SNP (Single Nucleotide Polymorphism) molecular marker related to cotton fiber length and application of SNP molecular marker

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to cotton fiber length and application thereof. The invention also discloses an SNP (Single Nucleotide Polymorphism) site related to the length of the cotton fiber, and the SNP site is positioned at the 95547325 site on the No. A05 chromosome in a cotton genome. Experiments prove that the length of the cotton fiber can be effectively identified by detecting the genotype of the SNP site based on the SNP molecular marker, and the identification method is simple to operate, contributes to identification and auxiliary selective breeding of the length of the cotton fiber, especially early identification and screening of the length of the cotton fiber, and has a good application prospect. The method is of great significance in breeding long-fiber cotton varieties and accelerating genetic improvement of cotton.

Description

Technical Field

[0001] The present invention relates to the technical field of cotton genetic breeding, and specifically relates to an SNP molecular marker related to cotton fiber length and its application. Background Art

[0002] Cotton is one of the important cash crops and also a necessity of life, playing a crucial role in national defense, medicine, industry and other fields. China is one of the major cotton producers and consumers in the world, with cotton output accounting for about one-fourth of the world's total. Improving cotton quality can reduce the cost of cotton cultivation and increase labor productivity, promoting the transformation of cotton production towards modern agriculture.

[0003] Cotton quality includes fiber length, strength, etc. Traditional genetic improvement methods for cotton fiber length rely on phenotypic selection, usually requiring waiting until the cotton is mature, and are easily affected by environmental factors. Molecular marker technology enables breeders to conduct genotype screening at an early stage, not only achieving precise genetic improvement, but also significantly reducing breeding costs, shortening the breeding cycle, and improving breeding efficiency. Therefore, developing molecular markers related to cotton fiber length is of great significance for accelerating the progress of cotton genetic breeding and the selection of excellent cotton varieties. Summary of the Invention

[0004] The object of the present invention is to provide an SNP molecular marker related to cotton fiber length and its application.

[0005] In the first aspect, the present invention claims a new use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome.

[0006] The present invention claims the application of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in identifying or assisting in identifying cotton fiber length.

[0007] The present invention also claims the application of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in preparing a product for identifying or assisting in identifying cotton fiber length.

[0008] The present invention also claims the application of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in screening or assisting in screening long-fiber cotton varieties.

[0009] The present invention also claims the application of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in preparing a product for screening or assisting in screening long-fiber cotton varieties.

[0010] The present invention also claims the application of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in the improvement of cotton varieties.

[0011] The present invention also claims the application of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in the preparation of products for cotton variety improvement.

[0012] The present invention also claims the application of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in cotton breeding.

[0013] The present invention also claims the application of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in the preparation of products for cotton breeding.

[0014] In the second aspect, the present invention claims a method for identifying or assisting in identifying the cotton fiber length.

[0015] The method for identifying or assisting in identifying the cotton fiber length claimed by the present invention includes the following steps: detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome, and identifying or assisting in identifying the cotton fiber length according to the genotype.

[0016] In the above method for identifying or assisting in identifying the cotton fiber length, the genotype is the AA genotype or the CC genotype.

[0017] The AA genotype is a homozygote in which both deoxyribonucleotides at position 95547325 on chromosome A05 in the cotton genome are A;

[0018] The CC genotype is a homozygote in which the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome is C.

[0019] The method for identifying or assisting in identifying the cotton fiber length according to the genotype may be that the fiber length of AA genotype cotton is greater than or candidate greater than that of CC genotype cotton.

[0020] In the third aspect, the present invention claims a method for screening or assisting in screening long-fiber cotton varieties.

[0021] The method for screening or assisting in screening long-fiber cotton varieties claimed by the present invention includes the following steps: detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome, and selecting cotton varieties with the AA genotype;

[0022] The AA genotype is a homozygous genotype in which the deoxyribonucleotides at the 95547325th position on chromosome A05 in the cotton genome are all A.

[0023] Fourthly, the present invention claims to protect a method for cotton variety improvement or cotton breeding.

[0024] The method for cotton variety improvement or cotton breeding claimed by the present invention includes the following steps: detecting the genotype of the deoxyribonucleotide at the 95547325th position on chromosome A05 in the cotton genome, and selecting cotton varieties with the AA genotype as parents for breeding.

[0025] In the above method for cotton variety improvement or cotton breeding, the indicators for variety improvement or breeding include fiber length.

[0026] The purpose of the variety improvement or breeding includes cultivating cotton with increased fiber length (cultivating long-fiber cotton varieties). The increase in fiber length means that the fiber length is greater than that of the parent.

[0027] In any of the above methods, the method for detecting the genotype of the deoxyribonucleotide at the 95547325th position on chromosome A05 in the cotton genome is direct sequencing or sequencing the PCR product containing the deoxyribonucleotide at the 95547325th position on chromosome A05 in the cotton genome.

[0028] In the present invention, the method of sequencing is not limited and can be sequencing methods classified by various classification methods, such as first-generation sequencing (i.e., Sanger sequencing), second-generation sequencing (i.e., NGS sequencing), third-generation sequencing (i.e., long-read sequencing) classified by technology generations, genomic sequencing, transcriptomic sequencing classified by application fields, and single-end sequencing, paired-end sequencing, etc. classified by sequencing methods.

[0029] The method for detecting the genotype of the deoxyribonucleotide at the 95547325th position on chromosome A05 in the cotton genome includes the following steps: using cotton genomic DNA as a template, performing PCR amplification with a primer pair composed of the single-stranded DNA shown in Sequence 1 and the single-stranded DNA shown in Sequence 2 to obtain a PCR product; sequencing the PCR product to obtain the cotton genotype.

[0030] In some embodiments, the system for PCR amplification is as follows: 0.5 μL of each primer, 1 μL of DNA template, 10 μL of TaqMix, and 8 μL of water.

[0031] In some embodiments, the program for PCR amplification is as follows: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 10 s, annealing at 55 °C for 30 s, extension at 72 °C for 40 s, 35 cycles; 72 °C for 10 min.

[0032] In a fifth aspect, the present invention claims protection for a product having at least one of the following functions 1)-4):

[0033] 1) Identifying or assisting in identifying the length of cotton fibers;

[0034] 2) Screening or assisting in screening long-fiber cotton;

[0035] 3) Improving cotton varieties;

[0036] 4) Cotton breeding.

[0037] The product having at least one of the above functions 1)-4) claimed by the present invention includes a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome.

[0038] The substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome as described above may be a reagent and / or instrument required to determine the polymorphism or genotype of the SNP locus indicated by the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome 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. Among them, 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.

[0039] Furthermore, the substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome may be any one of the following M1)-M4):

[0040] M1) A primer pair for amplifying a cotton genomic DNA fragment including the SNP locus indicated by the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome;

[0041] M2) A PCR reagent containing the primer pair;

[0042] M3) A kit containing the primer pair described in M1) or the PCR reagent described in M2);

[0043] M4) A chip containing the primer pair described in M1) or the PCR reagent described in M2).

[0044] Further, the primer pair may or may not be labeled with a label. The label refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Labels 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 by 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).

[0045] Furthermore, the primer pair consists of single-stranded DNA shown in Sequence 1 and single-stranded DNA shown in Sequence 2.

[0046] Any of the above-mentioned cotton genomes is the reference genome of upland cotton standard line TM-1.

[0047] Any of the above-mentioned cotton fiber lengths refers to the distance between the two ends when the cotton fiber is straightened.

[0048] In some embodiments, the fiber length can be measured by referring to the method in the document "China Fiber Inspection Bureau. GB1103.1-2012 'Cotton Part 1: Sawtooth Processed Fine Fiber Cotton' [S]. Beijing: China Standard Press, 2012."

[0049] Any of the above-mentioned cotton can be any cotton germplasm resource, variety, strain or individual plant common in the technical field of the present invention. In some embodiments, the cotton is the cotton variety in Table 1 below.

[0050] The present invention provides an SNP locus related to cotton fiber length, which is located at position 95547325 on chromosome A05 in the cotton genome, and the polymorphism of this locus is C / A. The present invention also develops an SNP molecular marker related to cotton fiber length based on this SNP locus. Experiments prove that: based on the SNP molecular marker of the present invention, the genotype of the SNP locus can be detected to effectively identify the cotton fiber length, and the identification method is simple to operate, which is helpful for the identification and assistant selection breeding of cotton fiber length, especially the early identification and screening of cotton fiber length, and has important significance for breeding long-fiber cotton varieties and accelerating cotton genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Based on the association analysis results of SNP locus genotypes and fiber length of 365 cotton germplasm materials. ** indicates P < 0.01. Specific implementation manners

[0052] The present invention will be further described in detail below in conjunction with specific 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.

[0053] 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 instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0054] The 365 cotton varieties in the following embodiments are recorded in Table S58 of Supplementary Table 1 (https: / / pmc.ncbi.nlm.nih.gov / articles / PMC8423627 / #_ad93_) of the literature "Ma, Zhiying, et al. "High-quality genome assembly and resequencing of modern cotton cultivars provide resources for crop improvement." Nature Genetics 53.9 (2021): 1385 - 1391.", and the public can obtain them from the applicant or the National Mid-term Genebank of Cotton Germplasm.

[0055] The reference genome sequence of upland cotton standard line TM-1 in the following embodiments has the GenBank number GCA_006980745.1 in NCBI.

[0056] Example 1. Discovery of SNP loci related to cotton fiber length and development of SNP molecular markers

[0057] I. Discovery of SNP loci related to cotton fiber length

[0058] In this invention, the fibers of upland cotton core germplasm resources 10 days after flowering were used as test materials. Total RNA was extracted using the SpectrumPlant TotalRNA Isolation Kit (Sigma, STRN250), and then a sequencing library was constructed using the IlluminaVAHTS Universal V6RNA-seq Library Prep Kit (Illumina, San Diego, CA, USA). Subsequently, transcriptome sequencing was performed on the illuminaNovaSeq 6000 platform to obtain high-quality clean data, with a data volume of 36.1 billion paired-end sequencing reads. The whole-genome resequencing data was aligned to the cotton reference genome using BWA-MEM (v.0.7.10-r789), and SNPs in the population were identified using the GATK4.0 software. The whole-genome resequencing data of the population was aligned to the reference genome to identify SNPs at unique loci. Subsequently, SNPs were filtered according to the following criteria: ① The deletion rate was less than 0.2; ② The minor allele frequency was greater than 0.05. A total of 1,971,744 SNPs were obtained. Among them, an SNP locus related to cotton fiber length was found, which was located at position 95,547,325 on chromosome A05 of the reference genome of the upland cotton standard line TM-1. The polymorphism of this locus was A / C, and its corresponding genotypes included the AA genotype and the CC genotype. Among them, the AA genotype was a homozygote in which the deoxyribonucleotides at position 95,547,325 on chromosome A05 of the cotton genome were all A; the CC genotype was a homozygote in which the genotype of the deoxyribonucleotides at position 95,547,325 on chromosome A05 of the cotton genome was C.

[0059] II. Development of SNP molecular markers related to cotton fiber length

[0060] PCR amplification primers for genotype detection were designed for the SNP locus obtained in Step I. The primer sequences are as follows:

[0061] F: 5’-CCATAAGCGAACTCGGACTC-3’ (Sequence 1);

[0062] R: 5’-GAGATTCGAGTTGAGATTGGGG-3’ (Sequence 2).

[0063] Example 2. Verification of SNP molecular markers related to cotton fiber length

[0064] Test materials: 365 cotton core germplasm resources, and the specific numbers are shown in Table 1.

[0065] I. Genotype detection

[0066] 1. Genomic DNA Extraction

[0067] Extract the genomic DNA of the test materials.

[0068] 2. PCR Amplification

[0069] Using the genomic DNA as a template, perform PCR amplification with the primers designed in Step 2 of Example 1 to obtain a PCR product.

[0070] The PCR amplification system is as follows: 0.5 μL of each primer, 1 μL of DNA template, 10 μL of Taq Mix, and 8 μL of water.

[0071] The PCR amplification program is as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 40 s, for 35 cycles; extension at 72°C for 10 min.

[0072] 3. Detection of PCR Products

[0073] Detect the PCR products using 1% agarose gel and sequence them to obtain the genotypes of the SNP sites obtained in Example 1.

[0074] II. Determination of Cotton Fiber Length

[0075] Plant the test materials in 2014 and 2015 according to the conventional method, and measure the fiber length at maturity using the fiber detection center platform of the Cotton Research Institute, Chinese Academy of Agricultural Sciences. The specific measurement method refers to "China Fiber Inspection Bureau. GB1103.1 - 2012 'Cotton - Part 1: Fine - lint Cotton Saw - ginned' [S]. Beijing: China Standards Press, 2012.".

[0076] III. Correlation Analysis between SNP Site Genotypes and Fiber Length

[0077] The results are shown in Table 1 and Figure 1 As shown, among the 365 materials, there are 219 materials with the genotype AA, and the average fiber length is 29.53 mm; there are 122 materials with the genotype CC, and the average fiber length is 28.86 mm. The cotton fiber length of the AA genotype materials is significantly greater than that of the CC genotype materials.

[0078] Table 1

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Note: - indicates missing, and M indicates heterozygous.

[0089] The present invention has been described in detail above. 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 modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made with conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. Use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in identifying or assisting in the identification of cotton fiber length; Or, use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in the preparation of a product for identifying or assisting in the identification of cotton fiber length.

2. Use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in screening or assisting in the screening of long-fiber cotton varieties; Or, use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in the preparation of a product for screening or assisting in the screening of long-fiber cotton varieties.

3. Use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in the improvement of cotton varieties; Or, use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in the preparation of a product for the improvement of cotton varieties.

4. Use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in cotton breeding; Or, use of a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome in the preparation of a product for cotton breeding.

5. The application according to any one of claims 1-4, characterized in that: The substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome is a primer pair composed of the single-stranded DNA shown in Sequence 1 and the single-stranded DNA shown in Sequence 2.

6. A method for identifying or assisting in the identification of cotton fiber length, comprising the following steps: detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome, and identifying or assisting in the identification of cotton fiber length according to the genotype.

7. A method for screening or assisting in the screening of long-fiber cotton varieties, comprising the following steps: detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome, and selecting cotton varieties with the AA genotype; The AA genotype is a homozygote in which both deoxyribonucleotides at position 95547325 on chromosome A05 in the cotton genome are A.

8. A method for cotton variety improvement or cotton breeding, comprising the following steps: detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome, and selecting cotton varieties with the AA genotype as parents for breeding; The AA genotype is a homozygote in which both deoxyribonucleotides at position 95547325 on chromosome A05 in the cotton genome are A.

9. The method according to any one of claims 6-8, characterized in that: The method for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome is direct sequencing or sequencing of the PCR product containing the deoxyribonucleotide at position 95547325 on chromosome A05 in the cotton genome.

10. A product having at least one of the following functions 1)-4), comprising a substance for detecting the genotype of the deoxyribonucleotide at position 95547325 on chromosome No. A05 in the cotton genome; 1) Identifying or assisting in identifying the cotton fiber length; 2) Screening or assisting in screening long-fiber cotton; 3) Improvement of cotton varieties; 4) Cotton breeding.

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