Molecular marker of actin cytoskeleton gene GoACT related to cotton fiber quality character and application of molecular marker

Abstract

The invention discloses an actin cytoskeleton gene GoACT molecular marker related to cotton fiber quality traits and application of the actin cytoskeleton gene GoACT molecular marker. SNP sites and InDel molecular markers related to the cotton fiber quality traits are screened out. Research finds that the cotton actin cytoskeleton gene GoACT has two haplotypes, and when the haplotype 1 gene sequence or the coded protein thereof exists in cotton, the allele or the protein thereof can reduce the micronaire value of cotton fibers, so that the cotton fiber micronaire value can be reduced, and the cotton fiber micronaire value can be reduced. Therefore, the gene haplotype 1 has important effects and application prospects in improvement of cotton fiber quality characters and cultivation of high-quality new varieties of cotton. The InDel molecular marker related to the cotton fiber quality character is a 7bp InDel difference at the 460bp position of a genome sequence located at the downstream of a termination codon of a haplotype 1 of an actin cytoskeleton GoACT gene. The InDel molecular marker can be used as a molecular marker for efficiently identifying high-quality cotton varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology application and relates to an actin cytoskeleton gene GoACT molecular marker related to cotton fiber quality traits and an application thereof. Background Art

[0002] In recent years, with the development of high-throughput sequencing, whole-genome association analysis has been widely used in the study of complex quantitative traits of various crops due to its unique advantages, and a large number of genetic loci associated with target traits have been identified. However, the QTL interval located by GWAS contains more genes, which has a greater impact on the prediction of causal genes of related traits. In addition, genomic variation does not necessarily cause changes in expression levels, and it may not affect the target traits or other biological processes. Using population transcriptome data, whole-transcriptome association analysis can establish the relationship between genetically regulated gene expression and traits, discover the causal genes of GWAS sites, and help to deeply explain the mechanism of trait variation.

[0003] Many genetic variations affect complex traits by regulating gene expression (Gusev et al., 2016). In recent years, more and more studies have used TWAS technology to discover causal genes for complex traits in crops. Ma et al. (2021) identified six high temperature response genes related to protein kinase activity through TWAS, of which three genes overlapped with the results of GWAS. Wang et al. (2022) integrated TWAS and colocalization analysis and found that the eQTL signal of gene Garb_05G040220 had a high colocalization probability with the fiber length QTL (FL6) at 4DPA and 8DPA, and this gene was also identified by TWAS at four fiber development stages (4DPA, 12DPA, 16DPA, 20DPA). This gene encodes a protein of the SGNH hydrolase superfamily.

[0004] Liu et al. (2022) measured the starch content of the sword leaf at the heading stage of 287 rice materials, and identified 25 key sites related to starch content using the GWAS method. Subsequently, 21 candidate genes were detected using TWAS analysis combined with transcriptome expression data and phenotypic data. Lin et al. (2022) integrated GWAS and TWAS analysis to identify 22 candidate genes that may regulate corn leaf epidermal conductivity, of which 4 were involved in cuticle precursor biosynthesis and export, 2 were involved in cell wall modification, 9 were involved in intracellular membrane transport, and 7 were involved in regulating cuticle development. These studies show that the application of TWAS technology can enable people to better understand the complex regulatory mechanisms of gene variation on various traits, and provide an effective means for mining key genes related to complex crop traits.

[0005] Actin is an essential substance for the formation of the cytoskeleton, and it plays an important role in the life activities of organisms, including cell division, cell movement, establishment and maintenance of cell morphology, endocytosis and exocytosis, etc. (Li Lan, 2014; Li Lifeng, 2015). In cotton, more and more studies have shown that the actin cytoskeleton is related to the elongation of cotton fiber cells and the synthesis of secondary walls (Wang et al., 2010; Han et al., 2013; Thyssen et al., 2017). Many actin and its binding protein-related genes are dominantly expressed in fiber cells and play an important role in the regulation of fiber development (Wang et al., 2010). Summary of the invention

[0006] The purpose of the present invention is to provide a GoACT molecular marker of an actin cytoskeleton gene related to cotton fiber quality traits and its application. Based on the phenotypic BLUP values ​​of fiber quality traits of natural populations of upland cotton for many years and multiple points, combined with the whole genome resequencing information of each material, a genotype-phenotype whole genome association analysis was performed on five fiber quality traits to locate the sites affecting fiber quality traits. We associated an important SNP mutation (G / T) located in the genome sequence of the GH_A10G0123 gene with ID number, starting from the 2467bp position of the start codon. The site is located in the exon region of the gene, causing changes in the amino acid sequence encoded by the gene, resulting in two different haplotypes (haplotype 1 and haplotype 2). In addition, there is an InDel site (TATTAAG / -) co-separated with the above two haplotypes at 460bp of the genome sequence downstream of the stop codon of haplotype 1 of the GH_A10G0123 gene. GH_A10G0123 encodes an actin cytoskeleton gene, named GoACT. Comparative analysis of fiber quality traits among different haplotypes of GoACT showed that haplotype 1 had a significant advantage over haplotype 2 in fiber micronaire trait.

[0007] The present invention also provides an InDel primer pair for detecting GoACT differential sites. At 460 bp of the genomic sequence downstream of the stop codon of the GoACT gene haplotype 1, there are two haplotypes with seven nucleotide insertion and deletion differences, which are co-segregated with the G / T haplotype. The differential site is detected, and the haplotype 1 amplification band type (TATTAAG) representing the 460 bp downstream of the gene with seven nucleotide insertions is selected, which is the cotton germplasm with high fiber quality traits.

[0008] The purpose of the present invention can be achieved through the following technical solutions:

[0009] In the first aspect, the present invention claims the use of a SNP site associated with cotton fiber quality traits or a substance for detecting the polymorphism or haplotype of the SNP site in any of the following:

[0010] (a1) screening or assisting in the screening of cotton germplasm with excellent fiber quality traits, or preparing products for screening or assisting in the screening of cotton germplasm with excellent fiber quality traits;

[0011] (a2) identifying or assisting in the identification of cotton fiber quality traits, or preparing products that identify or assist in the identification of cotton fiber quality traits;

[0012] (a3) improving the quality traits of cotton fibers, or preparing products for improving the quality traits of cotton fibers;

[0013] (a4) breeding new varieties of cotton with superior fiber quality, or preparing products for breeding new varieties of cotton with superior fiber quality;

[0014] The SNP site is located in the genomic sequence of the actin cytoskeleton GoACT gene with ID number GH_A10G0123, starting from the start codon position at the 2467bp of the genomic sequence shown in SEQ ID NO.2. The site is located in the exon region of the gene and has a termination mutation G / T, which causes the amino acid translation of the actin cytoskeleton GoACT gene to terminate prematurely.

[0015] Furthermore, when the SNP site is G, the actin cytoskeleton GoACT gene is haplotype 1, and when the SNP site is T, the actin cytoskeleton GoACT gene is haplotype 2, and the fiber quality traits of haplotype 1 are better than those of haplotype 2.

[0016] In a second aspect, the present invention claims the use of an InDel molecular marker co-separated with the above-mentioned SNP site or a substance for detecting the InDel molecular marker in any of the following:

[0017] (a1) screening or assisting in the screening of cotton germplasm with excellent fiber quality traits, or preparing products for screening or assisting in the screening of cotton germplasm with excellent fiber quality traits;

[0018] (a2) identifying or assisting in the identification of cotton fiber quality traits, or preparing products that identify or assist in the identification of cotton fiber quality traits;

[0019] (a3) improving the quality traits of cotton fibers, or preparing products for improving the quality traits of cotton fibers;

[0020] (a4) breeding new varieties of cotton with superior fiber quality, or preparing products for breeding new varieties of cotton with superior fiber quality;

[0021] (a5) identifying the haplotype of the cotton actin cytoskeleton GoACT gene, or preparing a product for identifying the haplotype of the cotton actin cytoskeleton GoACT gene;

[0022] The InDel molecular marker is the InDel difference TATTAAG / - located at the 460bp of the genome sequence downstream of the stop codon of the haplotype 1 of the actin cytoskeleton GoACT gene.

[0023] Furthermore, the InDel molecular marker was co-segregated with the two haplotypes of the SNP site, and haplotype 2 had a deletion of a 7bp nucleotide fragment TATTAAG at the 460bp of the genome sequence shown in SEQ ID NO.9 downstream of the GoACT gene stop codon compared to haplotype 1; the fiber quality traits of haplotype 1 were better than those of haplotype 2.

[0024] In a third aspect, the present invention claims protection for a product, which is or contains the above-mentioned substance for detecting the polymorphism or haplotype of the SNP site or / and the above-mentioned substance for detecting the InDel molecular marker, and is at least one of the following (b1)-(b5):

[0025] (b1) Products for screening or assisting in screening cotton germplasm with superior fiber quality traits;

[0026] (b2) Products that identify or assist in identifying the quality traits of cotton fiber;

[0027] (b3) Identification of products of cotton actin cytoskeleton GoACT gene haplotypes;

[0028] (b4) Products for improving cotton fiber quality traits;

[0029] (b5) Products used to breed new cotton varieties with superior fiber quality.

[0030] In a fourth aspect, the present invention claims a method, which is at least one of the following methods (c1)-(c5):

[0031] (c1) methods for selecting or assisting in selecting cotton germplasm for superior fiber quality traits;

[0032] (c2) Methods for identifying or assisting in the identification of cotton fiber quality traits;

[0033] (c3) Method for identifying haplotypes of cotton actin cytoskeleton GoACT gene;

[0034] (c4) Methods for improving cotton fiber quality traits;

[0035] (c5) Methods for breeding new cotton varieties with superior fiber quality;

[0036] The method is to detect the polymorphism of the SNP site or the haplotype or / and the InDel molecular marker, determine the haplotype of the actin cytoskeleton GoACT gene of the cotton germplasm material to be tested, the CDS sequence of haplotype 1 is shown as SEQ ID NO.1, and the genome sequence is shown as SEQ ID NO.2; the CDS sequence of haplotype 2 is shown as SEQ ID NO.4, and the genome sequence is shown as SEQ ID NO.5; haplotype 2 has a SNP (single nucleotide polymorphism) site for termination mutation G / T at the 2467bp starting from the start codon position in the genome sequence of the GoACT gene compared with haplotype 1, resulting in premature termination of amino acid translation; the InDel molecular marker is co-separated with the two haplotypes, and haplotype 2 has a SNP (single nucleotide polymorphism) site for termination mutation G / T at the 2467bp starting from the start codon position in the genome sequence of the GoACT gene compared with haplotype 1, as shown in SEQ ID There is a deletion of a 7bp nucleotide fragment TATTAAG at the 460bp position of the genome sequence shown in NO.9; the fiber quality traits of haplotype 1 are better than those of haplotype 2; the tested cotton germplasm material carrying haplotype 1 of the actin cytoskeleton GoACT gene is selected as the cotton germplasm with high fiber quality traits, which is used for cotton breeding to improve the cotton fiber quality traits.

[0037] Furthermore, in the above-mentioned applications, products and methods, the substance for detecting the InDel molecular marker is the following (d1) or (d2) or (d3) or (d4):

[0038] (d1) an InDel primer pair for detecting different haplotypes of the actin cytoskeleton GoACT gene, wherein the forward and reverse primers of the InDel primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8;

[0039] (d2) a reagent containing the InDel primer pair described in (d1);

[0040] (d3) a kit comprising the InDel primer pair described in (d1) or the reagent described in (d2);

[0041] (d4) A detection instrument comprising the InDel primer pair described in (d1), the reagent described in (d2) or the kit described in (d3).

[0042] Furthermore, in the above-mentioned applications, products and methods, the cotton fiber quality trait is the fiber micronaire value.

[0043] In a fifth aspect, the present invention seeks to protect the use of the cotton actin cytoskeleton GoACT gene, a substance that promotes the expression of the cotton actin cytoskeleton GoACT gene, or a substance that increases the activity or content of the protein encoded by the actin cytoskeleton GoACT gene in the following el) or e2):

[0044] e1) improving cotton fiber quality traits;

[0045] e2) Cultivate new germplasm with improved cotton fiber quality traits.

[0046] Furthermore, the substance that promotes the expression of the actin cytoskeleton GoACT gene or the substance that increases the activity or content of the protein encoded by the actin cytoskeleton GoACT gene is a biomaterial, and the biomaterial is any one of the following fl) to f6):

[0047] f1) an expression cassette containing the actin cytoskeleton GoACT gene;

[0048] f2) a recombinant vector containing the actin cytoskeleton GoACT gene, or a recombinant vector containing the expression cassette described in f1);

[0049] f3) a recombinant microorganism containing the actin cytoskeleton GoACT gene, or a recombinant microorganism containing the expression cassette described in f1), or a recombinant microorganism containing the recombinant vector described in f2);

[0050] f4) a transgenic plant cell line containing the actin cytoskeleton GoACT gene, or a transgenic plant cell line containing the expression cassette described in f1), or a transgenic plant cell line containing the recombinant vector described in f2);

[0051] f5) transgenic plant tissue containing the actin cytoskeleton GoACT gene, or transgenic plant tissue containing the expression cassette described in f1), or transgenic plant tissue containing the recombinant vector described in f2);

[0052] f6) a transgenic plant organ containing the actin cytoskeleton GoACT gene, or a transgenic plant organ containing the expression cassette of f1), or a transgenic plant organ containing the recombinant vector of f2).

[0053] Furthermore, the CDS sequence of the cotton actin cytoskeleton GoACT gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cotton actin cytoskeleton GoACT gene is shown as SEQ ID NO.3.

[0054] In the sixth aspect, the present invention claims protection for a method for improving cotton fiber quality traits, taking the actin cytoskeleton GoACT gene as the target gene, and through genetic engineering methods, overexpressing the actin cytoskeleton GoACT gene with a CDS sequence such as that shown in SEQID NO.1 in cotton or target plants, thereby improving cotton fiber quality traits or cultivating new germplasm with improved cotton fiber quality traits.

[0055] The present invention has found that there are two haplotypes of the actin cytoskeleton GoACT gene, the CDS sequence of haplotype 1 is shown in SEQ ID NO.1, the genome sequence is shown in SEQ ID NO.2, and the amino acid sequence encoded by it is shown in SEQ ID NO.3. The CDS sequence of haplotype 2 is shown in SEQ ID NO.4, the genome sequence is shown in SEQ ID NO.5, and the amino acid sequence encoded by it is shown in SEQ ID NO.6. Haplotype 2 (as shown in SEQ ID NO.5) has a single nucleotide polymorphism site that terminates the mutation G / T at the 2467bp starting from the start codon position in the genome sequence of the GoACT gene compared to haplotype 1 (as shown in SEQ ID NO.2), causing the amino acid translation to terminate prematurely. The site of the InDel molecular marker is co-separated with the two haplotypes, and haplotype 2 has a 7bp nucleotide fragment (TATTAAG) deletion at the 460bp of the genome sequence downstream of the stop codon of the GoACT gene compared to haplotype 1. The results of association analysis showed that haplotype 1 had an advantage over haplotype 2 in fiber quality traits.

[0056] The study showed that when the haplotype 1 gene sequence or the protein it encodes is present in cotton plants, the allele or its protein can promote the reduction of cotton fiber micronaire value, indicating that the gene haplotype 1 has an important role and application prospect in improving cotton fiber quality traits and breeding high-quality new cotton varieties. At the same time, the difference sequence between the two haplotypes can be used as a molecular marker for efficient identification of high-quality cotton varieties.

[0057] When the molecular markers screened by the present invention are used to assist in screening or identifying high fiber quality traits of cotton, the above-mentioned InDel primer pair is used to detect the differential sites of the actin cytoskeleton GoACT gene, and the haplotype 1 amplification band type representing the 460bp of the genome sequence downstream of the termination codon of the gene without seven nucleotide deletions is selected, which is the cotton germplasm with high fiber quality traits, thereby realizing the application of the molecular marker in screening high-quality cotton germplasm.

[0058] The beneficial effects of the present invention are:

[0059] 1. The present invention discovered an actin cytoskeleton gene GoACT that is significantly associated with cotton fiber micronaire trait through natural population resequencing, whole genome association analysis, whole transcriptome association analysis and co-localization analysis of cotton. A 10-fold deep resequencing of the upland cotton population identified 2,876,472 SNPs and 568,769 InDels. High-quality genotype data provides a guarantee for the accuracy of the association analysis sites.

[0060] 2. The expression level of GoACT in cotton fiber tissue at 10 days was obtained through transcriptome analysis. The expression of different haplotypes of this gene at 10 days of fiber development and elongation showed significant differences, indicating a close relationship between this gene and fiber quality traits.

[0061] 3. Develop an InDel primer pair to detect the differential site at the 460bp of the genomic sequence downstream of the stop codon of GoACT haplotype 1, and use PCR technology to distinguish and identify different haplotypes in the upland cotton population. According to the PCR results, the natural population can be divided into two categories, namely haplotype 1 and haplotype 2. Through statistical analysis, it was found that the fiber micronaire value of the population material carrying haplotype 1 was significantly different from that of the population material carrying haplotype 2, further proving the correlation between this gene and cotton fiber quality traits. This technology is simple and easy to operate, with high sensitivity and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 . GWAS Manhattan plot of fiber micronaire (Mic) in the DT-BLUP environment of upland cotton.

[0063] The horizontal axis represents chromosomes. The vertical axis represents the significance of SNP loci association, expressed as -log 10 (p) indicates.

[0064] Figure 2 .Local Manhattan plot (upper panel) and LD heat map (lower panel) of fiber micronaire values ​​on chromosome A10.

[0065] The X-axis in the local Manhattan plot is the marker position, and the Y-axis is the -log10(Pvalue). The inverted triangle is the LD heat map of the SNPs in the interval.

[0066] Figure 3 . Manhattan plot of eQTL of cotton GoACT (GH_A10G0123) gene at 10DPA fiber development stage.

[0067] The horizontal axis represents chromosomes. The vertical axis represents the significance of SNP loci association, expressed as -log 10 (p) indicates.

[0068] Figure 4.Gene structure of GH_A10G0123.

[0069] The black line represents the position of the mutation on the gene. A SNP site was identified in the variety population, which is located in the coding region of GoACT (GH_A10G0123), which encodes the actin cytoskeleton gene. Haplotype analysis found that there are two haplotypes of this gene. Haplotype 2 has a single nucleotide polymorphism site (G / T) that terminates the acquired mutation at the 2467bp starting from the start codon position in the genomic sequence of the GoACT gene, and causes changes in the encoded amino acid sequence.

[0070] Figure 5 .Comparative analysis of fiber micronaire traits among different haplotypes of GoACT.

[0071] The box plot represents the distribution of micronaire values ​​in natural populations of upland cotton. The horizontal axis represents different haplotype materials, and the vertical axis represents the corresponding micronaire values. The number of materials containing haplotype 1 (G) and haplotype 2 (T) were 124 and 201 respectively. The remaining 10 samples were not used for statistical analysis because their DNA quality did not meet the requirements. **** indicates significant differences at the 0.0001 level.

[0072] Figure 6 .Analysis of differential expression of different haplotypes of GoACT during the 10-day fiber development period.

[0073] The box plot represents the expression distribution of the natural population of upland cotton during the 10-day fiber development period. The horizontal axis represents different haplotype materials, and the vertical axis represents the corresponding gene expression value. The number of materials containing haplotype 1 (G) and haplotype 2 (T) was 122 and 201 respectively. The remaining 12 samples were not used for statistical analysis because the quality of the extracted fiber sample RNA did not meet the requirements. **** indicates a significant difference at the 0.0001 level.

[0074] Figure 7 .GoACT genome sequence alignment of different haplotypes.

[0075] A 7 bp nucleotide fragment deletion, namely TATTAAG, was found at the 460 bp of the genome sequence downstream of the stop codon of the GoACT gene haplotype 1 in the variety population. The red boxes are the forward and reverse primer positions of the InDel primer pair.

[0076] Figure 8 .PAGE gel typing analysis of the amplification products using InDel primers designed based on haplotype difference sites.

[0077] The picture shows that using this primer, the upland cotton population can be divided into two banding types. The amplified product of haplotype 1 has 7 more base differences than that of haplotype 2, which is co-segregated with the G / T SNP difference on the GoACT exon. M represents DNA Marker, lanes 3, 14, 15, 16, 23, 24, 25, 26, 32, 41, 60, 61, 64, 70, 72, 73, 74, 76, 78, 93, 94, 95 are the banding types of haplotype 1, and lanes 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 27, 28, 29, 30, 31, 33, 34, 35 ,36,37,38,39,40,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,62,63,65,66,67,68,69,71,75,77,79,80,81,82,83,84,85,86,87,88,89,90,91,92,96 are the banding patterns of haplotype 2. Haplotype 1: “TATTAAG”; haplotype 2:

[0079] Fig. 9 .InDel primers were used to identify haplotypes with excellent fiber quality.

[0080] The box plot represents the distribution of fiber micronaire in natural populations. The horizontal axis represents different haplotype materials. There are 77 and 153 varieties containing haplotype 1 (G or TATTAAG) and haplotype 2 (T or "-") respectively. **** indicates differences at the 0.0001 level. DETAILED DESCRIPTION

[0081] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0082] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0083] Example 1 Mining of actin cytoskeleton genes GoACT associated with cotton fiber quality traits

[0084] Whole genome resequencing was performed on 335 upland cotton germplasms introduced from different regions of China and multiple countries. After standard variation detection and genotype filtering, a total of 2,876,472 high-quality SNPs and 568,769 InDels (≤50bp) were detected. At the same time, we planted the population materials at the Nanjing Agricultural University Experimental Station in Dangtu, Anhui for many consecutive years (2018-2020), and conducted a detailed investigation of the five fiber quality traits of each material. Based on the BLUP values ​​of multiple environments, GWAS analysis of the five fiber quality traits was performed using GEMMA software, locating a candidate region (A10:966.85-999.70Mb) that was significantly associated with Mic-DT-BLUP ( Figure 1 , Figure 2 ), and a candidate gene in this region, GH_A10G0123, was identified in the fiber transcriptome at 10 days after flowering by TWAS and coloc analysis (Table 1). GH_A10G0123 has a stopgain variant in the coding region of the gene, which was also significantly associated with ( Figure 3 , Table 2). Based on the variation information of GH_A10G0123, this study divided it into two different types, haplotype 1 (G) and haplotype 2 (T) ( Figure 4 ), haplotype 2 has a single nucleotide polymorphism site that terminates the acquired mutation at the 2467bp (A10:985874) starting from the start codon position compared to haplotype 1 in the genomic sequence of GH_A10G0123 (as shown in SEQ ID NO.2 and SEQ ID NO.4), resulting in a change in the encoded amino acid sequence.

[0085] Table 1 TWAS and coloc analysis annotation information of GH_A10G0123

[0086]

[0087] Table 2 eQTL information of genetic variation of GH_A10G0123

[0088]

[0089] Example 2 Phenotypic Difference Analysis of Different Haplotypes of the Actin Cytoskeleton Gene GoACT

[0090] The distribution of micronaire values ​​in natural populations of upland cotton showed that, except for 10 materials with incomplete gene sequences and the variant sites that could not be confirmed, the remaining 325 materials contained 124 and 201 haplotypes of haplotype 1 (G) and haplotype 2 (T), respectively. Using the t-test statistical method, we calculated the phenotypic differences in fiber micronaire values ​​between the two haplotypes. The fiber micronaire values ​​of haplotype 1 (G) were significantly better than those of haplotype 2 (T) upland cotton germplasm ( Figure 5 ).

[0091] Example 3 Analysis of the difference in expression levels of different haplotypes of GoACT at 10 days of cotton fiber development

[0092] The present invention is based on 335 natural populations of upland cotton, and fiber samples were collected 10 days after flowering for transcriptome sequencing. Due to the lack of fiber samples of 12 materials, fiber transcriptome data were not obtained. The gene expression level is calculated by comparing the reads obtained by sequencing with the upland cotton genome, and the calculated expression level is expressed as the average expression level TPM of monomeric genes per million transcripts. Among the remaining 323 materials, the number of materials containing haplotype 1 (G) and haplotype 2 (T) were 122 and 201, respectively. Using the t-test statistical method, we calculated the difference in gene expression levels between the two groups of haplotypes. The gene expression level of haplotype 1 (G) was significantly higher than that of haplotype 2 (T) in upland cotton germplasm ( Figure 6 ), indicating the relevance of this gene to fiber quality.

[0093] Example 4 Application of two haplotypes of the GoACT gene in identifying high-quality cotton varieties

[0094] Sequence analysis revealed that there was a 7 bp nucleotide fragment (TATTAAG) indel at 460 bp of the genomic sequence shown in SEQ ID NO.9, located downstream of the stop codon of the actin cytoskeleton GoACT gene haplotype 1 (see Figure 7 ), and its genomic sequence, haplotype 1 (G) and haplotype 2 (T) SNP differences at the 2467bp starting from the start codon position co-segregated. Using this InDel site, we designed and developed InDel marker primers that effectively distinguish GoACT haplotypes, with sequences of SEQ ID NO.7 and SEQ ID NO.8. Compared with haplotype 2, haplotype 1 does not have seven nucleotide deletions at the 460bp of the genomic sequence downstream of the stop codon of the gene. The PCR reaction procedure is as follows: pre-denaturation at 95℃ for 5min; denaturation at 95℃ for 30sec, annealing at 58℃ for 30sec, extension at 72℃ for 20sec, 35 cycles; and finally extension at 72℃ for 5min. According to the amplification results of the molecular marker ( Figure 8), 77 haplotype 1 (G or TATTAAG) materials and 153 haplotype 2 (T or "-") materials were identified from natural populations. The names of materials carrying different haplotypes are shown in Table 3. The correlation of quality traits between the two haplotypes was calculated using the t-test statistical test method ( Fig. 9 The results showed that the fiber micronaire trait of haplotype 1 was significantly better than that of haplotype 2 cotton materials.

[0095] Table 3 Identification of haplotype 1 and haplotype 2 in population materials

[0096]

[0097]

[0098] The above results show that we discovered an actin cytoskeleton gene GoACT that is significantly associated with the micronaire value of upland cotton fiber based on natural population resequencing, whole genome association analysis, eQTL analysis, whole transcriptome association analysis and co-localization analysis. There are two haplotypes of the GoACT gene in natural populations, and different haplotypes have different expression levels in fiber tissue 10 days after flowering. Molecular markers were designed based on the haplotypes of the gene GoACT, and it was found that the marker can distinguish the different haplotypes carried by upland cotton germplasm, and can be used to effectively screen this locus in upland cotton materials from different sources. The GoACT gene and the InDel markers co-segregated with it play an important role and have application prospects in improving cotton quality traits and breeding new high-quality cotton varieties.

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Claims

1. Use of a SNP site associated with cotton fiber quality traits or a substance for detecting the polymorphism or haplotype of the SNP site in any of the following: (a1) screening or assisting in the screening of cotton germplasm with excellent fiber quality traits, or preparing products for screening or assisting in the screening of cotton germplasm with excellent fiber quality traits; (a2) identifying or assisting in the identification of cotton fiber quality traits, or preparing products that identify or assist in the identification of cotton fiber quality traits; (a3) improving the quality traits of cotton fibers, or preparing products for improving the quality traits of cotton fibers; (a4) breeding new varieties of cotton with superior fiber quality, or preparing products for breeding new varieties of cotton with superior fiber quality; The SNP site is located in the genomic sequence of the actin cytoskeleton GoACT gene with ID number GH_A10G0123, starting from the start codon position at the 2467bp of the genomic sequence shown in SEQ ID NO.

2. The site is located in the exon region of the gene and has a termination mutation G / T, which causes the amino acid translation of the actin cytoskeleton GoACT gene to terminate prematurely.

2. The application according to claim 1, characterized in that: When the SNP site is G, the actin cytoskeleton GoACT gene is haplotype 1, and when the SNP site is T, the actin cytoskeleton GoACT gene is haplotype 2. The fiber quality traits of haplotype 1 are better than those of haplotype 2.

3. Use of an InDel molecular marker co-separated with the SNP site in claim 1 or a substance for detecting the InDel molecular marker in any of the following: (a1) screening or assisting in the screening of cotton germplasm with excellent fiber quality traits, or preparing products for screening or assisting in the screening of cotton germplasm with excellent fiber quality traits; (a2) identifying or assisting in the identification of cotton fiber quality traits, or preparing products that identify or assist in the identification of cotton fiber quality traits; (a3) improving the quality traits of cotton fibers, or preparing products for improving the quality traits of cotton fibers; (a4) breeding new varieties of cotton with superior fiber quality, or preparing products for breeding new varieties of cotton with superior fiber quality; (a5) identifying the haplotype of the cotton actin cytoskeleton GoACT gene, or preparing a product for identifying the haplotype of the cotton actin cytoskeleton GoACT gene; The InDel molecular marker is the InDel difference TATTAAG / - located at the 460 bp of the genome sequence downstream of the termination codon of the actin cytoskeleton GoACT gene.

4. The use according to claim 1, characterized in that: The InDel molecular marker is co-segregated with the two haplotypes of the SNP site described in claim 1 or 2, and haplotype 2 has a deletion of a 7bp nucleotide fragment TATTAAG at the 460bp of the genome sequence shown in SEQ ID NO.9 downstream of the GoACT gene stop codon compared to haplotype 1; the fiber quality traits of haplotype 1 are better than those of haplotype 2.

5. A product, characterized in that The product is or contains the substance for detecting the polymorphism or haplotype of the SNP site as described in claim 1 or / and the substance for detecting the InDel molecular marker as described in claim 3, and is at least one of the following (b1)-(b5): (b1) Products for screening or assisting in screening cotton germplasm with superior fiber quality traits; (b2) Products that identify or assist in identifying the quality traits of cotton fiber; (b3) Identification of products of cotton actin cytoskeleton GoACT gene haplotypes; (b4) Products for improving cotton fiber quality traits; (b5) Products used to breed new cotton varieties with superior fiber quality.

6. A method, characterized in that The method is at least one of the following methods (c1) to (c5): (c1) methods for selecting or assisting in selecting cotton germplasm for superior fiber quality traits; (c2) Methods for identifying or assisting in the identification of cotton fiber quality traits; (c3) Method for identifying haplotypes of cotton actin cytoskeleton GoACT gene; (c4) Methods for improving cotton fiber quality traits; (c5) Methods for breeding new cotton varieties with superior fiber quality; The method is to detect the polymorphism of the SNP site or the haplotype described in claim 1 or 2 or / and detect the InDel molecular marker described in claim 3 or 4, and determine the haplotype of the actin cytoskeleton GoACT gene of the cotton germplasm material to be tested, the genome sequence of haplotype 1 is shown as SEQ ID NO.2; the genome sequence of haplotype 2 is shown as SEQ ID NO.5; haplotype 2 has a SNP site with a termination mutation G / T at the 2467bp starting from the start codon position in the genome sequence of the GoACT gene compared with haplotype 1, resulting in premature termination of amino acid translation; the InDel molecular marker is co-segregated with the two haplotypes, and haplotype 2 has a SNP site with a termination mutation G / T at the 2467bp starting from the start codon position in the genome sequence of the GoACT gene compared with haplotype 1, as shown in SEQ ID There is a deletion of a 7bp nucleotide fragment TATTAAG at the 460bp position of the genome sequence shown in NO.9; the fiber quality traits of haplotype 1 are better than those of haplotype 2; the tested cotton germplasm material carrying haplotype 1 of the actin cytoskeleton GoACT gene is selected as the cotton germplasm with high fiber quality traits, which is used for cotton breeding to improve the cotton fiber quality traits.

7. The use according to any one of claims 1 to 4, the product according to claim 5, the method according to claim 6, characterized in that: The substance for detecting the InDel molecule marker is the following (d1) or (d2) or (d3) or (d4): (d1) an InDel primer pair for detecting different haplotypes of the actin cytoskeleton GoACT gene, wherein the forward and reverse primers of the InDel primer pair are shown in SEQ ID NO.7 and SEQ ID NO.8; (d2) a reagent containing the InDel primer pair described in (d1); (d3) a kit comprising the InDel primer pair described in (d1) or the reagent described in (d2); (d4) A detection instrument comprising the InDel primer pair described in (d1), the reagent described in (d2) or the kit described in (d3).

8. The use according to any one of claims 1 to 4, the product according to claim 5, the method according to claim 6, characterized in that: The cotton fiber quality trait is fiber micronaire value.

9. Use of cotton actin cytoskeleton GoACT gene, a substance that promotes the expression of the cotton actin cytoskeleton GoACT gene, or a substance that increases the activity or content of the protein encoded by the actin cytoskeleton GoACT gene in the following el) or e2): e1) improving cotton fiber quality traits; e2) Cultivate new germplasm with improved cotton fiber quality traits.

10. The use according to claim 9, characterized in that: The substance that promotes the expression of the actin cytoskeleton GoACT gene or the substance that increases the activity or content of the protein encoded by the actin cytoskeleton GoACT gene is a biological material, and the biological material is any one of the following fl) to f6): f1) an expression cassette containing the actin cytoskeleton GoACT gene; f2) a recombinant vector containing the actin cytoskeleton GoACT gene, or a recombinant vector containing the expression cassette described in f1); f3) a recombinant microorganism containing the actin cytoskeleton GoACT gene, or a recombinant microorganism containing the expression cassette described in f1), or a recombinant microorganism containing the recombinant vector described in f2); f4) a transgenic plant cell line containing the actin cytoskeleton GoACT gene, or a transgenic plant cell line containing the expression cassette described in f1), or a transgenic plant cell line containing the recombinant vector described in f2); f5) transgenic plant tissue containing the actin cytoskeleton GoACT gene, or transgenic plant tissue containing the expression cassette described in f1), or transgenic plant tissue containing the recombinant vector described in f2); f6) a transgenic plant organ containing the actin cytoskeleton GoACT gene, or a transgenic plant organ containing the expression cassette of f1), or a transgenic plant organ containing the recombinant vector of f2).

11. The use according to claim 9 or 10, characterized in that: The CDS sequence of the cotton actin cytoskeleton GoACT gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cotton actin cytoskeleton GoACT gene is shown as SEQ ID NO.

3.

12. A method for improving the quality of cotton fibers, characterized in that: Taking the actin cytoskeleton GoACT gene as the target gene, the actin cytoskeleton GoACT gene with a CDS sequence as shown in SEQ ID NO.1 is overexpressed in cotton or target plants through genetic engineering methods to improve cotton fiber quality traits or cultivate new germplasm with improved cotton fiber quality traits.

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