Use of cotton fiber strength trait-associated gheif5a gene

Abstract

A use of a cotton fiber strength trait-associated GhEIF5A gene. In tetraploid Gossypium hirsutum TM-1, the cotton fiber strength trait-associated GhEIF5A gene has a cDNA sequence of SEQ ID NO: 1 and a genome sequence of SEQ ID NO: 2; the GhEIF5A gene is regulated by a neighboring SNP site, and a base at the SNP site is mutated from T to C; and the fiber strength of the mutated genotype is significantly higher than that of a wild-type genotype. The gene has important research value and application prospects in efficiently identifying high-fiber-strength Gossypium hirsutum varieties, improving cotton quality traits, and breeding novel high-fiber-strength cotton varieties.

Description

Application of GhEIF5A gene associated with cotton fiber strength traits Technical Field

[0001] The invention belongs to the field of biotechnology application and relates to the application of a gene GhEIF5A associated with cotton fiber strength traits. Background Art

[0002] Cotton (Gossypium spp.) is the primary source of natural textile fiber and plays a vital role in the national economy. Achieving high yield and high quality is the primary goal of cotton breeding. With technological advancements in the cotton textile industry, equipment upgrades, and increasing consumer demand for raw cotton fiber quality, the need to improve cotton fiber quality traits is becoming increasingly urgent. Cotton fiber quality traits are quantitative, and their phenotype is influenced by both genotype and environment. Cotton fiber strength is a key raw cotton quality indicator, determining yarn quality. Uncovering high-quality genes within cotton varieties is crucial for improving fiber strength and breeding high-quality cotton varieties.

[0003] Genome-wide association studies (GWAS), also known as linkage disequilibrium (LD) mapping, use phenotypic and genotypic variations within a species to identify the genetic basis of traits of interest, providing a systematic method for identifying complex traits. Currently, a large number of QTL and GWAS sites that control fiber quality have been identified in QTL and GWAS studies of cotton: Fang (Fang, L., Wang, Q., Hu, Y. et al. Genomic analyses in cotton identify signatures of selection and loci associated with fiber quality and yield traits. Nat Genet 49, 1089–1098 (2017)) identified 45 loci related to fiber quality in 258 upland cotton populations; Ma (Ma, Z., He, S., Wang, X., Sun, J., Zhang, Y., Zhang, G., et al. (2018). Resequencing a core collection of upland cotton identifies genomic variation and loci influencing fiber quality and yield.Nat.Genet.50,803–813.doi:10.1038 / s41588-018-0119-7) identified 6131 quality loci in 419 upland cotton yield; Yuan (Yuan, Y., Zhang, H., Wang, L., Xing, H., Mao, L., Tao, J., et al. (2019). Candidate quantitative trait loci and genes for fiber quality in Gossypium hirsutum l. detected using single- and multi-locus association mapping. Indust. Crops Prod. 134, 356–369) detected 41,815 SNPs in 196 cotton varieties and found 40 SNPs and 38 QTLs affecting fiber traits; Wang (Wang, P., He, S., Sun, G., Pan, Z., Sun, J., Geng, X., et al. (2021).Favorable pleiotropic loci for fiber yield and quality in upland cotton (Gossypium hirsutum). Sci. Rep. 11, 15935) used restriction site DNA sequencing (Rad-seq) and site-specific amplified fragment sequencing (SLAF-seq) from 316 cotton accessions to screen 231 single nucleotide polymorphisms (SNPs) in 27 genomic regions for association with fiber traits. Hu (Hu D, He S, Jia Y, Nazir MF, Sun G, Geng X, Pan Z, Wang L, Chen B, Li H, Ge Y, Pang B, Du X. Genome-wide association study for seedling biomass-related traits in Gossypium arboreum L. BMC Plant Biol. 2022 Jan 27;22(1):54) 215 cotton accessions were collected and 11 biomass-related traits, including seedling fresh weight, dry weight, water content, and root-to-shoot ratio, were studied. A genome-wide association study using 142,5003 high-quality single-nucleotide polymorphisms (SNPs) identified 83 significant associations and 69 putative candidate genes. Li (Yiqian Li, Zhanfeng Si, Guoping Wang, Zhuolin Shi, Jinwen Chen, Guoan Qi, Shangkun Jin, Zegang Han, Wenhao Gao, Yue Tian, ​​Yun Mao, Lei Fang, Yan Hu, Hong Chen, Xiefei Zhu, Tianzhen Zhang, Genomic Insights into the Genetic Basis of Cotton Breeding in China, Molecular Plant, 2023, ISSN 1674-2052, https: / / doi.org / 10.1016 / j.molp.2023.01.012) Sixteen upland cotton varieties with a significant historical role in Chinese cotton breeding were selected to construct a MAGIC population, identifying 54 genomic loci associated with lint yield and fiber quality. Genetic variation leads to phenotypic variation by inducing variation in gene expression. The numerous QTL and GWAS loci identified for fiber quality are typically located within large linkage disequilibrium regions of chromosomes. Expression quantitative trait loci (eQTL) association studies link gene expression to the genome, establishing correlations between genetic variation and gene expression, and enabling the localization of phenotypic variation to single genes.

[0004] EIF5A belongs to the eukaryotic translation initiation factor 5A (EIF5A) gene family. It is the only known protein in cells that contains a post-translational modification to form the amino acid derivative hypusine (Ne-(4-aminobutyl-2-hydroxybutyl)lysine). It plays a key role in sustained cell proliferation and has garnered widespread attention. Based on its protein synthesis initiation function, EIF5A promotes cell proliferation, aging, and cell death by promoting the transfer of specific mRNAs and the expression of related genes, and participates in plant responses and resistance to environmental stress.

[0005] Kemper (1976) first isolated EIF5A from the rabbit endoplasmic reticulum membrane. EIF5A deficiency in yeast is lethal. Research on plant EIF5A began relatively late. Thompson (2004) isolated three AtEIF5A genes from the model plant Arabidopsis thaliana. AtEIF5A1 is involved in the formation of secondary xylem. Transgenic Arabidopsis overexpressing AtEIF5A1 exhibits enlarged xylem. AtEIF5A2 is primarily expressed at high levels in mechanically damaged tissues, while AtEIF5A3 is primarily expressed in seeds, where cell division is highly active. Transgenic Arabidopsis overexpressing AtEIF5A3 exhibits syntrophic leaves, enlarged seeds, and the ability to respond to stress signals such as high salt concentration, drought, and osmotic stress.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to address the deficiencies of the existing technology and provide a gene associated with cotton fiber strength traits. Genome-wide association analysis and gene expression quantitative loci (eQTL) co-localization analysis show that the gene is a causal gene for cotton fiber strength.

[0008] Another object of the present invention is to provide applications of the gene.

[0009] The purpose of the present invention can be achieved through the following technical solutions:

[0010] The cDNA sequence of the eukaryotic initiation factor 5A gene GhEIF5A associated with the cotton fiber strength trait in tetraploid upland cotton TM-1 is: SEQ ID NO.1, and the genomic sequence is: SEQ ID NO.2; the expression of this gene is controlled by SNP (A03:65556479), and the base of this SNP site changes from T to C. The cotton fiber strength of the genotype after mutation is increased.

[0011] The invention relates to the use of the GhEIF5A gene associated with the cotton fiber strength trait in identifying high-fiber-strength upland cotton varieties.

[0012] The base type of the SNP site A03:65556479 controlling the GhEIF5A gene in the cotton genome was detected, and the cotton with the base C was a high fiber strength cotton variety.

[0013] The invention relates to the use of the GhEIF5A gene associated with the cotton fiber strength trait in improving cotton quality traits.

[0014] The present invention relates to the use of the GhEIF5A gene associated with the cotton fiber strength trait in breeding new cotton varieties with high fiber strength through genetic engineering. Specifically, a gene containing the high-yield haplotype GhEIF5A(C) can be introduced into cotton varieties through genetic engineering to increase cotton yield. Alternatively, a SNP site in the low-yield haplotype GhEIF5A(T) can be subjected to site-directed mutagenesis to transform it into a high-fiber strength haplotype, thereby breeding new high-quality cotton varieties.

[0015] A primer pair for detecting the SNP site, wherein the upstream primer is SEQ ID NO.3 and the downstream primer is SEQ ID NO.4.

[0016] The primer pair is used in screening high fiber strength cotton varieties.

[0017] A method for identifying high-fiber-strength cotton varieties comprises detecting the base type of the SNP site A03:65556479 controlling the GhEIF5A gene in the cotton genome, wherein cotton with the base C is a high-fiber-strength cotton variety.

[0018] The beneficial effects of the present invention are:

[0019] This study, using genome resequencing of cotton varieties and cotton fiber gene expression data from 20 days after anthesis, identified a gene, GhEIF5A, that is associated with the cotton quality trait fiber strength through genome-wide association analysis and co-localization of quantitative expression tags. GhEIF5A is regulated by a single nucleotide polymorphism (SNP) (A03:65556479). The genomic sequence containing the detected SNP provided by this study was obtained using PCR, a technique that requires minimal initial template, offers simple experimental steps, and exhibits high sensitivity.

[0020] The SNP genotypes of GhEIF5A in the relatively high fiber strength and low fiber strength cultivar populations were verified by PCR technology (Table 1), which is easy to operate, highly sensitive and accurate.

[0021] The variety populations can be divided into two major categories based on the different SNP genotypes that regulate GhEIF5A. Statistical analysis methods found that there were significant differences in fiber strength traits between the two groups (Figure 3), further proving the correlation between this gene and cotton fiber strength traits, and can be used to identify and transform high-fiber-strength cotton varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1. Association analysis results between cotton fiber strength traits and whole genome sequencing data in Anyang, 2008.

[0023] The horizontal axis represents the position on the chromosome, and the vertical axis represents the significance of the SNP site association, using -log 10 (P value) indicates that there are continuous SNP sites at chromosome A3 that are significantly associated with cotton fiber strength traits.

[0024] Fig. 2 Co-localization results of GhEIF5A relative expression level and fiber strength traits;

[0025] The horizontal axis represents the position on chromosome A3 (Mb), and the vertical axis represents the significance of the SNP site association, using -log 10 (P value). The top figure shows the significance of the association between the SNP loci and fiber strength traits, and the bottom figure shows the significance of the association between the SNP loci and the relative expression level of GhEIF5A. After 65 Mb, consecutive SNP loci showed high significance for the relative expression level of GhEIF5A, and nearby loci were also associated with cotton fiber strength traits.

[0026] Fig. 3 Comparative analysis of gene expression and fiber strength among different haplotypes of GhEIF5A.

[0027] Box plots represent the distribution of GhEIF5A expression and fiber strength across the cultivar populations. Expression represents the FPKM value of GhEIF5A in fibers at 20 days post-production (DPA) (normalized), indicating the relative expression level of GhEIF5A. There are 6 and 102 cultivars with CC and TT haplotypes, respectively. The horizontal line within the box represents the median of the trait distribution. The SNP site changes the base from T to C; the fiber strength of the mutant genotype is significantly higher than that of the wild type; the relative expression level of the GhEIF5A gene is increased. DETAILED DESCRIPTION

[0028] Example 1 Mining of the gene GhEIF5A associated with cotton strength traits:

[0029] From 2007 to 2009, a detailed survey of cotton-related traits was conducted in Anyang, Henan, Nanjing, Jiangsu, and Kuqa, Xinjiang for 258 modern varieties or lines. At the same time, whole-genome sequencing was performed on these 258 cotton varieties. The sequencing results were aligned with the genome sequence of upland cotton, and finally a whole-genome association analysis was performed with the phenotype. A SNP signal association site (A03:65556479) on chromosome A03 was found, as shown in Figures 1 and 2, which can be associated with cotton fiber strength traits. At the same time, this SNP is associated with the eukaryotic initiation factor 5A gene GhEIF5A in the GWAS signal region. The cDNA sequence of GhEIF5A in tetraploid upland cotton TM-1 is: SEQ ID NO.1, and the genomic sequence is: SEQ ID NO.2. This gene belongs to the eukaryotic initiation factor 5A gene family and is the only known protein (GH_A03G1210) to contain a post-translational modification to form the amino acid derivative hypusine (Ne-(4-aminobutyl-2-hydroxybutyl)lysine). The cDNA and genomic sequences of GhEIF5A (GH_A03G1210) were obtained from the Gossypium hirsutum genome. Full-length primers were designed from either end of the cDNA for PCR amplification. The primer sequences are F1: SEQ ID NO. 6 and R1: SEQ ID NO. 7. The PCR reaction protocol was as follows: 95°C denaturation for 5 minutes; 34 cycles of denaturation at 95°C for 15 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 3 minutes; and a final extension at 72°C for 5 minutes. The PCR amplified product was sequenced and compared with the cDNA to confirm sequence accuracy, resulting in the identification of the GhEIF5A gene.

[0030] Example 2 Identification of high fiber strength cotton varieties and application in improving quality traits:

[0031] Based on the location of the SNP (A03:65556479) on chromosome A03 and its adjacent sequence (SEQ ID NO. 5), genomic amplification primers were designed at either end: F2: SEQ ID NO. 3 and R2: SEQ ID NO. 4. This primer pair was used to perform PCR amplification and sequencing on DNA from 258 varieties. The PCR reaction protocol was as follows: 94°C denaturation for 5 minutes; 30 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 1 minute, and extension at 72°C for 45 seconds; and a final extension at 72°C for 7 minutes. The sequencing results were used to analyze the genotypes of each variety at this SNP. The SNP was found to change from a T to a C. Based on the base information of the two SNPs, the low-fiber-strength variety was labeled GhEIF5A(T), and the high-fiber-strength variety was labeled GhEIF5A(C). Based on the SNP genotypes, six accessions with the GhEIF5A(C) haplotype and 102 accessions with the GhEIF5A(T) haplotype were identified (Figure 3 and Table 1). The correlation between the fiber strength trait and the GhEIF5A gene expression levels between the two haplotypes was calculated using the t-test (Figure 3). The results showed that compared with GhEIF5A(T), accessions with GhEIF5A(C) had a 10.91% increase in fiber strength (P<0.001) and a higher GhEIF5A gene expression level.

[0032] These results demonstrate that the GhEIF5A gene holds significant research value in improving cotton quality traits and breeding new cotton varieties with high fiber strength. Molecular markers designed based on the two haplotypes of the regulatory SNP loci within the GhEIF5A gene can effectively identify cotton quality traits and have significant application value in the breeding of high-fiber-strength cotton varieties. Furthermore, genetic engineering can be used to introduce genes containing the high-yield haplotype GhEIF5A(C) into cotton varieties to increase yield. Alternatively, site-directed mutagenesis of the SNP loci within the low-yield haplotype GhEIF5A(T) can be performed to transform the haplotype into a high-fiber-strength haplotype, thereby breeding new high-quality cotton varieties.

[0033] Table 1 Identification of high fiber strength and low fiber strength haplotypes in population variety materials

[0034] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications derived therefrom remain within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting a SNP site in identifying a high fiber strength upland cotton variety, wherein the SNP site is located in A03:65556479 of the upland cotton genome, the base of the SNP site mutates from T to C, and the cotton fiber strength of the genotype after the mutation is increased; characterized in that, The application is specifically: The reagent for detecting SNP sites is used to detect the base type of the SNP site A03:65556479 that controls the GhEIF5A gene in the cotton genome, and the cotton with the base C is a high fiber strength cotton variety.

2. The use according to claim 1, characterized in that: The reagent for detecting the SNP site includes a primer pair: an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4.

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