GoWD40L, a class of WD40 family protein gene for improving cotton fiber quality and application thereof

Abstract

This invention discloses a WD40-like family protein gene, GoWD40L, for improving cotton fiber quality and its applications, belonging to the field of biotechnology applications. This WD40-like family protein gene, GoWD40L, exists in two haplotypes: haplotype 1 has a CDS sequence of SEQ ID NO.1; and haplotype 2 has a CDS sequence of SEQ ID NO.4. When the haplotype 2 gene sequence or its encoded protein is present in cotton plants, the allele or protein can promote an increase in cotton fiber length, fiber strength, fiber uniformity, and fiber elongation, and a decrease in micronaire value. This indicates that haplotype 2 of this gene plays an important role and has promising applications in improving cotton fiber quality traits and in breeding high-quality new cotton varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications and relates to a WD40-like family protein gene GoWD40L that improves cotton fiber quality and its application. Background Technology

[0002] Cotton is an important economic crop worldwide. Cotton fiber is a natural textile fiber with advantages such as being green, warm, breathable, and comfortable to wear. Upland cotton and Sea Island cotton, both allotetraploid cultivars, are two major cultivated varieties. They originated from a common ancestor and were domesticated independently. Upland cotton has high yields and strong adaptability, accounting for over 90% of the world's total cotton production, while Sea Island cotton has longer, finer, and stronger fibers, accounting for about 3% of the world's annual cotton production. Compared to many cotton varieties with high-yield characteristics, varieties with high-quality fibers are severely lacking. Therefore, cultivating superior varieties with both high-quality fibers and high yields is an urgent need to meet the demands of the modern textile industry and the growing health needs of the people.

[0003] Genome-wide association study (GWAS) is a research strategy based on linkage disequilibrium in a population. It utilizes genome-wide molecular markers to perform association analysis of whole-genome phenotypes and genotypes, locating gene loci influencing traits. It is an effective method for studying complex traits. With the development of genome sequencing technology and bioinformatics, GWAS has been applied to the study of important agronomic traits in cotton. Ma et al. (2018) performed resequencing GWAS analysis on 419 core upland cotton germplasms, identifying 7383 independent SNPs (Single Nucleotide Polymorphisms) and 4820 candidate genes significantly associated with fiber traits. Zhao et al. (2022) used 336 island cotton germplasms to discover 6241 independent SNPs associated with 15 traits, and further identified 5 candidate genes associated with disease resistance, fiber length, fiber strength, and lint percentage through gene expression analysis and VIGS (virus-induced gene silencing). Yu et al. (2021) used 240 Sea Island cotton accessions and, through GWAS analysis, identified three candidate genes related to fiber strength (GB_D11G3437, GB_D11G3460, and GB_D11G3471) and one candidate gene related to lint percentage (GB_A07G1034). These studies demonstrate that GWAS, with its high mapping accuracy and ability to map multiple traits at once, provides excellent QTLs and gene resources for cotton breeding, significantly accelerating breeding efficiency.

[0004] The WD40 protein, also known as the WD-repeat protein, is defined by a highly conserved specific WD40 motif. In plants, the WD40 gene is involved in regulating multiple biochemical and developmental pathways, including leaf epidermal hairs, root hairs, anthocyanin biosynthesis, meristem formation, seedling and flower development, etc. (Salih et al., 2018). WD40 proteins are widely distributed in eukaryotes and play important roles in various biological processes. Ma et al. (2022) showed through transcriptome analysis that GhWDR is widely expressed in various tissues and organs, and is transcribed at high levels in rapidly elongating fibers, suggesting that it may play a role in the regulation of cotton growth and development and fiber elongation. Summary of the Invention

[0005] The purpose of this invention is to provide a WD40-like family protein gene, GoWD40L, for improving cotton fiber quality and its application. Based on fiber quality phenotypic data and BLUP values ​​from two natural populations of Sea Island cotton over two years, combined with whole-genome resequencing information for each material, a 3VmrMLM model was used to perform genotype-phenotype genome-wide association analysis on five fiber quality traits, locating QTLs affecting fiber quality traits. Subsequently, important variants in the coding regions of candidate genes located within the QTLs were extracted and tested using local association analysis. An important InDel site was found in the exon region with ID number GB_D03G0092, causing an amino acid sequence change and generating two different haplotypes (haplotype 1 and haplotype 2). The GB_D03G0092 gene encodes a WD40-like family protein gene, named GoWD40L. Comparative analysis of quality traits among the different haplotypes of GoWD40L showed that haplotype 2 has significant advantages over haplotype 1 in fiber length, strength, micronaire value, uniformity, and elongation.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention seeks protection for a WD40 family protein gene GoWD40L, which has two haplotypes: haplotype 1 has a CDS sequence of SEQ ID NO.1, and haplotype 2 has a CDS sequence of SEQ ID NO.4.

[0008] Furthermore, haplotype 2 has a nucleotide deletion at 200 bp in the CDS sequence compared to haplotype 1, which causes premature termination of amino acid translation; association analysis results indicate that haplotype 2 has an advantage over haplotype 1 in terms of cotton quality traits.

[0009] Studies have shown that when the haplotype 2 gene sequence or its encoded protein is present in cotton plants, the allele or its protein can promote an increase in cotton fiber length, fiber strength, fiber uniformity, fiber elongation, and a decrease in micronaire value. This indicates that haplotype 2 of this gene plays an important role and has promising applications in improving cotton fiber quality traits and in breeding high-quality new cotton varieties.

[0010] Secondly, the present invention seeks protection for the protein encoded by the WD40 family protein gene GoWD40L, wherein the amino acid sequence of the protein encoded by haplotype 1 of the WD40 family protein gene GoWD40L is shown in SEQ ID NO.3; and the amino acid sequence of the protein encoded by haplotype 2 of the WD40 family protein gene GoWD40L is shown in SEQ ID NO.6.

[0011] Thirdly, the present invention seeks protection for haplotype 2 of the aforementioned WD40 family protein gene GoWD40L and the protein it encodes in the following (1) or (2):

[0012] (1) Improve the quality properties of cotton fibers;

[0013] (2) Application in promoting plant growth.

[0014] Furthermore, the above applications can be used to screen cotton germplasm materials carrying the WD40 family protein gene GoWD40L haplotype 2 for breeding purposes to improve cotton fiber quality traits, or to overexpress the WD40 family protein gene GoWD40L haplotype 2 in cotton.

[0015] Furthermore, in the above applications, overexpression of haplotype 2 of the WD40 family protein gene GoWD40L in Arabidopsis thaliana resulted in longer taproot length, root cell length, and hypocotyl length compared to the wild type.

[0016] Fourthly, this invention seeks to protect the use of reagents for detecting the GoWD40L sequence of WD40 family protein genes in test samples in screening or identifying cotton varieties with excellent fiber quality traits or in breeding new cotton varieties with high fiber quality.

[0017] The WD40 family protein gene GoWD40L has two haplotypes: haplotype 1 has a CDS sequence of SEQ ID NO.1, and haplotype 2 has a CDS sequence of SEQ ID NO.4. Haplotype 2 has a nucleotide deletion site at 200 bp in its CDS sequence compared to haplotype 1, which causes premature termination of amino acid translation. Association analysis results show that haplotype 2 has an advantage over haplotype 1 in terms of cotton fiber quality traits.

[0018] Furthermore, the above applications include detection at the gene level and / or protein level.

[0019] Furthermore, in the above applications, the test sample carrying the WD40 family protein gene GoWD40L haplotype 2 is a cotton germplasm with high fiber quality traits.

[0020] Fifthly, the present invention claims protection for a method for assisting in screening or identifying high fiber quality traits in cotton, which involves detecting the sequence of the aforementioned WD40 family protein gene GoWD40L in the sample to be tested, and selecting the sample to be tested carrying haplotype 2 of the WD40 family protein gene GoWD40L as cotton germplasm with high fiber quality traits.

[0021] The cotton fiber quality traits described in this invention are at least one of fiber length, specific strength, micronaire value, uniformity, and elongation.

[0022] The development process of the technical solution of this invention includes the following steps:

[0023] 1. Select natural populations of Sea Island cotton for multi-year, multi-location phenotypic data measurement and processing. Natural populations of Sea Island cotton were planted under multi-year, multi-location conditions, and fiber quality traits (fiber length, fiber strength, micronaire value, fiber uniformity, and fiber elongation) were investigated. Outliers were removed, and BLUP (Best Linear Unbiased Prediction) analysis was performed on the multi-year, multi-location phenotypic data.

[0024] 2. Next-generation resequencing (sequencing depth 10×) was performed on each germplasm in the sea island cotton population. Variation sites were detected using the sea island cotton Hai7124 genome as a reference genome to obtain genotype data. The genotype data filtering criteria were as follows: sites with a deletion rate greater than 0.1% and a minor allele frequency less than 0.05 were removed.

[0025] 3. Association analysis was performed using phenotypic and genotypic data to identify loci significantly associated with the quality traits of Sea Island cotton fibers. The CDS sequence of haplotype 1 of the WD40 family protein gene GoWD40L is SEQ ID NO.1, the genome sequence is SEQ ID NO.2, and the encoded amino acid sequence is SEQ ID NO.3. The CDS sequence of haplotype 2 is SEQ ID NO.4, the genome sequence is SEQ ID NO.5, and the encoded amino acid sequence is SEQ ID NO.6. Haplotype 2 has a nucleotide deletion at 200 bp in its CDS sequence compared to haplotype 1, resulting in premature termination of translation of the encoded amino acid. Comparative analysis of quality traits among different haplotypes of GoWD40L showed that haplotype 2 has advantages over haplotype 1 in fiber length, strength, micronaire value, uniformity, and elongation.

[0026] 4. This invention also constructed two transgenic Arabidopsis thaliana lines overexpressing different haplotypes. Compared to the wild type (WT), the transgenic Arabidopsis thaliana line overexpressing haplotype 2 exhibited longer taproot length, root cell length, and hypocotyl length, suggesting the elongation effect of this haplotype in cotton fiber development.

[0027] The advantages of this invention are as follows:

[0028] 1. This invention identified a WD40-like family protein gene, GoWD40L, significantly associated with cotton fiber quality traits through resequencing of natural cotton populations and genome-wide association analysis. Tenfold-depth resequencing of an island cotton population identified 3,780,162 SNPs and 449,380 InDels. High-quality genotypic data ensured the accuracy of the association analysis loci.

[0029] 2. The expression levels of GoWD40L in different tissues and organs of cotton were obtained through transcriptomic analysis. The gene was predominantly expressed at days 3, 5, and 10 of fiber development, indicating a close relationship between this gene and fiber quality traits.

[0030] 3. Based on the different InDel genotypes of GoWD40L, the population can be divided into two categories: haplotype 1 (C) and haplotype 2 (-). Statistical analysis revealed significant differences in fiber length, fiber strength, micronaire value, fiber uniformity, and fiber elongation among the populations corresponding to different haplotypes, further demonstrating the correlation between this gene and cotton fiber quality traits.

[0031] 4. Compared to the wild type (WT), the two overexpressed transgenic Arabidopsis lines of GoWD40L showed different differences. The transgenic Arabidopsis line of haplotype 2 showed longer taproot length, root cell length and hypocotyl length. Attached Figure Description

[0032] Figure 1 GWAS Manhattan plot of micronaire value (Mic) and fiber elongation (FE) for Sea Island cotton.

[0033] The horizontal axis represents chromosomes. The vertical axis represents the significance of SNP locus associations, expressed as -log. 10 (P-value) represents the threshold value. The dashed line represents the threshold value of LOD = 3.0.

[0034] Figure 2 CDS sequence alignment information of the cotton GoWD40L gene.

[0035] An InDel site was identified in the varietal population. This site is located in the coding region of GB_D03G0092, and the gene encodes a WD40 family protein gene. Haplotype analysis revealed that there are two haplotypes of this gene. Haplotype 2 has a deletion of a nucleotide (C) at 200 bp in the CDS sequence compared to haplotype 1, which causes premature termination of translation of the encoded amino acid.

[0036] Figure 3 Comparative analysis of the differences in fiber quality traits among different haplotypes of GoWD40L.

[0037] The box plot represents the distribution of quality traits in the natural population of Sea Island cotton. The horizontal axis represents different haplotype materials, and the vertical axis represents the corresponding quality trait values, namely fiber length, fiber strength, micronaire value, fiber uniformity, and fiber elongation. The number of materials containing haplotype 1 (C) and haplotype 2 (-) are 186 and 59, respectively. *** indicates a difference at the 0.001 level, and **** indicates a difference at the 0.0001 level.

[0038] Figure 4 Expression levels of .GoWD40L in different tissues and developmental stages of sea island cotton Hai7124.

[0039] The vertical axis represents gene expression levels (expressed as the average expression level (TPM) of a single gene per million transcripts). The horizontal axis represents different tissues, including roots, stems, leaves, floral organs, ovules, and fibers. Floral organ tissues include bracts, calyxes, receptacles, petals, anthers, and filaments; ovule tissues include -3, -1, 0, 1, 3, 5, 10, 20, and 25 DPA; and fiber tissues include 10, 20, and 25 DPA.

[0040] Figure 5 Subcellular localization of different haplotypes of GoWD40L in tobacco epidermal cells.

[0041] AtHTB2 is used as a nuclear marker, and AtPIP2 is used as a plasma membrane marker.

[0042] Figure 6 Phenotypic investigation of different haplotype transgenic Arabidopsis thaliana lines of GoWD40L.

[0043] Phenotypic analysis of taproot length in a, b, g, h WT and transgenic seedlings after 8 days of growth under long-day conditions. Scale bar shown in 1 cm. At least five biological replicates were used for each determination of mean taproot length. confocal images and root cell length analysis of root cells from WT and transgenic seedlings after 8 days of growth under long-day conditions (c, d, i, j). These images show the same region in the middle of the taproot. Scale bar shown in 50 μm. Three biological replicates were selected for each determination, and five cell lengths were measured in each replicate. Phenotypic analysis of hypocotyl length in e, f, k, l WT and transgenic seedlings after 8 days of growth under dark conditions. Scale bar shown in 1 cm. At least five biological replicates were used for each determination of mean hypocotyl length. Detailed Implementation

[0044] 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.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0046] Example 1: Mining of the GoWD40L gene, a WD40-like family protein associated with cotton fiber quality traits.

[0047] Whole-genome resequencing was performed on 269 Sea Island cotton germplasms from different regions of China and major cotton-producing countries worldwide. After standard variant detection and genotype filtering, a total of 3,780,162 high-quality SNPs and 449,380 InDels (≤50bp) were detected. Simultaneously, the population materials were planted at two different locations, Korla (Loc1) and Aksu (Loc2), for two consecutive years (2017 and 2018), and a detailed investigation of five fiber quality traits was conducted for each material. Based on the phenotypic data and BLUP values ​​from the two years and two locations, GWAS analysis was performed on the five fiber quality traits using a 3VmrMLM model, locating a QTL (D03:279866-1725076) on the D03 chromosome. Figure 1 An important InDel site was discovered located in the exon region of GB_D03G0092 (Table 1), which produces two different types, haplotype 1 (GB_D03G0092). B ) and haplotype 2 (GB_D03G0092) H ()( Figure 2 Haplotype 2 has a nucleotide deletion at 200bp (D03:662641) in the CDS sequence compared to haplotype 1, resulting in premature termination of the encoded amino acid.

[0048] Table 1. Mining of the WD40-like family protein gene GoWD40L associated with cotton fiber quality traits.

[0049]

[0050]

[0051] Example 2: Phenotypic Differences of Different Haplotypes of the WD40 Family Protein Gene GoWD40L

[0052] Using the t-test statistical method, we calculated the phenotypic differences in fiber quality traits between the two haplotype groups. Haplotype 2(-) showed significantly better fiber length, fiber strength, micronaire value, fiber uniformity, and fiber elongation than haplotype 1(C) Sea Island cotton. Figure 3 ).

[0053] Example 3: Analysis of GoWD40L expression levels in different tissues and developmental stages of cotton.

[0054] This invention collected transcriptome data from different tissues and developmental stages of the cotton variety Hai7124 from NCBI. Sample materials included roots, stems, leaves, floral organs, ovules, and fibers. Floral organ tissues included bracts, calyx, receptacle, petals, anthers, and filaments; ovule tissues included -3, -1, 0, 1, 3, 5, 10, 20, and 25 DPA; fiber tissues included 10, 20, and 25 DPA. Gene expression levels were calculated by comparing the sequenced reads with the Sea Island cotton genome, and the calculated expression level was expressed as the average expression level (TPM) of a single gene per million transcripts. Experimental results are as follows: Figure 4 As shown, this gene is predominantly expressed at 3, 5, and 10 DPA during fiber development, indicating its correlation with fiber quality traits. Figure 4 ).

[0055] Example 4: Subcellular localization analysis of two haplotypes of the GoWD40L gene

[0056] To verify the subcellular localization of the two haplotypes of GoWD40, we constructed the pBin-eGFP4 vector containing the target gene, which was fused with the target gene and eGFP protein (35S-target gene-eGFP-NOS) under the control of the 35S promoter of cauliflower mosaic virus (CaMV) (Liu et al. 2014. Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis. Nat Commun. 5:4686). The 35S-target gene-eGFP-NOS vector was injected into 5-week-old tobacco leaves using Agrobacterium. Three days after injection, fluorescence signals were detected and captured under a Zeiss LSM780 confocal microscope. Subcellular localization results showed haplotype 1 (GB_D03G0092). B ) is localized to the cell nucleus and plasma membrane, while haplotype 2 (GB_D03G0092) H It is only located in the plasma membrane. Figure 5 ).

[0057] Example 5: Functional analysis of two haplotypes of the GoWD40L gene

[0058] To further elucidate the functions of the two haplotypes of GoWD40L, different overexpression transgenic Arabidopsis lines were constructed: haplotype 1 (A4-1, A4-2, A4-3, A4-4, A4-5) and haplotype 2 (B4-1, B4-2, B4-4, B4-5). We selected three transgenic lines from each haplotype for phenotypic studies. Surface-sterilized seeds were sown in rows on 1 / 2 MS medium. One group of experiments was conducted under long-day conditions (24 / 22℃, 16h light / 8h dark) with vertical placement in a growth chamber; the other group was conducted under dark conditions (23℃) with vertical placement in a growth chamber. After 8 days, the taproot length and hypocotyl length of the wild-type (WT) and transgenic lines were measured. In addition, primary root cells were stained with propidium iodide (PI) and observed using a laser scanning confocal microscope (LSM780, Zeiss, Germany), and cell lengths of wild-type (WT) and transgenic lines were measured. Compared with wild-type (WT), haplotype 2 overexpression transgenic lines in Arabidopsis showed longer primary root and root cell lengths after 8 days of light culture and longer hypocotyl lengths after 8 days of dark culture, while haplotype 1 overexpression showed shorter primary root, root cell, and hypocotyl lengths. Figure 6 ).

[0059] The above results indicate that, based on cotton natural population resequencing and genome-wide association analysis, we identified a WD40-like protein gene, GoWD40L, which is significantly associated with cotton fiber length, strength, micronaire value, uniformity, and elongation. The GoWD40L gene is predominantly expressed during fiber development. Subcellular localization of the two haplotypes shows they are located in different positions. Transgenic experiments with different GoWD40L haplotypes in Arabidopsis thaliana revealed that the dominant haplotype 2 exhibited longer taproot length, root cell length, and hypocotyl length compared to the wild type (WT), suggesting its elongation role in cotton fiber development. Therefore, GoWD40L plays an important role and has promising applications in improving cotton quality traits and breeding high-quality cotton varieties.

Claims

1. A haplotype 2 of a WD40 family-like protein gene GoWD40L and the encoded protein thereof for use in (1) or (2) below: (1) improving fiber quality traits in cotton; (2) use in promoting primary root length, root cell length and hypocotyl length in Arabidopsis thaliana; The WD40 family protein gene GoWD40L The CDS sequence of haplotype 2 is shown in SEQ ID NO.

4.

2. Use according to claim 1, characterized in that, The WD40-like family protein gene GoWD40L The amino acid sequence of the protein encoded by haplotype 2 of the gene is shown as SEQ ID NO.

6.

3. Use according to claim 1, characterized in that, By screening for cotton germplasm materials carrying WD40-like family protein genes GoWD40L Cotton germplasm materials of haplotype 2, improved cotton fiber quality traits, or overexpression of WD40-like family protein genes in cotton GoWD40L Haplotype 2.

4. The use according to claim 1, characterized in that, Overexpression of the WD40-like family protein gene in Arabidopsis thaliana GoWD40L Haplotype 2, Arabidopsis thaliana showed longer primary root length, root cell length and hypocotyl length than wild type.

5. A reagent for detecting a WD40-like family protein gene in a sample to be tested GoWD40L The application of the sequence in screening or identifying cotton varieties with excellent fiber quality traits or breeding new cotton varieties with high fiber quality. The WD40-like family protein gene GoWD40L There are two haplotypes, the CDS sequence of haplotype 1 is SEQ ID NO. 1; the CDS sequence of haplotype 2 is SEQ ID NO. 4; haplotype 2 has a deletion site of one nucleotide at 200 bp of the CDS sequence compared with haplotype 1, and causes the premature termination of amino acid translation; the result of association analysis shows that haplotype 2 has an advantage over haplotype 1 in the fiber quality traits of cotton.

6. Use according to claim 5, characterized in that, including detection at the genetic level or / and protein level.

7. Use according to claim 5, characterized in that, Carrying wd40 family protein gene GoWD40L The sample to be tested of haplotype 2 is a cotton germplasm with high fiber quality traits.

8. A method of assisting in the selection or identification of high fiber quality traits in cotton, comprising, Detecting sequence of WD40-like family protein gene in sample to be tested GoWD40L Selecting sample to be tested carrying haplotype 2 of WD40-like family protein gene GoWD40L The sample to be tested carrying haplotype 2 of WD40-like family protein gene is a cotton germplasm with high fiber quality trait. The WD40-like family protein gene GoWD40L There are two haplotypes, the CDS sequence of haplotype 1 is SEQ ID NO. 1; the CDS sequence of haplotype 2 is SEQ ID NO. 4; haplotype 2 has a deletion site of one nucleotide at 200 bp of the CDS sequence compared with haplotype 1, and causes the premature termination of amino acid translation; the result of association analysis shows that haplotype 2 has an advantage over haplotype 1 in the fiber quality traits of cotton.

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