SNP molecular marker for identifying growth period of gossypium barbadense and application of SNP molecular marker

Abstract

The invention discloses an SNP molecular marker for identifying the growth period of gossypium barbadense and application of the SNP molecular marker. The invention belongs to the technical field of biology, and particularly relates to an SNP molecular marker for identifying the growth period of gossypium barbadense and application of the SNP molecular marker. The invention provides a method for identifying or assisting in identifying the growth period of sea island cotton, which comprises the following steps: detecting the genotype of an SNP site in a sea island cotton genome to be detected, and identifying or assisting in identifying the growth period of sea island cotton according to the genotype, the SNP site is an SNP site on a sea island cotton D07 chromosome, the nucleotide type is C or T, and the SNP site is the 811th nucleotide of a sequence 1 in a sequence table. The SNP molecular marker obviously associated with the growth period of the sea island cotton can be used for assistant breeding of early-maturing character molecules of the sea island cotton, the breeding efficiency is improved, and the SNP molecular marker has important significance for improving the economic benefits of the sea island cotton.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to SNP molecular markers for identifying the growth period of Gossypium barbadense and their applications. Background Art

[0002] Gossypium barbadense has extremely excellent fiber quality and is an indispensable raw material for high-end textiles. Xinjiang is the main planting area of Gossypium barbadense in China. With a relatively high latitude and a short frost-free period, planting early-maturing varieties is more conducive to improving the stable yield of cotton. The most important characteristic of early-maturing cotton is its short growth period. Developing molecular markers for identifying the length of the growth period of Gossypium barbadense is of great significance for breeding early-maturing Gossypium barbadense varieties and promoting the stable development of China's high-end cotton industry. Currently, there are few molecular markers available for identifying the growth period of Gossypium barbadense. Summary of the Invention

[0003] The main problem to be solved by the present invention is how to identify the length of the growth period of Gossypium barbadense.

[0004] To solve the above problems, the present invention provides a method for identifying or assisting in identifying the growth period of Gossypium barbadense.

[0005] The method for identifying or assisting in identifying the growth period of Gossypium barbadense provided by the present invention includes detecting the genotype of the SNP locus in the genome of the Gossypium barbadense to be tested, and identifying or assisting in identifying the growth period of Gossypium barbadense according to the genotype. The SNP locus is an SNP locus on chromosome D07 of Gossypium barbadense, and its nucleotide type is C or T, which is the 811th nucleotide of Sequence 1 in the sequence listing.

[0006] The SNP molecular marker is located at the 15803388th position of the nucleotide sequence of chromosome D07 of the Gossypium barbadense reference genome version Gossypium barbadense (AD2)_'3-79'_HAU_v2.0.

[0007] The present invention also provides a method for breeding Gossypium barbadense. The method includes detecting the genotype of the SNP described above in the genome of Gossypium barbadense, and selecting Gossypium barbadense with the genotype TT of the SNP as a parent for breeding Gossypium barbadense with a short growth period, where TT is the homozygous type of the SNP being T.

[0008] Furthermore, the method further includes detecting the genotype of the SNP described above in the genome of Gossypium barbadense, and selecting Gossypium barbadense with the genotype CC of the SNP as a parent for breeding Gossypium barbadense with a long growth period, where CC is the homozygous type of the SNP being C.

[0009] The application of the method described above in the breeding of Gossypium barbadense also belongs to the scope of protection required by the present invention.

[0010] The present invention also provides the use of a substance for detecting the polymorphism or genotype of SNPs in the genome of Gossypium barbadense L. in any of the following aspects: (1) Identifying or assisting in identifying the growth period of Gossypium barbadense L.; (2) Breeding Gossypium barbadense L.; (3) Preparing a product for identifying or assisting in identifying the growth period of Gossypium barbadense L.; (4) Preparing a product for breeding Gossypium barbadense L.; The SNP locus is an SNP locus on chromosome D07 of Gossypium barbadense L., and its nucleotide type is C or T, which is the 811th nucleotide of Sequence 1 in the sequence listing.

[0011] In the above applications, the genotype of the SNP is CC or TT. The CC is the homozygous type where the SNP is C, and the TT is the homozygous type where the SNP is T; the growth period of the Gossypium barbadense L. to be tested with the genotype of CC of the SNP is longer or higher than that of the Gossypium barbadense L. to be tested with the genotype of TT of the SNP.

[0012] The present invention also provides a product, which contains the substance for detecting the polymorphism or genotype of the SNPs in the genome of Gossypium barbadense L. described above, and the product is any one of the following: C1) A product for detecting the single nucleotide polymorphism or genotype related to the growth period of Gossypium barbadense L.; C2) A product for identifying or assisting in identifying the growth period of Gossypium barbadense L.; C3) A product for breeding Gossypium barbadense L.

[0013] In the above applications, methods and products, the substance can be the reagents and / or instruments required to determine the polymorphism or genotype of the SNP locus 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 chip. Among them, the SNP chip includes a chip based on nucleic acid hybridization reaction, a chip based on single-base extension reaction, a chip based on allele-specific primer extension reaction, a chip based on "one-step" reaction, a chip based on primer ligation reaction, a chip based on restriction endonuclease reaction, a chip based on protein-DNA binding reaction, and a chip based on fluorescent molecule-DNA binding reaction.

[0014] In the above applications and products, the substance can be any of the following D1), D2), or D3): D1) The substance is a primer composition for amplifying a genomic DNA fragment of Gossypium barbadense L. including the SNP locus; 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).

[0015] In the above applications, methods, and products, the primer composition may or may not be labeled with a labeling agent. The labeling agent refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Labeling agents include, but are not limited to, dyes; radioactive labels such as 32P; binding moieties such as biotin; haptens such as digoxin (DIG); luminescent, phosphorescent, or fluorescent moieties; and individual fluorescent dyes or fluorescent dyes combined with moieties that can inhibit or shift the emission spectrum through fluorescence resonance energy transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moiety (positive or negative charge) or, optionally, can be charge-neutral. The label can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence containing the label is detectable. In some embodiments, the nucleic acid is directly detected without a label (e.g., directly reading the sequence).

[0016] The present invention also provides a DNA molecule, the nucleotide sequence of which is Sequence 1 in the Sequence Listing.

[0017] The present invention also provides the use of the DNA molecule described above in any of the following: (1) Identifying or assisting in identifying the growth period of Gossypium barbadense L.; (2) Breeding Gossypium barbadense L.; (3) Preparing a product for identifying or assisting in identifying the growth period of Gossypium barbadense L.; (4) Preparing a product for breeding Gossypium barbadense L.

[0018] Optionally, in the above applications, the DNA molecule serves as a detection target.

[0019] Substances for detecting the polymorphism and genotype of the SNP locus can be combined with other substances (such as substances for detecting the single nucleotide polymorphism or genotype of other molecular markers related to the growth period of Gossypium barbadense L.) to prepare a product for identifying Gossypium barbadense L. varieties with long / short growth periods.

[0020] In this article, the purpose of the breeding may include cultivating Gossypium barbadense L. varieties with long growth periods or Gossypium barbadense L. varieties with short growth periods. The cotton can be a pure line or an inbred line.

[0021] The present invention locates a SNP locus (Gbar_D07_15803388) that is extremely significantly associated with the growth period of Gossypium barbadense using the method of genome-wide association study (GWAS). This SNP locus is located at the 15,803,388th position on chromosome D07 of the Gossypium barbadense genome (referring to the AD2_HAU_v2.0 genome of the Gossypium barbadense variety 3-79). The nucleotide types of this SNP locus are C or T, which is the 811th nucleotide of Sequence 1 in the sequence listing. The Gossypium barbadense variety carrying the CC genotype is a late-maturing type with a long growth period, and the Gossypium barbadense variety carrying the TT genotype is an early-maturing type with a short growth period. The present invention uses this SNP molecular marker to accurately genotype 336 Gossypium barbadense germplasm resources.

[0022] Compared with the prior art, the SNP molecular marker significantly associated with the growth period of Gossypium barbadense excavated by the present invention can be used for molecular-assisted breeding of the growth period traits of Gossypium barbadense, which is of great significance for molecular breeding to improve the maturity of Gossypium barbadense and increase the economic benefits of Gossypium barbadense. Description of the Drawings

[0023] Figure 1 It is a frequency distribution histogram of the growth period phenotypes of a Gossypium barbadense population surveyed in nine field environments in Xinjiang, China. Among them, a and b are the frequency distribution histograms of the growth period (Growth period, GP, unit: days) phenotypes of 336 Gossypium barbadense materials grown in the fields of Awati (abbreviated as A) in Xinjiang, China in 2018 (abbreviated as 18) and 2019 (abbreviated as 19) respectively; c is the frequency distribution histogram of the growth period phenotypes of 336 Gossypium barbadense materials grown in the field of Baotou Lake (abbreviated as B) in Xinjiang in 2019; d-i are the frequency distribution histograms of the growth period phenotypes of 336 Gossypium barbadense materials grown in the fields of Korla (abbreviated as K) in Xinjiang, China in 2013 (abbreviated as 13), 2014 (abbreviated as 14), 2015 (abbreviated as 15), 2016 (abbreviated as 16), 2018 and 2019 respectively.

[0024] Figure 2Manhattan plot (left) and quantile - quantile (QQ) plot (right) for mapping the growth stage - associated loci of Gossypium barbadense in nine groups of environments in Xinjiang, China. Among them, a and b are the Manhattan plot and QQ plot for mapping the growth stage - associated loci of 336 Gossypium barbadense materials grown in the fields of Awati, Xinjiang, China in 2018 and 2019 respectively; c is the Manhattan plot and QQ plot for mapping the growth stage - associated loci of 336 Gossypium barbadense materials grown in the fields of Baotou Lake, Xinjiang in 2019; d to i are the Manhattan plot and QQ plot for mapping the growth stage - associated loci of 336 Gossypium barbadense materials grown in the fields of 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 indicates the SNP locus (Gbar_D07_15803388) that was mapped to be extremely significantly associated with the growth stage.

[0025] Figure 3 Local Manhattan plot (upper) and linkage disequilibrium (LD) heatmap (lower) for the SNP locus (Gbar_D07_15803388) that was mapped to be extremely significantly associated with the growth stage of Gossypium barbadense. Among them, a is the local Manhattan plot for mapping the growth stage of the Gossypium barbadense population, and the arrow indicates the SNP locus (Gbar_D07_15803388) that was mapped to be extremely significantly associated with the growth stage of Gossypium barbadense; b is the local LD heatmap near the SNP locus (Gbar_D07_15803388).

[0026] Figure 4 Phenotypes of the growth stage of Gossypium barbadense varieties carrying two major genotypes (CC and TT) and the heterozygous genotype (CT) of the SNP locus (Gbar_D07_15803388). n Indicates the number of Gossypium barbadense varieties carrying a specific genotype of the SNP (Gbar_D07_15803388). Detailed implementation manners

[0027] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0028] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0029] Unless otherwise specified, in the following quantitative tests of the embodiments, three repeated experiments are set, and the results are averaged.

[0030] The 336 Gossypium barbadense cultivars in the following examples have been documented in: Zhao N, et al. Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China and selection for fibre length, lint percentage and Fusarium wilt resistance. Plant Biotechnol J. 2022;20(4):691-710. The public can obtain this biological material from the applicant. This biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0031] In the following examples, the IBM SPSS_Statistics_25 statistical software was used to process the data. The experimental results are expressed as averages. The T-Test was used for testing. P < 0.05 (*) indicates significant differences, P < 0.01 (**) indicates extremely significant differences, and P < 0.001 (***) indicates extremely significant differences.

[0032] Example 1. Investigation of the phenotypic data of the growth period of Gossypium barbadense cultivars The 336 Gossypium barbadense materials jointly preserved by the Joint Laboratory of Crop Molecular Breeding of China Agricultural University and the Institute of Cash Crops of Xinjiang Academy of Agricultural Sciences were planted in the fields of Awati, Xinjiang, China in 2018 and 2019, Baotou Lake, Xinjiang, China in 2019, and Korla, Xinjiang, China from 2013 to 2016 and from 2018 to 2019, respectively. A total of nine groups of phenotypic data of the growth period were investigated. One plant row was planted for each Gossypium barbadense cultivar. When the cotyledons of 70% of the single plants in the plant row were flattened, it was recorded as the start of the emergence period of the Gossypium barbadense cultivar. The time from the start of emergence to the boll opening of the first cotton boll in the plant row was defined as the growth period of the Gossypium barbadense cultivar. The growth periods of all Gossypium barbadense cultivars were statistically analyzed.

[0033] The results showed that: among the nine groups of phenotypic data, the growth periods of Gossypium barbadense all showed obvious quantitative genetic characteristics of normal distribution ( Figure 1 in a-i), which are applicable to genome-wide association analysis to identify significantly associated SNPs.

[0034] Example 2. Whole-genome resequencing and SNP detection of Gossypium barbadense cultivars Take 336 single-plant leaf samples of Gossypium barbadense varieties, extract genomic DNA, and obtain raw data through quality inspection, library construction, and sequencing. After quality control and filtering, high-quality clean data is obtained. Align it to the Gossypium barbadense reference genome 3-79 (http: / / cotton.hzau.edu.cn / EN / download.php) using the BWA (version 0.7.8) software (parameters: mem -t 4 -k 32 -M). Use the GATK (version v3.1) software to perform population SNP detection by the UnifiedGenotyper method, filter to obtain high-quality SNPs, and generate a vcf file.

[0035] Use the genome-wide efficient mixed model association software GEMMA 0.94.1 (http: / / www.xzlab.org / software.html) to perform genome-wide association analysis of the growth period traits of Gossypium barbadense using the mixed linear model. The parameter settings of the GEMMA software are "gemma -bfile file -k kinship -lmm 1 -o outfile -miss 0.2 -maf 0.05 -c covariates (GCTA: PCA)". Use the R package qqman to make Manhattan plots and QQ plots. By F -test to detect the effect values of gene markers, and use Bonferroni correction to correct multiple detections. Screen SNPs significantly associated with the growth period through the criterion (-log 10 (P-value)>6).

[0036] In eight of the nine groups of environments, the same SNP locus (Gbar_D07_15803388) that is extremely significantly associated with the growth period of Gossypium barbadense was located. This SNP locus is located at the 15,803,388th position on chromosome D07. The highest -log 10 P value is 19.61 ( Figure 2 in a-i and Figure 3 in a-b). This SNP locus (Gbar_D07_15803388) is the 811th position of SEQ ID No:1, and its nucleotide type is C or T. The nucleotide sequence of SEQ ID No:1 is the sequence of the physical position 15,803,200 bp - 15,804,198 bp on chromosome D07 of Gossypium barbadense. In SEQ ID No:1, y represents c or t.

[0037] Example 3: Identify the growth period of Gossypium barbadense using the SNP molecular marker Gbar_D07_15803388 DNA of young leaves was extracted from 336 Gossypium barbadense materials of the natural population of the present invention, and genotyping was performed for the SNP molecular marker Gbar_D07_15803388.

[0038] The sequencing results showed that among the 336 Gossypium barbadense materials, 149 Gossypium barbadense materials were homozygous for nucleotide C at the SNP locus Gbar_D07_15803388, and the genotype was CC ; 169 Gossypium barbadense materials were homozygous for nucleotide T at the SNP locus Gbar_D07_15803388, and the genotype was TT ; 4 Gossypium barbadense materials were heterozygous for nucleotides C and T at the SNP locus Gbar_D07_15803388, and the genotype was CT (Table 1). In addition, 14 Gossypium barbadense materials did not detect a specific nucleotide type at this locus due to sequencing reasons.

[0039] Table 1. Genotypes and average growth periods of SNP molecular markers (Gbar_D07_15803388) in Gossypium barbadense materials

[0040] Statistical analysis was performed on the data in Table 1. The average growth period of Gossypium barbadense materials carrying the genotype CC was 135 days, which was longer than that of Gossypium barbadense materials carrying the genotype TT (average growth period was 126 days), and the difference between the two reached an extremely significant level (P = 4.33E-74, that is, P < 0.001); the average growth period of Gossypium barbadense materials carrying the genotype CC was longer than that of Gossypium barbadense materials carrying the genotype CT (average growth period was 131 days), and the difference between the two reached a significant level (P = 2.82E-02, that is, P < 0.05) (Table 2 and Figure 4 ).

[0041] Table 2. Statistical analysis of SNP (Gbar_D07_15803388) genotypes and growth period phenotypes in Gossypium barbadense

[0042] In summary, SNP (Gbar_D07_15803388) is a genetically stable SNP molecular marker locus that is significantly associated with the growth period of Gossypium barbadense, and can be used to identify or assist in identifying the length of the growth period of Gossypium barbadense, and can be used in the breeding of Gossypium barbadense. For example, Gossypium barbadense with the genotype of the SNP (Gbar_D07_15803388) locus is used as a parent to breed varieties with a long growth period, or Gossypium barbadense with the genotype of the SNP (Gbar_D07_15803388) locus is used as a parent to breed varieties with a short growth period, so as to shorten the breeding time of the target Gossypium barbadense variety. CC In summary, SNP (Gbar_D07_15803388) is a genetically stable SNP molecular marker locus that is significantly associated with the growth period of Gossypium barbadense, and can be used to identify or assist in identifying the length of the growth period of Gossypium barbadense, and can be used in the breeding of Gossypium barbadense. For example, Gossypium barbadense with the genotype of the SNP (Gbar_D07_15803388) locus is used as a parent to breed varieties with a long growth period, or Gossypium barbadense with the genotype of the SNP (Gbar_D07_15803388) locus is used as a parent to breed varieties with a short growth period, so as to shorten the breeding time of the target Gossypium barbadense variety. TT In summary, SNP (Gbar_D07_15803388) is a genetically stable SNP molecular marker locus that is significantly associated with the growth period of Gossypium barbadense, and can be used to identify or assist in identifying the length of the growth period of Gossypium barbadense, and can be used in the breeding of Gossypium barbadense. For example, Gossypium barbadense with the genotype of the SNP (Gbar_D07_15803388) locus is used as a parent to breed varieties with a long growth period, or Gossypium barbadense with the genotype of the SNP (Gbar_D07_15803388) locus is used as a parent to breed varieties with a short growth period, so as to shorten the breeding time of the target Gossypium barbadense variety.

[0043] The above has described the present invention in detail. For those skilled in the art, without departing from the spirit and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art.

Claims

1. A method for identifying or assisting in identifying the growth period of Sea Island Cotton, characterized in that: The method includes detecting the genotype of the SNP site in the genome of the island cotton to be tested, and identifying or assisting in identifying the growth period of the island cotton according to the genotype. The SNP site is a SNP site on the D07 chromosome of the island cotton, and its nucleotide type is C or T, which is the 811th nucleotide of sequence 1 in the sequence table.

2. A method for breeding sea island cotton, characterized in that: The method comprises detecting the genotype of the SNP in claim 1 in the genome of sea island cotton, selecting sea island cotton with the genotype of the SNP being TT as a parent for breeding of short-growing sea island cotton, wherein the TT is the homozygous type of the SNP being T.

3. A method for breeding sea island cotton, characterized in that: The method comprises detecting the genotype of the SNP in claim 1 in the genome of sea island cotton, and selecting sea island cotton with the genotype of the SNP being CC as a parent for breeding sea island cotton with a long growth period, wherein CC is the homozygous type of the SNP being C.

4. Application of the method according to any one of claims 1 to 3 in breeding of sea island cotton.

5. Application of a substance for detecting polymorphism or genotype of SNP in the genome of sea island cotton in any of the following: (1) Identify or assist in identifying the growth period of Sea Island cotton; (2) Sea Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the growth period of sea island cotton; (4) Preparing products of sea island cotton breeding; The SNP site is a SNP site on the chromosome of Gossypium barbadense D07, the nucleotide type of which is C or T, and is the 811th nucleotide of sequence 1 in the sequence list.

6. The use according to claim 5, characterized in that: The genotype of the SNP is CC or TT, the CC is the homozygous type of the SNP being C, and the TT is the homozygous type of the SNP being T; the growth period of the tested sea island cotton whose genotype is CC is longer than or higher than that of the tested sea island cotton whose genotype is TT.

7. The product is characterized by: The product contains the substance described in claim 5 or 6, and the product is any one of: C1) Products for detecting single nucleotide polymorphisms or genotypes related to the growth period of Sea Island cotton; C2) Identify or assist in identifying products during the Sea Island cotton growth period; C3) Products used for sea island cotton breeding.

8. The use according to claim 5 or 6 or the product according to claim 7, characterized in that: The substance is as follows (D1), (D2) or (D3): D1) the substance is a primer composition for amplifying a DNA fragment of the Gossypium barbadense genomic DNA including the SNP site; D2) the substance is a PCR reagent containing the primer combination 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 in that: The nucleotide sequence of the DNA molecule is sequence 1 in the sequence list.

10. Use of the DNA molecule according to claim 9 in any of the following: (1) Identify or assist in identifying the growth period of sea island cotton; (2) Sea Island cotton breeding; (3) Prepare products for identification or auxiliary identification of the growth period of sea island cotton; (4) Prepare products of sea island cotton breeding.

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