Pair of non-synonymous SNPs molecular markers for identifying sea island cotton wilt resistance on D03 chromosome and application of non-synonymous SNPs molecular markers

Abstract

The invention discloses a pair of non-synonymous SNPs molecular markers for identifying sea island cotton wilt resistance on a D03 chromosome and application of the non-synonymous SNPs molecular markers. The invention belongs to the technical field of biology, and particularly relates to an SNPs molecular marker for identifying gossypium barbadense wilt resistance and application of the SNPs molecular marker. The invention provides a method for identifying or assisting in identifying sea island cotton wilt resistance, which comprises the following steps: detecting genotypes of SNPs sites in a sea island cotton genome to be detected, and identifying or assisting in identifying the sea island cotton wilt resistance according to the genotypes, the SNPs sites are a pair of non-synonymous SNPs sites on a sea island cotton D03 chromosome, and the SNPs sites are a pair of non-synonymous SNPs sites on a sea island cotton D03 chromosome. The nucleotides are located at the 67th site and the 105th site of the sequence 1 in the sequence table. The SNPs molecular marker disclosed by the invention can be used for molecular assisted breeding of disease-resistant sea island cotton, is beneficial to solving the problem of insufficient disease resistance of sea island cotton varieties in current cotton breeding, and also has great significance in overall improvement of quality and yield of sea island cotton.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a pair of non-synonymous SNPs molecular markers on chromosome D03 used to identify resistance to wilt disease in sea island cotton and their applications. Background Technology

[0002] Cotton is an important economic crop in my country, produced by the wilt-specific strain of Fusarium oxysporum (Fusarium oxysporum). Fusarium oxysporum f. sp. vasinfectum Wilt disease caused by ) is a limiting factor for the growth of island cotton ( Gossypium barbadense This disease is one of the most devastating diseases for sustainable production, often causing severe yield losses and a decline in fiber quality. Traditional field methods for assessing disease resistance are time-consuming, highly susceptible to environmental conditions, and easily confused with other soil-borne diseases, resulting in low efficiency and difficulty in guaranteeing accuracy.

[0003] With the development of molecular biology, marker-assisted selection technology has provided a new, efficient, and accurate approach for disease-resistant breeding. Although studies have reported quantitative trait loci associated with resistance to Fusarium wilt in sea island cotton, developing practical molecular markers that co-segregate with resistance genes and can be used for breeding remains a challenge. In particular, single nucleotide polymorphism (SNP) markers that can directly cause gene function alterations and have clear biological significance are especially scarce. Therefore, identifying key SNP molecular markers closely linked to resistance to Fusarium wilt in sea island cotton and possessing functional effects is of great significance for achieving early, efficient, and precise breeding of disease-resistant varieties. Summary of the Invention

[0004] The main problem this invention aims to solve is how to determine the level of resistance to Fusarium wilt in island cotton.

[0005] To address the aforementioned problems, this invention provides a method for identifying or assisting in the identification of wilt resistance in sea island cotton.

[0006] The method for identifying or assisting in the identification of wilt resistance in sea island cotton provided by the present invention includes detecting the genotype of SNP sites in the genome of the sea island cotton to be tested, and identifying or assisting in the identification of wilt resistance in sea island cotton based on the genotype.

[0007] The SNPs are two SNP sites, SNP1 and SNP2, on the D03 chromosome of cotton sea island. SNP1 is the 67th nucleotide of SEQ ID No:1 in the sequence listing, and its nucleotide type is G or A. SNP2 is the 105th nucleotide of SEQ ID No:1 in the sequence listing, and its nucleotide type is G or A.

[0008] The SNP molecular markers are located at positions 1601160 and 1601122 of the nucleotide sequence of chromosome D03 in the reference genome version of Gossypium barbadense (AD2)_3-79_HAU_v2.0.

[0009] This invention also provides a method for breeding sea island cotton, the method comprising detecting the genotypes of the SNPs mentioned above in the sea island cotton genome, and selecting the genotypes of the SNPs as... AAAA The island cotton was used as a parent for disease-resistant island cotton breeding. AAAA SNP1 is AA Genotype and the SNP2 is AA genotype.

[0010] Furthermore, the method also includes detecting the genotypes of the aforementioned SNPs in the genome of sea island cotton, and selecting the genotypes of the SNPs as... GGGG The sea island cotton was used as a parent for breeding susceptible sea island cotton. GGGG SNP1 is GG Genotype and the SNP2 is GG genotype.

[0011] The application of the methods described above in the breeding of island cotton also falls within the scope of protection claimed by this invention.

[0012] This invention also provides the application of a substance for detecting SNP polymorphisms or genotypes in the genome of sea island cotton in any of the following: (1) To identify or assist in the identification of Wilt resistance in Island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of resistance to Fusarium wilt in sea island cotton; (4) Prepare products for sea island cotton breeding; The SNPs are two SNP sites, SNP1 and SNP2, on the D03 chromosome of cotton sea island. SNP1 is the 67th nucleotide of SEQ ID No:1 in the sequence listing, and its nucleotide type is G or A. SNP2 is the 105th nucleotide of SEQ ID No:1 in the sequence listing, and its nucleotide type is G or A. In SEQ ID No:1, r represents G or A.

[0013] In the above applications, the genotypes of the SNPs are: GGGG or AAAA The GGGG SNP1 is GG Genotype and the SNP2 is GG Genotype, the AAAA SNP1 is AA Genotype and the SNP2 is AA Genotype; the genotype of the SNPs is GGGG The wilt disease incidence rate of the tested island cotton was higher than that of the genotypes of the SNPs mentioned above. AAAA The island cotton to be tested.

[0014] The present invention also provides a product containing the substance described above for detecting the polymorphism or genotype of the SNP in the genome of sea island cotton, wherein the product is any one of the following: C1) Products that detect single nucleotide polymorphisms or genotypes related to resistance to Fusarium wilt in sea island cotton; C2) Products used to identify or assist in identifying resistance to Fusarium wilt in island cotton; C3) is a product used for breeding island cotton.

[0015] In the above applications, methods, and products, the substance may be a reagent and / or instrument required to determine the polymorphism or genotype of the SNP site by at least one of the following methods: DNA sequencing, restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chips. The SNP chips include chips based on nucleic acid hybridization reactions, chips based on single-base extension reactions, chips based on allele-specific primer extension reactions, chips based on one-step reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule-DNA binding reactions.

[0016] In the above applications and products, the substance may be D1), D2), or D3). D1) The substance described is a primer composition for amplifying genomic DNA fragments of sea island cotton, including the SNP site described above; D2) The substance is a PCR reagent containing the primer composition described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

[0017] In the above applications, methods, and products, the primer composition may or may not 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 linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, 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 whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, optionally, charge-neutral. The marker can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without labeling (e.g., direct sequence reading).

[0018] The present invention also provides a DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing.

[0019] This invention also provides for the use of the DNA molecule described above in any of the following: (1) To identify or assist in the identification of wilt resistance in sea island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of resistance to Fusarium wilt in sea island cotton; (4) Prepare products for island cotton breeding.

[0020] Optionally, in the above applications, the DNA molecule serves as a detection target.

[0021] The substance that detects the SNP site polymorphism and genotype can be combined with other substances (such as substances that detect single nucleotide polymorphisms or genotypes of other molecular markers associated with resistance to Fusarium wilt in sea island cotton) to prepare a product for identifying sea island cotton varieties that are resistant to / susceptible to Fusarium wilt.

[0022] In this article, the breeding objectives may include developing wilt-resistant or wilt-susceptible island cotton varieties. The cotton may be a pure line or an inbred line.

[0023] This invention utilizes genome-wide association analysis (GWAS) to locate a pair of non-synonymous SNPs (Gbar_D03_1601160 and Gbar_D03_1601122) that are highly associated with resistance to Fusarium wilt in sea island cotton. These SNPs are located at positions 1601160 and 1601122 on chromosome D03 of the sea island cotton genome (reference genome for sea island cotton variety 3-79_HAU, https: / / yanglab.hzau.edu.cn / CottonMD / blast.1). The first non-synonymous SNP (Gbar_D03_1601160) has a nucleotide type of G or A and is located at nucleotide 67 of sequence 1 in the sequence listing. The second non-synonymous SNP (Gbar_D03_1601122) also has a nucleotide type of G or A and is located at nucleotide 105 of sequence 1 in the sequence listing. These two non-synonymous SNPs form two variant combinations, GG or AA, corresponding to the following genotypes: GGGG or AAAA .carry GGGG The genotype of Sea Island cotton varieties is susceptible to the disease and carries it. AAAA The genotype of Sea Island cotton is disease-resistant. This invention utilizes this SNP molecular marker to accurately genotype 195 Sea Island cotton germplasm resources.

[0024] Compared with existing technologies, the SNP molecular markers discovered in this invention that are significantly associated with wilt resistance in sea island cotton can be used for molecular-assisted breeding of wilt resistance traits in sea island cotton, helping to select disease-resistant cotton varieties in an early, efficient and precise manner, and greatly accelerating the molecular design breeding process of disease-resistant sea island cotton. Attached Figure Description

[0025] Picture 1 This is a histogram showing the frequency distribution of Fusarium wilt incidence phenotypes in two field environmental survey groups of sea island cotton in Korla, Xinjiang, China. Table a represents the frequency distribution of these phenotypes. Show Histogram of the frequency distribution of Fusarium wilt incidence (Disease percentage, DP, unit: %) phenotype in 195 field-grown Sea Island cotton accessions in Korla (K), Xinjiang, China, in 2015 (abbreviated as 15). b represents the histogram of the frequency distribution of Fusarium wilt incidence (DP) phenotype in 195 field-grown Sea Island cotton accessions in Korla (K), Xinjiang, China, in 2016 (abbreviated as 16).

[0026] Picture 2Local Manhattan plots and linkage disequilibrium (LD) heatmaps were used to locate non-synonymous SNPs that were significantly associated with the incidence of Fusarium wilt in sea island cotton. In plot a, the local Manhattan plot shows the non-synonymous SNPs significantly associated with the incidence of Fusarium wilt in sea island cotton; the light gray arrows mark the two non-synonymous SNPs (Gbar_D03_1601160 and Gbar_D03_1601122) that were located and significantly associated with the incidence of Fusarium wilt in sea island cotton. Plot b shows the local LD ​​heatmap near the SNPs.

[0027] Picture 3 For two genotypes carrying two non-synonymous SNP loci (Gbar_D03_1601160 and Gbar_D03_1601122) GGGG and AAAA The incidence of Fusarium wilt in two environmental groups of island cotton varieties were compared. Each dot represents an island cotton variety carrying a specific genotype. 'a' represents the incidence of Fusarium wilt (DP) in two genotypes of island cotton materials carrying two non-synonymous SNP loci grown in the field in Korla, Xinjiang, China in 2015 (15). 'b' represents the incidence of Fusarium wilt (DP) in two genotypes of island cotton materials carrying two non-synonymous SNP loci grown in the field in Korla, Xinjiang, China in 2016 (16). Detailed Implementation

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0031] The 195 Sea Island cotton cultivars in the following examples are described in: Zhao N, et al. Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadensein China and selection for fiber length, lint percentage and Fusarium wilt resistance. Plant Biotechnol J. 2022;20(4):691-710. The biological material is available to the public from the applicant and is intended solely for the purpose of repeating experiments of this invention and may not be used for any other purpose.

[0032] The following examples use statistical software to process the data. The experimental results are expressed as average values. T-tests are used. * indicates that the difference is statistically significant (P < 0.05), ** indicates that the difference is highly statistically significant (P < 0.01), and *** indicates that the difference is extremely statistically significant (P < 0.001).

[0033] Example 1: Survey of Wilt Resistance Phenotypic Data in Sea Island Cotton Populations In 2015 and 2016, 195 sea island cotton materials preserved by the Cotton Research Institute of the Xinjiang Academy of Agricultural Sciences were planted in the field in Korla, Xinjiang, China. The incidence of wilt disease was investigated during the seedling stage (3-4 leaf stage). The incidence of wilt disease was recorded as the percentage of individual plants showing symptoms of wilt disease such as chlorosis, yellowing, wilting, and dwarfing, and the percentage of diseased and dead plants out of the total number of plants. Two groups of phenotypic data on the incidence of wilt disease were investigated.

[0034] The results showed that in both sets of phenotypic data, the incidence of Fusarium wilt in Island cotton exhibited a continuous normal distribution, and the phenotypic variability of Fusarium wilt incidence in Island cotton populations was relatively large. Picture 1 (a and b).

[0035] Example 2: Whole-genome resequencing and SNP detection of sea island cotton population 195 leaf samples from individual plants of the Sea Island cotton variety were collected, and genomic DNA was extracted. The raw data was obtained through quality control, library construction, and sequencing. a Data. After quality control and filtering, high-quality clean data was obtained. Alignment to the reference genome 3-79 of *Cotton Island* was performed using BWA (version 0.7.8) software (parameters: mem -t 4 -k 32 -M). Population SNP detection was performed using the UnifiedGenotyper method with GATK (version v3.1) software, and high-quality SNPs were obtained through filtering, generating a vcf file.

[0036] Genome-wide association analysis (GEMMA) of resistance to Fusarium wilt in sea island cotton was performed using the efficient genome-wide association modeling software GEMMA 0.94.1 (http: / / www.xzlab.org / software.html) and a mixed linear model. The GEMMA software parameters were set to "gemma -bfile file -k kinship -lmm 1 -o outfile -miss 0.2 -maf0.05 -c covariates (GCTA: PCA)". Manhattan and QQ plots were generated using the R package qqman. F -test detects the effect value of gene markers and uses Bonferroni correction to correct multiple tests, using a standard (-log) 10 (P)>6) Screening for SNPs that are highly significantly associated with the incidence of Fusarium wilt.

[0037] In both environmental groups, a pair of non-synonymous SNP sites (Gbar_D03_1601160 and Gbar_D03_1601122) were located that were significantly associated with the incidence of Fusarium wilt in sea island cotton. Picture 2 (a and b). The first non-synonymous mutation SNP, Gbar_D03_1601160, is located at position 1601160 on chromosome D03. In 2015, the -log mutation of this SNP was located in Korla. 10 The p-value was 6.2, and the -log value of this SNP site was located in Korla in 2016. 10 The (P) value is 6.3, and its nucleotide type in the coding strand is either G or A (nucleotide 67 of sequence 1 in the sequence listing). The second nonsynonymous mutation SNP site, Gbar_D03_1601122, is located at position 1601122 on chromosome D03. In 2015, a -log sequence of this SNP site was located in Korla. 10 The p-value was 6.3, and the -log value of this SNP site was located in Korla in 2016. 10 With a p-value of 6.5, its nucleotide type in the coding strand is either G or A (nucleotide 105 of sequence 1 in the sequence listing). They form two haplotypes, GG and AA, corresponding to genotypes GG and AA, respectively. GGGG and AAAA It is speculated that these two non-synonymous SNP sites are significantly associated with the wilt resistance of sea island cotton. The nucleotide sequence of sequence 1 in the sequence listing is the sequence of the D03 chromosome of sea island cotton at the physical location of 1601226 bp - 1601027 bp.

[0038] Example 3: Identification of Wilt resistance in sea island cotton using a combination of two non-synonymous SNP molecular markers (Gbar_D03_1601160 and Gbar_D03_1601122). DNA was extracted from young leaves of 195 sea island cotton materials, and genotyping was performed using two non-synonymous SNP molecular markers (Gbar_D03_1601160 and Gbar_D03_1601122, also referred to as SNP1 and SNP2 in this paper).

[0039] Sequencing results revealed that among 195 Sea Island cotton accessions, 175 accessions showed homozygous G and G nucleotides at two non-synonymous SNP sites, SNP1 and SNP2 (Gbar_D03_1601160 and Gbar_D03_1601122), with genotypes of [missing information]. GGGG (GGGG is the SNP1 mentioned above) GG Genotype and the SNP2 is GG Genotype); 20 Sea Island cotton materials showed homozygous A and A nucleotides at two nonsynonymous SNP sites (Gbar_D03_1601160 and Gbar_D03_1601122), with genotypes of [missing information]. AAAA (AAAA is the SNP1 mentioned above) AA Genotype and the SNP2 is AA Genotypes (Table 1).

[0040] Table 1. Genotypes and average wilt incidence of two non-synonymous SNP molecular markers (Gbar_D03_1601160 and Gbar_D03_1601122) in Sea Island cotton materials

[0041] Statistical analysis was performed on the data in Table 1, and the carrier genotype was... GGGG The average incidence of wilt disease in island cotton was 39.4%, higher than that of cotton carrying the genotype [missing information]. AAAA The sea island cotton material (with an average wilt resistance of 1.7%) showed a highly significant difference between the two (P = 3.3E-17, i.e., P < 0.001) (Table 2 and). Picture 3 (a and b).

[0042] Table 2. Statistical analysis of the genotypes of two non-synonymous SNP molecular markers (Gbar_D03_1601160 and Gbar_D03_1601122) in sea island cotton and the phenotypic relationship with the average incidence of Fusarium wilt.

[0043] In summary, the two non-synonymous SNP molecular markers (Gbar_D03_1601160 and Gbar_D03_1601122) are SNP molecular marker loci that are highly significantly associated with the incidence of Fusarium wilt in sea island cotton. They can be used to identify or assist in identifying the level of resistance to Fusarium wilt in sea island cotton and can be applied to sea island cotton breeding, such as using the genotypes of the SNPs (Gbar_D03_1601160 and Gbar_D03_1601122) as... AAAA Sea Island cotton was used as a parent to breed varieties with high resistance to Fusarium wilt, or the genotypes at the SNPs (Gbar_D03_1601160 and Gbar_D03_1601122) were selected. GGGG The goal is to use sea island cotton as a parent to breed varieties with low resistance to Fusarium wilt, thereby shortening the breeding time of the target sea island cotton variety.

[0044] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for identifying or assisting in the identification of wilt resistance in sea island cotton, characterized in that: This includes detecting the genotype of SNP sites in the genome of the cotton sea island to be tested, and identifying or assisting in the identification of wilt resistance in cotton sea island based on the genotype. The SNP sites are two SNP sites, SNP1 and SNP2, on the D03 chromosome of cotton sea island. SNP1 is the 67th nucleotide of SEQ ID No:1 in the sequence listing, and its nucleotide type is G or A. SNP2 is the 105th nucleotide of SEQ ID No:1 in the sequence listing, and its nucleotide type is G or A.

2. A method for breeding island cotton, characterized by: The method includes detecting the genotype of the SNPs described in claim 1 in the genome of sea island cotton, and selecting the genotype of the SNPs as... AAAA The island cotton was used as a parent for disease-resistant island cotton breeding. AAAA The SNP1 is genotype AA and the SNP2 is genotype AA.

3. A method for breeding island cotton, characterized by: The method includes detecting the genotype of the SNPs described in claim 1 in the genome of sea island cotton, and selecting the genotype of the SNPs as... GGGG The sea island cotton was used as a parent for breeding susceptible sea island cotton. GGGG SNP1 is of the GG genotype and SNP2 is of the GG genotype.

4. The application of the method according to any one of claims 1-3 in the breeding of island cotton.

5. Application of substances used to detect SNP polymorphisms or genotypes in the genome of sea island cotton in any of the following: (1) To identify or assist in the identification of Wilt resistance in Island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of resistance to Fusarium wilt in sea island cotton; (4) Preparation of products for sea island cotton breeding; The SNPs are two SNP sites, SNP1 and SNP2, on the D03 chromosome of cotton sea island. SNP1 is the 67th nucleotide of SEQ ID No:1 in the sequence listing, and its nucleotide type is G or A. SNP2 is the 105th nucleotide of SEQ ID No:1 in the sequence listing, and its nucleotide type is G or A.

6. The application according to claim 5, characterized in that: The genotypes of the SNPs are GGGG or AAAA The GGGG SNP1 is GG Genotype and the SNP2 is GG Genotype, the AAAA Yes, SNP1 is... AA Genotype and the SNP2 is AA genotype; The genotypes of the SNPs are GGGG The wilt disease incidence rate of the tested island cotton was higher than that of the genotype of the SNP. AAAA The island cotton to be tested.

7. The product, characterized in that: The product contains the substance described in claim 5 or 6, wherein the product is any one of the following: C1) Products that detect single nucleotide polymorphisms or genotypes related to resistance to Fusarium wilt in sea island cotton; C2) Products used to identify or assist in identifying resistance to Fusarium wilt in island cotton; C3) is a product used for breeding island cotton.

8. The application according to claim 5 or 6, or the product according to claim 7, characterized in that: The substance is either D1), D2), or D3). D1) The substance described is a primer composition for amplifying genomic DNA fragments of sea island cotton, including the SNP site described above; D2) The substance is a PCR reagent containing the primer composition described in D1); D3) The substance is a kit containing the primer composition described in D1) or the PCR reagent described in D2).

9. A DNA molecule, characterized by: The nucleotide sequence of the DNA molecule is sequence 1 in the sequence listing.

10. The use of the DNA molecule of claim 9 in any of the following: (1) To identify or assist in the identification of wilt resistance in sea island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of resistance to Fusarium wilt in sea island cotton; (4) Prepare products for island cotton breeding.

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