A cysteine-rich receptor protein kinase GhCRK25 gene associated with cotton lint content and its application

Abstract

The present invention provides a cysteine-rich receptor protein kinase gene (GhCRK25) associated with cotton lint percentage traits and its application, belonging to the field of biotechnology applications, the cDNA sequence of the GhCRK25 gene in tetraploid upland cotton TM-1 is: SEQ ID NO.1, the sequence of the encoded amino acid is: SEQ ID NO.2, and the DNA sequence is SEQ ID NO.3; the GhCRK25 gene is regulated by the adjacent SNP (A06:116611278) site, and the base of the SNP site mutates from cytosine (C) to thymine (T). The present invention provides a method for identifying high lint percentage cotton varieties, and at the same time, the present invention also provides an application for cultivating new cotton varieties with high lint percentage by genetic engineering means.

Description

Technical Field

[0001] The present invention relates to genes associated with cotton lint content and applications, in particular to cysteine-rich receptor protein kinase GhCRK25 genes associated with cotton lint content and applications, belonging to the field of biotechnology applications. Background Art

[0002] Cotton is the most important fiber economic crop in my country, and its main product, cotton fiber, is the raw material for the textile industry and industry. Cotton fiber is a single cell differentiated from the epidermal cells of the outer integument of the ovule. Its development process can be artificially divided into four stages: initial differentiation, rapid elongation, secondary wall synthesis, and mature dehydration (Tian Y, Zhang T. MIXTAs and phytohormones orchestrate cotton fiber development[J]. Current Opinion in Plant Biology, 2021, 59:101975.). The growth and development process of cotton fiber cells is accompanied by the formation of cotton fiber yield, which is crucial to improving the economic value of cotton. Therefore, studying and analyzing the genes related to the formation of cotton fiber yield has important application value for cotton breeding.

[0003] Cotton fiber yield is generally expressed as lint yield, and the components of lint yield are the total number of bolls per unit area, the average boll weight per plant, and the lint percentage (Zhang Z, Li J, Jamshed M, et al. Genome-wide quantitative trait loci reveal the genetic basis of cotton fibre quality and yield-related traits in a Gossypium hirsutum recombinant inbred line population[J]. Plant Biotechnology Journal, 2020, 18(1):239-253.). Cotton yield is not only affected by genetic genes, but also by environmental interference. Therefore, discovering and analyzing genes related to cotton fiber yield formation has become a great challenge in cotton breeding.

[0004] In recent years, with the development of sequencing technology, bioinformatics has been rapidly applied in breeding. Genome-wide association study (GWAS) has been widely used in the mining of genes for complex traits in cotton due to its rapidity, high efficiency and low cost. In 2017, Zhang Tianzhen's team and others resequenced 318 upland cotton materials, and then conducted GWAS analysis on 258 varieties, obtaining a total of 119 loci associated with multiple traits such as fiber yield, fiber quality and resistance to Verticillium wilt. Among them, two gene loci related to the ethylene pathway were found to be significantly associated with yield (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[J]. Nature Genetics, 2017, 49(7):1089-1098. ). In 2021, Ma Zhiying's team deeply resequenced the genomes of 1,081 large-scale upland cotton variety resources, and conducted GWAS analysis based on multiple phenotypic data such as fiber length, specific strength, micronaire, lint percentage, and 401 Verticillium wilt resistance data. They found 346 structural variations related to fiber quality, 97 structural variations significantly related to yield, and 3 structural variations related to Verticillium wilt resistance, and functionally verified the candidate gene GhNCS related to Verticillium wilt resistance (Ma Z, Zhang Y, Wu L, et al. High-quality genome assemblyand resequencing of modern cotton cultivars provide resources for cropimprovement[J]. Nature Genetics, 2021, 53(9):1385-1391.).Zhao et al. conducted GWAS analysis on 336 sea island cotton materials based on multiple traits such as fiber yield and quality, and further found that there was a gene GbLP2 on A05. The expression level of this gene in long-fiber materials was significantly lower than that in short-fiber varieties. Further experimental verification showed that this gene may negatively regulate fiber length (Zhao N, Wang W, Grover CE, et al. Genomic and GWAS analyses demonstrate phylogenomic relationships of Gossypium barbadense in China and selection for fibre length, lint percentage and Fusarium wilt resistance[J]. Plant Biotechnology Journal, 2022, 20(4):691-710.). The above results show that GWAS analysis has a high positioning accuracy for genes associated with complex traits. Using populations with large differences in traits for association analysis can improve the efficiency of mining and screening target genes, thereby promoting the rapid development of precision breeding.

[0005] Cysteine-rich receptor-like protein kinases (CRKs) are a type of receptor-like protein kinase. Their structural characteristics are: they contain two copies of the unknown function 26 (DUF26) domain in the extracellular region. In addition, disulfide bonds can be formed between four conserved cysteine ​​residues (Wrzaczek M, Brosché M, Salojärvi J, et al. Transcriptional regulation of the CRK / DUF26 group of receptor-like protein kinases by ozone and plant hormones in Arabidopsis[J]. BMC Plant Biology, 2010, 10(1):95). Many studies have shown that CRKs play an important role in plant disease resistance and programmed cell death (Acharya, BR; Raina, S.; Maqbool, SB, et al. Overexpression of CRK13; anArabidopsis cysteine-rich receptor-like kinase; results in enhancedresistance to Pseudomonas syringae [J]. Plant Journal, 2007, 50(1): 488-499;Ederli, L.; Madeo, L.; Calderini, O., et al. The Arabidopsis thalianacysteine-rich receptor-like kinase CRK20 modulates host responses toPseudomonas syringae pv. tomato DC3000 infection[J]. Plant Physiology, 2011,168(1): 1784-1794.). Verticillium wilt is a common disease of cotton. In mild cases, it can significantly reduce cotton yield, and in severe cases, it can cause the death of cotton.In sea island cotton, silencing of GbCRK18 reduced the resistance of sea island cotton to Verticillium wilt, indicating that this gene can enhance cotton's resistance to Verticillium wilt and thus increase cotton yield (Li TG, Zhang DD, Zhou L, et al. Genome-Wide Identification and Functional Analyses of the CRK Gene Family in Cotton Reveals GbCRK18 Confers Verticillium Wilt Resistance in Gossypiumbarbadense[J]. Front Plant Science, 2018, 9:1266.).Other studies have found that the loss of AtCRK28 in Arabidopsis increases the mitotic activity of the primary roots, leading to longer main roots, more lateral roots, and an increase in the number of epidermal hairs, suggesting that this gene is involved in the growth of Arabidopsis roots and the differentiation of epidermal hairs (Pelagio-Flores R, Muñoz-Parra E, et al. The cysteine-rich receptor-like protein kinase CRK28 modulatesArabidopsis growth and development and influences abscisic acid responses[J]. Planta, 2019, 251(1):2. ); Arabidopsis epidermal trichomes and cotton fibers are both non-glandular single-cell structures, and their developmental mechanisms are very similar and have common regulatory mechanisms (Evolution, expression and functional analysis of cultivated allotetraploid cotton DIR genes[J]. BMC Plant Biology, 2021, 21(1):89; Guan XY, Pang MX, Nah G, et al. miR828 and miR858 Therefore, we speculate that the homologous gene of AtCRK28 in cotton may be involved in cotton fiber development and affect cotton yield formation. In-depth research on the function and molecular mechanism of CRKs in cotton fiber development may provide theoretical support for improving cotton yield traits and promote the progress of cotton breeding. Summary of the invention

[0006] In view of the above problems, the present invention provides a gene associated with cotton lint percentage trait and its application.

[0007] To achieve the above-mentioned purpose, the technical solution of the present invention is: the cysteine-rich receptor protein kinase gene GhCRK25 associated with the cotton lint content trait has a cDNA sequence of GhCRK25 as shown in SEQ ID NO:1; the protein encoded by it has an amino acid sequence as shown in SEQ ID NO.2, and the DNA sequence of the gene is SEQ ID NO.3.

[0008] A SNP marker on a gene containing the above-mentioned gene GhCRK25 sequence is significantly correlated with the cotton lint percentage trait, the marker site of the SNP marker is located in the same association analysis interval as the gene GhCRK25 and is located downstream of the gene GhCRK25, and the gene GhCRK25 is regulated by the marker site; the marker site of the SNP marker is the base type of A06:116611278, wherein the cotton with base C is a high lint percentage cotton variety, and the cotton with base T is a low lint percentage cotton variety.

[0009] The above-mentioned SNP markers are used in the breeding process of new cotton varieties. A gene containing the gene GhCRK25 and the base of the marker site of the SNP marker is C is transferred into a cotton variety to cultivate a new cotton variety with a high lint percentage, or the marker site T on the gene containing the gene GhCRK25 and the base of the marker site of the SNP marker is T is site-specifically mutated to C through genetic engineering to cultivate a new cotton variety with a high lint percentage.

[0010] A primer pair for detecting the marker site of the above-mentioned SNP marker, wherein the upstream primer sequence of the primer pair is shown as SEQ ID NO.7, and the downstream primer sequence is shown as SEQ ID NO.8.

[0011] A kit comprises the above-mentioned primer pair.

[0012] An application of the above-mentioned kit, wherein the kit is used to identify the lint percentage trait of cotton and to screen and cultivate new varieties of cotton with high lint percentage.

[0013] The beneficial effects of the cysteine-rich receptor protein kinase GhCRK25 gene associated with cotton lint percentage and its application of the present invention are:

[0014] 1. The present invention mines a cysteine-rich receptor protein kinase gene GhCRK25 associated with cotton lint content through genome-wide association analysis. The cysteine-rich receptor protein kinase gene GhCRK25 of the present invention is found in the genome-wide association analysis ( Figure 1 ), expression level analysis of different tissues and developmental stages of cotton ( Figure 2 ) and regression analysis of relative expression level and lint percentage trait level ( Figure 3 ) are closely related to the characteristics of cotton clothing.

[0015] 2. The breed population can be divided into two categories according to the different genotypes of the SNP regulating GhCRK25. Statistical analysis methods found that there were significant differences in the lint percentage between the two groups ( Figure 4 ), further proving the correlation between this gene and cotton yield traits.

[0016] 3. The SNP genotype of GhCRK25 in relatively high and low lint percentage varieties can be verified by PCR technology, which has the advantages of easy operation, high sensitivity and good accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 1 This is the result of the association analysis between cotton lint content and the whole genome; in the figure:

[0019] The horizontal axis represents the position on the chromosome (Mb), and the vertical axis represents the significance of the association between the SNP locus and the agronomic trait, expressed as -log 10 (P value) indicates. Arrows indicate SNP variant sites associated with agronomic traits.

[0020] Figure 2 This is a statistical diagram of the gene expression level of GhCRK25 in different tissues and developmental stages of cotton; in the figure:

[0021] The horizontal axis represents different tissues, namely root, stem, leaf, ovule, and fiber. The ovule tissue includes 3 days before flowering, 1 day before flowering, the day of flowering, and 1 to 35 days after flowering. The fiber tissue includes 5 days after flowering, 10 days after flowering, 20 days after flowering, and 25 days after flowering. The vertical axis represents FPKM, which is a standard for measuring the relative expression of genes.

[0022] Figure 3 This is the result of regression analysis between the relative expression level of GhCRK25 and the lint percentage trait level; in the figure:

[0023] Different scattered points represent different variety groups, the horizontal axis represents the standardized gene expression (FPKM), and the vertical axis represents the yield trait lint percentage. The R in the legend in the upper left corner represents the regression coefficient, and p represents the significance level, which is used to evaluate the statistical significance of the fitting curve.

[0024] Figure 4 This is a graph showing the results of differential analysis of expression and lint percentage between different genotypes of GhCRK25 in a population; in the graph:

[0025] The left figure represents the difference analysis of expression between different genotypes in the population, and the right figure represents the difference analysis of lint percentage between different genotypes in the population. The box plot represents the distribution of lint percentage traits in the variety population. There are 111 and 14 varieties with CC and TT genotypes, respectively. The horizontal line in the box represents the median of the trait distribution. DETAILED DESCRIPTION

[0026] The technical scheme in the embodiment of the present invention is described clearly and completely below in conjunction with the accompanying drawings in the embodiment of the present invention. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0027] A cysteine-rich receptor protein kinase gene GhCRK25 associated with cotton lint content has a cDNA sequence of GhCRK25 as shown in SEQ ID NO: 1; the protein encoded by it has an amino acid sequence as shown in SEQ ID NO.2, and the DNA sequence of the gene is SEQ ID NO.3.

[0028] The gene GhCRK25 described in the present invention is regulated by a downstream genetic site (SNP: A06:116611278). The base of the SNP site mutates from C to T. In the population, the material with the base of the SNP site being C has a higher expression of GhCRK25, which is manifested as a higher lint percentage; the material with the base of the SNP site being T has a lower expression of GhCRK25, which is manifested as a lower lint percentage.

[0029] The application of the gene GhCRK25 described in the present invention in identifying high lint percentage upland cotton varieties is specifically: using the specific primer pair for detecting the SNP site A06:116611278, SEQ ID NO.7 and SEQ ID NO.8, to detect the base type of the SNP site controlling the cysteine-rich receptor protein kinase gene GhCRK25 in the cotton genome, wherein the cotton with the base being cytosine (C) is a high lint percentage cotton variety.

[0030] The application of the gene GhCRK25 of the present invention in breeding new cotton varieties with high lint percentage by genetic engineering means is as follows: the SNP site in the low-yield genotype GhCRK25 (T) can be site-directed mutated into (C) by genetic engineering means, thereby breeding new cotton varieties with high lint percentage.

[0031] Example 1 Mining of the cysteine-rich receptor protein kinase gene GhCRK25 associated with cotton lint content:

[0032] From 2007 to 2009, a detailed survey of cotton yield traits (lint percentage, seed index, etc.) was conducted in Anyang, Henan, Nanjing, Jiangsu, and Kuqa, Xinjiang for 258 modern varieties or strains. At the same time, the whole genome of these 258 cotton varieties was sequenced. The sequencing data was compared with the reference genome of the standard upland cotton line TM-1, and then the software samtools was used to identify SNPs on the basis of the whole genome level, and the mined genetic variation sites and cotton phenotypic data were subjected to whole genome association analysis. The results are as follows Figure 1 As shown, a SNP signal association site (A06:116611278) on chromosome A06 can be associated with cotton lint percentage traits. At the same time, this SNP is associated with the cysteine-rich receptor protein kinase gene GhCRK25 (GH_A06G1696) in the GWAS signal region. It is preliminarily judged that this gene may be involved in the formation of cotton lint percentage.

[0033] RNA samples from different tissues and different developmental stages of the TM-1 upland cotton variety were collected for transcriptome sequencing. Sample materials included roots, stems, leaves, ovules, and fibers. Ovule tissue included 3 and 1 days before flowering, the day of flowering, and 1 to 35 days after flowering. Fiber tissue included 5 to 25 days after flowering. In the results, the GhCRK25 gene was dominantly expressed in ovule tissue 3 days before flowering and the day of flowering ( Figure 2 ), that is, it is dominantly expressed in the initial stage of fiber development, indicating that this gene is related to the constituent factors of cotton lint content traits.

[0034] The relative expression level of GhCRK25 and the level of lint percentage trait were regressed. The ordinate represents the lint percentage level, and the abscissa represents the FPKM value of GhCRK25 (after normal centering), which represents the relative expression level of GhCRK25. The R in the legend in the upper left corner represents the regression coefficient, and p represents the significance level, which is used to evaluate the statistical significance of the fitting curve. The results of the regression analysis show that the GhCRK25 gene positively regulates the lint percentage trait ( Figure 3 ).

[0035] The three elements of cotton yield are the number of bolls per plant, the weight of bolls per plant, and the lint percentage. Therefore, by studying the function of GhCRK25 in regulating the lint percentage of cotton, it is possible to provide a genetic basis for the improvement of high-yield cotton varieties, thereby promoting the process of precision breeding of cotton. The cDNA sequence of GhCRK25 in tetraploid upland cotton TM-1 is shown in SEQ ID NO.1, the amino acid sequence of the encoded protein is shown in SEQ ID NO.2, and the genomic DNA sequence is shown in SEQ ID NO.3. The cDNA sequence and DNA sequence of GhCRK25 were obtained from the upland cotton genome sequence, and the full-length primers of the gene were designed according to the two ends of the cDNA for PCR amplification. The primer sequences were F1: SEQ ID NO.5 and R1: SEQ ID NO.6. The PCR reaction procedure was as follows: 94℃ pre-denaturation for 2min; 98℃ denaturation for 10sec; 55℃ annealing for 5sec; 68℃ extension for 10sec, 35 cycles; and finally 68℃ extension for 5min. The PCR amplification product is sequenced and further compared with the cDNA to determine the accuracy of the sequence, and the GhCRK25 gene can be obtained.

[0036] Example 2 Method for identifying high lint percentage cotton varieties:

[0037] Based on the position of the SNP site (A06:116611278) on chromosome A06 and the sequence nearby (SEQ ID NO.4, the SNP site is at position 51 in the SEQ ID NO.4 sequence), genomic amplification primers were designed at both ends of the SNP site. The primer sequences were F2: SEQ ID NO.7 and R2: SEQ ID NO.8. This pair of primers was used to perform PCR amplification and sequencing in the DNA of 258 varieties. The PCR reaction procedure was as follows: pre-denaturation at 94°C for 2min; denaturation at 98°C for 10sec; annealing at 55°C for 5sec; extension at 68°C for 10sec, 35 cycles; and finally extension at 68°C for 5min. The variety population was divided into two categories according to the different genotypes of the regulatory SNP site: CC represents a high lint percentage genotype, and the SNP site is a homozygous site; TT represents a low lint percentage genotype, and the SNP site is a homozygous mutation. As shown in Tables 1 and Figure 4As shown, the number of varieties with the two genotypes is 111 and 14 respectively (a total of 258 varieties were tested, and the results of classification by genotype are 4 types, one is the homozygous type, namely the CC, one is the homozygous mutation, namely the TT; another is the heterozygous mutation, the genotype is CT, and the remaining one is the deletion type, that is, the SNP site is missing at this position. In the actual analysis process, the deletion type is generally not considered, and the homozygous mutation is more valuable than the heterozygous mutation. Therefore, the statistical analysis of the present invention only compares the homozygous type CC and the homozygous mutation TT, and the number of varieties with the two genotypes is 111 and 14 respectively). Using the statistical analysis method, it was found that there were significant differences between the homozygous site and the homozygous mutation population, whether between the population genotype and the gene expression level, or between the population genotype and the lint percentage trait level, proving the correlation between the SNP site and the cotton lint percentage trait.

[0038] The above results show that the cysteine-rich receptor protein kinase gene GhCRK25 has important research value in improving lint percentage and breeding new varieties with high lint percentage. Molecular markers can be designed based on the two genotypes of the SNP site controlling the cysteine-rich receptor protein kinase gene GhCRK25, which can effectively identify the lint percentage trait of cotton and have good application value in the research of breeding high lint percentage cotton varieties.

[0039] Table 1 Identification of high lint percentage and low lint percentage genotypes in population cultivar materials

[0040]

[0041] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. Application of a reagent for detecting SNP markers in identification of cotton lint content traits, characterized in that: The SNP marker is used to identify the lint percentage trait of cotton and distinguish between high lint percentage cotton varieties and low lint percentage cotton varieties; The marker site of the SNP marker is located in the same association analysis interval as the gene GhCRK25 and is located downstream of the gene GhCRK25, and the gene GhCRK25 is regulated by the marker site; the marker site of the SNP marker is the 51st genotype as shown in the sequence of SEQ ID NO:4, wherein the cotton with the genotype CC is a high lint percentage cotton variety, and the cotton with the genotype TT is a low lint percentage cotton variety; The cDNA sequence of the gene GhCRK25 is shown in SEQ ID NO:

1. The gene GhCRK25 is a cysteine-rich receptor protein kinase gene associated with cotton lint percentage traits.

2. The use of a reagent for detecting SNP markers according to claim 1 in identifying cotton lint content traits, characterized in that: The upstream primer sequence of the primer pair for detecting the marker site of the SNP marker is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.8.

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