Actin cytoskeleton gene goact molecular marker related to cotton fiber quality traits and application thereof

Abstract

The application discloses a molecular marker of an actin cytoskeleton gene GoACT related to a cotton fiber quality trait and application thereof, and screens out an SNP site and an InDel molecular marker related to the cotton fiber quality trait. Research finds that two haplotypes of the cotton actin cytoskeleton gene GoACT exist, when the gene sequence of the haplotype 1 or the protein coded by the gene sequence exists in cotton, the allele or the protein can reduce the micronaire value of the cotton fiber, which indicates that the haplotype 1 of the gene has important roles and application prospects in improving the cotton fiber quality trait and cultivating a new cotton variety with high quality. The InDel molecular marker related to the cotton fiber quality trait is a 7bp InDel difference at the 460bp of the genomic sequence downstream of the termination codon of the actin cytoskeleton GoACT gene haplotype 1. The InDel molecular marker can be used as a molecular marker for efficiently identifying a high-quality cotton variety.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications and relates to a GoACT molecular marker for the actin cytoskeleton gene, which is related to cotton fiber quality traits, and its application. Background Technology

[0002] In recent years, with the development of high-throughput sequencing, genome-wide association studies (GWAS) have been widely applied to the study of complex quantitative traits in various crops, identifying a large number of genetic loci associated with target traits. However, GWAS-mapped QTL regions contain a large number of genes, significantly impacting the prediction of causal genes related to traits. Furthermore, genomic variations do not necessarily lead to changes in expression levels, and therefore may not affect the target trait or other biological processes. Using population transcriptome data, GWAS can establish the relationship between genetically regulated gene expression and traits, uncover causal genes at GWAS loci, and contribute to a deeper understanding of the mechanisms underlying trait variation.

[0003] Many genetic variations influence complex traits by regulating gene expression (Gusev et al., 2016). In recent years, there has been an increasing number of studies using 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 using TWAS, three of which overlapped with GWAS results. Wang et al. (2022) integrated TWAS and colocalization analysis and found that the eQTL signal of the gene Garb_05G040220 had a high colocalization probability with the fiber length QTL (FL6) at 4DPA and 8DPA. This gene was also identified by TWAS at four fiber development stages (4DPA, 12DPA, 16DPA, 20DPA), and it encodes an SGNH hydrolase-like esterase superfamily protein.

[0004] Liu et al. (2022) measured the starch content of flag leaves at the heading stage in 287 rice materials. Using GWAS, they identified 25 key loci associated with starch content, and subsequently used TWAS analysis combined with transcriptome expression and phenotypic data to detect 21 candidate genes. Lin et al. (2022) integrated GWAS and TWAS analyses to identify 22 candidate genes that may regulate the epidermal conductivity of maize leaves. Among them, 4 are involved in cuticle precursor biosynthesis and export, 2 are involved in cell wall modification, 9 are involved in intracellular membrane transport, and 7 are involved in regulating cuticle development. These studies indicate that the application of TWAS technology can help us better understand the complex regulatory mechanisms of gene variation on various traits, providing an effective means for identifying key genes related to complex traits in crops.

[0005] Actin is an essential component of the cytoskeleton and plays a vital role in biological life activities, including cell division, cell movement, cell morphology establishment and maintenance, and endocytosis and exocytosis (Li Lan, 2014; Li Lifeng, 2015). In cotton, increasing research has shown that the actin cytoskeleton is associated with cotton fiber cell elongation and secondary wall synthesis (Wang et al., 2010; Han et al., 2013; Thyssen et al., 2017). Many actin and its binding protein-related genes are predominantly expressed in fiber cells and play important roles in the regulation of fiber development (Wang et al., 2010). Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker for the actin cytoskeleton gene GoACT, which is associated with cotton fiber quality traits, and its application. Based on the BLUP values ​​of fiber quality phenotypes from multi-year, multi-location natural populations of upland cotton, combined with whole-genome resequencing information for each material, genotype-phenotype genome-wide association analysis was performed on five fiber quality traits to locate the sites influencing fiber quality traits. We associated an important SNP mutation (G / T) located at 2467 bp from the start codon position in the genomic sequence of the gene with ID number GH_A10G0123. This site is located in the exon region of this gene, causing a change 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 / -) cosegregating with the above two haplotypes at 460 bp downstream of the stop codon of haplotype 1 in the genomic sequence of GH_A10G0123. GH_A10G0123 encodes the actin cytoskeleton gene, named GoACT. Comparative analysis of fiber quality traits among different haplotypes in GoACT showed that haplotype 1 had a significant advantage over haplotype 2 in fiber micronaire value.

[0007] This invention also provides an InDel primer pair for detecting differentially expressed sites in the GoACT gene. At a genomic sequence position 460 bp downstream of the stop codon of haplotype 1 in the GoACT gene, two haplotypes with a seven-nucleotide insertion / deletion difference exist, co-segregating with the G / T haplotype. By detecting these differentially expressed sites, the amplified band of haplotype 1 (TATTAAG) representing the seven-nucleotide insertion at 460 bp downstream of this gene is selected, indicating cotton germplasm with high fiber quality traits.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] In a first aspect, the present invention seeks protection for the use of SNP sites related to cotton fiber quality traits or substances for detecting polymorphisms or haplotypes of said SNP sites in any of the following:

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

[0011] (a2) To identify or assist in the identification of cotton fiber quality traits, or to prepare products for the identification or assistance in the identification of cotton fiber quality traits;

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

[0013] (a4) To cultivate new cotton varieties with excellent fiber quality, or to prepare products for cultivating new cotton varieties with excellent fiber quality;

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

[0015] Furthermore, when the SNP site is G, the actin cytoskeleton GoACT gene is haplotype 1; when the SNP site is T, the actin cytoskeleton GoACT gene is haplotype 2; and the fiber quality trait of haplotype 1 is superior to that of haplotype 2.

[0016] Secondly, the present invention seeks protection for the use of InDel molecular markers co-separated from the aforementioned SNP sites or substances for detecting said InDel molecular markers in any of the following:

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

[0018] (a2) To identify or assist in the identification of cotton fiber quality traits, or to prepare products for the identification or assistance in the identification of cotton fiber quality traits;

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

[0020] (a4) To cultivate new cotton varieties with excellent fiber quality, or to prepare products for cultivating new cotton varieties with excellent fiber quality;

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

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

[0023] Furthermore, the InDel molecular marker co-segregates with the two haplotypes of the SNP site. Haplotype 2 has a deletion of a 7bp nucleotide fragment TATTAAG at position 460bp downstream of the GoACT gene stop codon, as shown in SEQ ID NO.9, compared to haplotype 1. The fiber quality trait of haplotype 1 is superior to that of haplotype 2.

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

[0025] (b1) Products that screen or assist in screening cotton germplasm with superior fiber quality traits;

[0026] (b2) Products used to identify or assist in the identification of cotton fiber quality traits;

[0027] (b3) Products that identify the GoACT gene haplotype of cotton actin cytoskeleton;

[0028] (b4) Products used to improve the quality properties of cotton fibers;

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

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

[0031] (c1) Methods for screening or assisting in screening cotton germplasm with superior fiber quality traits;

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

[0033] (c3) A method for identifying the GoACT gene haplotype of cotton actin cytoskeleton;

[0034] (c4) Methods for improving the quality traits of cotton fibers;

[0035] (c5) Methods for cultivating new cotton varieties with excellent fiber quality;

[0036] This method involves detecting the aforementioned SNP polymorphisms or haplotypes and / or detecting the aforementioned InDel molecular markers to determine the haplotype of the GoACT gene, the actin cytoskeleton of the cotton germplasm material to be tested. The CDS sequence of haplotype 1 is shown in SEQ ID NO.1, and the genome sequence is shown in SEQ ID NO.2; the CDS sequence of haplotype 2 is shown in SEQ ID NO.4, and the genome sequence is shown in SEQ ID NO.5. Compared to haplotype 1, haplotype 2 has a G / T termination gain mutation SNP (single nucleotide polymorphism) at position 2467 bp from the start codon in the GoACT gene genome sequence, causing premature termination of amino acid translation. The InDel molecular marker co-segregates with the two haplotypes. Compared to haplotype 1, haplotype 2 has a termination gain mutation G / T SNP at position 2467 bp downstream of the stop codon in the GoACT gene, as shown in SEQ ID NO.5. The genome sequence shown in NO.9 contains a 7bp deletion of the nucleotide fragment TATTAAG at position 460bp; haplotype 1 exhibits superior fiber quality traits compared to haplotype 2; cotton germplasm materials carrying the GoACT gene of the actin cytoskeleton were selected as cotton germplasm with high fiber quality traits for cotton breeding to improve cotton fiber quality traits.

[0037] Furthermore, in the above applications, products, and methods, the substance used to detect the InDel molecular label is one of the following (d1) or (d2) or (d3) or (d4):

[0038] (d1) InDel primer pairs used to detect different haplotypes of the actin cytoskeleton GoACT gene, wherein the forward and reverse primers of the InDel primer pairs 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 containing the InDel primer pair described in (d1) or the reagent described in (d2);

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

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

[0043] Fifthly, the present invention claims protection for the use of the cotton actin cytoskeleton GoACT gene, substances that promote the expression of the cotton actin cytoskeleton GoACT gene, or substances that increase the activity or content of the protein encoded by the actin cytoskeleton GoACT gene in the following e1) or e2):

[0044] el) Improves the quality traits of cotton fibers;

[0045] e2) Develop new germplasm for improving cotton fiber quality traits.

[0046] Furthermore, the substance that promotes the expression of the GoACT gene in the actin cytoskeleton or increases the activity or content of the protein encoded by the GoACT gene in the actin cytoskeleton is a biological material, and the biological material is any one of the following f1) to f6):

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

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

[0049] f3) Recombinant microorganisms containing the GoACT gene of the actin cytoskeleton, or recombinant microorganisms containing the expression cassette of f1), or recombinant microorganisms containing the recombinant vector of f2);

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

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

[0052] f6) A transgenic plant organ containing the GoACT gene of the actin cytoskeleton, 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 in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cotton actin cytoskeleton GoACT gene is shown in SEQ ID NO.3.

[0054] Sixthly, the present invention claims protection for a method for improving cotton fiber quality traits, using the actin cytoskeleton GoACT gene as the target gene, and through genetic engineering methods, overexpressing the actin cytoskeleton GoACT gene with a CDS sequence as shown in SEQ ID NO.1 in cotton or target plants to improve cotton fiber quality traits or to cultivate new germplasm with improved cotton fiber quality traits.

[0055] This invention reveals that the GoACT gene, the actin cytoskeleton, exists in two haplotypes. Haplotype 1 has the CDS sequence shown in SEQ ID NO.1, the genome sequence shown in SEQ ID NO.2, and its encoded amino acid sequence shown in SEQ ID NO.3. Haplotype 2 has the CDS sequence shown in SEQ ID NO.4, the genome sequence shown in SEQ ID NO.5, and its encoded amino acid sequence shown in SEQ ID NO.6. Haplotype 2 (as shown in SEQ ID NO.5) has a single nucleotide polymorphism (SNP) at position 2467 bp (start codon) in the GoACT gene genome sequence compared to haplotype 1 (as shown in SEQ ID NO.2), resulting in premature termination of amino acid translation. The InDel molecular marker site co-segregates with both haplotypes. Haplotype 2 has a 7 bp nucleotide fragment (TATTAAG) deletion at position 460 bp downstream of the GoACT gene stop codon, compared to haplotype 1. The correlation analysis results show that haplotype 1 has an advantage over haplotype 2 in terms of fiber quality traits.

[0056] Studies have shown that when haplotype 1 gene sequence or its encoded protein is present in cotton plants, the allele or protein can promote a decrease in the micronaire value of cotton fibers, indicating that haplotype 1 plays an important role and has promising applications in improving cotton fiber quality traits and breeding high-quality new cotton varieties. Furthermore, the differential sequences between the two haplotypes can be used as molecular markers for the efficient identification of high-quality cotton varieties.

[0057] When using the molecular markers selected in this invention to assist in screening or identifying high fiber quality traits in cotton, the aforementioned InDel primer pair is used to detect the differential sites of the GoACT gene in the actin cytoskeleton. The haplotype 1 amplification band, which represents the genomic sequence downstream of the stop codon of this gene without the deletion of seven nucleotides at position 460 bp, is selected as cotton germplasm with high fiber quality traits, thus realizing the application of this molecular marker in screening high-quality cotton germplasm.

[0058] The beneficial effects of this invention are as follows:

[0059] 1. This invention identified a GoACT gene, an actin cytoskeleton gene, significantly associated with the micronaire value trait in cotton fibers, through resequencing of natural cotton populations, genome-wide association analysis (GWAA), transcriptome-wide association analysis (TMA), and colocalization analysis. Tenfold depth resequencing of upland cotton populations identified 2,876,472 SNPs and 568,769 InDels. High-quality genotypic data ensured the accuracy of the association analysis loci.

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

[0061] 3. An InDel primer pair was developed to detect the differential site at 460 bp downstream of the stop codon of GoACT haplotype 1 in the genome. Different haplotypes were distinguished and identified in upland cotton populations using PCR technology. Based on the PCR results, the natural population was divided into two categories: haplotype 1 and haplotype 2. Statistical analysis revealed a significant difference in fiber micronaire value between populations carrying haplotype 1 and those carrying haplotype 2, further demonstrating the correlation between this gene and cotton fiber quality traits. This technique is simple to operate, highly sensitive, and accurate. Attached Figure Description

[0062] Figure 1 GWAS Manhattan plot of micronaire value (Mic) of upland cotton under DT-BLUP conditions.

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

[0064] Figure 2 Local Manhattan plot (top) and LD heatmap (bottom) of fibrillary maclon values ​​on chromosome A10.

[0065] In the local Manhattan plot, the X-axis represents the marker location, and the Y-axis represents the -log10 (P-value) value. The inverted triangle is the LD heatmap of the SNP within the interval.

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

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

[0068] Figure 4The gene structure of .GH_A10G0123.

[0069] The black line represents the location of the mutation on the gene. A SNP site was identified in the varietal population, located in the coding region of GoACT (GH_A10G0123), a gene encoding the actin cytoskeleton. Haplotype analysis revealed two haplotypes of this gene. Haplotype 2, compared to haplotype 1, has a single nucleotide polymorphism (G / T) at position 2467 bp from the start codon in the GoACT gene genome sequence, resulting in a change in the encoded amino acid sequence.

[0070] Figure 5 Comparative analysis of the differences in micronaire values ​​among different haplotypes of GoACT.

[0071] Box plots represent the distribution of micronaire values ​​in natural upland cotton populations. The horizontal axis represents different haplotypes, and the vertical axis represents the corresponding micronaire values. The number of samples containing haplotype 1 (G) and haplotype 2 (T) were 124 and 201, respectively. The remaining 10 samples were not included in the statistical analysis due to insufficient DNA quality. **** indicates a significant difference at the 0.0001 level.

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

[0073] Box plots represent the expression distribution of upland cotton in natural populations during the 10-day fiber development stage. The horizontal axis represents different haplotypes, and the vertical axis represents the corresponding gene expression values. The number of samples containing haplotype 1 (G) and haplotype 2 (T) were 122 and 201, respectively. The remaining 12 samples were not included in the statistical analysis because the RNA quality of the extracted fiber samples did not meet the requirements. **** indicates a significant difference at the 0.0001 level.

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

[0075] A 7 bp nucleotide deletion, TATTAAG, was found at position 460 bp downstream of the stop codon 1 in the GoACT gene haplotype of the cultivar population. The red boxes indicate the positions of the forward and reverse primers for the InDel primer pair.

[0076] Figure 8 PAGE gel typing analysis of InDel primers amplified based on haplotype differential sites.

[0077] The image shows that using this primer, upland cotton populations can be divided into two banding patterns. Haplotype 1 has 7 more bases in its amplification product than haplotype 2, and it co-segregates with the G / T SNP difference on the GoACT exon. M represents the 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 represent the banding pattern of haplotype 1. 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 The numbers 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, and 96 represent the banding pattern of haplotype 2. Haplotype 1: “TATTAAG”; Haplotype 2: “-”.

[0078] Figure 9 InDel primers are used to identify haplotypes with superior fiber quality.

[0079] Box plots represent the distribution of fiber micronaire values ​​in natural populations. The horizontal axis represents different haplotypes. There are 77 varieties containing haplotype 1 (G or TATTAAG) and 153 varieties containing haplotype 2 (T or "-"). **** indicates differences at the 0.0001 level. Detailed Implementation

[0080] To make the technical problems solved by the present invention, the technical solutions and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments.

[0081] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0082] Example 1: Mining of the actin cytoskeleton gene GoACT associated with cotton fiber quality traits

[0083] Whole-genome resequencing was performed on 335 upland cotton germplasms introduced from different regions of China and multiple countries. After standard variant detection and genotype filtering, a total of 2,876,472 high-quality SNPs and 568,769 InDels (≤50bp) were detected. Simultaneously, the population materials were continuously cultivated for several years (2018-2020) at the Nanjing Agricultural University Experimental Station in Dangtu County, Anhui Province, and a detailed investigation was conducted on five fiber quality traits for each material. Based on multi-environment BLUP values, GWAS analysis was performed on the five fiber quality traits using GEMMA software, locating a candidate region significantly associated with Mic-DT-BLUP (A10: 966.85-999.70Mb). Figure 1 , Figure 2 The candidate gene GH_A10G0123 in the fiber transcriptome 10 days post-flowering was identified using TWAS and coloc analyses (Table 1). GH_A10G0123 has a stopgain variant in its coding region, which was also significantly associated with eQTL analysis. Figure 3 (Table 2). Based on the variation information of GH_A10G0123, this study classifies it into two different types, haplotype 1 (G) and haplotype 2 (T). Figure 4 Haplotype 2 has a single nucleotide polymorphism (SNP) with a termination gain mutation at 2467 bp (A10:985874) from the start codon position in the genomic sequence GH_A10G0123 (as shown in SEQ ID NO.2 and SEQ ID NO.4), compared to haplotype 1, resulting in a change in the encoded amino acid sequence.

[0084] Table 1. TWAS and coloc analysis annotation information for GH_A10G0123

[0085]

[0086] Table 2 eQTL information for genetic variation in GH_A10G0123

[0087]

[0088] Example 2: Phenotypic Differences Among Different Haplotypes of the Actin Cytoskeleton Gene GoACT

[0089] The distribution of micronaire value in natural upland cotton populations showed that, except for 10 materials where the gene sequence was incomplete and the variant site could not be identified, the remaining 325 materials contained haplotypes 1 (G) and 201 haplotypes 2 (T). Using a t-test, we calculated the phenotypic difference in micronaire value between the two haplotype groups. The micronaire value of haplotype 1 (G) was significantly better than that of upland cotton germplasm with haplotype 2 (T). Figure 5 ).

[0090] Example 3: Analysis of the difference in expression levels of different GoACT haplotypes at 10 days of cotton fiber development

[0091] This invention used fiber samples collected 10 days after flowering from 335 natural upland cotton populations for transcriptome sequencing. Due to the absence of fiber samples from 12 materials, no fiber transcriptome data were obtained. Gene expression levels were calculated by comparing the sequenced reads with the upland cotton genome, and the calculated expression level was expressed as the average expression level (TPM) of haplotypes per million transcripts. Of the remaining 323 materials, 122 contained haplotype 1 (G) and 201 contained haplotype 2 (T). Using a t-test, we calculated the difference in gene expression levels between the two haplotype groups. The gene expression level of haplotype 1 (G) was significantly higher than that of haplotype 2 (T) in upland cotton germplasm. Figure 6 This indicates the correlation between the gene and fiber quality.

[0092] Example 4: Application of two haplotypes of the GoACT gene in identifying high-quality cotton varieties

[0093] Sequence analysis revealed a 7 bp nucleotide fragment (TATTAAG) insertion / deletion at position 460 bp of the genomic sequence shown in SEQ ID NO.9, downstream of the stop codon of haplotype 1 of the GoACT gene, which is part of the actin cytoskeleton. Figure 7 The InDel site, located at 2467 bp from the start codon, showed co-separation of haplotypes 1 (G) and 2 (T) with its genomic sequence. Using this InDel site, we designed and developed InDel marker primers to effectively distinguish GoACT haplotypes, with sequences SEQ ID NO.7 and SEQ ID NO.8. Compared to haplotype 2, haplotype 1 does not have a seven-nucleotide deletion at 460 bp downstream of the stop codon in this gene. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 58℃ annealing for 30 sec, 72℃ extension for 20 sec, 35 cycles; final extension at 72℃ for 5 min. Based on the amplification results of this molecular marker ( Figure 8) 77 haplotype 1 (G or TATTAAG) materials and 153 haplotype 2 (T or "-") materials were identified from the natural population. The names of materials carrying different haplotypes are shown in Table 3. The correlation of quality traits between the two haplotype groups was calculated using the t-test statistical method. Figure 9 The results showed that the micronaire value of haplotype 1 fibers was significantly better than that of haplotype 2 cotton materials.

[0094] Table 3 Identification of haplotypes 1 and 2 in pluripotent materials

[0095]

[0096]

[0097] The above results indicate that, based on resequencing of natural upland cotton populations, genome-wide association analysis (GWAA), eQTL analysis, transcriptome-wide association analysis (TQTL), and co-localization analysis, we identified a GoACT actin cytoskeleton gene significantly associated with the micronaire value of upland cotton fibers. Two haplotypes of the GoACT gene exist in the natural population, and these different haplotypes exhibit varying expression levels in fiber tissue 10 days post-flowering. Molecular markers designed based on the GoACT gene haplotypes were found to distinguish different haplotypes carried by upland cotton germplasm, enabling effective screening of upland cotton materials from different sources at this locus. The GoACT gene and its co-segregated InDel marker play an important role and hold significant application potential in improving cotton quality traits and breeding high-quality cotton varieties.

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Claims

1. The use of SNP sites related to cotton fiber quality traits, or substances for detecting polymorphisms or haplotypes of said SNP sites, in any of the following: (a1) Screening cotton germplasm with excellent fiber quality traits; (a2) Identifying the quality traits of cotton fibers; The SNP site is located in the actin cytoskeleton with ID number GH_A10G0123. GoACT The gene's genomic sequence, corresponding to position 2467 bp as shown in SEQ ID NO.2, is located in the exon region of the gene and contains a termination gain mutation G / T, resulting in the actin cytoskeleton. GoACT The premature termination of amino acid translation in genes; The cotton fiber quality trait is the fiber micronaire value; the cotton is upland cotton.

2. The application according to claim 1, characterized in that, When the SNP site is G, the actin cytoskeleton... GoACT When the gene is haplotype 1 and the SNP site is T, the actin cytoskeleton... GoACT The gene is haplotype 2, and the fiber quality trait of haplotype 1 is superior to that of haplotype 2.

3. The use of an InDel molecular marker co-separated from the SNP site described in claim 1, or a substance for detecting the InDel molecular marker, in any of the following: (a1) Screening cotton germplasm with excellent fiber quality traits; (a2) Identifying the quality traits of cotton fibers; (a3) Identification of cotton actin cytoskeleton GoACT Haplotype of a gene; The InDel molecular marker is located in the actin cytoskeleton. GoACT The InDel difference at position 460 bp downstream of the gene stop codon, as shown in SEQ ID NO.9, is TATTAAG / -. The cotton fiber quality trait is the fiber micronaire value; the cotton is upland cotton.

4. The application according to claim 3, characterized in that, The InDel molecular marker described herein co-segregates with two haplotypes of the SNP site described in claim 2, with haplotype 2 being more segregated than haplotype 1. GoACT A 7 bp nucleotide fragment TATTAAG is deleted at position 460 bp downstream of the gene stop codon, as shown in SEQ ID NO. 9; haplotype 1 has better fiber quality traits than haplotype 2.

5. A method, characterized in that, The method is at least one of the following (c1)-(c3): (c1) Methods for screening cotton germplasm with superior fiber quality traits; (c2) Methods for identifying the quality traits of cotton fibers; (c3) Identification of cotton actin cytoskeleton GoACT Methods for identifying gene haplotypes; This method involves detecting SNP site polymorphisms or haplotypes as described in claim 1 or 2, and / or detecting the InDel molecular marker as described in claim 3 or 4, to determine the actin cytoskeleton of the cotton germplasm material to be tested. GoACT The genome sequences of haplotypes are as follows: haplotype 1 is shown in SEQ ID NO.2; haplotype 2 is shown in SEQ ID NO.5; haplotype 1 exhibits superior fiber quality traits compared to haplotype 2; selection was made for those carrying the actin cytoskeleton. GoACT The cotton germplasm material with haplotype 1 was used as cotton germplasm with high fiber quality traits for cotton breeding to improve cotton fiber quality traits. The cotton fiber quality trait is the fiber micronaire value; the cotton is upland cotton.

6. The application according to any one of claims 3-4, or the method according to claim 5, characterized in that, The substances that are detected by the InDel molecular label are (d1) or (d2) or (d3) or (d4) as follows: (d1) used to detect actin cytoskeleton GoACT InDel primer pairs for different haplotypes of the gene, the forward and reverse primers of the InDel primer pairs 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 containing the InDel primer pair described in (d1) or the reagent described in (d2); (d4) A detection instrument containing the InDel primer pair described in (d1), the reagent described in (d2), or the kit described in (d3).

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