SNP (Single Nucleotide Polymorphism) molecular marker for identifying initial fruit branch position of gossypium barbadense and application thereof

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker for identifying an initial fruit branch knot position of gossypium barbadense and application of the SNP molecular marker. The invention belongs to the field of nucleic acid detection, and particularly relates to an SNP molecular marker for identifying an initial fruit branch knot position of gossypium barbadense and application of the SNP molecular marker. The invention provides a method for identifying or assisting in identifying an initial fruit branch knot position of gossypium barbadense, which comprises the following steps: detecting the genotype of an SNP (Single Nucleotide Polymorphism) site in a gossypium barbadense genome to be detected, and identifying or assisting in identifying the initial fruit branch knot position of gossypium barbadense according to the genotype, the SNP site is an SNP site on a gossypium barbadense D07 chromosome, and the nucleotide type is C or T; the nucleotide is the 811th nucleotide of the sequence 1 in the sequence table. The SNP molecular marker obviously related to the beginning fruit branch knot position of the sea island cotton can be used for sea island cotton early-maturing character molecule auxiliary breeding, high-efficiency screening of early-maturing cotton varieties is assisted, and the economic value of the sea island cotton is improved.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid detection, specifically relating to SNP molecular markers for identifying the nodes of the initial fruiting branches of sea island cotton and their applications. Background Technology

[0002] Sea Island cotton ( Gossypium barbadense As the allotetraploid cotton cultivar with the best fiber quality, its fiber is an important raw material for high-end textiles and is of strategic significance for the upgrading of the textile industry. Xinjiang, as my country's only large-scale sea island cotton planting area, has unique light and heat conditions that are more suitable for the cultivation of early-maturing varieties. It is worth noting that early-maturing varieties typically have a lower initial fruiting node. Therefore, developing molecular markers for accurately identifying the initial fruiting node is crucial for accelerating the breeding of early-maturing sea island cotton varieties and supporting the development of my country's high-end cotton textile industry.

[0003] However, the key bottleneck restricting the breeding efficiency of early-maturing island cotton is the extreme scarcity of molecular markers that can be used for genetic identification of the first fruiting branch nodes of island cotton. Traditional breeding methods that rely on phenotypic selection have problems such as long cycles and low efficiency, making it difficult to meet the industry's urgent demand for early-maturing and high-quality island cotton varieties. Summary of the Invention

[0004] The main problem this invention aims to solve is how to determine the height of the first fruiting branch node in sea island cotton.

[0005] To address the aforementioned problems, this invention 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 first fruiting branch node of sea island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the first fruiting branch nodes of sea island cotton; (4) Preparation of products for sea island cotton breeding; The SNP site is an SNP site on the D07 chromosome of sea island cotton, and its nucleotide type is C or T, which is the 811th nucleotide of sequence 1 in the sequence listing.

[0006] In the above application, the genotype of the SNP is CC or TT The CC It is the homozygous type where the SNP is C, the TT The SNP is homozygous for T; the genotype of the SNP is CC The genotype of the sea island cotton whose initial fruiting branch node is higher than the SNP is... TT The island cotton to be tested.

[0007] This invention also provides a method for identifying or assisting in the identification of the node position of the first fruiting branch of sea island cotton.

[0008] The method for identifying or assisting in the identification of the initial fruiting branch node of sea island cotton provided by this invention includes detecting the genotype of a SNP site in the genome of the sea island cotton to be tested, and identifying or assisting in the identification of the initial fruiting branch node of the sea island cotton based on the genotype. The SNP site is a SNP site on the D07 chromosome of sea island cotton, and its nucleotide type is C or T, specifically the 811th nucleotide of Sequence 1 in the sequence listing. In Sequence 1, y represents C or T.

[0009] The SNP molecular marker is located in the reference genome version of cotton sea island. Gossypium barbadense (AD2) _3-79_HAU_v2.0, position 15803388 of the D07 chromosome nucleotide sequence.

[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... TT The island cotton was used as a parent for breeding island cotton with low fruiting branch nodes. TT It is a homozygous form of the SNP being T.

[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... CC The island cotton was used as a parent for breeding island cotton with high fruiting branch positions. CC It is a homozygous form of the SNP being C.

[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] 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 the nodes of the first fruiting branches of sea island cotton; C2) Products used for identification or auxiliary identification of the first fruiting branch node of sea island cotton; C3) 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] 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).

[0017] The present invention also provides a DNA molecule whose nucleotide sequence is sequence 1 in the sequence listing.

[0018] This invention provides for the use of the DNA molecule described above in any of the following: (1) To identify or assist in the identification of the first fruiting branch node of island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the first fruiting branch nodes of sea island cotton; (4) Prepare products for island cotton breeding.

[0019] Optionally, in the above applications, the DNA molecule serves as a detection target.

[0020] 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 the fruiting branch node of Sea Island cotton) to prepare a product for identifying Sea Island cotton varieties with high / low fruiting branch node.

[0021] In this article, the breeding objectives may include developing island cotton varieties with high or low initial fruiting branch nodes. The cotton may be a pure line or an inbred line.

[0022] This invention utilizes genome-wide association analysis (GWAS) to locate a SNP locus (Gbar_D07_15803388) that is highly significantly associated with the initial fruiting branch node of *Cotton Island cotton*. This SNP locus is located at position 15803388 on chromosome D07 of the *Cotton Island cotton* genome (referencing *Cotton Island cotton* variety 3-79 genome AD2_HAU_v2.0). The nucleotide type of this SNP locus is C or T, and it is nucleotide 811 of sequence 1 in the sequence listing. CC The genotype of Sea Island cotton is a late-maturing type with high fruiting branch nodes, carrying... TT The genotype of island cotton is an early-maturing type with low fruiting branch nodes. This invention utilizes this SNP molecular marker to accurately genotype 322 island cotton germplasm resources.

[0023] Compared with existing technologies, the SNP molecular markers discovered in this invention that are significantly associated with the node position of the first fruiting branch of sea island cotton can be used for molecular-assisted breeding of the node position trait of the first fruiting branch of sea island cotton, which is of great significance for the molecular breeding of early-maturing sea island cotton. Attached Figure Description

[0024] Figure 1This is a histogram showing the frequency distribution of the node of first fruit branch (NFFB) phenotype in nine field environmental surveys of island cotton materials in Xinjiang, China. a and b are the frequency distribution histograms of the node of first fruit branch (NFFB) phenotype in Awati (A), Xinjiang, China, in 2018 (abbreviated as 18) and 2019 (abbreviated as 19), respectively; c is the frequency distribution histogram of the node of first fruit branch (NFFB) phenotype in Baotou Lake (B), Xinjiang, China, in 2019; and di is the frequency distribution histogram of the node of first fruit branch (NFFB) phenotype in Korla (K), Xinjiang, China, in 2013 (abbreviated as 13), 2014 (abbreviated as 14), 2015 (abbreviated as 15), 2016 (abbreviated as 16), 2018, and 2019.

[0025] Figure 2 Manhattan plots (left) and quantile-quantile (QQ) plots (right) for locating the primary fruiting node association sites of *Cotton Island Humboldt* in nine environmental groups in Xinjiang, China. A and b are the Manhattan and QQ plots for locating the primary fruiting node association sites of *Cotton Island Humboldt* grown in the field in Awat, Xinjiang, China in 2018 and 2019, respectively; c is the Manhattan and QQ plot for locating the primary fruiting node association sites of *Cotton Island Humboldt* grown in the field in Baotouhu, Xinjiang, China in 2019; d to i are the Manhattan and QQ plots for locating the primary fruiting node association sites of *Cotton Island Humboldt* grown in the field in Korla, Xinjiang, China in 2013, 2014, 2015, 2016, 2018, and 2019. The horizontal dashed line represents the significance threshold, i.e., -log 10 (P) is 6.00. The arrow marks the SNP site (Gbar_D07_15803388) that is highly associated with the node of the fruiting branch.

[0026] Figure 3 To carry the SNP locus (Gbar_D07_15803388) and two main genotypes ( CC and TT ) and heterozygous genotype ( CT The phenotype of the first fruiting branch node of the island cotton variety. n This indicates the number of island cotton varieties carrying the specific genotype of SNP (Gbar_D07_15803388). Detailed Implementation

[0027] 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.

[0028] 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.

[0029] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0030] The sea island cotton material in the following examples is described in: Zhao N, et al. Genomic and GWASanalyses 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.

[0031] The following examples use IBM SPSS_Statistics_25 statistical software to process the data. The experimental results are expressed as mean values. T-tests are used, with P < 0.05 indicating statistical significance, P < 0.01 indicating highly statistical significance, and P < 0.001 indicating extremely significant statistical significance.

[0032] Example 1: Phenotypic Data Survey of the First Fruiting Branch Nodes in Sea Island Cotton Population Thirty-two accessions of sea island cotton, preserved by the Cotton Research Institute of the Xinjiang Academy of Agricultural Sciences, were planted in fields in Awat, Xinjiang, China in 2018 and 2019, Baotou Lake, Xinjiang, China in 2019, and Korla, Xinjiang, China from 2013 to 2016 and 2018 to 2019. A total of nine sets of phenotypic data on the first fruiting branch node were investigated. In this invention, the first fruiting branch node of the cotton plant is defined as the main stem node from the bottom to the top after the sea island cotton has budded.

[0033] The results showed that in the nine groups of phenotypic data, the node position of the first fruiting branch of sea island cotton all showed a clear normal distribution, which is a typical quantitative trait. Figure 1 (i). Genome-wide association analysis can be used to locate SNP sites that are significantly associated with the nodes of the fruiting branches of sea island cotton.

[0034] Example 2: Whole-genome resequencing and SNP detection of Sea Island cotton materials Genomic DNA was extracted from single leaf samples of 322 sea island cotton materials. Raw data was obtained through quality control, library construction, and sequencing. High-quality clean data was obtained after quality control and filtering. Data was aligned to sea island cotton reference genome 3-79 (https: / / www.cottongen.org / cottongen_downloads / Gossypium_barbadense / HAU_v2.0_AD2genome / assembly / Gbarbadense_genome.fasta.gz) using BWA software (version 0.7.8) (parameters: mem -t 4 -k 32 -M). SNPs were detected using the UnifiedGenotyper method with GATK software (version v3.1), and high-quality SNPs were obtained through filtering, generating a vcf file.

[0035] Genome-wide association analysis (GEMMA) of the first fruiting branch node trait 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 significantly associated with the node position of the first fruiting branch.

[0036] A single SNP locus (Gbar_D07_15803388) significantly associated with the fruiting branch node of *Cotton Island Tree* was identified in nine environmental studies. This SNP locus is located at position 15803388 on chromosome D07, with a nucleotide type of G or A in the negative strand and C or T in the coding strand. Notably, in 2018, the -log nucleotide of this SNP locus was located in Awati. 10 The p-value is 13.83. Figure 2 (a) In 2019, the -log sequence of this SNP locus was located in Awat. 10 The (P) value is 9.04 ( Figure 2 (b) In 2019, the -log of this SNP locus was located in Baotou Lake. 10 The (P) value is 14.66 ( Figure 2 (c) In 2013, the -log of this SNP locus was located in Korla. 10 The p-value was 21.90. Figure 2 (d) In 2014, the -log of this SNP locus was located in Korla. 10 The value of (P) is 6.15 ( Figure 2 (e), in 2015, the -log of this SNP site was located in Korla. 10 The p-value was 20.59. Figure 2 (f), in 2016, the -log of this SNP site was located in Korla. 10 The value of (P) is 15.01. Figure 2 (g), in 2018, the -log of this SNP locus was located in Korla. 10 The p-value was 26.83. Figure 2 (h), in 2019, the -log of this SNP site was located in Korla. 10 The value of (P) is 23.38 ( Figure 2 i).

[0037] This shows that the nine environmental localizations to this SNP site were -log 10 (P) values ​​all exceeded 6.00, with the highest being -log 10 The p-value reached 26.83 (Table 1), indicating that this SNP site is likely a major-effect site regulating the position of the fruiting branch node in sea island cotton, possessing high reliability and genetic stability. This SNP site (Gbar_D07_15803388) is position 811 of SEQ ID No:1, and its nucleotide type is C or T. The nucleotide sequence of SEQ ID No:1 is the sequence at the physical location 15803200 bp - 15804198 bp on the D07 chromosome of sea island cotton. In SEQ ID No:1, y represents c or t.

[0038] Table 1. Key SNPs (Gbar_D07_15803388) at the initial fruiting branch nodes of nine environmentally positioned branches. 10 (P) value

[0039] Example 3: Identification of the first fruiting branch node in sea island cotton using the SNP molecular marker Gbar_D07_15803388 DNA was extracted from young leaves of 322 sea island cotton materials, and genotyping was performed using the SNP molecular marker Gbar_D07_15803388.

[0040] Sequencing results revealed that among the 322 Sea Island cotton accessions, 149 accessions were homozygous for nucleotide C at SNP site Gbar_D07_15803388, with the following genotype: CC 169 Sea Island cotton materials showed a homozygous T nucleotide sequence at SNP site Gbar_D07_15803388, with the genotype being [missing information]. TT Four Sea Island cotton materials showed a heterozygous C / T nucleotide sequence at SNP site Gbar_D07_15803388, with the following genotype: CT (Table 2).

[0041] Table 2. Genotypes and average first fruiting branch nodes of SNP molecular marker (Gbar_D07_15803388) in Sea Island cotton.

[0042] Statistical analysis was performed on the data in Table 2, and the carrier genotype was... CC The average first fruiting branch node of the island cotton material is 5 nodes, which is higher than that of the genotype carrying the above. TT The sea island cotton material (average fruiting branch node is 3 nodes) showed a highly significant difference between the two (P = 1.1E-100, i.e., P < 0.001); the carrying genotype was CT The average first fruiting node of the island cotton material is 5 nodes, which is higher than that of the genotype carrying the average fruiting node. TTThe difference between the two sea island cotton materials reached a highly significant level (P = 6.1E-08, i.e., P < 0.001) (Table 3 and Figure 3 ).

[0043] Table 3. Statistical analysis of the genotype and average fruiting branch node position of SNP (Gbar_D07_15803388) in Sea Island cotton.

[0044] In summary, SNP (Gbar_D07_15803388) is a genetically stable molecular marker locus that is highly significantly associated with the position of the first fruiting branch in sea island cotton. It can identify or assist in identifying the position of the first fruiting branch in sea island cotton and can be used for sea island cotton breeding. For example, the genotype of the SNP (Gbar_D07_15803388) locus can be used as... CC and CT Sea island cotton was used as a parent to breed varieties with high fruiting branch nodes, or the genotype at the SNP (Gbar_D07_15803388) locus was used as a reference. TT Sea island cotton was selected as a parent to breed varieties with low fruiting branch nodes, in order to shorten the breeding time of the target sea island cotton variety.

[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. 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 first fruiting branch node of sea island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the first fruiting branch nodes of sea island cotton; (4) Prepare products for sea island cotton breeding; The SNP site is an SNP site on the D07 chromosome of sea island cotton, and its nucleotide type is C or T, which is the 811th nucleotide of sequence 1 in the sequence listing.

2. The application according to claim 1, characterized in that: The SNP has a genotype of CC or TT, where CC is the homozygous form of SNP C and TT is the homozygous form of SNP T; the fruiting branch node of the tested Sea Island cotton with the SNP genotype CC is higher than that of the tested Sea Island cotton with the SNP genotype TT.

3. The application according to claim 1 or 2, 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).

4. A method for identifying or assisting in the identification of the node position of the first fruiting branch of island cotton, characterized in that: This includes detecting the genotype of the SNP site in the genome of the cotton island to be tested, and identifying or assisting in the identification of the fruiting branch node of the cotton island based on the genotype. The SNP site is an SNP site on chromosome D07 of the cotton island, and its nucleotide type is C or T, which is the 811th nucleotide of sequence 1 in the sequence listing.

5. 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, selecting sea island cotton with the genotype TT of the SNP as the parent for breeding sea island cotton with low fruiting branch nodes, wherein TT is the homozygous type of the SNP with the genotype T.

6. 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, selecting sea island cotton with the genotype CC of the SNP as a parent for breeding sea island cotton with high fruiting branch nodes, wherein CC is the homozygous type of the SNP being C.

7. The application of the method according to any one of claims 4-6 in the breeding of island cotton.

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

9. The use of the DNA molecule of claim 9 in any of the following: (1) To identify or assist in the identification of the first fruiting branch node of island cotton; (2) Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the first fruiting branch nodes of sea island cotton; (4) Prepare products for island cotton breeding.

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