T / C.

2. The SNP molecular marker as described in claim 1, characterized in that, The disease-resistant dominant alleles of the SNP molecular markers Lsnp1-Lsnp10 are, in order, AA, CC, TT, CC, TT, TT, TT, TT, CC, TT.

3. A method for detecting resistance to Verticillium wilt in upland cotton, characterized in that, The disease resistance of upland cotton can be determined by detecting the genotype of the SNP molecular marker as described in claim 1. The physical location information of the 10 SNP molecular markers is as follows: The physical location information of SNP molecular markers was determined based on the Gossypiumhirsutum(AD1)'TM-1'genome version ZJU-improved_v2.1_a1 of the Texas Marker-1 upland cotton standard line.

4. The method as described in claim 3, characterized in that, The criterion for judging the resistance of upland cotton to Verticillium wilt is: the more disease-resistant genotypes there are, the stronger the resistance.

5. A primer set for amplifying the nucleotide sequence of the SNP molecular marker of claim 1, characterized in that, The primer sequences for amplifying the 10 SNP molecular markers Lsnp1-Lsnp10 are shown in SEQ ID NO.11-30.

6. The application of the SNP molecular marker as described in claim 1 or the primer set as described in claim 5 in the early prediction, screening or breeding of resistance to Verticillium wilt in upland cotton.

7. A primer set for detecting resistance to Verticillium wilt in upland cotton, characterized in that, The primer set is used to amplify nucleotide fragments within a 400bp range upstream and downstream of the SNP molecular marker described in claim 1. The primer sequences corresponding to the 10 SNP molecular markers Lsnp1-Lsnp10 are shown in SEQ ID NO.31-50, respectively.

8. The primer set as described in claim 7, characterized in that, A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.31-32 indicates a susceptible genotype, and the annealing temperature is 54-57℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.33-34 indicates a susceptible genotype, and the annealing temperature is 50-55℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.35-36 indicates a disease-resistant genotype, and the annealing temperature is 65-67℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.37-38 indicates a disease-resistant genotype, and the annealing temperature is 50-52℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.39-40 indicates a disease-resistant genotype, and the annealing temperature is 50-55℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.41-42 indicates a susceptible genotype, and the annealing temperature is 50-55℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.43-44 indicates a disease-resistant genotype, and the annealing temperature is 50-53.5℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.45-46 indicates a disease-resistant genotype, and the annealing temperature is 55-57℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.47-48 indicates a susceptible genotype, and the annealing temperature is 58-60℃; A positive PCR amplification result for the primer set corresponding to sequence SEQ ID NO.49-50 indicates a disease-resistant genotype, and the annealing temperature is 55-58℃.

9. The application of the primer set as described in claim 7 in the early prediction, screening, or breeding of resistance to Verticillium wilt in upland cotton.

10. A reagent kit for detecting resistance to Verticillium wilt in upland cotton, characterized in that, The kit contains the primer set as described in claim 5 or 7.

Images