SNP molecular marker GbarD031878030 for identifying fusarium wilt resistance of gossypium barbadense and application of SNP molecular marker GbarD031878030

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker GbarD031878030 for identifying fusarium wilt resistance of gossypium barbadense and application of the SNP molecular marker GbarD031878030. The invention belongs to the technical field of biology, and particularly relates to an SNP (Single Nucleotide Polymorphism) molecular marker for identifying gossypium barbadense wilt resistance and application of the SNP molecular marker. The invention provides a method for identifying or assisting in identifying sea island cotton wilt resistance, which comprises the following steps: detecting the genotype of an SNP (Single Nucleotide Polymorphism) site in a sea island cotton genome to be detected, and identifying or assisting in identifying the sea island cotton wilt resistance according to the genotype, the SNP site is located in a sea island cotton D03 chromosome, the nucleotide variety of the SNP site in a coding sequence is T or C, and the nucleotide variety of the SNP site in the coding sequence is T or C; the nucleotide is the 388th nucleotide of the sequence 1 in the sequence table. The SNP molecular marker disclosed by the invention can be used for rapidly and efficiently identifying the variety of the fusarium wilt-resistant sea island cotton in an early stage, assists in molecular assisted breeding of the sea island cotton, and has great significance in increasing the proportion of the sea island cotton in the whole textile industry and increasing the international influence of the sea island cotton in the whole textile industry.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the SNP molecular marker Gbar_D03_1878030 for identifying resistance to Fusarium wilt in sea island cotton and its application. Background Technology

[0002] Cotton is a vital economic crop and raw material for the textile industry in my country and globally, and its stable production is crucial for ensuring national food and cotton security. Island cotton (…) Gossypium barbadense L.) has long fibers, high strength, and excellent quality, making it extremely valuable economically. Cotton wilt is a devastating soil-borne vascular disease that severely restricts the yield and quality improvement of Sea Island cotton, causing reduced yields or even total crop failure, resulting in huge economic losses for cotton production.

[0003] Currently, the most economical, effective, and environmentally friendly strategy for controlling cotton wilt is to cultivate and plant disease-resistant varieties. Traditionally, the identification of cotton wilt resistance mainly relies on field nurseries or greenhouse inoculation, but this process is time-consuming, requires a large amount of land and manpower, and is easily affected by various factors such as environmental conditions, inoculation concentration, differentiation of pathogen physiological races, and plant growth stage, which seriously delays the process of disease-resistant breeding.

[0004] Marker-assisted selection (MAS) technology provides a powerful tool for crop genetic improvement. Single nucleotide polymorphism (SNP) markers have become the core of third-generation molecular marker technology. Functional SNPs (non-synonymous SNPs) closely linked to resistance to Fusarium wilt in sea island cotton, especially those located in the gene coding region and capable of directly causing amino acid changes, and thus potentially participating in the disease resistance response, are extremely scarce.

[0005] Therefore, identifying and verifying key SNP sites that are highly significantly associated with resistance to Fusarium wilt in sea island cotton and may themselves participate in regulating resistance, and developing their corresponding functional molecular markers, is of great scientific significance and application value for achieving early, rapid, and precise breeding of Fusarium wilt-resistant varieties of sea island cotton and breaking through breeding technology bottlenecks. Summary of the Invention

[0006] The main problem this invention aims to solve is how to determine the level of resistance to Fusarium wilt in island cotton.

[0007] To address the aforementioned problems, this invention provides a method for identifying or assisting in the identification of wilt resistance in sea island cotton.

[0008] 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. The SNP site is the 388th nucleotide of sequence 1 in the sequence listing, and its nucleotide type is T or C.

[0009] The SNP molecular marker is located at position 388 of the nucleotide sequence of chromosome D03 in the reference genome version of Gossypium barbadense (AD2)_3-79_HAU_v2.0, and is a non-synonymous SNP site on chromosome D03 of Gossypium barbadense.

[0010] This invention also provides a method for breeding sea island cotton, the method comprising detecting the genotype of the SNPs mentioned above in the sea island cotton genome, and selecting the genotype of the SNPs as... CC Sea island cotton was used as a parent to breed sea island cotton resistant to Fusarium wilt. CC It is a homozygous form of the SNP being C.

[0011] Furthermore, the method also includes detecting the genotype of the aforementioned SNP in the genome of sea island cotton, and selecting the genotype of the SNP as... TT The sea island cotton was used as a parent to breed sea island cotton susceptible to Fusarium wilt. TT It is a homozygous form of the SNP being T.

[0012] The application of the methods described above in the breeding of island cotton also falls within the scope of protection claimed by this invention.

[0013] 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) Preparation of products for sea island cotton breeding; The SNP site is the 388th nucleotide of sequence 1 in the sequence listing, and its nucleotide type is T or C.

[0014] In this invention, the genotype of the SNP is TT or CC The TT It is the homozygous form of the SNP being T, the CC It is the homozygous type of the SNP being C; the SNP genotype is TT The incidence of wilt in the tested island cotton was higher than that of the genotype of the SNP. CC The island cotton to be tested.

[0015] 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.

[0016] 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.

[0017] 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).

[0018] 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).

[0019] The present invention also provides a DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing.

[0020] 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.

[0021] In the above applications, the DNA molecule serves as a detection target.

[0022] 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 wilt resistance in sea island cotton) to prepare a product for identifying sea island cotton varieties that are resistant to / susceptible to wilt.

[0023] 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.

[0024] This invention utilizes genome-wide association analysis (GWAS) to locate a non-synonymous SNP locus (Gbar_D03_1878030) that is highly significantly associated with resistance to Fusarium wilt in sea island cotton. This SNP locus is located at position 1878030 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). In the coding strand, its nucleotide type is T or C, and it is nucleotide 388 of sequence 1 in the sequence listing. The genotype formed by this non-synonymous SNP locus is... TT or CC .carry TT The genotype of Sea Island cotton varieties is susceptible to the disease and carries it. CC The genotype of Sea Island cotton is disease-resistant. This invention utilizes this SNP molecular marker to accurately genotype 197 Sea Island cotton germplasm resources.

[0025] Compared with existing technologies, the SNP molecular markers discovered in this invention that are significantly associated with resistance to Fusarium wilt in sea island cotton have outstanding advantages such as rapid detection and accurate results. They can achieve early and direct prediction of resistant sea island cotton materials, break through the bottleneck of traditional breeding, provide key technical support for the breeding of stable disease-resistant cotton varieties, and have significant industrial application value. Attached Figure Description

[0026] Figure 1 This is a frequency distribution histogram of the average incidence rate (Disease percent, DP, %) of wilt disease in four field environmental surveys of sea island cotton populations in Xinjiang, China.

[0027] Figure 2 Local 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 that were significantly associated with the incidence of Fusarium wilt in sea island cotton, with light gray arrows marking the located non-synonymous SNPs (Gbar_D03_1878030) that were significantly associated with the incidence of Fusarium wilt in sea island cotton. Plot b shows the local LD ​​heatmap near the SNPs.

[0028] Figure 3 To carry two genotypes of nonsynonymous SNP locus (Gbar_D03_1878030) TT and CC The average incidence of Fusarium wilt in four environmental groups for sea island cotton varieties. Each dot represents a sea island cotton variety carrying a specific genotype. Detailed Implementation

[0029] 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.

[0030] 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.

[0031] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0032] The 197 Sea Island cotton cultivars in the following examples are described in: Zhao N, et al. Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China and selection for fiber length, lint percentage and Fusarium wiltresistance. 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.

[0033] 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).

[0034] Example 1: Survey of Wilt Resistance Phenotypic Data in Sea Island Cotton Populations 197 sea island cotton materials preserved by the Cotton Research Institute of Xinjiang Academy of Agricultural Sciences were planted in the field in Korla, Xinjiang, China in 2015, 2016, 2018, and 2019. 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. A total of four groups of wilt disease incidence phenotypic data were collected, and the average incidence of wilt disease (%) of the four groups of raw data was calculated.

[0035] The results showed that, in the average wilt incidence phenotype of the four sets of raw data, the wilt incidence of sea island cotton ranged from 0 to 86.0%, exhibiting a large range of variation and displaying typical characteristics of quantitative genetic traits, namely a continuous normal distribution. This makes it suitable for locating disease resistance-related loci using genome-wide association analysis. Figure 1 ).

[0036] Example 2: Whole-genome resequencing and SNP detection of sea island cotton population 197 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.

[0037] 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.

[0038] In five datasets (four original environments and average values), three datasets jointly located a non-synonymous SNP locus (Gbar_D03_1878030) that was highly significantly associated with the incidence of Fusarium wilt in sea island cotton. Figure 2 The SNP locus is located at position 1878030 on chromosome D03. In 2015, the -log sequence of this SNP locus was located in Korla. 10 The p-value was 7.7, and the -log value of this SNP site was located in Korla in 2016. 10 The p-value is 7.4, and the -log value of the SNP locus is located in the mean dataset of the four sets of original data. 10 The (P) value is 6.1, and its nucleotide type in the coding strand is either T or C (the 388th nucleotide of sequence 1 in the sequence listing), forming the genotype. TT and CC , speculation Should Nonsynonymous SNP sites were significantly associated with wilt resistance in Sea Island cotton. The nucleotide sequence of Sequence 1 in the sequence listing is the sequence at the physical location 1877643 bp - 1878140 bp on the D03 chromosome of Sea Island cotton. In Sequence 1, y represents T or C.

[0039] Example 3: Identification of Wilt Resistance in Island Cotton Using Non-Synonymous SNP Molecular Marker (Gbar_D03_1878030) DNA was extracted from young leaves of 197 sea island cotton materials and genotyped using non-synonymous SNP molecular marker (Gbar_D03_1878030).

[0040] Sequencing results revealed that among 197 Sea Island cotton accessions, 172 accessions showed a homozygous T nucleotide sequence at the non-synonymous SNP site (Gbar_D03_1878030), with the genotype being [missing information]. TT 25 Sea Island cotton materials were homozygous for C nucleotides at the non-synonymous SNP site (Gbar_D03_1878030), with the following genotype: CC (Table 1).

[0041] Table 1. Genotypes of non-synonymous SNP molecular markers (Gbar_D03_1878030) and average wilt disease incidence rates in four environmental groups in Sea Island cotton materials.

[0042] Statistical analysis was performed on the data in Table 1, and the carrier genotype was... TT The average incidence of wilt disease in island cotton materials was 40.3%, higher than that of materials carrying the genotype [missing information]. CC The sea island cotton material (with an average wilt resistance of 2.1%) showed a highly significant difference between the two (P = 7.4E-18, i.e., P < 0.001) (Table 2 and). Figure 3 ).

[0043] Table 2. Statistical analysis of the genotypes of non-synonymous SNP molecular markers (Gbar_D03_1878030) in sea island cotton and the average incidence of Fusarium wilt.

[0044] In summary, the non-synonymous SNP molecular marker (Gbar_D03_1878030) is a SNP molecular marker locus that is highly significantly associated with the incidence of Fusarium wilt in sea island cotton. It can identify or assist in identifying the level of resistance to Fusarium wilt in sea island cotton and can be used in sea island cotton breeding, such as using the genotype of the SNP (Gbar_D03_1878030) locus as... CCSea Island cotton was used as a parent to breed wilt-resistant Sea Island cotton varieties, or the genotype at the SNP (Gbar_D03_1878030) locus was used as the parent. TT The aim is to use sea island cotton as a parent to breed sea island cotton varieties susceptible to Fusarium wilt, thereby shortening the breeding time for the target sea island cotton varieties.

[0045] 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 site is the 388th nucleotide of sequence 1 in the sequence listing, and its nucleotide type is T or C.

2. A method for breeding island cotton, characterized by: The method includes detecting the genotype of the SNP in claim 1 in the genome of sea island cotton, and selecting the genotype of the SNP as... CC Sea island cotton was used as a parent to breed sea island cotton resistant to Fusarium wilt. CC It is a homozygous form of the SNP being C.

3. A method for breeding island cotton, characterized by: The method includes detecting the genotype of the SNP in claim 1 in the genome of sea island cotton, and selecting the genotype of the SNP as... TT The sea island cotton was used as a parent to breed sea island cotton susceptible to Fusarium wilt. TT It is a homozygous form of the SNP being T.

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 SNP site is an SNP site on the D03 chromosome of cotton sea island, and its nucleotide type in the coding sequence is T or C, which is the 388th nucleotide of sequence 1 in the sequence listing.

6. The method according to claim 1, and the application according to claim 5, characterized in that: The genotype of the SNP is TT or CC The TT It is the homozygous form of the SNP being T, the CC It is the homozygous type of the SNP being C; the genotype of the SNP is TT The incidence of wilt in the tested island cotton was higher than that of the genotype of the SNP. CC 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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