CN116904482A - Kinesin gene affecting upland cotton fiber properties and SNP marker thereof - Google Patents
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Abstract
The application combines upland cotton (G.hirsutum L.) association mapping to identify Kinesin genes which play an important role in cotton fiber and cotton boll development, and association analysis identifies that one of Kinesin-7 subfamily genes (Ghir_D04G 017880) is obviously related to fiber strength. The Kinesin gene family plays an important role in cotton fiber and cotton boll development, and the identification of the Kinesin gene with functions and related molecular markers is beneficial to the genetic improvement of fiber quality and yield.
Description
Technical Field
The application belongs to the technical field of molecular biology, and particularly relates to a key Kinesin gene affecting upland cotton fiber properties and SNP markers thereof.
Background
Because of the high cellulose content in cotton, it is used as the most important fiber crop planting in the world. Meanwhile, cotton fibers belong to single cells, and are an excellent model for researching cellulose formation. There is a reported class of molecular motile proteins that play a critical role in the directional deposition of cellulose microfibrils and the mechanical strength of secondary walls. This motor protein is designated as Kinesin, which is responsible for intracellular organelle movement, signal transduction, mitosis and meiosis, and cytoskeletal construction. Kinesin kinesins convert chemical energy into mechanical energy by hydrolyzing adenosine triphosphate (adenosine triphosphate, ATP) as the driving force. Typically such kinesins can specifically bind to microtubules and move on linear microtubule rails, thereby completing specific physiological activities; in addition, kinesins have highly conserved kinesin domains that contain a globular domain of 360 residues, known as the catalytic core, and thus genes encoding such proteins are known as the kinesin gene family. The Kinesin gene family can be divided into 14 subfamilies, i.e., kinesin-1 to Kinesin-14 subfamilies, depending on the specific Kinesin domain.
Extensive studies of the Kinesin gene family have been conducted in animals, where the Kinesin-13 subfamily has been demonstrated to play a key role in regulating mitosis, cilia assembly and depolymerization. In plants, moscateli et al (1988) first identified Kinesin motor proteins in tobacco pollen tubes, and subsequently identified that the Kinesin gene functions significantly in maize, arabidopsis, tomato, etc. plants. Of the Kinesin superfamily, the Kinesin-13 subfamily has been shown to be involved in plant growth and development, and in moss (Physcomitrella Patens), kineins-13 and kineins-8 have been shown to affect cell division and cell growth (Leong et al, 2020). In addition, the Kinesin-13 subfamily in Arabidopsis thaliana and rice (Oryza spp.) has also been studied, and researchers found Kinesin-13 protein in the Golgi apparatus of Arabidopsis thaliana, and it was shown by T-DNA mediated gene knockout experiments that Kinesin-13 affects the morphology of leaf epidermis trichomes; furthermore, knock-out and over-expression experiments confirm that Kinesin-13 plays a key role in secondary cell wall synthesis. Meanwhile, in rice, kinesin-13 genes for regulating the grain length of the rice are identified through map cloning, and Wu and the like verify that the Kinesin-13 genes affect the seed length and plant height of the rice through map cloning and transgenic experiments.
Preuss et al found that the proteins associated with the Kinesin driver gene family were involved in the development of cotton fibroblasts. Furthermore, the kinesin gene GHKCH2 was observed to interact with microtubules and microfilaments in developing cotton fibers. Li et al report that although the GHKIS13A1 gene is at 6Y
Relatively low expression levels were maintained in cotton fibers, but overexpression of GHKIS13A1 in arabidopsis resulted in branching of the epidermal hair.
Cotton fibers are an ideal model for studying plant cell elongation and cell wall synthesis. Thus, it is necessary to study the Kinesin gene family throughout the genome and to identify key Kinesin genes involved in the cotton development process. In recent years, a large number of Quantitative Trait Loci (QTLs) have been mined based on high quality cotton reference genome and whole genome association analysis (GWAS). Thus, binding gene families and association analysis are viable strategies for mining functional genes of a gene family of interest.
Disclosure of Invention
Aiming at the bottleneck problems of improving the quality and the yield of the fiber faced by the prior upland cotton, the application aims to: the upland cotton whole genome Kinesin gene is identified by using upland cotton reference genome, upland cotton population phenotype data and resequencing data, and important Kinesin genes which can influence the upland cotton fiber phenotype are mined so as to be applied to genetic improvement of the quality and yield of the upland cotton fiber in the future.
The application aims at providing a Gh_Kinesin7 gene affecting the fiber properties of upland cotton, and the sequence of the gene is shown as SEQ ID NO. 1.
Further, the full-length sequence of the Gh_Kinesin7 gene is shown as SEQ ID NO. 2.
Further, the genes can be used to improve cotton agronomic traits.
Further, the cotton is upland cotton and the agronomic trait is a fiber trait.
The second object of the application is to provide a method for improving the agronomic characters of cotton, which comprises the step of highly expressing the Gh_Kinesin7 gene in cotton varieties, wherein the Gh_Kinesin7 gene sequence is shown as SEQ ID NO. 1.
Further, the cotton variety is upland cotton and the agronomic trait is a fiber trait.
The application further aims to provide a SNP marker related to cotton fiber properties, wherein the polymorphism of the SNP marker is T/C, and the SNP marker is positioned at the 5869bp position of the sequence shown in SEQ ID NO. 2.
Further, the individual fiber properties of genotype TT are significantly stronger than the CC genotype.
The fourth object of the present application is to provide a primer set for detecting the SNP marker, wherein the primer set is as follows: gh_Kin7 pre-primer as shown in SEQ ID NO. 3: GTGCTTGCTTCTGATCCGTC; gh_Kin7 post primer as shown in SEQ ID NO. 4: CTCTCTGCCCCCACCTGAAA.
The fifth object of the present application is to provide a kit comprising the above-mentioned detection primer pair.
The application aims at providing a method for identifying the fiber properties of cotton materials, which comprises the step of detecting the cotton materials by adopting the primer pair and/or the kit, wherein the fiber properties are good when the SNP marker genotype is TT.
The beneficial effects of the application are as follows: the application combines the correlation mapping of upland cotton (G.hirsutum L.) to identify Kinesin genes with important roles in cotton fiber and cotton boll development, and the correlation analysis identifies that one of the Kinesin-7 subfamily genes (Ghir_D04G 017880) is obviously related to fiber strength, the Kinesin gene family has important roles in cotton fiber and cotton boll development, and the identification of the Kinesin-7 genes with functions and related molecular markers based on the genes are beneficial to genetic improvement of fiber quality and yield.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a whole genome identification of the Kinesin Gene family in example 1
FIG. 2 is a genome-wide SNPs and population structure analysis of natural populations of upland cotton of 205 materials in example 2
FIG. 3 is 3 fiber traits of the natural population of upland cotton in 8 field test cells in example 2
FIG. 4 is a correlation analysis of upland cotton fiber strength and candidate gene analysis in example 3.
Detailed Description
The technical scheme of the present application will be clearly and completely described in the following in connection with specific embodiments. The following examples are preferred embodiments of the present application, but are not intended to limit the scope of the present application in any way. The substitution of simple parameters in the embodiments of the present application cannot be described in detail in the examples, but the present application is not limited thereto, and any other modifications, substitutions, combinations, and simplifications made without departing from the theoretical spirit and principles of the present application should be considered as equivalent substitution modes, and are included in the scope of the present application.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 identification of upland cotton Whole genome Kinesin Gene
A hidden Markov model of the Kinesin protein conserved domain was obtained from the Pfam database (http:// Pfam. Xfam. Org /) (Pfam Number: PF 00225). Upland cotton reference genomes and annotation files were obtained from the cottonGen website (https:// www.cottongen.org /). The Kinesin gene (P < 1E-20) containing a conserved Kinesin protein domain was identified by HMMSearch software (Wheeler and Eddy, 2013), and 170 Kinesin members were identified in upland cotton by looking up the Kinesin gene family conserved domain in the upland cotton genome. And further analyzing the protein sequence of the Kinesin genes by an online tool CDD (http:// www.ncbi.nlm.nih.gov/CDD /), and confirming the existence of the conserved domain of the Kinesin protein genes. In addition, 161 Kinesin genes containing conserved domains were identified in NCBI by conserved domain search screening; two kinesin genes were filtered out, and the evolutionary tree could not be constructed because there was a large genetic difference between the two genes. Thus, 159 Kinesin genes were obtained in upland cotton. Carrying out multi-sequence comparison on the protein sequence of the Kinesin gene by using Mega-X software to obtain a phylogenetic relationship; the phylogenetic tree was then drawn using on-line Itol software (https:// Itol. Embl. De /) (fig. 1).
Example 2 acquisition of upland cotton Natural population genotype and phenotype data
In order to locate the Kinesin functional gene associated with gossypium hirsutum fiber development, candidate gene association analysis was performed on the 3 fiber traits and whole genome genetic variation of the natural population of gossypium hirsutum. The upland cotton natural population comprises 205 materials, and early studies were planted in 4 cotton ecological areas in 2012 and 2013 respectively, so that phenotype data were collected in 8 test cells, and the 8 test points were named: BJ2012 (north region 2012), BJ2013 (north region 2013), NJ2012 (south region 2012), NJ2013 (south region 2013), CJ2012 (Yangtze river basin 2012), CJ2013 (Yangtze river basin 2013), HH2012 (yellow river basin 2012) and HH2013 (yellow river basin 2013). And carrying out association analysis and identification on the genome-wide Kinesin gene by utilizing 3 upland cotton fiber traits, wherein the three fiber traits are respectively Fiber Length (FL), fiber Strength (FS) and coat-Length (LW). Natural population Fiber Length (FL) mean performs best in CJ2012 cells; the Xinjiang area generally exhibits better FL and FS quality than the Yangtze river basin and the yellow river basin; the coefficient of variation of LW is higher than FL and FS (FIG. 2) compared to FL and FS.
The natural population was resequenced approximately 5-fold deep using whole genome resequencing methods at the early stage of the study. Sequencing data was aligned with the TM-1 reference genome using BWA0.7.10 software. Identification of SNP genetic variation in BAM files by GATK 3.1.1 software, genetic variation was filtered by VCFTools software, and 3,203,378 genetic variations were finally identified in total from the resequencing of natural populations of upland cotton (fig. 3 a). The subspecies of the natural population were estimated using Admix 1.3.0, and the principal component analysis of the natural population was calculated using TASSEL 5.0 and high quality SNP. Analysis of the population structure showed that the population could be divided into 4 subspecies (fig. 3 b), with Principal Component Analysis (PCA) substantially consistent with this result (fig. 3 c).
Example 3 analysis of the association of Kinesin Gene with phenotype, the association of a phenotypic trait with Whole genome Kinesin Gene
To study the relationship between the Kinesin gene and fiber development, we performed candidate gene association mapping between actin genes and 3 fiber traits. Genetic variation of all Kinesin genes was extracted using TASSEL 5.0, and 1318 genetic variations were extracted from the whole genome genetic variation of 159 Kinesin genes of upland cotton. Candidate gene association localization was performed on 3 fiber traits and SNPs in 159 Kinesin genes using the MLM (PCA+K) model of TASSEL 5.0 software, and the threshold was set to-log (1/N, N is the number of markers).
Correlation analysis results show that 19 Kinesin genes are obviously related to FL, and the phenotype variation interpretation (PVE) range of the related markers is 5.981% -15.524%; 8 Kinesin genes are obviously related to FS, and PVEs of related markers range from 5.836% to 13.352%; the 3 Kinesin genes were significantly associated with LW, with correlation-tagged PVEs ranging from 6.149% to 16.195% (as shown in table 1).
TABLE 1 analysis of candidate Gene correlation between genetic variation and Whole genome Kinesin Gene
A gene of the Kinesin-7 subfamily (Ghir_D04G 017880) was identified by years of multi-point candidate gene association analysis as being significantly associated with FS, which gene was located between 53421041-53430230bp of the D04 chromosome and was designated Gh_Kinesin7 (Table 1 and FIG. 4 a). Gh_Kinesin7 full length 9190 bp) (shown as SEQ ID NO. 2) comprises 27 exons and 26 introns, the full length of the coding sequence is 3000bp, and 999 amino acids are coded. The conserved domain analysis shows that the Kinesin motor domain of Gh_Kinesin7 is located between 103 and 420 amino acids. 15 genetic variations were identified in total for gh_kinesin7, with 11 genetic variations for gh_kinesin7 being a significant marker for candidate gene association localization and 5 genetic variations in the Kinesin motor domain. Two genetic variations resulted in mutations in the amino acids of the Kinesin motor domain, with the SNP of D04:53423182 inducing aspartic acid to serine and the SNP of D04:53424009 inducing alanine to threonine (FIG. 4 e).
A significantly associated SNP marker (SD 04: 53426909) was located at the 5869bp position of the gh_kinesin7 gene, which could explain the 13.35% variation in fiber strength in NJ2012 (table 4 a), the FL average (average=29.50 mm) for the TT genotype group was significantly higher than the CC genotype group (average=26.91 mm) (P value= 0.00848, fig. 4 b), and only homozygous genotypes were present in the natural population. The following Gh_Kin7 primer tags were designed for this SNP variation:
Gh_Kin7 pre-primer (5 '. Fwdarw.3'): GTGCTTGCTTCTGATCCGTC (SEQ ID NO: 3);
Gh_Kin7 post primer (5 '. Fwdarw.3'): CTCTCTGCCCCCACCTGAAA (SEQ ID NO: 4).
Gh_Kinesin7 was preferentially expressed in 10-DPA fibers, and RT-PCR confirmed the expression of Gh_Kinesin7 in cotton varieties (FIGS. 4 c-d).
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
1. The Gh_Kinesin7 gene affecting the fiber property of upland cotton is characterized in that the sequence of the gene is shown as SEQ ID NO. 1.
2. The gene according to claim 1, wherein the full length sequence of the Gh_Kinesin7 gene is shown in SEQ ID NO. 2.
3. Use of a gene according to any one of claims 1-2 for improving agronomic traits in cotton.
4. The use according to claim 3, wherein the cotton is upland cotton and the agronomic trait is a fiber trait.
5. A method for improving the agronomic characters of cotton is characterized by comprising the step of high expressing Gh_Kinesin7 gene in cotton varieties, wherein the Gh_Kinesin7 gene sequence is shown as SEQ ID NO. 1.
6. A SNP marker related to cotton fiber characteristics is characterized in that polymorphism of the SNP marker is T/C, and the SNP marker is positioned at a 5869bp position of a sequence shown in SEQ ID NO. 2.
7. The SNP marker according to claim 6, wherein the individual fiber property of genotype TT is significantly stronger than that of CC genotype.
8. A primer pair for detecting the SNP marker as set forth in claim 6, characterized in that the primer pair is: gh_Kin7 pre-primer as shown in SEQ ID NO. 3: GTGCTTGCTTCTGATCCGTC; gh_Kin7 post primer as shown in SEQ ID NO. 4: CTCTCTGCCCCCACCTGAAA.
9. A kit comprising the detection primer pair of claim 7.
10. A method for identifying the fiber properties of cotton materials, comprising detecting cotton materials using the primer set of claim 7 and/or the kit of claim 8, wherein the fiber properties are indicated to be good when the SNP marker genotype is TT.
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