An arginyl-tRNA-protein transferase gene for improving upland cotton fiber length and its application

Abstract

This invention relates to an arginine-tRNA-protein transferase gene for improving upland cotton fiber length and its application. Through population resequencing and genome-wide association analysis (GWAS) of upland cotton varieties, this invention identified an arginine-tRNA-protein transferase gene, ATE, associated with upland cotton fiber length. GWAS results show that this gene is closely related to fiber length. The presence of genotype A at the 1283 bp site of the arginine-tRNA-protein transferase gene indicates that the cotton plant is of high fiber length.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to an arginyl-tRNA-protein transferase gene for improving the fiber length of upland cotton and its application. Background Technology

[0002] Cotton (Gossypium spp.), as one of my country's key economic crops, has a significant impact on agriculture and national economic development. Cotton fiber originates from a single cell differentiated from the epidermal cells of the ovule's outer integument. Its development goes through four stages: initial differentiation, rapid elongation, secondary cell wall synthesis, and maturation and dehydration, ultimately determining the fiber's quality. As a core raw material for the textile industry, the quality of cotton fiber, especially fiber length, plays a decisive role in spinning processes, yarn properties, and the texture, luster, and durability of the final textiles. Although my country ranks first globally in cotton production, consumption, textile production, and exports, the quality of domestically produced cotton cannot fully meet the diversified needs of the textile industry, leading some cotton textile enterprises to rely on imported cotton to produce high-grade cotton yarn. Therefore, improving the length and quality of domestically produced cotton fiber is crucial for ensuring the supply of raw materials for the textile industry and enhancing international competitiveness. Discovering superior genetic variations related to cotton fiber length and breeding high-quality cotton varieties is of great significance for improving the competitiveness and economic benefits of my country's cotton industry, providing a clear direction and research value for cotton molecular breeding.

[0003] Early research on cotton quality traits primarily focused on quantitative trait locus (QTL) analysis. Genome-wide association analysis (GWAS) is a high-throughput method that utilizes whole-genome resequencing technology to identify molecular markers (such as SNPs and CNVs) in genetically diverse populations and combines this with phenotypic data for association analysis. This method uses statistical methods to determine the association between mutation sites and traits, rapidly locating chromosomal segments or gene loci affecting phenotypic phenotypes. With advancements in sequencing technology and computational methods, GWAS has become an important tool for identifying natural variations in complex traits in crops. It can mine genes associated with agronomic traits on a large scale, without the need for pre-selected candidate genes, and boasts high detection capabilities and accuracy, making it a focal point of molecular breeding. GWAS has been successfully applied in various crops, including rice, mustard, soybean, and cotton, with significant results.

[0004] Li et al. (2024) studied the phenotypic variation of leaf drop traits in 383 upland cotton (Gossypium hirsutum L.) germplasms and conducted genome-wide association studies (GWAS), linkage disequilibrium (LD) genotyping, and functional identification. The study found that defoliants significantly increased leaf drop rate with little impact on yield and fiber quality. 174 significant SNPs were identified, and two loci significantly associated with relative leaf drop rate (RDR7 on chromosome A02 and RDR13 on chromosome A13) were located. Key candidate genes GhLRR and GhCYCD3;1 were also identified. The study found that the combination of two favorable haplotypes increased sensitivity to defoliants, and the frequency of these favorable haplotypes generally increased in high-latitude regions of China, reflecting adaptation to the local environment. Tian et al. (2024) identified a major site (PH1) controlling plant height in upland cotton (Gossypium hirsutum) using a genome-wide association study (GWAS). This site encodes gibberellin 2-oxidase 1A (GhPH1), and a related structural variant (PAVPH1) was also discovered. The study revealed that the GhGARF transcription factor recognizes and binds to specific sequences in the GhPH1 promoter and PAVPH1, thereby downregulating GhPH1 expression. Furthermore, a direct interaction between DWARF53 (D53) and GhGARF was found, inhibiting its binding to the target site. These findings reveal a novel gibberellin-brassicin cross-regulatory mechanism that regulates upland cotton plant height through the GhD53-GhGARF-GhPH1 / PAVPH1 module, providing valuable insights for breeding semi-dwarf cotton varieties. Zhao et al. (2021) resequencing 336 Gossypium barbadense accessions, identifying 16 million SNPs, and conducted a genome-wide association study (GWAS) based on 15 agronomic traits, including fiber quality, yield, disease resistance, maturity, and plant structure. The study revealed two main gene pools and one mixed subpopulation, as well as the results of geographical dispersal and hybridization. Through gene expression analysis and VIGS transgenic experiments, five candidate genes regulating four key traits were identified: disease resistance, fiber length, fiber strength, and seed cotton percentage. The study also demonstrated the geographical and temporal considerations for the selection and improvement of Gossypium barbadense in China, and how pedigree selection breeding increased resistance to Fusarium wilt and improved fiber quality and yield. Li et al. (2020) used an automated phenotyping platform (APP) to systematically evaluate 119 image-based digital traits (i-traits) of 200 representative upland cotton varieties under drought stress at the seedling stage. 390 genetic loci were identified through genome-wide association studies (GWAS) that combined 56 morphological and 63 texture i-traits.The study identified candidate genes associated with drought response, including GhRD2, GhNAC4, GhHAT22, and GhDREB2. Further analysis revealed that Gh_A04G0377 and Gh_A04G0378 act as negative regulators in cotton drought response. Combining digital phenotype, GWAS analysis, and transcriptome data, the study concluded that the phenotypic dataset provides an excellent resource for characterizing key genetic loci, which can inform future genome-based improvement of cotton drought resistance. Du et al. (2018) resequencing 243 *Gossypium* accessions (including *Gossypium chinense* and *Gossypium herbaceum*) to generate genomic variation maps and found that they were similarly differentiated from *Gossypium raymondii*. Through a genome-wide association study (GWAS) of *Gossypium chinense*, 98 significant association peaks associated with 11 important agronomic traits were identified, including nonsynonymous mutations in the GaKASIII gene regulating fatty acid composition and activation of the GaGSTF9 gene, which is associated with cotton Verticillium wilt resistance. These findings provide an important step towards understanding the evolution of the cotton A genome and improving key agronomic traits for cotton fiber production. These results clearly demonstrate that genome-wide association analysis (GWAS) has high mapping accuracy, down to the individual gene level. By utilizing the functional markers associated with target traits, effective screening for these traits can be achieved, significantly accelerating the breeding process and improving breeding efficiency, thus promoting the rapid development of precision crop breeding. This method provides new strategies and tools for crop improvement and has significant application prospects.

[0005] Arginine-tRNA-protein transferases are key enzymes involved in post-translational modifications of proteins, transferring arginine from arg-tRNA to the N-terminus of the acceptor protein. This process is crucial for protein degradation and various intracellular metabolic pathways. These transferases typically contain one or more additional domains, particularly a C-terminal domain, which may be involved in regulating enzyme activity and interacting with other proteins (Shrader TE et al., 1993). Arabidopsis thaliana ATE1 (AT5G05700) possesses an arginine-tRNA-protein transferase (PF04377) domain and is believed to encode an arginyl-tRNA-protein transferase that plays a crucial role in the N-end rule pathway of proteins. This gene is highly expressed in the roots and leaves of Arabidopsis thaliana, and its loss of function leads to various growth defects, such as abnormal root development and inhibited seed germination (Zhang et al., 2015; Willi Riber et al., 2015). Furthermore, mutations in the ATE1 gene delay leaf senescence, indicating its important role in plant senescence regulation (Yoshida S et al., 2002). In bryophytes (Physcomitrella patens), deletion of the ATE1 gene results in severe developmental defects, affecting plant morphogenesis and physiological functions (Schuessele C et al., 2016). ATE1 has a significant impact on seed germination and seedling growth in wheat (Shao et al., 2017). Therefore, we hypothesize that the homolog of AT5G05700 in cotton may be involved in cotton fiber development. Further research into the function and molecular mechanisms of ATEs in cotton fiber development may provide theoretical support for improving cotton fiber length and advance cotton breeding. Summary of the Invention

[0006] The purpose of this invention is to identify an arginine-tRNA-protein transferase gene (ATE) associated with upland cotton fiber length through population resequencing and genome-wide association analysis. Genome-wide association analysis results indicate that this gene is closely related to the fiber length trait.

[0007] Specifically, an arginyl-tRNA-protein transferase gene for improving the fiber length of upland cotton, the sequence of which is shown in SEQ ID NO.2.

[0008] And the application of the aforementioned arginyl-tRNA-protein transferase gene for improving upland cotton fiber length in the identification of high fiber length upland cotton.

[0009] The aforementioned application of the arginyl-tRNA-protein transferase gene for improving upland cotton fiber length in improving the upland cotton fiber length trait.

[0010] The aforementioned arginyl-tRNA-protein transferase gene for improving upland cotton fiber length is applied in the breeding of new upland cotton varieties with high fiber length through genetic engineering.

[0011] A method for screening upland cotton varieties with high fiber length involves detecting the genotype at the 1283bp position of the arginyl-tRNA-protein transferase gene in upland cotton. Upland cotton with genotype A is considered to be high fiber length upland cotton.

[0012] The primers for genotype detection are as follows: the upstream primer is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6.

[0013] The advantages of this invention are as follows:

[0014] (1) The genome of allotetraploid cultivated cotton is relatively complex, and research on the exploration and breeding of upland cotton resources is not in-depth. Based on high-quality upland cotton genome sequences, this invention uses population genome resequencing and genome-wide association analysis (GWAs) to identify the gene GhATE, which is closely associated with the fiber length trait of upland cotton. The arginyl-tRNA-protein transferase gene GhATE of this invention was identified in the GWAs analysis (…). Figure 1 Analysis of expression levels in different tissues and developmental stages of cotton () Figure 2 All of these are closely related to the length property of cotton fibers.

[0015] (2) The SNP genotypes of GhATE in a population of varieties with relatively high fiber length were verified by PCR technology, which is easy to operate, highly sensitive and accurate.

[0016] (3) Based on different SNP genotypes of GhATE, the varietal population can be divided into two major groups, and there are significant differences in cotton fiber length between these two groups. This result further demonstrates the correlation between this gene and the cotton fiber length trait. Figure 3 ). Attached Figure Description

[0017] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0018] Figure 1 This is the GWAS correlation analysis result of the cotton fiber length trait of this invention;

[0019] In this context, the highest point on chromosome A10 represents the SNP locus associated with the quality trait; the horizontal axis represents the location on the chromosome (Mb), and the vertical axis represents the significance of the SNP locus association, expressed as -log10 (P value).

[0020] Figure 2 This invention represents the expression levels of GhATE in different tissues and developmental stages of cotton.

[0021] The horizontal axis represents different tissues, including root, stem, leaf, ovule, and fiber; ovule tissue includes 3 days before flowering, 1 day before flowering, flowering day, and 1 to 35 days after flowering; fiber tissue includes 5 to 25 days after flowering; the vertical axis represents the relative expression level FPKM, which is a standard for measuring the relative expression level of genes.

[0022] Figure 3 For the sequence information of GhATE and the identification of different haplotypes in this invention;

[0023] Among them, a non-synonymous SNP site was detected in the GhATE sequence in the variety population; the base of this SNP site in the genome changed from A to G; the variety population was divided into different haplotypes, labeled as GhATE L and GhATE S; L represents long fiber and S represents short fiber.

[0024] Figure 4 A comparative analysis of fiber lengths among different haplotypes of the GhATE of this invention;

[0025] The box plot represents the distribution of fiber length in the varietal population; there are 190 and 22 varieties containing haplotypes AA and GG, respectively; the gray box plot represents the distribution of fiber length traits of haplotypes AA and GG; the horizontal line in the box represents the median of the trait distribution; * indicates a difference at the 0.05 level. Detailed Implementation

[0026] 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.

[0027] I. Discovery of arginyl-tRNA-protein transferase genes associated with cotton fiber length trait:

[0028] From 2011 to 2013, a detailed survey of cotton fiber length traits was conducted on 234 modern cotton varieties or lines in Xinxiang and Anyang, Henan Province, and Korla, Xinjiang. Simultaneously, whole-genome resequencing was performed on these 234 cotton varieties, yielding 4.54 Tb of sequencing data. These sequences were aligned to the genome sequence of the upland cotton genetic standard line TM-1 (V2.1). Single nucleotide polymorphism (SNP) markers were identified using Samtools software, uncovering 5,566,352 high-quality SNPs (minimum gene frequency > 0.05) for subsequent analysis. Genome-wide association analysis was performed using EMMAx software, and the results were then evaluated based on p < 1 × 10⁻⁶. -6 SNP-associated signal loci were screened, and 111 loci associated with cotton yield and fiber quality traits were ultimately obtained. Among them, one trait-associated locus (A10Gh:114152259) was identified on chromosome A10, which was significantly associated with cotton fiber length. Figure 1 The candidate gene in the LD block region where this site is located is the arginyl-tRNA-protein transferase gene GhATE (GH_A10G2601), whose cDNA sequence and genome sequence are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0029] II. Obtaining the arginyl-tRNA-protein transferase gene GhATE:

[0030] The GhATE gene (GH_A10G2601) was obtained from the upland cotton genome sequence. Full-length primers were designed based on both ends of the cDNA for PCR amplification. The primers are shown in Table 1. The PCR reaction program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 sec, 58℃ annealing for 5 sec, 68℃ extension for 25 sec, 34 cycles; final extension at 68℃ for 5 min. The PCR amplification product was sequenced, and the sequence was compared with the cDNA to determine the accuracy of the sequence. The arginyl-tRNA-protein transferase gene GhATE was obtained.

[0031] Table 1. Primer sequences for PCR amplification

[0032]

[0033]

[0034] III. Analysis of GhATE expression levels in different tissues and developmental stages of cotton:

[0035] This experiment used RNA samples from roots, stems, leaves, ovules, fibers, and different developmental stages for transcriptome sequencing. The average read length for each sample reached 25,530,023. The sequenced reads were aligned to the upland cotton genome TM-1 using TopHat 2.1.1 software. The transcriptome data were quantified using Cufflinks 2.2.1 software, and expression levels were expressed as FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values. Figure 2 The experimental results showed that the gene GhATE was highly expressed at different stages of cotton ovule development, indicating that this gene is related to the formation of fiber length traits. This result demonstrates that GhATE does indeed have a close relationship with factors influencing cotton fiber length and has a significant effect on increasing cotton fiber length.

[0036] IV. Application of arginyl-tRNA-protein transferase gene in the identification of high fiber length cotton:

[0037] GhATE sequences contain non-synonymous SNP sites in the population, such as Figure 3 As shown, at position 1283 bp in the genome sequence, the base changes from A to G, and the corresponding position on the transcribed cDNA changes from A to G, resulting in an amino acid change from Asp to Ser. Based on the location of this SNP site on chromosome A10 (A10Gh:114152259), amplification primers were designed at both ends (Table 2) for PCR amplification and sequencing. The PCR reaction program was as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 10 sec, 57℃ annealing for 5 sec, 68℃ extension for 10 sec, 34 cycles; and a final extension at 68℃ for 5 min.

[0038] Table 2 PCR amplification primer sequences

[0039]

[0040] Based on the base information and sequencing results of this SNP locus (A10:114152259), the genotypes of each variety population at this SNP locus were analyzed, identifying 190 haplotype AA materials and 22 haplotype GG materials (Table 2). Combining GWAS association results and phenotypic survey data, the AA haplotype varieties were labeled as GhATE L, and the GG haplotype varieties were labeled as GhATE S. Figure 3 L represents high fiber length; S represents low fiber length.

[0041] The correlation of cotton fiber length between the two haplotypes was calculated using the t-test method. Figure 4The results showed that, compared with GhATES(G), the haplotype GhATEL(A) increased the cotton fiber length by 2.90%;

[0042] The results above demonstrate that the GhATE gene has significant research value in improving cotton fiber length in new cotton varieties. On one hand, molecular markers can be designed based on the two haplotypes of the GhATE gene to effectively identify cotton fiber length traits, demonstrating excellent application value in the breeding research of high-fiber-length cotton varieties. On the other hand, taking the breeding of high-fiber-length cotton varieties as an example, genetic engineering techniques can be used to transfer the gene containing the high-fiber-length haplotype GhATE(A) into cotton varieties to increase fiber length. Alternatively, site-directed mutagenesis can be performed on the SNP sites in the low-fiber-length haplotype GhATE(G) to transform it into a high-fiber-length haplotype, thereby breeding new cotton varieties with high fiber length.

[0043] Table 2. Distribution of the two genotypes in various varieties.

[0044]

[0045]

[0046] Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. The application of an arginyl-tRNA-protein transferase gene in the identification of high-fiber-length upland cotton, characterized in that: The sequence of the arginyl-tRNA-protein transferase gene is shown in SEQ ID NO.

2. The bases at position 1283 bp of the arginyl-tRNA-protein transferase gene are detected. The bases at position 1283 bp that are A are long-fiber upland cotton, and the bases that are G are short-fiber upland cotton.

2. The application according to claim 1, characterized in that, The primers for detecting the genotype at position 1283 bp of the arginyl-tRNA-protein transferase gene are as follows: the upstream primer is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6.

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