A cell wall-associated kinase gene associated with cotton fiber length and its application

Abstract

The application relates to a cell wall associated kinase gene associated with cotton fiber length and application thereof, and belongs to the field of biotechnology and crop genetic breeding. The gene is GhWAK, the cDNA sequence of which is shown as SEQ ID NO. 1, the protein sequence of the coded protein is shown as SEQ ID NO. 2, and the corresponding genomic DNA sequence is shown as SEQ ID NO. 3. The gene GhWAK is regulated by a SNP site (A09:4676444) on the cDNA sequence, and after the base of the site is changed from adenine (A) to thymine (T), the cotton material carrying the mutation genotype shows significant fiber length improvement. Based on the above finding, the gene GhWAK can be used to establish an efficient molecular marker assisted selection system, and provides important technical support for identification, genetic improvement and excellent new variety breeding of high fiber length cotton germplasm, and has wide scientific research and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and crop genetics and breeding, specifically relating to a cell wall-associated kinase gene associated with cotton fiber length and its application. Background Technology

[0002] Cotton (Gossypium spp.) is an important economic crop worldwide, and its natural fibers are a core raw material for the textile industry. Among the various indicators for measuring cotton fiber quality, fiber length plays a crucial role. It not only directly affects the strength and uniformity of yarn but is also one of the core factors determining the quality of the final textiles. This trait is a typical quantitative trait controlled by multiple genes, with a complex genetic mechanism, making improvement through conventional breeding methods difficult and time-consuming. Therefore, identifying the key genes controlling fiber length and analyzing their functions is of great significance for promoting molecular design breeding of cotton and improving fiber quality.

[0003] In the process of elucidating the genetic basis of quantitative traits, QTL mapping is a commonly used research method. This method typically utilizes segregating populations (such as F2 populations or recombinant inbred lines) constructed from parental crosses. By building genetic maps and analyzing the association between markers and traits, the chromosomal segments controlling the traits are preliminarily identified. However, due to the limited number of recombination events within the population, traditional QTL mapping results often suffer from excessively large intervals and a large number of genes, making subsequent fine cloning and functional verification of genes extremely difficult. In contrast, genome-wide association studies (GWAS) utilize the vast genetic variation accumulated by natural populations over long-term evolution and historically frequent recombination events, enabling more precise identification of genetic loci significantly associated with target traits, providing a more effective approach for discovering key genes.

[0004] In recent years, with the improvement of the cotton genome map and the popularization of SNP chips and resequencing technology, genome-wide association analysis (GWAS) has played an increasingly important role in studying the genetic basis of cotton fiber quality and has been successfully applied to the identification of a large number of key genes. Taking the study of Ma et al. (2018) as an example, the team performed whole-genome resequencing on 419 representative cotton germplasms and constructed a genotype dataset containing approximately 3.66 million single nucleotide polymorphisms. Through association analysis, they identified several previously unreported genes, which regulate multiple traits such as flowering days, fiber length, and fiber strength (Ma Z, He S, Wang X, et al. Resequencing a core collection of uplandcotton identifies genomic variation and loci influencing fiber quality and yield[J]. Nature Genetics, 2018, 50(6): 803-813.). Liu et al. (2020) identified 42 SNPs and 31 QTLs significantly associated with fiber quality traits in 276 diverse upland cotton germplasms from different ecological regions. RNA-seq and RT-qPCR analyses identified Gh_D09G2376 and Gh_D06G1908 as candidate genes controlling fiber quality traits. (Liu W, Song C, Ren Z, et al. Genome-wide association study reveals the genetic basis of fiber quality traits in upland cotton) Gossypium hirsutum L.)[J]. BMC Plant Biology, 2020, 20(1): 395.). These studies collectively demonstrate that genome-wide association analysis (GWAS) can effectively overcome the inherent limitations of traditional quantitative trait locus (QTL) mapping methods, directly linking to genomic loci and even candidate genes with breeding value at a higher resolution. It is a key technology for achieving the leap from "locating intervals" to "discovering genes".

[0005] Wall-associated kinase family proteins (WAKs) belong to the plant receptor kinase family. These proteins are located in the cell membrane, with extracellular domains that can bind to cell wall pectin components and intracellular segments containing serine / threonine kinase domains. They are important molecular hubs mediating cell wall-cytoplasmic signaling communication (Kohorn BD, Kohorn SL. The cell wall-associated kinases, WAKs, as pectin receptors[J]. Frontiers in Plant Science, 2012, 3: 88.). Existing research has confirmed that WAK family members are widely involved in plant cell elongation, cell wall remodeling, hormone signal transduction, and biotic and abiotic stress responses (Wagner TA, Kohorn BD. Wall-associated kinases are expressed throughout plant development and are required for cell expansion[J]. The Plant Cell, 2001, 13(2): 303-318.). In cotton, some WAK family genes have been reported to participate in the regulation of Verticillium wilt resistance and salt tolerance (Zhang Z, Ma W, Ren Z, et al. Characterization and expression analysis of wall-associated kinase (WAK) and WAK-like family in cotton[J]. International Journal of Biological Macromolecules, 2021, 187: 867-879.); transcriptome data also show that multiple WAK genes are predominantly expressed during the initiation and elongation stages of cotton fibers, suggesting that members of this family may be involved in the development of cotton fibers (Dou L, Wang S, Liu X, et al. Genome-wide characterization of the WAK genefamily and expression analysis under plant hormone treatment in cotton[J]. BMC Genomics, 2021, 22(1): 85.).

[0006] Although some studies have provided preliminary insights into the evolutionary characteristics of the cotton WAK family and the stress resistance functions of some members, existing technologies still have significant shortcomings: First, most studies focus on elucidating the function of WAK genes in cotton's stress response, with very little attention paid to the regulation of agronomic traits related to cotton fiber quality by WAK family genes; second, existing publicly available technologies have not yet identified specific WAK functional genes that can stably regulate upland cotton fiber length, and have failed to discover natural allelic variations that are significantly associated with fiber length traits; third, there is a lack of specific molecular markers developed based on this WAK gene, making it difficult to directly apply them to high-throughput screening and molecular breeding of superior long-fiber cotton germplasm.

[0007] Therefore, identifying the key WAK genes that regulate fiber length in upland cotton, analyzing the regulatory effect of allelic variations on fiber length phenotype, and developing molecular markers suitable for breeding selection are of significant theoretical value and promising prospects for the cultivation of high-quality long-staple cotton varieties. Summary of the Invention

[0008] To address the aforementioned problems, this invention aims to provide a cell wall-associated kinase gene related to cotton fiber length and its applications. The cell wall-associated kinase gene is GhWAK, which was identified based on resequencing and genome-wide association analysis of a cotton population.

[0009] The objective of this invention can be achieved through the following technical solutions:

[0010] On the one hand, the present invention provides an application of a reagent for detecting SNP molecular markers in the identification or screening of cotton varieties with high fiber length, wherein the SNP molecular marker is located at base A09:4676444 in the upland cotton reference genome TM-1 V2.1.

[0011] Furthermore, the population was divided into two haplotypes based on the SNP genotype in the variety population: the SNP site base of low fiber length cotton material is A; the SNP site base of high fiber length cotton material is T, corresponding to the genotypes TT, TA, and AA. Among them, cotton with the TT genotype is a high fiber length cotton variety.

[0012] Therefore, the aforementioned SNP sites can serve as markers for rapid identification of cotton varieties with high fiber length. The base type of this SNP in upland cotton plants is detected; cotton with base A is a low-fiber-length cotton plant, while cotton with base T is a high-fiber-length cotton plant.

[0013] Furthermore, the SNP molecular marker is located at nucleotide 950 of the GhWAK gene cDNA sequence, the cDNA sequence of the GhWAK gene is shown in SEQ ID NO.1, the encoded protein sequence is shown in SEQ ID NO.2, and the corresponding genomic DNA sequence is shown in SEQ ID NO.3. The GhWAK gene is regulated by an internal SNP (A09:4676444), and a mutation from A to T at this SNP site significantly increases cotton fiber length.

[0014] Furthermore, the reagent for detecting SNP molecular markers includes an upstream primer as shown in SEQ ID NO.6 and a downstream primer as shown in SEQ ID NO.7.

[0015] On the other hand, the present invention provides a method for cultivating high fiber length cotton, wherein the gene GhWAK carrying the T allele is introduced into recipient cotton material through genetic transformation. The T allele refers to the gene whose 950th nucleotide of the GhWAK gene cDNA sequence is T, and the cDNA sequence of the GhWAK gene is shown in SEQ ID NO.1.

[0016] On the other hand, the present invention provides a method for increasing the length of cotton fibers by using gene editing technology to mutate nucleotide A to T at position 950 of the cDNA sequence of the GhWAK gene, the cDNA sequence of which is shown in SEQ ID NO.1.

[0017] On the other hand, the present invention provides a kit for identifying or screening high fiber length cotton, comprising an upstream primer as shown in SEQ ID NO. 6 and a downstream primer as shown in SEQ ID NO. 7.

[0018] Furthermore, the kit also includes DNA polymerase, dNTPs, and buffer.

[0019] The beneficial effects of this invention are as follows:

[0020] (1) In view of the complex genome structure of allotetraploid cotton, this invention relies on a high-quality reference genome and combines population resequencing and genome-wide association analysis to successfully identify the key gene GhWAK, which is closely related to fiber length. This method has been widely used in gene mining of various crops and has a reliable technical basis.

[0021] (2) The genome-wide association analysis confirmed that the gene GhWAK is significantly associated with cotton fiber length, indicating that it plays an important role in fiber development regulation.

[0022] (3) The cDNA and genomic sequence of the GhWAK gene can be obtained by PCR technology. This technology has the advantages of high specificity, high sensitivity, speed and simplicity, and strong repeatability, which can ensure the accuracy of the sequence.

[0023] (4) Using PCR technology, this invention verified the SNP genotype of the GhWAK gene in varieties with relatively high and low fiber lengths. It is fast, simple, accurate and reliable.

[0024] (5) Based on the different SNP genotypes of the GhWAK gene, the varietal population can be divided into two major categories, and these two groups show significant differences in fiber length. This result further confirms the close association between the GhWAK gene and cotton fiber length. Attached Figure Description

[0025] Figure 1 The figure shows the results of a fiber length trait association analysis conducted using whole-genome sequencing data based on cotton populations in Xinjiang. The horizontal axis represents the physical location of each chromosome (unit: Mb), and the vertical axis represents the significance level of the association between SNP loci and fiber length, expressed as -log 10 Presented in (P-value) format, the horizontal line represents the significance threshold for association analysis (-log). 10 (Pvalue)=5), the arrow indicates the genomic location of the SNP site that is significantly associated with fiber length.

[0026] Figure 2 To divide the population into two haplotype groups based on different SNP genotypes of the GhWAK gene, box plots were used to reflect the differences in fiber length between the two haplotype groups using a t-test. The number of materials containing the AA and TT genotypes were 252 and 85, respectively. The box plot on the left represents the fiber length distribution of haplotype AA, and the box plot on the right represents the phenotypic distribution of haplotype TT. The horizontal line in the box represents the median of the fiber length distribution. *** indicates that the difference between the two groups is statistically significant at the P < 0.001 level. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art can make similar extensions without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] The high fiber length cotton described in this invention is for carrying GhWAKTT genotype cotton, whose cDNA sequence has a nucleotide T at position 950, has a significantly higher fiber length (average length of the upper half) than AA genotype cotton. Specifically, high fiber length cotton refers to TT genotype cotton with a fiber length of not less than 30.0 mm.

[0029] (1) Identification process of the gene GhWAK

[0030] This embodiment systematically determined the cotton fiber length trait in 370 modern cotton varieties (lines) at an experimental base in Xinjiang. Simultaneously, whole-genome resequencing was performed on all 370 materials, with an average sequencing depth of 20×. The obtained sequencing data were compared with the upland cotton reference genome TM-1 V2.1. Single nucleotide variants (SNPs) were identified using GATK software, and genome-wide association analysis was performed using EMMAX software, with a p-value of <10. -5 Screening for SNP-associated signal sites identified a site significantly associated with cotton fiber length, namely nucleotide 4676444 on chromosome A09 (A09:4676444). Figure 1 (As indicated by the arrow). This SNP is located at nucleotide 950 of the cDNA sequence of the cell wall-associated kinase gene GhWAK, and it is speculated that it may be involved in regulating the elongation process of cotton fibers. The cDNA sequence, encoding amino acid sequence, and genomic sequence of the gene GhWAK are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0031] (2) Cloning and verification of the GhWAK gene

[0032] Based on the annotated genomic sequence information, specific primers were designed to amplify the complete coding region of the GhWAK gene. The primers used included upstream primer F1 as shown in SEQ ID NO.4 and downstream primer R1 as shown in SEQ ID NO.5 (see Table 1 for details). The amplification reaction program was set as follows: 95℃ pre-denaturation for 3 minutes; followed by 34 cycles of 95℃ denaturation for 15 seconds, 57℃ annealing for 15 seconds, and 72℃ extension for 45 seconds; and a final extension at 72℃ for 5 minutes. The obtained PCR products were sequenced, and the sequencing results were compared with the expected cDNA sequence to confirm sequence accuracy, thus completing the acquisition of the gene.

[0033] Table 1. Primer sequences for PCR amplification

[0034] (3) Application of the GhWAK gene in identifying high fiber length cotton:

[0035] Specific amplification primers F2 (SEQ ID NO. 6) and R2 (SEQ ID NO. 7) were designed for the SNP locus (A09:4676444) on chromosome A09 of the upland cotton reference genome TM-1 V2.1 and its flanking genomic sequences (200 bp upstream and downstream of the SNP locus), as shown in SEQ ID NO. 8 (see Table 2 for details). Genomic DNA from 370 cotton materials was amplified by PCR using the aforementioned specific amplification primers F2 and R2. The reaction program was as follows: 94℃ pre-denaturation for 2 minutes; 98℃ denaturation for 10 seconds, 55℃ annealing for 5 seconds, and 68℃ extension for 10 seconds, for a total of 35 cycles; and a final extension at 68℃ for 5 minutes. The amplified products were sequenced and used for genotyping, thereby achieving effective identification of cotton with different fiber lengths.

[0036] Table 2 PCR amplification primer sequences

[0037] Genotypes of each variety population at this SNP locus were analyzed based on sequencing peak diagrams or sequence alignment results. The AA type was identified as a homozygous genotype with shorter fiber length, while the TT type was identified as a homozygous mutant genotype with longer fiber length. The results are shown in Table 3. There were 252 AA type haplotypes, 85 TT type haplotypes, and 33 heterozygous AT haplotypes (not shown in the table). Statistical analysis of fiber length traits between the two haplotypes was performed using a t-test, and the results are as follows: Figure 2 The results showed that the fiber length of the TT genotype population was significantly better than that of the AA genotype population (P < 0.001), indicating that this locus is highly significantly associated with the fiber length trait.

[0038] Table 3. Identification results of high fiber length and low fiber length genotypes in population varietal materials.

[0039] The above results indicate that the GhWAK gene has significant application potential in cotton fiber length improvement and new variety breeding. Specifically: First, molecular markers developed based on different haplotypes of this gene can provide efficient identification tools for early selection of cotton fiber length traits, significantly improving breeding selection efficiency. Second, in breeding practice, the superior allele GhWAK(T) can be introduced into recipient materials through transgenic technology, or the target site in AA-type materials can be precisely modified to TT-type using gene editing technology, thereby directionally improving fiber length traits. These two strategies provide new technical pathways for breeding high-yield and high-quality cotton varieties.

[0040] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of a reagent for detecting SNP molecular markers in the identification or screening of high-fiber-length cotton varieties, characterized in that, The SNP molecular marker is located at base A09:4676444 of the upland cotton reference genome TM-1 V2.1, with polymorphisms of T or A, corresponding to genotypes TT, TA, and AA. Among them, cotton with the TT genotype is a cotton variety with high fiber length.

2. The application according to claim 1, characterized in that, The SNP molecular marker is located at nucleotide 950 of the GhWAK gene cDNA sequence, and the GhWAK gene cDNA sequence is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The reagents for detecting SNP molecular markers include an upstream primer as shown in SEQ ID NO. 6 and a downstream primer as shown in SEQ ID NO.

7.

4. A method for cultivating high-fiber-length cotton, characterized in that, The GhWAK gene carrying the T allele was introduced into recipient cotton material through genetic transformation. The T allele refers to the gene whose 950th nucleotide of the GhWAK gene cDNA sequence is T. The cDNA sequence of the GhWAK gene is shown in SEQ ID NO.

1.

5. A method for increasing cotton fiber length, characterized in that, Using gene editing technology, nucleotide A at position 950 of the cDNA sequence of the GhWAK gene was mutated to T. The cDNA sequence of the GhWAK gene is shown in SEQ ID NO.

1.

6. A reagent kit for identifying or screening cotton with high fiber length, characterized in that, It contains an upstream primer as shown in SEQ ID NO.6 and a downstream primer as shown in SEQ ID NO.

7.

7. The reagent kit according to claim 6, characterized in that, It also includes DNA polymerase, dNTPs, and buffer solutions.

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