Cotton first fruit branch height trait associated kinesin domain-containing motor protein gene ghffbph and application

Abstract

This invention belongs to the field of biotechnology applications and discloses a gene containing a kinesin domain that is associated with the height trait of the first fruiting branch of cotton. GhFFBPH And its applications. This invention provides the cDNA sequence (SEQ ID NO.1) and genome sequence (SEQ ID NO.2) of this gene in tetraploid upland cotton TM-1, and the gene... GhFFBPH The gene contains a non-synonymous SNP located at 353 bp in the coding region, where the base changes from A to T, and the corresponding amino acid changes from Gln to Leu. The gene provided in this invention was obtained from cotton variety resequencing and genome-wide association analysis, and is significantly associated with the height trait of the first fruiting branch in cotton. Two haplotypes of this gene are used to distinguish the height phenotype of the first fruiting branch: the AA haplotype corresponds to the tall phenotype, and the TT haplotype corresponds to the short phenotype. This invention... GhFFBPH Genes have significant research value and application prospects in improving the height of the first fruiting branch of cotton and in breeding new varieties of cotton with high first fruiting branch height.

Description

Technical Field

[0001] This invention relates to the field of biotechnology applications, specifically to a kinesin gene containing a Kinesin domain that is associated with the height trait of the first fruiting branch of cotton. GhFFBPH and its applications. Background Technology

[0002] Cotton, as an important economic crop and textile raw material in my country and worldwide, has a plant architecture that is a key factor affecting the efficiency and yield of mechanized production. Among these factors, the first fruiting branch node position—the node on the main stem where the first fruiting branch grows and the height of that first fruiting branch—is a crucial component of plant architecture, directly related to cotton's early maturity, suitable planting density, and the feasibility of mechanized harvesting. This trait needs to be maintained within an appropriate range. If the first fruiting branch node position is too low or the first fruiting branch height is too short, it easily leads to twig hanging and soil contamination during mechanized harvesting, affecting the quality of the raw cotton; conversely, if it is too high, it may reduce the number of bolls per plant, limiting yield potential. Therefore, breeding varieties with suitable first fruiting branch node position and height is crucial for coordinating high yield, high quality, and efficient mechanized production. However, the height of the first fruiting branch is a complex quantitative trait controlled by multiple genes, and its molecular regulatory mechanism is not yet fully understood. Identifying key genes closely linked to this trait or with direct regulatory functions, and then using molecular marker-assisted selection or genetic engineering for targeted genetic improvement, has become an important strategy for accelerating the breeding process and achieving precise plant architecture design.

[0003] Genome-wide association study (GWAS) is a research strategy that analyzes the statistical associations between millions of single nucleotide polymorphism (SNP) markers and phenotypes across the entire genome to elucidate the genetic basis of complex traits. With the development and cost reduction of genome sequencing technology, combined with advancements in bioinformatics methods, GWAS has become a mainstream method for identifying key genes for complex human diseases and important agronomic traits in crops. This method typically does not require pre-defined candidate genes and features strong detection capabilities and high localization accuracy. It plays a crucial role in linkage marker development, target gene mining, and genetic analysis of complex traits, and has attracted significant attention in modern molecular breeding. In crop science, the application of GWAS has yielded many important results. For example, Belo et al. (2008) analyzed 553 maize inbred lines using 8,950 SNPs to locate genetic loci affecting oleic acid content, which is considered the first standardized GWAS in maize research. Huang et al. (2011) resequencing 517 local rice varieties, obtaining a massive amount of SNP data, and then performing association analysis on 14 agronomic traits, successfully identifying 80 related loci. Subsequent research by the same team (Huang et al., 2012) further uncovered several known genes controlling flowering time and yield traits through resequencing a larger population (950 accessions). In tomatoes, Lin et al. (2014) performed whole-genome resequencing on 360 tomato germplasm accessions from around the world. Through population differentiation analysis, they discovered for the first time a key variation site determining the color of the pinkish pericarp, namely… SlMYB12 A 603 bp deletion in the promoter region of a gene inhibits its expression, preventing the accumulation of flavonoids in the skin of mature pink tomatoes, thus causing differences between fresh and processed tomatoes. Zhou et al. (2015) identified 96 overlapping quantitative trait loci (QTLs) in 302 soybean materials through resequencing and GWAS, and newly identified loci associated with traits such as oil content and plant height. Furthermore, Zhang et al. (2016) used GWAS to analyze SNPs related to starch and protein content in wheat grains under high-temperature stress, screening for varieties with good heat stability; Fang et al. (2017) detected 245 genetic loci and 14 oil synthesis-related genes in 809 soybean varieties across 84 agronomic traits via GWAS; Huang et al. (2024) found that rice... Wx Genes are key sites that influence the taste and quality of rice.

[0004] GWAS has also played a crucial role in cotton genetic improvement research. With the completion of the upland cotton reference genome, this technology has provided strong support for gene mining of traits such as fiber quality, disease resistance, early maturity, and yield. A series of studies have made significant progress using association populations of different sizes: Fang et al. (2017) resequencing 318 upland cotton accessions identified 119 significantly associated loci, including 71 loci associated with yield, 45 with fiber quality, and 3 with Verticillium wilt resistance. Ma et al. (2018), based on resequencing data from 419 core upland cotton germplasms, discovered 7383 SNPs significantly associated with target traits and conducted in-depth analysis of some candidate genes controlling flowering and fiber traits. Regarding disease resistance and stress tolerance, Li et al. (2018) performed GWAS on 299 accessions and identified genes that exert Verticillium wilt resistance through the jasmonic acid pathway. GhTNL1 Liu et al. (2021) identified the major active site Fov7 and key genes for resistance to Fusarium wilt. GhGLR4.8 Regarding yield and quality traits, Li et al. (2020) screened for flowering-related candidate genes associated with early maturity. GhTOC1 and GhAGL8 Zhang et al. (2021) discovered candidate genes that are significantly associated with fiber length. GhF2KP Yang et al. (2023) identified candidate genes affecting lignin synthesis and Verticillium wilt resistance. GhMYB78Li et al. (2023) selected 16 cotton varieties that hold an important place in the history of cotton breeding in China as parents. Through four generations of hybridization and seven generations of self-pollination, they established a multiparent advanced generation intercross (MAGIC) population of upland cotton consisting of 920 recombinant inbred lines. Whole-genome sequencing was performed on the 16 parents and 920 recombinant inbred lines, yielding 5.6 TB of resequencing data. Sequence alignment and variant identification yielded 4,774,181 high-quality variants across the entire genome. Genome-wide association analysis was conducted using multi-year, multi-location phenotypic data and the high-quality variants, identifying 54 QTLs significantly associated with complex traits, 25 of which exhibited pleiotropic effects. Du et al. (2024) further identified 5 stable QTLs associated with lint percentage and boll weight, as well as 25 candidate genes. In summary, genome-wide association analysis (GWAS) can achieve high-precision gene localization and efficiently uncover natural allelic variations controlling important agronomic traits. It has become a core tool in the field of crop molecular genetics and breeding, and is of great significance for shortening breeding cycles and accelerating molecular design breeding. Functional markers closely linked to target traits developed using this strategy can significantly improve the efficiency and accuracy of breeding selection. However, how to transform the large number of association signals identified by GWAS into key genes with clear breeding value and elucidate their functional mechanisms remains a major challenge. Against this backdrop, this study aims to use GWAS to discover and validate a gene directly related to the height of the first fruiting branch in cotton, encoding a kinesin-containing kinesin protein. GhFFBPH This aims to provide new genetic resources and theoretical support for cotton plant type improvement.

[0005] Kinesin-domain kinesin proteins are a class of microtubule-dependent molecular motor proteins widely distributed in eukaryotic cells. They utilize energy generated from ATP hydrolysis to directionally transport various "cargo" along the microtubule "tracks" of the cytoskeleton, acting as core executors of intracellular substance transport and organelle localization. In plant cells, the functions of these proteins are particularly unique and crucial because plant cells have fixed cell walls, and intracellular transport is essential for cell morphogenesis and environmental responses. Kinesin-domain kinesin proteins participate in a variety of important biological processes within the cell, including vesicle transport, organelle localization, spindle formation, and cell elongation. Summary of the Invention

[0006] The purpose of this invention is to provide a gene encoding a kinesin-containing kinesin protein. GhFFBPH Its applications. Genome-wide association analysis results showed that this gene was closely related to the height trait of the first fruiting branch in cotton.

[0007] The objective of this invention can be achieved through the following technical solutions: This invention provides a gene containing a kinesin domain kinesin protein. GhFFBPH The cDNA sequence in the allotetraploid upland cotton TM-1 is shown in SEQ ID NO.1, and the genome sequence is shown in SEQ ID NO.2; among 920 lines, based on the GWAS association site A05:15959491 for the first fruiting branch height trait, the gene... GhFFBPH The gene contains a non-synonymous SNP site located at position 353 bp in the coding region (SEQ ID NO.1), where the base changes from A to T, and the corresponding amino acid changes from Gln to Leu. Two haplotypes of this gene are used to distinguish the height phenotype of the first fruiting branch: the AA haplotype corresponds to the tall phenotype, and the TT haplotype corresponds to the short phenotype.

[0008] This invention also provides a gene containing a kinesin domain kinesin. GhFFBPH Application in identifying the height of the first fruiting branch in upland cotton varieties. Primer sequences were designed for the SNP locus of this gene. By amplifying the genotype of this SNP locus in the upland cotton population, the population was divided into two haplotype groups. Statistical analysis of the first fruiting branch height trait between the two groups revealed a significant difference in the height of the first fruiting branch between the two haplotype groups.

[0009] This invention also provides a gene containing a kinesin domain kinesin. GhFFBPH Application in improving the height trait of the first fruiting branch in cotton.

[0010] This invention also provides a gene containing a kinesin domain kinesin. GhFFBP Application of H in the breeding of new cotton varieties with high first fruiting branch height through genetic engineering.

[0011] The present invention also provides a primer pair for detecting the SNP site, wherein the upstream primer is SEQ ID NO.5 and the downstream primer is SEQ ID NO.6; the application of the primer pair in screening cotton varieties with high first fruiting branch height.

[0012] The present invention also provides a method for screening cotton varieties with high first fruiting branch height, wherein a SNP site is detected, and cotton with base A at position 353 bp of the coding region sequence is selected as the cotton variety with high first fruiting branch height.

[0013] The beneficial effects of this invention are as follows: (1) This study discovered a novel gene highly associated with the first fruiting branch, a key plant architecture trait in cotton, through resequencing and genome-wide association analysis of cotton MAGIC (multi-parent high-generation recombinant self-pollinated population). Using resequencing data and precise phenotypic identification from natural cotton populations, the gene encoding a kinesin-containing kinesin protein was successfully identified and located. GhFFBPH The discovery of this gene provides a direct key molecular target for understanding and regulating the height of the first fruiting branch in cotton, a core trait that determines adaptability to mechanized harvesting.

[0014] (2) The present invention provides GhFFBPH Gene cDNA and genomic sequences can be stably obtained using conventional PCR techniques. This technique has the advantages of requiring a small amount of starting template, having simple experimental steps, high sensitivity, and good specificity, making it convenient to complete gene cloning and sequence verification under ordinary laboratory conditions, thus laying a reliable technical foundation for subsequent functional research and application development.

[0015] (3) This invention confirms that GhFFBPH Different genotypes of the gene can divide the MAGIC population into two major categories. Statistical analysis revealed a significant difference in the height of the first node of the fruiting branch between these two groups, further demonstrating the correlation between this gene and cotton quality traits. Attached Figure Description

[0016] Figure 1 Candidate genes for GWAS sites GhFFBPH Haplotype association analysis and expression profiles; where the horizontal axis represents the location on the chromosome (Mb), and the vertical axis represents the significance of the SNP site association, expressed as -log10 (P value).

[0017] Figure 2 for GhFFBPH Expression levels in different tissues and developmental stages of cotton; the horizontal axis represents different tissues, including leaf, stem, root, ovule, and fiber; root tissue includes 24 to 120 hours of root growth. Ovule tissue includes 3 and 1 days before flowering, on the day of flowering (day 0), and 1 to 35 days after flowering; fiber tissue includes 5 to 25 days after flowering.

[0018] Figure 3 for GhFFBPH Sequence information and identification of different haplotypes; detected in the variety population GhFFBPH There is a non-synonymous SNP site in the exon region of the sequence, located at position 353 bp in the coding region. The base at this SNP site changes from A to T, and the corresponding amino acid changes from Gln to Leu.

[0019] Figure 4 For genes GhFFBPH SNP haplotype association analysis; the x-axis represents different haplotypes and sample size, and the y-axis represents the height of the first fruit branch; there are 613 and 125 varieties containing haplotypes AA and TT, respectively; **** indicates a difference at the 0.0001 level; ns indicates no significant difference. Detailed Implementation

[0020] Example 1 Genes containing kinesin domains that are associated with the height trait of the first fruiting branch in cotton. GhFFBPH The excavation.

[0021] A multi-parental, high-generation recombinant self-pollination population (MAGIC population) was constructed from 920 cotton lines and planted in Shihezi and Kuitun, Xinjiang in 2024. Three replicates of each line were planted in the field to conduct a detailed survey of the height of the fruiting branch initiation node. Simultaneously, whole-genome resequencing was performed on these 920 lines, yielding 4.4 Tb of sequencing data with an average sequencing depth of 3.5×. These sequences were aligned to the genome sequence of cotton upland cotton TM-1 (TM-1_V2.1), and bioinformatics software was used to identify whole-genome variations. A total of 4,774,181 high-quality variations (minimum gene frequency >0.05) were identified for subsequent analysis.

[0022] First, perform a genome-wide association analysis, then based on P < 1 × 10⁻⁶. -6 Associated signaling loci were screened. Analysis of these associated loci revealed a signaling locus (A05:15959491) on chromosome A05 that was significantly associated with the height of the fruiting branch's first node. Figure 1 This associated site is located precisely in the exon region of the gene and causes a change in the amino acid sequence. This associated site is located within the Kinesin domain of the kinesin protein. GhFFBPH For this study, RNA samples from different tissues and developmental stages of the cotton TM-1 variety were collected for transcriptome sequencing. Sample materials included roots, stems, leaves, ovules, and fibers. Ovule tissue samples included samples from 3 and 1 days before flowering, on the day of flowering, and 1 to 25 days after flowering. Fiber tissue samples included samples from 5 to 25 days after flowering. Transcriptome sequencing was performed using the Illumina HiSeq 2500 platform, with an average sequencing depth of 6 Gb per sample. GhFFBPH The gene expression level is calculated by comparing the reads obtained from sequencing with the upland cotton genome. The calculated expression level is expressed as the number of sequencing fragments (FPKM) per thousand transcripts per million sequencing bases.

[0023] Experimental results are as follows Figure 2As shown, this gene is predominantly expressed in ovules 3 and 1 days before flowering, seeds 1, 3, 5, 10, and 20 days after flowering, and roots 24, 48, 72, 96, and 120 hours after flowering. This indicates that the gene is associated with the factors that constitute the height of the first fruiting branch and is a gene that simultaneously improves the height of the first fruiting branch in cotton.

[0024] Example 2 Genes containing kinesin domains that are associated with the height trait of the first fruiting branch in cotton. GhFFBPH The acquisition.

[0025] The genome sequence of upland cotton was obtained. GhFFBPH The cDNA and genome sequences are shown in SEQ ID NO.1 and SEQ ID NO.2. Full-length primers were designed based on both ends of the cDNA for PCR amplification. The primer sequences are F1 (shown in SEQ ID NO.3) and R1 (shown in SEQ ID NO.4). The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 60℃ annealing for 1 min, 72℃ extension for 1 min, 30 cycles; and a final extension at 72℃ for 10 min. The PCR amplification products were sequenced and further compared with the cDNA to determine sequence accuracy.

[0026] Example 3 Genes containing kinesin domains GhFFBPH Application in identifying cotton varieties with first fruiting branch height and improving the first fruiting branch height trait.

[0027] Based on the location of the SNP locus (A05:15959491) on chromosome A05, genome amplification primers were designed at both ends of the locus. The primer sequences are F2: SEQ ID NO.5 and R2: SEQ ID NO.6. Using these primers, PCR amplification and sequencing were performed on the DNA of 920 varieties. The PCR reaction program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 sec, 58℃ annealing for 1 min, 72℃ extension for 45 sec, 30 cycles; and a final extension at 72℃ for 10 min. The genotype of each variety population at this SNP locus was analyzed based on the sequencing results.

[0028] Confirmed GhFFBPH The sequence contains a nonsynonymous SNP site located at position 353 bp in the coding region. The base at this SNP site changes from A to T, corresponding to an amino acid change from Gln to Leu. Based on the base information of this SNP site, modern upland cotton varieties (lines) are divided into two haplotypes: AA and TT. Figure 3 ).

[0029] This embodiment detected SNP sites in 738 materials and determined the phenotype of each sample. The results are as follows: Figure 4 As shown, there are 613 and 125 materials containing haplotypes AA and TT, respectively (as shown in Table 1, which lists 738 materials).

[0030] The results of the embodiments of the present invention illustrate that genes GhFFBPH It has important research value in improving the height trait of the first fruiting branch in cotton and in breeding new cotton varieties. On the one hand, it can be based on genes. GhFFBPH The design of haplotype molecular markers can effectively identify the height trait of the first fruiting branch in cotton, which has great application value in the breeding research of high-quality fiber cotton varieties.

[0031] Obviously, those skilled in the art can make various modifications and variations to this technical solution without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this technical solution and their equivalents, this technical solution also intends to include these modifications and variations.

[0032] Table 1. Identification of haplotypes of first fruiting branch height and first fruiting branch low in population materials.

Claims

1. A gene containing a kinesin domain that is associated with the height trait of the first fruiting branch in cotton. GhFFBPH Its characteristics are, The cDNA sequence of the gene in tetraploid upland cotton TM-1 is shown in SEQ ID NO.1, and the genome sequence is shown in SEQ ID NO.

2.

2. The gene according to claim 1 GhFFBPH Its characteristics are, The gene GhFFBPH The SNP site contains a nonsynonymous mutation located at 15959491 bp on chromosome A05 of the upland cotton TM-1 genome; the base of the SNP site is "A" or "T"; the base of the SNP site is "A", which encodes the amino acid Gln; the base of the SNP site is "T", which encodes the amino acid Leu.

3. The gene containing a kinesin domain as described in claim 1 GhFFBPH Application in identifying upland cotton varieties with the height of the first fruiting branch.

4. The gene containing a kinesin domain as described in claim 1 GhFFBPH Application in improving the height trait of the first fruiting branch.

5. The gene containing a kinesin domain kinin protein according to claim 1 GhFFBPH Application in the development of new cotton varieties with high first fruiting branch height through genetic engineering.

6. A method for detecting the height of the first fruiting branch of cotton, characterized in that, By detecting the SNP sites described in claim 2.

7. The method according to claim 6, characterized in that, The method specifically involves using primer pairs to detect the SNP sites in upland cotton, with the AA haplotype corresponding to the tall phenotype on the first fruiting branch and the TT haplotype corresponding to the dwarf phenotype on the first fruiting branch.

8. The method according to claim 6, characterized in that, The upstream primer sequence of the primer pair is shown in SEQ ID NO. 5, and the downstream primer sequence is shown in SEQ ID NO. 6.

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