SNP molecular marker for identifying plant height of gossypium barbadense and identification method and application thereof

Abstract

This invention discloses SNP molecular markers for identifying the height of Island cotton plants, along with their identification methods and applications. This invention belongs to the field of biotechnology, specifically relating to SNP molecular markers for identifying the height of Island cotton plants, along with their identification methods and applications. This invention provides a method for identifying or assisting in the identification of Island cotton plant height, including detecting the genotype of an SNP locus in the genome of the Island cotton to be tested, and identifying or assisting in the identification of the Island cotton plant height based on the genotype. The SNP locus is a single SNP site on chromosome A05 of the Island cotton, with nucleotide type T or A, and is the 100th nucleotide of sequence 1 in the sequence listing. The SNP molecular markers of this invention, significantly associated with Island cotton plant height, can be used to assist in the breeding of Island cotton varieties with suitable plant height, leading to the efficient development of new cotton varieties with suitable plant height for machine harvesting.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to SNP molecular markers for identifying the height of island cotton plants, their identification methods, and applications. Background Technology

[0002] Sea Island cotton ( Gossypium barbadense L. (Lysimachia christinae) fiber is of exceptionally high quality, making it an irreplaceable high-end natural raw material for the textile industry. However, Sea Island cotton plants are generally tall during their natural growth, making them highly susceptible to lodging in the later stages of growth and hindering mechanized harvesting. Plant height is a core agronomical trait that determines the plant structure, yield, and lodging resistance of Sea Island cotton. Precise improvement of plant height is crucial for achieving high yields, high quality, and mechanized harvesting of Sea Island cotton.

[0003] Traditional breeding methods mainly rely on manual phenotypic observation during the field growing season. This is not only time-consuming and inefficient, but also highly susceptible to environmental factors (such as temperature, light, water, fertilizer, and planting density), leading to poor selection accuracy. With the rapid development of molecular breeding technology, marker-assisted selection (MAS) using single nucleotide polymorphism (SNP) markers has provided an efficient technical means for early and precise improvement of the plant type of Sea Island cotton.

[0004] However, current technologies for finely locating control sites for plant height in sea island cotton are relatively limited, and there is a lack of high-throughput, high-specificity, and cost-effective SNP molecular markers. This greatly restricts the clinical application of molecular marker-assisted breeding for sea island cotton plant type. Therefore, identifying stable SNP sites significantly associated with plant height in sea island cotton and developing corresponding identification methods and applications is of significant theoretical and practical value for accelerating the breeding process for ideal plant type in sea island cotton and promoting mechanized harvesting. Summary of the Invention

[0005] The main problem this invention aims to solve is how to determine the plant height of sea island cotton.

[0006] To address the above problems, this invention provides a method for identifying or assisting in the identification of island cotton plant height.

[0007] This invention first provides a method for breeding sea island cotton, the method comprising detecting the genotype of SNPs in the sea island cotton genome, wherein the SNP molecular marker is located at position 12525419 of the nucleotide sequence of chromosome A05 of the sea island cotton reference genome version Gossypium barbadense (AD2)_3-79_HAU_v2.0, and its nucleotide type is T or A, and is the 100th nucleotide of sequence 1 in the sequence listing; The genotype of the SNP can be selected as TT Using sea island cotton as a parent, high-plant-height sea island cotton breeding was carried out. TT It is a homozygous form of the SNP being T.

[0008] 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... AA Using sea island cotton as a parent, low-plant-height sea island cotton breeding was carried out. AA It is a homozygous form of the SNP being A.

[0009] The method for identifying or assisting in the identification of island cotton plant height provided by the present invention includes detecting the genotype of the aforementioned SNP locus in the genome of the island cotton to be tested, and identifying or assisting in the identification of island cotton plant height based on the genotype.

[0010] The application of the identification method described above in the breeding of island cotton also falls within the scope of protection claimed by this invention.

[0011] 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 the plant height of island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the plant height of sea island cotton; (4) Preparation of products for sea island cotton breeding; The SNP site is an SNP site on chromosome A05 of sea island cotton, and its nucleotide type in the coding strand is T or A, which is the 100th nucleotide of sequence 1 in the sequence listing.

[0012] In the above methods or applications, the genotype of the SNP is: TT or AA The TT It is the homozygous form of the SNP being T, the AA It is the homozygous type of the SNP being A; the genotype of the SNP is TT The plant height of the sea island cotton to be tested was higher than that of the genotype of the SNP mentioned above. AA The island cotton to be tested.

[0013] 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: T1) Products that detect single nucleotide polymorphisms or genotypes related to plant height in sea island cotton; T2) Products used for identifying or assisting in the identification of island cotton plant height; T3) is a product used for breeding island cotton.

[0014] 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.

[0015] 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).

[0016] The present invention also provides a DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing. Optionally, in the above applications, the DNA molecule serves as a detection target.

[0017] This invention also provides for the use of the DNA molecule described above in any of the following: (1) To identify or assist in identifying the plant height of island cotton; (2) Breeding of island cotton plant height; (3) Prepare products for identification or auxiliary identification of the plant height of sea island cotton; (4) Prepare products for breeding of island cotton plant height.

[0018] 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 related to the plant height of Island cotton) to prepare a product for identifying tall / low Island cotton varieties.

[0019] In this article, the breeding objectives may include developing tall or short-planted island cotton varieties. The cotton may be a pure line or an inbred line.

[0020] This invention utilizes genome-wide association study (GWAS) to preliminarily locate a SNP locus (Gbar_A05_12525419) that is significantly associated with plant height in *Cotton Island cotton*. This SNP locus is located at position 12525419 on chromosome A05 of the *Cotton Island cotton* genome (reference genome: *Cotton Island cotton* 3-79_HAU, https: / / yanglab.hzau.edu.cn / CottonMD / blast.1). It is significantly associated with plant height, contains either T or A nucleotides, is the 100th nucleotide of sequence 1 in the sequence listing, and is located in the upstream promoter region of the gene. It regulates gene function by influencing functional expression. TT The genotype of sea island cotton varieties is tall and carries... AA The genotype of Sea Island cotton is characterized by low plant height. This SNP molecular marker can be used to accurately genotype 295 Sea Island cotton germplasm resources.

[0021] Compared with existing technologies, the SNP molecular markers discovered in this invention that are significantly associated with the plant height of Island cotton can assist in the breeding of Island cotton varieties with suitable plant height, help break through the current bottleneck in breeding Island cotton with suitable machine-harvested plant types, and enhance the commercial value of Island cotton genetic resources in the application of machine-harvested long-staple cotton breeding. Attached Figure Description

[0022] Figure 1 This is a histogram showing the frequency distribution of plant height phenotype in a field environmental survey conducted in Korla, Xinjiang Uygur Autonomous Region, China in 2019. 2019 is abbreviated as 19. Korla is abbreviated as K. Plant height is abbreviated as PH, in centimeters.

[0023] Figure 2 Manhattan plots and quantile-quantile (QQ) plots are shown for locating plant height-related loci in the Korla environment of Xinjiang Uygur Autonomous Region, China, in 2019. In plot 'a', the Manhattan plot shows the location of plant height-related loci in 336 field-grown cotton accessions in Korla, Xinjiang Uygur Autonomous Region, China, in 2019. The horizontal dashed line represents the significance threshold, i.e., -log[…]. 10 (P) is 6.0. Arrows mark SNP sites that are significantly associated with plant height (Gbar_A05_12525419); b is a QQ plot of plant height-associated sites in 336 sea island cotton accessions grown in the field in Korla, Xinjiang Uygur Autonomous Region, China in 2019.

[0024] Figure 3 Two genotypes carrying the SNP locus (Gbar_A05_12525419) TTand (AA) Plant height phenotype of island cotton varieties. n This indicates the number of island cotton varieties carrying a specific genotype. Detailed Implementation

[0025] 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.

[0026] Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0029] The 336 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 inChina 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.

[0030] The 295 samples of Sea Island cotton described in the following examples are documented in: Zhao N, et al. Genomic and GWASanalyses demonstrate phylogenomic relationships of Gossypium barbadense inChina 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.

[0031] The following examples use IBM SPSS Statistical 25 software to process the data. Experimental results are expressed as mean values, and T-tests are used. This indicates that the difference is statistically significant (P < 0.05). This indicates that the difference is highly statistically significant (P < 0.01). This indicates that the difference is statistically significant (P < 0.001).

[0032] Example 1: Discovery of the SNP locus (Gbar_A05_12525419) that is highly significantly associated with plant height of sea island cotton. 1. Survey of plant height phenotypic data of sea island cotton population materials In 2019, 336 sea island cotton materials preserved by the Cotton Research Institute of the Xinjiang Academy of Agricultural Sciences in China were planted in the field in Korla, Xinjiang Uygur Autonomous Region, China. Plant height phenotypic data were investigated, and the height (cm) from the cotyledon node to the top of the main stem was measured.

[0033] The results showed that the plant height of sea island cotton exhibited a clear normal distribution. Figure 1 This is suitable for locating genetic loci that are significantly associated with the height of island cotton plants using genome-wide association analysis.

[0034] 2. Whole-genome resequencing and SNP detection of sea island cotton population Genomic DNA was extracted from 336 leaf samples of individual plants of the Sea Island cotton variety. Raw data was obtained through quality control, library construction, and sequencing. After quality control and filtering, high-quality clean data was obtained. Data was aligned to the Sea Island cotton reference genome (3-79) using BWA (version 0.7.8) software (parameters: mem -t 4 -k 32 -M). Population SNPs were detected using the UnifiedGenotyper method with GATK (version v3.1) software, and high-quality SNPs were obtained through filtering, generating a vcf file.

[0035] Genome-wide association analysis (GEMMA) of plant height 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 -maf 0.05 -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, via -log. 10 Standard screening for SNPs with (P)>6 and a highly significant association with plant height.

[0036] In 2019, a SNP locus (Gbar_A05_12525419) significantly associated with plant height of sea island cotton was located in a field environment in Korla, Xinjiang Uygur Autonomous Region, China. This SNP locus is located at position 12525419 on chromosome A05, and the -log value of this SNP locus was determined. 10 The value of (P) is 6.8 ( Figure 2 (a and b), whose nucleotide type is T or A (located at position 100 of sequence 1 in the sequence listing). The nucleotide sequence of sequence 1 in the sequence listing is the sequence at the physical location 12525320 bp - 12525519 bp on chromosome A05 of Cotton Island.

[0037] The specific nucleotide sequence of Sequence 1 is: 5'-agttgggcctttttaaaatttttttatagatttatatgaaaatattggtcggccgaatgaaaagtgaaaacccgattttgtaacttttgctctttttaawgtatatttcatacgaatgagatggaccaatcaatttttttaaagaaacataaatttgataaatatttccaatttttttagtaaaaatatttttatacatcc-3'. In Sequence 1, w represents T or A.

[0038] Example 2: Identification of plant height of Sea Island cotton using SNP molecular marker (Gbar_A05_12525419) DNA was extracted from young leaves of 295 sea island cotton materials, and genotyping was performed using the SNP molecular marker (Gbar_A05_12525419). This invention obtains the genotype of the SNP molecular marker through sequencing. Given the nucleotide sequence of Sequence 1, the reagents and / or instruments required to determine the polymorphism or genotype of the SNP site in Sequence 1 can also be determined using at least one of the following methods: restriction fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, or SNP microarray, etc.

[0039] The sequencing results in Table 1 revealed that among the 295 Sea Island cotton accessions, 58 accessions showed a homozygous T nucleotide sequence at the SNP site (Gbar_A05_12525419), indicating a genotype of [missing information]. TT 237 Sea Island cotton materials showed homozygous nucleotides of A at the SNP locus (Gbar_A05_12525419), indicating that their genotype was... AA .

[0040] Table 1. Genotypes and plant height of SNP molecular marker (Gbar_A05_12525419) in Sea Island cotton materials

[0041] Statistical analysis was performed on the data in Table 1 (Table 2 and...). Figure 3 ), carrying the genotype TT The average plant height of the island cotton material was 92.8 cm, which is higher than that of the material carrying the genotype. AA The sea island cotton material (average plant height 75.2 cm) showed a highly significant difference between the two. P = 7.5E-13, i.e., P<0.001) level.

[0042] Table 2. Statistical analysis of SNP molecular marker (Gbar_A05_12525419) genotype and plant height phenotype in Sea Island cotton.

[0043] In summary, the SNP molecular marker (Gbar_A05_12525419) is a SNP molecular marker locus significantly associated with the height of Island cotton plants. It can identify or assist in identifying the height of Island cotton plants and can be used for Island cotton breeding, for example, by using the genotype of the SNP (Gbar_A05_12525419) locus as... TT Sea island cotton was used as a parent to breed tall varieties, or the genotype at the SNP (Gbar_A05_12525419) locus was used as a reference. AA Sea island cotton was used as a parent to breed varieties with low plant height, in order to shorten 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 breeding island cotton, characterized by: The method includes detecting the genotype of SNP sites in the genome of sea island cotton, wherein the SNP sites are located on chromosome A05 of sea island cotton, and the nucleotide type is T or A, which is the 100th nucleotide of sequence 1 in the sequence listing; Select the genotype of the SNP as TT Using sea island cotton as a parent, high-plant-height sea island cotton breeding was carried out. TT It is a homozygous form of the SNP being T.

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... AA Using sea island cotton as a parent, low-plant-height sea island cotton breeding was carried out. AA It is a homozygous form of the SNP being A.

3. A method for identifying or assisting in the identification of island cotton plant height, characterized in that: This includes detecting the genotype of the SNP locus described in claim 1 in the genome of the cotton island to be tested, and identifying or assisting in the identification of cotton island plant height based on the genotype.

4. The application of the method described in claim 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 the plant height of island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the plant height of sea island cotton; (4) Preparation of products for sea island cotton breeding; The SNP site is an SNP site on chromosome A05 of sea island cotton, and its nucleotide type in the coding strand is T or A, which is the 100th nucleotide of sequence 1 in the sequence listing.

6. The method according to any one of claims 1-3, and the application according to claim 5, characterized in that: The genotype of the SNP is TT or AA The TT It is the homozygous form of the SNP being T, the AA It is the homozygous type of the SNP being A; the genotype of the SNP is TT The plant height of the sea island cotton to be tested was higher than that of the genotype of the SNP mentioned above. AA The island cotton to be tested.

7. A nucleotide sequence is the application of sequence 1 of the sequence listing in any of the following ways: (1) To identify or assist in identifying the plant height of island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the plant height of sea island cotton; (4) Prepare products for island cotton breeding.

8. The product, characterized in that: The product contains the substance described in claim 5, wherein the product is any one of the following: T1) Products that detect single nucleotide polymorphisms or genotypes related to plant height in sea island cotton; T2) Products used for identifying or assisting in the identification of island cotton plant height; T3) is a product used for breeding island cotton.

9. The application according to claim 5 or 6, or the product according to claim 8, 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).

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