Method for identifying or assisting in identifying length of cotton fiber and application thereof
By detecting the genotypes of specific sites in the cotton genome and using PCR technology to identify cotton fiber lengths, the problem of difficulty in quickly screening long fiber cotton varieties in the existing technology is solved, and rapid and accurate fiber length identification and breeding efficiency improvement are achieved.
Patent Information
- Application Number
- CN202510562544.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-08-08
AI Technical Summary
It is difficult for the prior art to quickly and effectively identify and screen long fiber cotton varieties, which affects cotton breeding and quality improvement.
By detecting the deoxyribonucleotide genotype at 9092066 on chromosome A05 in the cotton genome, using AA genotype and TT genotype to identify or assist in identifying cotton fiber length, and determining cotton genotype through PCR amplification and sequencing technology, long fiber cotton varieties were screened out.
It has achieved rapid and accurate identification of cotton fiber length, improved the efficiency of cotton breeding, especially in the early identification and screening of long fiber cotton varieties, and promoted the genetic improvement of cotton.
Smart Images
Figure BDA0005385013140000061 
Figure BDA0005385013140000071 
Figure BDA0005385013140000081
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cotton genetic breeding, and in particular to a method for identifying or assisting in identifying cotton fiber length and an application thereof. Background Art
[0002] Cotton is a major cash crop and a daily necessity, playing a crucial role in defense, medicine, industry, and other fields. China is one of the world's major cotton producers and consumers, accounting for approximately a quarter of global cotton production. Improving cotton quality can reduce cotton planting costs and increase labor productivity, driving the transition of cotton production to modern agriculture.
[0003] Cotton fiber length is a key indicator for evaluating cotton quality, directly impacting spinning process design and yarn quality. The longer and more uniform the fibers, the better the yarn's strength, uniformity, and surface finish. With the textile industry's increasing demand for high-quality cotton fiber, the ability to quickly identify cotton fiber length has become increasingly important. Summary of the Invention
[0004] The technical problem to be solved by the present invention is how to identify the length of cotton fibers.
[0005] In order to solve the above technical problems, the present invention first provides a new use of a substance for detecting the genotype of the deoxyribonucleotide at position 9092066 on chromosome A05 in the cotton genome.
[0006] The present invention provides the use of a substance for detecting the genotype of the deoxyribonucleotide at position 9092066 on chromosome A05 in the cotton genome in any of the following A1)-A4):
[0007] A1) Identify or assist in identifying cotton fiber length;
[0008] A2) Screening or assisting in the selection of long-fiber cotton varieties;
[0009] A3) Cotton breeding;
[0010] A4) Cotton variety improvement.
[0011] The present invention also provides the use of a substance for detecting the genotype of the deoxyribonucleotide at position 9092066 on chromosome A05 in the cotton genome in any of the following B1) to B4):
[0012] B1) preparing products for identifying or assisting in identifying cotton fiber length;
[0013] B2) preparing products for screening or assisting in screening long-fiber cotton varieties;
[0014] B3) preparing cotton breeding products;
[0015] B4) Preparation of products of improved cotton varieties.
[0016] In order to solve the above technical problems, the present invention further provides a method for identifying or assisting in identifying the length of cotton fibers.
[0017] The method for identifying or assisting in identifying cotton fiber length provided by the present invention comprises the following steps: detecting the 9092066th deoxyribonucleotide genotype on chromosome A05 in the cotton genome, and identifying or assisting in identifying the cotton fiber length according to the genotype.
[0018] In the above method for identifying or assisting in identifying cotton fiber length, the genotype is an AA genotype or a TT genotype.
[0019] The AA genotype is a homozygous body in which the deoxyribonucleotides at positions 9092066 on chromosome A05 in the cotton genome are all A;
[0020] The TT genotype is a homozygous body in which the 9092066th deoxyribonucleotide genotype on chromosome A05 in the cotton genome is T.
[0021] According to the method for identifying or assisting in identifying the fiber length of cotton based on the genotype, the fiber length of cotton with TT genotype is greater than or may be greater than that of cotton with AA genotype.
[0022] In order to solve the above technical problems, the present invention also provides a method for screening or assisting in screening long-fiber cotton varieties.
[0023] The method for screening or assisting in screening long-fiber cotton varieties provided by the present invention comprises the following steps: detecting the genotype of the deoxyribonucleotide at position 9092066 on chromosome A05 in the cotton genome, and selecting a cotton variety with the TT genotype;
[0024] The TT genotype is a homozygous body in which the deoxyribonucleotides at positions 9092066 on chromosome A05 in the cotton genome are all T.
[0025] In order to solve the above technical problems, the present invention also provides a method for cotton breeding or cotton variety improvement.
[0026] The cotton breeding or cotton variety improvement method provided by the present invention comprises the following steps: detecting the 9092066th deoxyribonucleotide genotype on chromosome A05 in the cotton genome, and selecting a cotton variety with the TT genotype as a parent for breeding.
[0027] In the above-mentioned cotton breeding or cotton variety improvement method, the breeding or variety improvement index includes fiber length.
[0028] The purpose of breeding or variety improvement includes cultivating cotton with increased fiber length (cultivating long-fiber cotton varieties). The increased fiber length means that the fiber length is greater than that of the parent.
[0029] In any of the above methods, the method for detecting the genotype of deoxyribonucleotide No. 9092066 on chromosome A05 in the cotton genome is direct sequencing or sequencing a PCR product containing deoxyribonucleotide No. 9092066 on chromosome A05 in the cotton genome.
[0030] In the present invention, the sequencing method is not limited and can be a sequencing method classified by various classification methods, such as first-generation sequencing (i.e., Sanger sequencing), second-generation sequencing (i.e., NGS sequencing), and third-generation sequencing (i.e., long-read sequencing) classified according to technology generations, genome sequencing and transcriptome sequencing classified according to application fields, and single-end sequencing and double-end sequencing classified according to sequencing methods, etc.
[0031] In some embodiments, the method for detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome comprises the following steps: using cotton genomic DNA as a template, performing PCR amplification using a primer pair consisting 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.
[0032] In some embodiments, the PCR amplification system is as follows: 0.5 μl of each primer, 1 μl of DNA template, 10 μl of TaqMix, and 8 μl of water.
[0033] In some embodiments, 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 50 s, 35 cycles; 72°C for 10 min.
[0034] In order to solve the above technical problems, the present invention finally provides a product having at least one of the following functions C1) to C4):
[0035] C1) Identify or assist in identifying cotton fiber length;
[0036] C2) screening or assisting in screening long-fiber cotton;
[0037] C3) Cotton breeding;
[0038] C4) Cotton variety improvement.
[0039] The product provided by the invention comprises a substance for detecting the genotype of the deoxyribonucleotide at position 9092066 on chromosome A05 in the cotton genome.
[0040] Any of the aforementioned substances for detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome may be reagents and / or instruments required for determining the polymorphism or genotype of the SNP site represented by deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome by at least one of the following methods: DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chip. 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.
[0041] Furthermore, the substance for detecting the genotype of the deoxyribonucleotide at position 9092066 on chromosome A05 in the cotton genome may be any one of the following A1) to A4):
[0042] A1) contains a primer pair for amplifying a cotton genomic DNA fragment including the SNP site indicated by deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome;
[0043] A2) PCR reagents containing the primer pair;
[0044] A3) a kit containing the primer pair described in A1) or the PCR reagent described in A2);
[0045] A4) A chip containing the primer pair described in A1) or the PCR reagent described in A2).
[0046] Further, the primer pair 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 attached to a nucleic acid. Markers include, but are not limited to, dyes; radioactive labels, 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 that can inhibit or shift the emission spectrum by fluorescence resonance energy transfer (FRET). The marker can provide a signal that can be detected by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzymatic activity, etc. The marker can be a charged moiety (positive or negative charge) or, alternatively, can be charge neutral. The marker can include a nucleic acid or protein sequence or a combination thereof, as long as the sequence comprising the marker is detectable. In some embodiments, the nucleic acid is directly detected (e.g., directly reading the sequence) without a marker.
[0047] Furthermore, the primer pair consists of the single-stranded DNA shown in sequence 1 and the single-stranded DNA shown in sequence 2.
[0048] Any of the above-mentioned cotton genomes is the reference genome of the upland cotton standard line TM-1.
[0049] The length of any of the above-mentioned cotton fibers refers to the distance between the two ends of the cotton fiber when it is straightened.
[0050] In some embodiments, the fiber length can be measured with reference to the method in the document “China Fiber Inspection Bureau. GB1103.1-2012 “Cotton Part 1: Sawtooth Processed Fine-staple Cotton” [S]. Beijing: China Standards Press, 2012.”
[0051] Any of the aforementioned cotton can be any cotton germplasm resource, variety, strain or plant commonly used in the art. In some embodiments, the cotton is a cotton variety listed in Table 1 below.
[0052] The present invention provides a method for identifying or assisting in the identification of cotton fiber length. The method comprises the following steps: detecting the genotype of deoxyribonucleotides at positions 9092066 on chromosome A05 in the cotton genome, and identifying or assisting in the identification of cotton fiber length based on the genotype. Experiments have demonstrated that the method provided by the present invention can effectively identify cotton fiber length, is simple to operate, and can be used for identifying cotton fiber length and assisting in selective breeding, particularly for early identification and screening of cotton fiber length. The method has important application value in breeding long-fiber cotton varieties and accelerating cotton genetic improvement. BRIEF DESCRIPTION OF THE DRAWINGS
[0053] Figure 1Results of association analysis between SNP genotypes and fiber length based on 365 cotton germplasm accessions. ** indicates P < 0.01. DETAILED DESCRIPTION
[0054] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.
[0055] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.
[0056] The 365 cotton varieties in the following examples are recorded in Table S58 in Supplementary Table 1 (https: / / pmc.ncbi.nlm.nih.gov / articles / PMC8423627 / #_ad93_) of the document "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 are available to the public from the applicant or the National Cotton Germplasm Interim Bank.
[0057] The reference genome sequence of the upland cotton standard line TM-1 in the following examples has a GenBank number of GCA_006980745.1 in NCBI.
[0058] Example 1. Discovery of SNP loci associated with cotton fiber length and development of SNP molecular markers
[0059] 1. Discovery of SNPs associated with cotton fiber length
[0060] The present invention uses fibers from upland cotton core germplasm resources 10 days after development and flowering as test materials, uses the SpectrumPlant TotalRNA Isolation Kit (Sigma, STRN250) to extract total RNA, and then uses the IlluminaVAHTS Universal V6RNA-seq Library Prep Kit (Illumina, San Diego, CA, USA) to construct a sequencing library. Then, transcriptome sequencing is performed on the Illumina NovaSeq 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 is aligned to the cotton reference genome using BWA-MEM (v.0.7.10-r789), and GATK4.0 software is used to identify SNPs in the population. The whole-genome resequencing data of the population is aligned to the SNPs of the reference genome identifying unique sites. Subsequently, the SNPs are filtered according to the following criteria: ① the missing rate is less than 0.2; ② the minimum allele frequency is greater than 0.05, and a total of 1,971,744 SNPs are obtained. Among them, a SNP site associated with cotton fiber length was discovered. It is located at position 9092066 on chromosome A05 of the reference genome of the upland cotton standard line TM-1. The polymorphism of this site is A / T, and its genotypes include AA genotype and TT genotype. Among them, the AA genotype is a homozygous body in which the deoxyribonucleotide genotype at position 9092066 on chromosome A05 of the cotton genome is all A; the TT genotype is a homozygous body in which the deoxyribonucleotide genotype at position 9092066 on chromosome A05 of the cotton genome is T.
[0061] 2. Development of SNP molecular markers associated with cotton fiber length
[0062] Design PCR amplification primers for genotype detection based on the SNP sites obtained in step 1. The primer sequences are as follows:
[0063] F: 5'-ACAGGCACGTAAATGCATCA-3' (SEQ ID NO: 1).
[0064] R: 5'-CGCAGGATCGTCGAAAGG-3' (SEQ ID NO: 2).
[0065] Example 2: Verification of SNP Molecular Markers Related to Cotton Fiber Length
[0066] Test materials: 365 cotton core germplasm resources, specific numbers see Table 1.
[0067] 1. Genotype testing
[0068] 1. Genomic DNA Extraction
[0069] Extract genomic DNA from the test materials.
[0070] 2. PCR amplification
[0071] Using the genomic DNA as a template, PCR amplification was performed using the primers designed in step 2 of Example 1 to obtain a PCR product.
[0072] The PCR amplification system was as follows: 0.5 μl of each primer, 1 μl of DNA template, 10 μl of Taq Mix, and 8 μl of water.
[0073] The PCR amplification program was 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 50 s, 35 cycles; and 72°C for 10 min.
[0074] 3. PCR product detection
[0075] The PCR product was detected using 1% agarose gel and sequenced to obtain the genotype of the SNP site obtained in Example 1.
[0076] 2. Cotton fiber length determination
[0077] The test materials were planted according to conventional methods in 2014 and 2015, and the fiber length was measured at maturity using the fiber testing center platform of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences. The specific measurement method was based on the "China Fiber Inspection Bureau. GB1103.1-2012 "Cotton Part 1: Sawtooth Processed Fine-staple Cotton" [S]. Beijing: China Standards Press, 2012."
[0078] 3. Association analysis between SNP genotype and fiber length
[0079] The results are shown in Table 1 and Figure 1 The results showed that among the 365 materials, 53 were of the AA genotype, with an average fiber length of 28.33 mm; 289 were of the TT genotype, with an average fiber length of 29.44 mm. The cotton fiber length of the TT genotype materials was significantly longer than that of the AA genotype materials.
[0080] Table 1
[0081]
[0082]
[0083]
[0084]
[0085]
[0086]
[0087]
[0088]
[0089]
[0090] Note: - indicates deletion, W indicates heterozygous.
[0091] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.
Claims
1. Use of a substance for detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome in any of the following A1) to A4): A1) Identify or assist in identifying cotton fiber length; A2) Screening or assisting in the selection of long-fiber cotton varieties; A3) Cotton breeding; A4) Cotton variety improvement.
2. Use of a substance for detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome in any of the following B1) to B4): B1) preparing products for identifying or assisting in identifying cotton fiber length; B2) preparing products for screening or assisting in screening long-fiber cotton varieties; B3) preparing cotton breeding products; B4) Preparation of products of improved cotton varieties.
3. The use according to claim 1 or 2, characterized in that: The substance for detecting the genotype of the deoxyribonucleotide at position 9092066 on chromosome A05 in the cotton genome is a primer pair consisting of the single-stranded DNA shown in sequence 1 and the single-stranded DNA shown in sequence 2.
4. A method for identifying or assisting in the identification of cotton fiber length, comprising the following steps: detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome, and identifying or assisting in the identification of cotton fiber length based on the genotype.
5. A method for screening or assisting in screening long-fiber cotton varieties, comprising the following steps: detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome, and selecting cotton varieties with the TT genotype; The TT genotype is a homozygous body in which the deoxyribonucleotides at positions 9092066 on chromosome A05 in the cotton genome are all T.
6. A cotton breeding method comprising the following steps: detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome, and selecting a cotton variety with the TT genotype as a parent for breeding.
7. A method for improving cotton varieties, comprising the following steps: detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome, and selecting a cotton variety with the TT genotype as a parent for breeding.
8. The method according to any one of claims 4 to 7, characterized in that: The method for detecting the genotype of the 9092066th deoxyribonucleotide on chromosome A05 in the cotton genome is direct sequencing or sequencing a PCR product containing the 9092066th deoxyribonucleotide on chromosome A05 in the cotton genome.
9. A product comprising a substance for detecting the genotype of deoxyribonucleotide position 9092066 on chromosome A05 in the cotton genome; the product having at least one of the following functions C1) to C4): C1) Identify or assist in identifying cotton fiber length; C2) screening or assisting in screening long-fiber cotton; C3) Cotton breeding; C4) Cotton variety improvement.
10. The product according to claim 9, characterized in that: The substance for detecting the genotype of the deoxyribonucleotide at position 9092066 on chromosome A05 in the cotton genome is a primer pair consisting of the single-stranded DNA shown in sequence 1 and the single-stranded DNA shown in sequence 2.