Cotton gene GhFBH1 and application thereof in identifying height of first fruit branch of cotton

Abstract

The invention discloses a cotton gene GhFBH1 and application thereof in identifying the height of a first fruit branch of cotton, and belongs to the field of biotechnology application. The cDNA (complementary deoxyribonucleic acid) sequence of the gene GhFBH1 associated with the first fruit branch height character of the cotton is as shown in SEQ ID NO. 1, and the genome sequence is as shown in SEQ ID NO. 2. The GhFBH1 is regulated and controlled by an SNP site on the gene, and after the SNP site is mutated, the height of a first fruit branch is obviously lower than that of a wild type. The gene has important research value and application prospect in efficient identification of cotton varieties with low first fruit branch height and improvement and breeding of excellent cotton varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a cotton gene GhFBH1 and its application in identifying the height of the first fruiting branch of cotton. Background Technology

[0002] Cotton is one of the world's most important economic crops and a source of natural fiber, and its yield and quality directly affect agricultural economic benefits. First fruiting branch height (FBH) is one of the important agronomic traits of cotton, significantly affecting plant type, maturity date, planting density, and adaptability to mechanized harvesting. Generally, a lower first fruiting branch height is conducive to forming a compact plant type, improving the light energy utilization rate of the canopy, and adapting to high-density planting and mechanized management, and is one of the important goals of modern cotton breeding.

[0003] Traditional breeding methods for improving first-degree hybrid rice (FBH) are time-consuming and inefficient. Molecular marker-assisted selection (MAS) provides an effective way to accelerate the breeding process. Genome-wide association analysis (GWAS) is an effective method based on the principle of population linkage disequilibrium (LD) to identify genes / QTLs controlling complex quantitative traits by analyzing the associations between genetic markers and target traits in natural populations. In recent years, GWAS has been widely used for the genetic analysis of complex agronomic traits in various crops such as rice, maize, soybean, and cotton.

[0004] N-linked glycosylation is a common and important post-translational modification of proteins in eukaryotes, crucial for proper protein folding, stability, subcellular localization, and function. Oligosaccharyl transferase (OST) complexes are key enzymes catalyzing this core step, responsible for transferring oligosaccharide chains (typically Glc3Man9GlcNAc2) synthesized pre-synthesized on the endoplasmic reticulum membrane from their lipid carrier, dolicolpyrophosphate, to specific asparagine residues in the nascent polypeptide chain (Asn in the Asn-X-Ser / Thr sequence motif).

[0005] The OST complex is typically composed of multiple distinct subunits that exhibit a degree of conservation across different species, working synergistically to ensure efficient and precise glycosylation. For example, the STT3 subunit is considered the core catalytic subunit of the OST complex. Other subunits may be involved in substrate recognition and binding (glycan donors and protein acceptors), or in maintaining the complex's structural integrity and regulating its activity. The OST complex in plants is also indispensable for normal cellular physiological functions. Proteins modified by N-glycosylation are widely involved in various aspects of plant growth and development, including pathogen defense, cell survival, and glycosylation influences hormone signaling and cell wall synthesis. Therefore, dysfunction of any subunit in the OST complex can lead to alterations in plant growth and development phenotypes by affecting the maturation and function of a range of important proteins. Although the fundamental role of oligosaccharide transferases in protein N-glycosylation modification is widely recognized, and N-glycosylation affects many aspects of plant growth and development, current research on whether and how specific OST complex subunits (especially GhFBH1 and its encoded subunit, which are the focus of this invention) directly participate in the genetic mechanism regulating the important agronomic trait of cotton first fruiting branch height is still insufficient, and direct genetic and molecular biological evidence is lacking.

[0006] Therefore, in order to overcome the limitations of existing technologies in accurately analyzing the genetic basis of cotton FBH and to provide new gene resources and molecular tools for breeding cotton with ideal plant type, it is of great theoretical value and practical application prospect to discover key genes closely related to cotton FBH. Summary of the Invention

[0007] The purpose of this invention is to provide a cotton gene GhFBH1 and its application in identifying the height of the first fruiting branch in cotton. Through resequencing and genome-wide association analysis of the YM8 variety population, this gene was found to be closely associated with the height of the first fruiting branch in cotton. The specific technical solution is as follows:

[0008] First, this invention provides a cotton gene GhFBH1. The cDNA sequence of the cotton gene GhFBH1 in the tetraploid YM8 is SEQ ID NO.1, and the genomic sequence is SEQ ID NO.2. The genomic sequence is transcribed in the forward direction. The gene is regulated by a SNP site on the gene, and mutation of this SNP results in a significantly shorter first fruiting branch height in cotton compared to the wild type.

[0009] Second, this invention provides the application of the cotton gene GhFBH1 in identifying the height of the first fruiting branch of cotton.

[0010] This invention provides a single-nucleotide polymorphic nipple (SNP) marker associated with the height of the first fruiting branch in cotton, and the application of reagents for detecting this SNP site in identifying cotton varieties with low first fruiting branch height. The population is divided into two haplotype groups based on the genotype of this SNP: cotton materials with base A have lower first fruiting branch height, while cotton materials with base C have higher first fruiting branch height. Therefore, this SNP, located at the 96th bp position of the GhFBH1 gene (YM8 genome A05:16783268), can serve as a marker for rapid identification of cotton varieties with low first fruiting branch height. Detection of the base at the 96th bp position of the GhFBH1 gene in upland cotton plants reveals that cotton materials with base A at the 96th bp position have lower first fruiting branch height, while those with base C at the 96th bp position have higher first fruiting branch height.

[0011] In addition, the reagents for detecting SNP sites include primer pairs: the upstream primer is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.6.

[0012] A further kit is provided and its application in identifying the height trait of the first fruiting branch of cotton. The kit contains a primer pair: an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.6.

[0013] Third, this invention provides the application of the GhFBH1 gene in improving the height of the first fruit branch, and the application of the GhFBH1 gene in breeding new varieties with low first fruit branch height through genetic engineering.

[0014] The applications include: cloning the cotton gene GhFBH1 of the low first fruiting branch height haplotype and introducing it into cotton varieties, or performing site-directed mutation on the base located at the 96th bp position of the cotton gene GhFBH1 in the high first fruiting branch height haplotype.

[0015] Fourth, this invention provides a method for improving the height of the first fruiting branch in cotton. This involves editing the 96th bp site of the cotton gene GhFBH1 described in this invention from C to A using a CRISPR / Cas9 system, or screening for genotypes with the A site using SNP marker-assisted selection, thereby reducing the height of the first fruiting branch in cotton. Specific steps include: constructing a gRNA expression vector targeting this site using a CRISPR / Cas9 system; transforming cotton callus tissue using Agrobacterium-mediated transformation; obtaining transgenic plants through screening and regeneration; verifying the genotype through sequencing; and evaluating the fruiting branch height phenotype in the field. The nucleotide sequence of the cotton gene GhFBH1 is shown in SEQ ID NO.2.

[0016] Fifth, a method for breeding cotton varieties with low first fruiting branch height, using genetic engineering techniques, with materials carrying the cotton gene GhFBH1 allele described in this invention as donors, the allele is introduced into the target variety through backcrossing or self-crossing, genotyping is performed using the above-mentioned SNP markers, and combined with field phenotypic identification, new cotton varieties with shortened growth period are selected.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) Based on a high-quality upland cotton genome sequence, this invention successfully identified a gene closely related to the height of the first fruiting branch of cotton using population genome resequencing and genome-wide association analysis (GWAS). This gene is GhFBH1, and the GhFBH1 gene in this invention was significantly associated with the height of the first fruiting branch in GWAS. The cDNA and genome sequence of the GhFBH1 gene were obtained by PCR technology, which has the advantages of high specificity, high sensitivity, speed, simplicity, and high reproducibility, ensuring the accuracy of the sequence.

[0019] (2) Using PCR technology, this invention verified the genotype of the GhFBH1 gene in varieties with relatively low first fruiting branch height and relatively high first fruiting branch height. It can be used as an efficient method to identify the height of the first fruiting branch of cotton, with the advantages of being fast, simple, accurate and reliable.

[0020] (3) Based on the different genotypes of the GhFBH1 gene, the varietal population can be divided into two major categories, and these two groups show significant differences in the height of the first fruiting branch. This result further confirms the close association between the GhFBH1 gene and the height of the first fruiting branch in cotton. Attached Figure Description

[0021] Figure 1 This is a schematic diagram showing the association analysis results between the height of the first fruiting branch of cotton and whole-genome sequencing data, as well as the sequence information of the GhFBH1 gene. Among them, (A) shows the GWAS association analysis results of the first fruiting branch height trait, with the horizontal axis representing chromosomes and the vertical axis representing the significance of the SNP site association, expressed as -log10 (P value); (B) shows the location of the SNP site on the chromosome, with the horizontal axis representing the location on the chromosome (Mb) and the vertical axis representing the significance of the SNP site association, expressed as -log10 (P value).

[0022] Figure 2This is a comparative analysis of the height of the first fruiting branch among different haplotypes of the GhFBH1 gene; the box plots represent the distribution of the height of the first fruiting branch in the varietal population, in centimeters. The red box plot (left) represents the phenotypic distribution of haplotype A / A, and the blue box plot (right) represents the phenotypic distribution of haplotype C / C; the horizontal lines in the boxes represent the median of the phenotypic distribution; *** indicates a difference at the 0.001 level. Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] Example 1: Discovery and acquisition of the GhFBH1 gene, which is highly associated with the first fruiting branch of cotton.

[0025] A detailed investigation of the first fruiting branch height trait was conducted on 370 modern cotton varieties or lines. Simultaneously, whole-genome resequencing was performed on these 370 cotton varieties, with an average sequencing depth of 20X. These sequences were aligned to the genome sequence of upland cotton YM8, and genome-wide association analysis was performed with phenotypes using EMMAX software, with a p-value < 10⁻⁶. -5 SNP-associated signal sites were screened, and a site highly significantly associated with the first fruiting branch of cotton (A05:16783268) was identified. Figure 1 The SNP is located on an exon of the candidate gene GhFBH1 in the LD block region, and its cDNA sequence and genomic sequence are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0026]

[0027]

[0028] GhFBH1 was obtained from the genome sequence. A pair of full-length primers (Table 1) were designed based on both ends of its cDNA sequence, such as the upstream primer F1 for cDNA amplification shown in SEQ ID NO.3 and the downstream primer R1 for cDNA amplification shown in SEQ ID NO.4, for subsequent PCR amplification. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 57℃ annealing for 15 sec, 72℃ extension for 45 sec, 34 cycles; and a final extension at 72℃ for 5 min. The PCR amplification products were sequenced and compared with the cDNA sequence to determine the sequence accuracy. Through the above steps, the GhFBH1 gene was obtained.

[0029] Example 2: Application of the GhFBH1 gene in identifying cotton with low first fruiting branch height.

[0030] The GhFBH1 sequence contains non-synonymous SNP sites in the population, such as... Figure 1 As shown, the base changes from C to A at position 96 bp in the genome sequence. Based on the location of this SNP site on chromosome A05 (A05:16783268), amplification primers were designed at both ends (Table 2), such as the upstream primer F2 for SNP detection shown in SEQ ID NO.5 and the downstream primer R2 for SNP detection shown in SEQ ID NO.6. PCR amplification and sequencing were performed. The PCR reaction program was as follows: 98℃ pre-denaturation for 3 min; 95℃ denaturation for 15 sec, 57℃ annealing for 15 sec, 72℃ extension for 30 sec, 34 cycles; and a final extension at 72℃ for 5 min.

[0031] Based on the base information and sequencing results of this SNP locus (A05:16783268), the genotypes of each variety population at this SNP locus were analyzed, and 249 haplotype GhFBH1 (C / C) materials and 24 haplotype GhFBH1 (A / A) materials were identified. Figure 2 (See Table 3). Based on the GWAS association results and phenotypic survey data, the haplotype with higher first fruit branch height was labeled as GhFBH1. SNP- Haplotypes with lower first fruiting branch height were labeled GhFBH1. SNP+ ( Figure 2 SNP- indicates that the first fruiting branch is at a relatively high height, and SNP+ indicates that the first fruiting branch is at a relatively low height.

[0032] Meanwhile, the correlation between the height of the first fruiting branch of cotton between the two haplotypes was calculated using the t-test method. Figure 2The results show that, compared to GhFBH1 SNP- (C / C), haplotype GhFBH1 SNP+ The height of the first fruiting branch (A / A) decreased by 17.2%, which was significantly negatively correlated with the height of the first fruiting branch (P<0.001).

[0033] In summary, the above results all demonstrate that the GhFBH1 gene has significant research value in improving the height of the first fruiting branch in cotton and in the breeding of new cotton varieties. Firstly, molecular markers developed based on two different haplotypes of the GhFBH1 gene can be used to accurately identify the first fruiting branch height trait in cotton, which has extremely high practical value for cotton variety selection. Secondly, in the process of breeding cotton varieties with low first fruiting branch height, genetic engineering techniques can be used to directly introduce GhFBH1 genes containing low first fruiting branch height. SNP+ The (A / A) gene was introduced into cotton varieties to reduce the height of the first fruiting branch. Simultaneously, the haplotype GhFBH1, which exhibits high first fruiting branch height, could also be used. SNP- Mutating the SNP sites in (C / C) to transform them into haplotypes with low first fruit branch height contributes to the breeding of new cotton varieties in two ways.

[0034] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto. Any simple changes or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the scope of the technology disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A cotton gene GhFBH1, characterized in that, The nucleotide sequence of the cotton gene GhFBH1 is shown in SEQ ID NO.

2.

2. The application of the cotton gene GhFBH1 as described in claim 1 in identifying the height of the first fruiting branch of cotton.

3. The application of a reagent for detecting SNP sites in determining the height of the first fruiting branch of cotton, characterized in that, The SNP site is located at the 96th bp position of the cotton gene GhFBH1 according to claim 1, and the nucleotide sequence of the cotton gene GhFBH1 is shown in SEQ ID NO.2; the application is specifically as follows: using a reagent for detecting SNP sites to detect the nucleotide sequence at the 96th bp position, wherein cotton plants with a base of A at the 96th bp position are cotton plants with low first fruiting branch height, and cotton plants with a base of C at the 96th bp position are cotton plants with high first fruiting branch height.

4. The application according to claim 3, characterized in that, The reagents for detecting SNP sites include primer pairs: the upstream primer is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.

6.

5. A reagent kit, characterized in that, The kit contains primer pairs: the upstream primer is shown in SEQ ID NO.5, and the downstream primer is shown in SEQ ID NO.

6.

6. The application of the kit according to claim 5 in identifying the height trait of the first fruiting branch of cotton.

7. A method for improving the height of the first fruiting branch of cotton, characterized in that, The 96th bp site of the cotton gene GhFBH1 described in claim 1 was edited from C to A using the CRISPR / Cas9 system, or the genotype with the site being A was selected using SNP marker-assisted selection.

8. A method for breeding cotton varieties with low first fruiting branch height, characterized in that, Using genetic engineering techniques, materials carrying the cotton gene GhFBH1 allele as described in claim 1 are used as donors. The allele is introduced into the target variety through backcrossing or self-crossing. Genotype screening is performed using the aforementioned SNP markers, and combined with field phenotypic identification, to breed new cotton varieties with shortened growth periods.

Images