Small peptide protein gene GhSP1 molecular marker related to verticillium wilt resistance of cotton and application of small peptide protein gene GhSP1 molecular marker

Abstract

The invention discloses a small peptide protein gene GhSP1 molecular marker related to cotton verticillium wilt resistance and application of the small peptide protein gene GhSP1 molecular marker. The small peptide protein gene GhSP1 has two haplotypes in an upstream promoter region, and compared with a haplotype 2, the haplotype 1 has insertion of a 5bp nucleotide fragment (GATCT) at a promoter sequent-591bp. An InDel functional marker capable of remarkably distinguishing the haplotype 1 and the haplotype 2 is further developed. Compared with the cotton small peptide protein gene GhSP1 corresponding to the haplotype 1, the cotton small peptide protein gene GhSP1Gb corresponding to the haplotype 2 has the advantages that the expression level, the verticillium wilt induced expression level and the disease resistance of the cotton small peptide protein gene GhSP1Gb corresponding to the haplotype 2 are superior to those of the haplotype 1. The haplotype 2 has important effects and application prospects in improvement of verticillium wilt resistance of cotton and cultivation of new varieties of cotton with high verticillium wilt resistance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology applications and relates to a molecular marker for GhSP1, a small peptide protein gene associated with resistance to cotton Verticillium wilt, and its application. Background Technology

[0002] Cotton is an important economic crop and a major strategic resource, holding an irreplaceable position in my country's national economy. Verticillium wilt is a significant factor limiting cotton yield and quality, causing losses of billions of yuan in severe years and constituting one of the major obstacles to sustainable cotton production in my country (Lin et al., 2014; Yu, 2018). Verticillium wilt pathogen is a soil-borne vascular tissue pathogen with a wide host range, rapid spread, long survival time, and high mutation frequency, making it difficult to control in the field (Zhang et al., 2022). Currently, in major cotton-growing areas such as Xinjiang, the severity of Verticillium wilt is increasing, and chemical pesticide control is ineffective and pollutes the environment. Therefore, it is urgent to combine resistant cotton germplasm resources to deeply analyze the molecular genetic basis of cotton resistance to Verticillium wilt, explore the interaction mechanism between cotton and Verticillium wilt pathogen, and discover key resistance genes.

[0003] Sea island cotton and upland cotton are currently the two main cultivated varieties. Upland cotton has high yield and wide adaptability, and is widely planted globally, accounting for over 90% of the planted area and approximately 97% of global production. However, it generally lacks resistance to Verticillium wilt. Sea island cotton, on the other hand, has high resistance to Verticillium wilt and has long been considered an ideal material for discovering cotton disease resistance genes and elucidating disease resistance mechanisms. By hybridizing sea island cotton and upland cotton and performing multiple generations of backcrossing, combined with Verticillium wilt resistance trait screening, superior substitution lines carrying chromosomal segments related to sea island cotton disease resistance can be obtained, also known as Chromosome Segment Introgression Lines (CSILs). These introgression lines have a single genetic background against the upland cotton genome. Combined with techniques such as marker-assisted selection or transcriptome analysis, it is easy to accurately locate disease resistance loci and discover key disease resistance genes in sea island cotton, which is beneficial for rapidly improving the disease resistance improvement process in cotton breeding (Benbouza et al., 2010).

[0004] In recent years, with the release and continuous updating of cotton genome data, comparative genomics has provided strong support for the development of novel genetic markers. Among them, insertion / deletion (InDel) markers, as a novel type of molecular marker based on nucleotide sequence structural variations, have shown significant value in population genetic structure analysis, important trait gene localization, and marker-assisted breeding due to their outstanding advantages such as co-dominance, high stability, and convenient detection. In cotton Verticillium wilt resistance breeding research, developing InDel markers based on genomic differences between marine and upland species has become an efficient and precise strategy. van Deynze et al. (2009) identified 279 interspecific InDel loci by comparing the EST sequences of island cotton and upland cotton, of which as many as 200 were successfully integrated into the genetic map, laying an important foundation for subsequent gene localization. Lü et al. (2010) analyzed the EST-SSR distribution of cotton based on the Sea Island cotton EST database, identifying an InDel marker system suitable for detecting intraspecific subgroup differentiation and interspecific variation. This system can be used to detect polymorphism between Sea Island and Upland cotton, laying the foundation for constructing saturated genetic maps and comparative genomics studies among cotton species. Because InDel markers can accurately identify genotypes and avoid environmental interference, InDel marker-assisted selection technology is gradually becoming one of the core methods for breeding disease-resistant cotton varieties. Closely linking these markers with resistance genes can achieve early and efficient screening of disease-resistant materials, significantly improving breeding efficiency and providing key technical support for the genetic improvement of cotton disease resistance.

[0005] Small peptides are formed by amino acids linked together by peptide bonds, typically consisting of fewer than 100 amino acids, and possess important biological functions (Tavormina et al., 2015). The first compound to be extracted from the animal gastrointestinal tract that stimulated pancreatic juice secretion was named secretin, marking the first discovery of small peptides in organisms (Bayliss and Starling, 1902). Subsequently, numerous small peptide molecules were identified, widely involved in animal growth, development, and disease resistance (Li et al., 2006). Research on small peptides in plants is still relatively preliminary. Systemin was the first small peptide discovered in tomato, comprising 18 amino acid residues processed from the 200-amino acid precursor of protosysteminase, and mainly involved in plant responses to pests and diseases (Ryan et al., 1998; Pearce et al., 2003). Small peptides play an important role in intercellular information sensing and transmission after plant stress responses. Most small peptides are encoded by precursor genes, which typically possess family-specific conserved sequences at their C-terminus. These precursors can be cleaved by proteases and modified post-translationally to produce mature functional small peptides (Ogawa-Ohnishi et al., 2022). Most mature small peptides can be transported to the cell membrane via secretion, where they are recognized and bound by the extracellular domain of receptor kinases located on the plasma membrane surface. Some small peptides are secreted extracellularly, then transported over long distances to bind to receptors, inducing activation of intracellular kinase domains. This activation leads to phosphorylation of downstream signal transduction genes such as the MAPK cascade or transcription factors, regulating plant tolerance to abiotic stress (Hohmann et al., 2017; Fletcher et al., 2020). Small peptide protein genes often exhibit highly conserved domains across different plants, and their functions have been reported in various plants. For example, in tomato, the small peptide PSK interacts with the calcium-dependent protein kinase CPK28 via its receptor PSKR1, phosphorylating downstream proteins to promote plant defense responses (Ding et al., 2022). RGF7 peptide is a novel damage-associated molecular pattern that can be recognized by RGI4 / 5 receptors and, together with co-receptors SERK4 and BAK1, induces an immune response in Arabidopsis thaliana (Wang et al., 2021). Exogenous application of the potato peptide StPIP1 can increase the expression of reactive oxygen species-related genes in leaves and internodes, and overexpression of StPIP1 can induce activation of the plant's immune response, enhancing resistance to Potato Virus Y (Combest et al., 2021). Numerous studies have demonstrated the potential application value of small peptide signaling molecules in the genetic improvement of crop disease resistance. Summary of the Invention

[0006] The purpose of this invention is to provide a molecular marker for the small peptide protein gene GhSP1, which is associated with resistance to Verticillium wilt in cotton, and its application.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] In a first aspect, the present invention seeks protection for the use of an InDel molecular marker associated with the cotton resistance to Verticillium wilt trait or a substance for detecting the InDel molecular marker, wherein the InDel molecular marker is an insertion of a 5 bp nucleotide fragment GATCT at -591 bp of the promoter sequence of the cotton small peptide protein gene GhSP1, as shown in SEQ ID NO.1.

[0009] The application is at least one of the following (a1)-(a5):

[0010] (a1) Screening or assisting in the screening of cotton germplasm with different resistance to Verticillium wilt, or preparing products for screening or assisting in the screening of cotton germplasm with different resistance to Verticillium wilt;

[0011] (a2) To identify or assist in the identification of cotton resistance to Verticillium wilt, or to prepare products for the identification or assistance in the identification of cotton resistance to Verticillium wilt;

[0012] (a3) Identify the promoter haplotype of cotton small peptide protein gene GhSP1, or prepare a product for identifying the promoter haplotype of cotton small peptide protein gene SP1;

[0013] (a4) Improve cotton’s resistance to Verticillium wilt, or prepare products for improving cotton’s resistance to Verticillium wilt;

[0014] (a5) Breeding of cotton with high resistance to Verticillium wilt, or preparing products for breeding of cotton with high resistance to Verticillium wilt.

[0015] Furthermore, in the above application, the promoter region of the cotton peptide protein gene GhSP1 has two haplotypes. The genomic nucleotide sequence of haplotype 1 is shown in SEQ ID NO.1, and the genomic nucleotide sequence of haplotype 2 is shown in SEQ ID NO.4. Haplotype 1 has a 5 bp nucleotide fragment GATCT inserted at -591 bp in the promoter sequence compared to haplotype 2. Compared with the cotton peptide protein gene GhSP1 corresponding to haplotype 1, the cotton peptide protein gene GhSP1 corresponding to haplotype 2 has a different nucleotide sequence. Gb The expression level in plant roots, the expression level induced by Verticillium wilt, and the disease resistance were all superior to haplotype 1.

[0016] Secondly, the present invention claims protection for a product containing the aforementioned substance for detecting InDel molecular markers, and which is at least one of the following (b1)-(b5):

[0017] (b1) Products for screening or assisting in screening cotton germplasm with different Verticillium wilt resistance;

[0018] (b2) Products used to identify or assist in the identification of cotton resistance to Verticillium wilt;

[0019] (b3) Products for identifying the promoter haplotype of the cotton small peptide protein gene GhSP1;

[0020] (b4) Products used to improve cotton's resistance to Verticillium wilt;

[0021] (b5) Products used in the breeding of cotton with high resistance to Verticillium wilt.

[0022] Thirdly, the present invention claims protection for a method, which is at least one of the following methods (c1)-(c5):

[0023] (c1) Methods for screening or assisting in the screening of cotton germplasm with high resistance to Verticillium wilt;

[0024] (c2) Methods for identifying or assisting in the identification of cotton Verticillium wilt resistance;

[0025] (c3) A method for identifying the promoter haplotype of the cotton small peptide protein gene GhSP1;

[0026] (c4) Methods to improve cotton's resistance to Verticillium wilt;

[0027] (c5) Methods for breeding cotton with high resistance to Verticillium wilt;

[0028] This method involves using a substance that detects InDel molecular markers to determine whether the promoter region of the cotton small peptide protein gene GhSP1 contains the InDel molecular markers as described above, thereby identifying the haplotype of the GhSP1 promoter. The GhSP1 promoter region contains two haplotypes. Haplotype 1 has a 5 bp nucleotide fragment GATCT insertion at -591 bp in the promoter sequence, unlike haplotype 2. Haplotype 2 exhibits superior resistance to Verticillium wilt. Cotton germplasm carrying haplotype 2 is selected as cotton germplasm with high resistance to Verticillium wilt for cotton breeding to improve cotton resistance to the disease.

[0029] In a specific embodiment of the present invention, PCR amplification is performed using InDel molecular marker primers, and different haplotypes are identified based on the presence or absence of the target band in the amplification product. Haplotype 1 is the one that amplifies a product with a molecular weight of 464 bp using InDel molecular marker primers, while haplotype 2 is the one for which the target band cannot be obtained.

[0030] In the technical solution of this invention, the substance used to detect the InDel molecular marker is one of the following (d1) or (d2) or (d3) or (d4):

[0031] (d1) Contains in vitro nucleic acid amplification primers as shown in SEQ ID NO.7 and SEQ ID NO.8 that specifically amplify the InDel molecular marker;

[0032] (d2) In vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primers described in (d1);

[0033] (d3) A kit containing the in vitro nucleic acid amplification primers described in (d1) or the in vitro nucleic acid amplification reagents described in (d2);

[0034] (d4) A detection instrument containing the in vitro nucleic acid amplification primers described in (d1), the in vitro nucleic acid amplification reagents described in (d2), or the kit described in (d3).

[0035] In the technical solution of this invention, the trait of resistance to Verticillium wilt in cotton is at least one of the disease phenotype and the incidence rate.

[0036] Fourthly, the present invention seeks protection for the small peptide protein gene GhSP, whose nucleotide sequence is shown in SEQ ID NO.2, or the small peptide protein gene GhSP1, as shown in SEQ ID NO.5. Gb Promote the small peptide protein gene GhSP1 or GhSP1 Gb The expressed substance or the enhancement of the small peptide protein gene GhSP1 or GhSP1 Gb The application of substances encoding protein activity or abundance in the following e1) or e2) :

[0037] el) Improve cotton resistance to Verticillium wilt or cultivate new germplasm with improved resistance to Verticillium wilt;

[0038] e2) Improve the disease resistance of target plants.

[0039] This invention demonstrates that overexpression of the small peptide protein genes GhSP1 and GhSP1 in Arabidopsis thaliana can effectively improve the efficacy of this invention. Gb Compared to the wild type (WT), the overexpressed Arabidopsis thaliana strains showed stronger resistance to Verticillium wilt.

[0040] Furthermore, in the above applications, the small peptide protein gene GhSP1 or GhSP1 is promoted. Gb The expressed substance or the small peptide protein gene GhSP1 or GhSP1 Gb The substance encoding the activity or abundance of a protein is a biological material, and the biological material is any one of the following (fl) to (f6):

[0041] f1) Contains the small peptide gene GhSP1 or GhSP1 Gb Expression box;

[0042] f2) Contains the small peptide gene GhSP1 or GhSP1 Gb The recombinant vector, or the recombinant vector containing the expression cassette described in f1);

[0043] f3) Contains the small peptide gene GhSP1 or GhSP1 Gb Recombinant microorganisms, or recombinant microorganisms containing the expression cassette described in f1), or recombinant microorganisms containing the recombinant vector described in f2);

[0044] f4) Contains the small peptide gene GhSP1 or GhSP1 Gb The transgenic plant cell line, or the transgenic plant cell line containing the expression cassette described in f1), or the transgenic plant cell line containing the recombinant vector described in f2);

[0045] f5) Contains the small peptide gene GhSP1 or GhSP1 Gb The transgenic plant tissue, or the transgenic plant tissue containing the expression cassette described in f1), or the transgenic plant tissue containing the recombinant vector described in f2);

[0046] f6) Contains the small peptide gene GhSP1 or GhSP1 Gb The transgenic plant organ, or the transgenic plant organ containing the expression cassette described in f1), or the transgenic plant organ containing the recombinant vector described in f2).

[0047] Furthermore, the amino acid sequence of the protein encoded by the small peptide gene GhSP1 is shown in SEQ ID NO.3. Gb The amino acid sequence encoding the protein is shown in SEQ ID NO.6.

[0048] Fifthly, this invention claims protection for a method to improve resistance to Verticillium wilt in cotton, using the small peptide protein gene GhSP1 or GhSP1 Gb The target gene is the small peptide protein gene GhSP1 with the nucleotide sequence shown in SEQ ID NO.2 or the small peptide protein gene GhSP1 with the nucleotide sequence shown in SEQ ID NO.5, which is overexpressed in cotton through genetic transformation. Gb To improve cotton's resistance to Verticillium wilt.

[0049] In the early stages of this invention, a partial genomic fragment from the island cotton Hai7124 was introgressed into upland cotton TM-1 through multiple rounds of crosses, constructing a set of introgression lines. Seedling resistance identification to Verticillium wilt identified an important resistant material, IL64, and further investigation was conducted to uncover key resistance-related genes and molecular markers. Genome resequencing results showed that a genomic fragment from Hai7124 was introgressed into the A09 chromosome 63-71 Mb region of IL64. Combined transcriptome sequencing and RT-qPCR analysis of Hai7124 and TM-1, a novel small peptide gene, SP1, was identified. SP1 encodes 66 amino acids, with a signal peptide consisting of 28 amino acids at the N-terminus, a transmembrane domain consisting of 23 amino acids at the C-terminus, and a 15-amino acid extracellular domain located on the outer membrane. The expression level of SP1 in Hai7124 roots and its expression level induced by Verticillium wilt were significantly higher than that in TM-1. In Hai7124 and TM-1, the SP1 protein differs only in the 17th amino acid position by an S / A ratio, but this does not affect the protein's intracellular localization. SP1 is localized within the inner membrane system composed of the cell membrane, endoplasmic reticulum, and nucleus, and cannot be secreted into the apoplast. In Hai7124, inhibiting SP1 gene expression significantly weakens the plant's resistance to Verticillium wilt, while silencing SP1 in TM-1 has no effect on resistance. We named the SP1 genes derived from TM-1 and the TM-1-based infiltration line IL64 GhSP1 and GhSP1, respectively. Gb GhSP1 Gb The homologous gene was named GbSP1 in Hai7124. Overexpression of GhSP1 and GhSP1 in Arabidopsis thaliana... Gb Both significantly improved the resistance of Arabidopsis thaliana plants to Verticillium wilt. Promoter analysis revealed that, compared to the introgressive IL64 of Hai7124, a five-base GATCT insertion was present 591 bp upstream of the GhSP1 gene in TM-1. This insertion was present in all 335 natural populations of upland cotton, while the SP1 promoter region lacked the GATCT sequence in all 269 natural populations of sea island cotton. The sequence type of the differentially expressed region of the GhSP1 gene in the promoter of TM-1 was named haplotype 1, and the GhSP1 gene with the Hai7124 genotype in IL64 was identified as haplotype 1. Gb The sequence type in the differentially expressed region of the promoter of the gene is named haplotype 2. This applies to GhSP1 and GhSP2. Gb A functional molecular marker was developed using a 5 bp insertion / deletion site between two haplotypes on the promoter, and the accuracy of the marker was validated in populations of Sea Island cotton and Upland cotton. GhSP1 in IL64 GbThe transcriptional activity of the gene promoter was significantly higher in TM-1 both before and after Verticillium wilt infection. GATCt is a conserved binding element of GATA transcription factors. Three GATA transcription factors, GATA8 / 9 / 10, are upregulated in cotton by Verticillium wilt, with induction levels similar to SP1. Yeast one-hybrid and gel migration assays demonstrated that GATA10 can bind to the promoter sequence of GhSP1 in TM-1, but not to GhSP1 in IL64. Gb The promoter sequence was obtained; in addition, based on the LUC reporter gene expression system, it was demonstrated that GATA10 in upland cotton can inhibit the transcriptional activity of the GhSP1 promoter, and the results revealed the important role of the GATCt sequence in regulating SP1 expression and cotton disease resistance.

[0050] Using the functional molecular markers screened in this invention, differential sites of the small peptide protein gene SP1 among different cotton haplotypes were detected using marker-specific primers. Samples carrying haplotype 2 of the small peptide protein gene SP1 were selected as cotton germplasm possessing high resistance to Verticillium wilt. The sequences of the marker-specific forward and reverse primers are shown in Table 4. All detections described herein were performed at the DNA level. This study shows that when haplotype 2 is present in cotton plants, the allele promotes Verticillium wilt resistance, indicating that haplotype 2 of this gene plays an important role and has promising applications in improving cotton resistance to Verticillium wilt and breeding new disease-resistant varieties.

[0051] The development process of the technical solution of this invention specifically includes the following steps:

[0052] 1. Based on the Hai7124-upland cotton TM-1 infiltration line population, seedling resistance to Verticillium wilt was identified, and the IL64 strain with significantly improved disease resistance was screened.

[0053] 2. Using genome resequencing, a fragment from Hai7124 was identified as introgressed into the 63-71 Mb region of chromosome A09 in the IL64 strain. Combined with transcriptome analysis of tissues, organs, and roots of both marine and terrestrial materials, the small peptide protein gene SP1 was identified as being highly expressed only in Hai7124 and significantly upregulated after Verticillium wilt induction. The SP1 genes originating from TM-1 and from the TM-1-based marine-terrestrial introgression line IL64 were named GhSP1 and GhSP1, respectively. Gb GhSP1 Gb In the context of Hai7124, it is also known as GbSP1.

[0054] 3. Gene cloning and sequence analysis revealed that SP1 differs from TM-1 by only one amino acid, but this did not affect the protein's localization in the endometrial system.

[0055] 4. Specific silencing of SP1 in Hai7124 significantly weakened plant resistance to Verticillium wilt, while silencing in TM-1 had no effect; overexpression of GhSP1 and GhSP1 in Arabidopsis thaliana... Gb After (GbSP1) treatment, the disease resistance of the plants was significantly improved.

[0056] 5. Regarding GhSP1 and GhSP1 Gb Cloning and alignment of the promoter 1271 bp upstream of the (GbSP1) gene revealed an insertion of five GATCT bases 591 bp upstream of the GhSP1 gene in TM-1. The promoter type of the small peptide protein gene SP1 in TM-1 was named haplotype 1, and the promoter type in IL64 (Hai7124 introgression) was named haplotype 2. The genomic sequence of GhSP1 for haplotype 1 is SEQ ID NO.1, the CDS sequence is SEQ ID NO.2, and the encoded amino acid sequence is SEQ ID NO.3. The GhSP1 sequence for haplotype 2... Gb The genome sequence of (GbSP1) is SEQ ID NO.4, the CDS sequence is SEQ ID NO.5, and the encoded amino acid sequence is SEQ ID NO.6. Haplotype 1 has a 5 bp nucleotide insertion 591 bp upstream of the ATG compared to haplotype 2.

[0057] 6. In GhSP1 and GhSP1 Gb Functional molecular markers were developed near the 5 bp difference in the promoter region, and the R primers of the markers were placed in the difference region to distinguish between the two haplotypes. Genotyping of this locus was performed using natural populations of sea island cotton and upland cotton to confirm the accuracy of the markers.

[0058] 7. Promoter activity analysis showed that GbSP1 (named GhSP1 in IL64) originated from Hai7124. Gb The GATA10 promoter exhibited stronger transcriptional activity before and after induction by Verticillium wilt. Combined with inducible expression analysis, yeast single-hybrid assays, EMSA, and LUC luciferase reporter assays, it was found that the GATA10 transcription factor binds to the GATCt element in the promoter region and inhibits GhSP1 expression and plant resistance in upland cotton TM-1. This demonstrates the important role of the GATCt sequence marker in cotton Verticillium wilt resistance.

[0059] The advantages of this invention are as follows:

[0060] 1. This invention, based on the highly resistant Verticillium wilt (Verticillium wilt) sea island cotton Hai7124-upland cotton TM-1 introgression line, combined with tissue, organ, and Verticillium wilt-induced transcriptome data, identified a gene encoding a small peptide 1 (SP1), GH_A09G1406. Its homologous gene is highly expressed in the roots of Hai7124 and induced by Verticillium wilt, but its expression is low and almost uninduced in TM-1, making it an important candidate gene for Verticillium wilt resistance. The SP1 genes derived from TM-1 and the TM-1-based sea-upland introgression line IL64 are named GhSP1 and GhSP1, respectively. Gb GhSP1 Gb In the context of sea 7124, it is also known as GbSP1.

[0061] 2. Based on the differences in the promoter sequence of SP1 in different cotton materials, upland cotton and sea island cotton can be clearly divided into two categories: upland cotton is haplotype 1, and IL64 and sea island cotton are haplotype 2. Haplotype 2 exhibits better resistance to Verticillium wilt than haplotype 1.

[0062] 3. SP1 is located in the inner membrane system, and its disease resistance function differences between marine and land cotton materials are caused by differences in promoter sequence and gene transcription level.

[0063] 4. A functional molecular marker was developed using a 5 bp insertion / deletion site between two haplotypes of SP1 to distinguish between the two haplotypes of the small peptide protein gene SP1. This marker can be used as a molecular marker to assist in screening or identifying whether a sample contains haplotype 2 of the aforementioned small peptide protein gene SP1.

[0064] 5. Inhibiting GbSP1 expression in Hai7124 weakens plant disease resistance, while overexpression of GbSP1 / GhSP1 in Arabidopsis increases plant disease resistance, indicating that SP1 positively regulates cotton Verticillium wilt resistance.

[0065] 6. The key transcription factor GATA10 was identified as inhibiting gene expression by binding to the GATCt element of the GhSP1 promoter in upland cotton, clarifying the reason why haplotype 2 has higher resistance to Verticillium wilt than haplotype 1, and providing an important theoretical basis for improving the disease resistance trait of cotton. Attached Figure Description

[0066] Figure 1 Disease resistance phenotype analysis and identification of key disease resistance candidate genes in the marine-terrestrial infiltration line IL64.

[0067] The study included: a) observation of disease phenotype in the Hai7124-TM-1 infiltrative line IL64 18 and 25 days after inoculation; b) detection of the average diseased leaf rate of IL64 infiltrative line plants; cd) observation of stem vascular tissue and analysis of fungal recovery ability 10 days after inoculation; e) identification of the infiltrative fragment in the 63-71 Mb region on chromosome A09 based on resequencing; f) analysis of disease resistance candidate genes with high expression levels in roots and induced by Verticillium wilt, with green boxes showing the expression and induced expression characteristics of the SP1 gene in different tissues of Hai7124; g) expression levels of the dominant disease resistance candidate gene SP1 in Hai7124 in the transcriptome and RT-qPCR results, with bar charts showing the RT-qPCR results and error bars representing the standard deviation of three biological replicates; and line graphs showing the average of three biological replicates in the transcriptome data, with both results showing consistent trends.

[0068] Figure 2 Structural and subcellular localization analysis of GbSP1 derived from Hai7124 and GhSP1 derived from TM-1.

[0069] The analysis included: a) amino acid sequence alignment and protein structure analysis of GbSP1 and GhSP1, with red boxes indicating signal peptides, green boxes indicating transmembrane domains, yellow boxes indicating extracellular structures, and asterisks indicating differentially expressed amino acids; b) co-localization analysis of GbSP1-GFP and GhSP1-GFP fusion proteins with endoplasmic reticulum-labeled AtWAK2-RFP red fluorescent protein in tobacco leaf cells; c) co-localization analysis of GbSP1-GFP and GhSP1-GFP fusion proteins with cell membrane-labeled AtPIP2A-RFP red fluorescent protein; and d) co-localization analysis of GbSP1-GFP and GhSP1-GFP fusion proteins with cell membrane-labeled AtPIP2A-RFP red fluorescent protein after plasmolysis of tobacco leaves.

[0070] Figure 3 Phenotypic analysis of Verticillium wilt resistance in plants after GbSP1 and GhSP1 gene silencing.

[0071] Among them, ab, GbSP1 and GhSP1 genes were specifically silenced using the VIGS method, and the silencing efficiency of GbSP1 and GhSP1 was verified by RT-qPCR. Compared with the control TRV:00 plants, both were significantly silenced; cd, Phenotypic data were recorded by photographing GbSP1 and GhSP1 at different time points after specific silencing; ef, Disease leaf rate of GbSP1 and GhSP1 silenced plants was investigated, and the error bar represents the standard deviation of three biological replicates; gh, The degree of browning of vascular tissue at 1 cm above the cotyledon node on the stem of the plant was detected 10 days after inoculation with Verticillium wilt.

[0072] Figure 4Molecular identification and disease resistance phenotype analysis of transgenic Arabidopsis thaliana overexpressing GbSP1 and GhSP1.

[0073] The study included: a) PCR detection of genomic DNA in GbSP1 and GhSP1 transgenic plants, with P representing the positive control Plasmid, WT representing the non-transgenic negative control, and OEs indicating different transgenic clonal lines; b) RT-qPCR detection of gene transcription levels in GbSP1 and GhSP1 transgenic lines; and c) Arabidopsis thaliana AtUbq5 (AT3G62250) as an internal reference gene. d) Four-week-old Arabidopsis thaliana plants were inoculated with Verticillium wilt pathogens. Two weeks after infection, the phenotypes of transgenic Arabidopsis thaliana and control plants were observed and photographed. e) The severity of disease in transgenic and control plants was statistically analyzed, using a grading method from grade 0 (healthy plants, no symptoms), grade 1 (0-25% leaf disease), grade 2 (25-50% leaf disease), grade 3 (50-75% leaf disease), and grade 4 (75-100% leaf disease) to record the severity of the disease. The GbSP1 sequence is the same as GhSP1. Gb .

[0074] Figure 5 Alignment diagram of the promoter sequences of the GbSP1 and GhSP1 genes.

[0075] The area shown in the green box represents the 5 bp insertion / deletion fragment.

[0076] Figure 6 Electrophoretic banding of the SP1 gene promoter functional markers in island cotton and upland cotton materials.

[0077] Where a and M represent DNA Markers, the first to sixth bands on the left after the Marker are the band patterns of the standard reference system TM-1, and the seventh to twelfth bands are the band patterns of the standard reference system Hai7124, each containing 6 biological replicates; b, 96 natural population materials of sea island cotton and 96 natural population materials of upland cotton were randomly selected to verify the accuracy of the markers.

[0078] Figure 7 Identification and regulatory role analysis of GATA10 transcription factor.

[0079] The study included: a) Analysis of the activity of GbSP1 and GhSP1 promoters under normal and Verticillium wilt-induced conditions; b) Analysis of the expression pattern of GATA transcription factors in cotton under Verticillium wilt-induced conditions, with the red box showing GATA8 / 9 / 10 significantly upregulated after Verticillium wilt induction; c) Prediction of conserved binding elements of GATA8 / 9 / 10 to downstream gene promoter regions using PlantPAN 4.0; d) Yeast one-hybrid experiments of GATA8 / 9 / 10 transcription factors binding to the promoters of GbSP1 and GhSP1, showing that GhGATA10 can bind to the promoter sequence of GhSP1, and the addition of 40 mM 3-amino-1,2,4-triazole (3-AT) to inhibit the self-activation of the bait vector; e) Detection of the binding of GhGATA10 to the core promoter sequence of GhSP1 using EMSA, selecting promoters containing the GATCT element. The bp fragment was used as a probe and treated with biotin, while unlabeled primers served as a competitive control; f) Transcriptional activation analysis using the LUC reporter system showed that GhGATA10 could bind to the promoter of GhSP1 and inhibit the expression of downstream LUC genes; g) The LUC / REN ratio of GbSP1 / GhSP1 promoter co-injected with empty vector or GhGATA10. LUC activities were normalized to REN. Detailed Implementation

[0080] To make the technical problems, solutions, and beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the embodiments.

[0081] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0082] Example 1: Based on the Hai7124-TM-1 marine-terrestrial infiltration line, combined with tissue, organ, and Verticillium wilt induced expression transcriptome data, a small peptide protein gene (Small peptide 1, SP1, GH_A09G1406) was identified in cotton.

[0083] Based on a previously configured population of Sea Island cotton (Hai7124) and upland cotton (TM-1) infiltrating lines, a Verticillium wilt resistance identification experiment was conducted in a temperature- and humidity-controlled culture room. Each material contained at least 30 plants, with three biological replicates. After inoculation with Verticillium wilt pathogen, the disease incidence rate was investigated. Compared to the control TM-1 plants, some infiltrating lines showed significant resistance phenotypes; 25 days after inoculation, the disease incidence rate of TM-1 reached 96.2%, with leaves almost completely yellowing and falling off, while one IL64 line showed only 65.3%, indicating significantly improved Verticillium wilt resistance. Figure 1ab). Further, approximately 10 days into the initial infection period, fungal recovery experiments were conducted on the stem and stalk to observe the colonization of the pathogen in the vascular tissue. It was found that the IL64 strain showed a significant reduction in the degree of browning in the vascular tissue and the amount of pathogen accumulation. Figure 1 (cd), the results showed that the IL64 strain had high resistance to Verticillium wilt.

[0084] Based on genome resequencing analysis, chromosome fragments derived from Hai7124 were identified in the IL64 introgression line. Using a criterion of more than 3000 SNPs within a 1 Mb region, a large introgression fragment of approximately 63-71 Mb was identified on chromosome A09. Combined with transcriptome sequencing analysis of tissues, organs, and Verticillium wilt-induced roots, 41 of these fragments were found to be highly expressed in cotton roots and induced by Verticillium wilt. Figure 1 The gene SP1, which encodes a small peptide protein, was expressed at a significantly higher level in the roots and stems of island cotton Hai7124 than in upland cotton TM-1. Furthermore, its expression was significantly upregulated in the roots of Hai7124 after Verticillium wilt induction, while no significant change was observed in the roots of TM-1. Figure 1 f). Specific primers for gene amplification were designed using Beacon Designer 7 software, and RT-qPCR detection experiments were performed using the AB7500 Real-Time PCR system. The CT values ​​of the cotton internal control primer and the quantitative primer were read, the relative gene expression levels were calculated, and a significant difference analysis was performed. The experimental results were consistent with the transcriptome results. Figure 1 g). Therefore, SP1 was selected as an important candidate gene for resistance to Verticillium wilt and its functional role was studied. It was named GbSP1 in Hai7124 and GhSP1 in TM-1, respectively. The SP1 gene originating from the TM-1-based infiltration line IL64 was named GhSP1. Gb GbSP1 and GhSP1 Gb These are the names of the same gene in different genetic contexts. The primers used in the study are listed in Table 1.

[0085] Table 1: Primers used for amplification

[0086]

[0087] Example 2: Sequence and localization analysis of cotton small peptide protein SP1

[0088] First, using the released genome data of island cotton Hai7124 and upland cotton TM-1 as references, primers SP1 F / R for SP1 gene amplification were designed after sequence alignment. Using cDNA from root tissues of Hai7124 and TM-1 as templates, PCR amplification was performed using DNA polymerase. The products were subjected to agarose gel electrophoresis, the target band was recovered and ligated into a T vector, transformed into Escherichia coli strain DH5α, and the detected positive strains were sent to Nanjing Qingke Company for sequencing. The results showed that SP1 encodes a small protein of 66 amino acids, with a sequence conserved in cotton and no homologous genes. It is only present in some dicotyledonous plants; no homologous genes of this gene were found in the model plants Arabidopsis thaliana and tobacco, or major crops such as rice, wheat, and maize. Amino acid structure analysis showed that the N-terminal 28 amino acids of SP1 form a signal peptide, the middle 15 form an extracellular domain, and the remaining 23 amino acids at the C-terminus form a transmembrane domain. Due to its short length and signal peptide characteristics, this small peptide segment, consisting of only 15 amino acids besides the signal peptide and transmembrane domain, was initially named SP1 (Small peptide 1). Sequence alignment analysis revealed that the coding regions of GbSP1 in sea island cotton and GhSP1 in upland cotton differed by only one amino acid, S / A. Figure 2 a). Recombinant primers were designed using CE Design software. The vector was digested with Kpn I and BamHI restriction sites. Using the recombinant primer SP1-GFP F / R, the complete ORF sequence of GbSP1 / GhSP1 was constructed into the pBinGFP4 vector fused with GFP expression protein, and transformed into Agrobacterium strain GV3101 (Shang et al., 2022). The GFP vector, along with HDEL-RFP and AtPIP2-RFP (red fluorescent protein fused with endoplasmic reticulum and cell membrane localization marker genes), was injected into tobacco leaves via Agrobacterium-mediated transformation, and the localization of green fluorescence was observed. Three days after Agrobacterium infection, confocal microscopy revealed that the green fluorescent signal of GbSP1 / GhSP1-GFP could co-localize with the endoplasmic reticulum and cell membrane marker genes. A significant fluorescent signal was also observed in the cell nucleus. After plasmolysis, the green fluorescence remained in the cell membrane and cytoplasm and could not be secreted extracellularly. Figure 2 (bd). The results showed that both GbSP1 and GhSP1 were localized in the endomembrane system, which consists of the cell membrane, endoplasmic reticulum, and nucleus. A variation of one non-synonymous amino acid did not affect their intracellular localization. The primers used in the study are listed in Table 2.

[0089] Table 2: Primers used for amplification

[0090]

[0091] Example 3: Expression of the small peptide protein gene SP1 promotes plant resistance to Verticillium wilt.

[0092] Virus-mediated gene silencing (VIGS) technology was used to specifically silence GbSP1 / GhSP1 in Hai7124 and TM-1 to clarify their disease resistance function. Recombinant primers TRV:SP1 F / R were designed to construct the full-length 198 bp coding region into the VIGS vector. The TRV2 vector was digested with BamHI and EcoRI restriction enzymes, and homologous recombination was performed. The vector was then transformed into *E. coli*, and positive strains were detected and sequenced to complete the construction of the TRV:SP1 vector. The empty TRV:00 vector served as a control (Wang et al., 2014). The constructed vector was injected into the cotyledons of one-week-old cotton seedlings using Agrobacterium-mediated transformation, with uninjected seedlings serving as controls. To verify the silencing efficiency of the target gene, root RNA was extracted from both the TRV:SP1 silencing and control lines two weeks after bacterial infection, and RT-qPCR was used to verify the gene silencing efficiency. Compared with the TRV:00 empty vector control, the expression levels of GbSP1 / GhSP1 in the TRV:SP1 silencing line were significantly reduced (P<0.01). Figure 3 ab). Simultaneously, the plants were assessed for resistance to Verticillium wilt, with 1×10⁻⁶ plants... 7 25 mL of a spore suspension of Verticillium wilt fungus was used for inoculation using the root-tear method. At least 30 cotton seedlings were used for each material, with three replicates. Hai7124 and TM-1 served as controls. Inhibition of GbSP1 expression in Hai7124 significantly reduced plant resistance, while silencing GhSP1 in TM-1 showed no significant change in plant resistance compared to the control. Figure 3 Similar results were obtained from the observation of fungal accumulation in the vascular tissue of the stem and stalk. After GbSP1 silencing, more melanin and microsclerotia accumulated in the stem, resulting in severe browning. Figure 3 gh).

[0093] Furthermore, we created transgenic Arabidopsis lines overexpressing GbSP1 / GhSP1. Recombinant primers 121-SP1 F / R were designed using BamHI and SacI restriction sites to ligate the complete GbSP1 / GhSP1 ORF sequence into the overexpression vector pBI121, forming a constitutive expression element driven by the 35S promoter (Wang et al., 2020). Then, 8 μL of the pBI121-35S::GbSP1 / GhSP1 overexpression vector plasmid was transformed into Agrobacterium. Using wild-type Arabidopsis (Col-0) as the recipient, positive strains were selected for flower infection. Finally, nine independent transgenic clones overexpressing GbSP1 / GhSP1 were obtained. DNA was extracted from leaves of each strain. DNA quality was assessed using primers (S2777F / R) for the Arabidopsis ubiquitinase gene AtUBQ5. An F primer was used in the promoter region of the pBI121-35S::GbSP1 / GhSP1 overexpression vector, and an R primer (Y668F / R) was used in the coding region to detect the transformation band of the target gene. Figure 4 a). For the T3 generation homozygous transgenic lines, root tissue was further extracted and RNA was obtained under greenhouse conditions. The transcriptional levels of GbSP1 / GhSP1 were detected by RT-qPCR to clarify the expression levels of GbSP1 / GhSP1 in the plants. Figure 4 bc). Two transgenic clones with high GbSP1 / GhSP1 expression levels (OE6b, OE9b, OE6h, and OE9h) were selected for subsequent Verticillium wilt resistance analysis. Verticillium wilt pathogens were inoculated using the root-dipping method, and the disease severity was recorded two weeks later. Wild-type WT plants with water-dipped roots grew normally and served as the control group. All four transgenic clones with GbSP1 / GhSP1 overexpression showed stronger disease resistance phenotypes, especially OE9b and OE6h with higher expression levels, exhibiting significantly reduced leaf yellowing and wilting. Figure 4 (de). The results showed that the expression of the SP1 gene was positively correlated with resistance to Verticillium wilt in cotton, and increased expression levels contributed to plant resistance. The primers used in the study are listed in Table 3.

[0094] Table 3: Primers used for amplification

[0095]

[0096] Example 4: Promoter analysis and functional marker development of the SP1 gene

[0097] The SP1 gene showed significant expression differences in Hai7124 and TM-1 cotton varieties, as well as differences in expression induced by Verticillium wilt. Primers qSP1 F / R were designed to clone the GbSP1 / GhSP1 gene upstream of the 1271 bp promoter sequence in both cotton species. Comparison revealed a 5-base difference at 591 bp upstream of the gene. In TM-1, GhSP1 contains the GATCT sequence, while Hai7124 lacks this 5 bp (…). Figure 5 To address this 5 bp insertion / deletion difference, we developed a functional molecular marker to distinguish between haplotypes 1 and 2 of TM-1 and Hai7124. The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 sec, 58℃ annealing for 30 sec, 72℃ extension for 30 sec, 28 cycles; and a final extension at 72℃ for 5 min. The amplified products were detected by PAGE gel electrophoresis. This specific primer effectively distinguished between the two haplotypes, TM-1 and Hai7124. Haplotype 1 was amplified to a molecular weight of 464 bp using the marker primer, while haplotype 2 was not amplified. Figure 6 a). Furthermore, based on whole-genome resequencing data from 335 upland cotton natural populations and 269 island cotton natural populations, analysis revealed that all upland cotton materials possessed 5 bases of haplotype 1, while the island cotton population lacked them. Further genetic verification of the upland and island population materials was performed using functional markers. Ninety-six random samples from each population were amplified by PCR combined with electrophoresis. It was clearly observed that the island cotton materials all possessed the characteristics of haplotype 2, lacking 5 bp, while upland cotton had 5 bp, and both could be amplified into bands. Figure 6 b). The primers used in the study are listed in Table 4.

[0098] Table 4: Primers used for amplification

[0099]

[0100] Example 5: Differences in GATCT sequences in the promoter region affect SP1 gene expression in different cotton materials

[0101] To investigate the transcriptional activation activity of the GbSP1 / GhSP1 gene promoter, a luciferase (LUC) reporter gene assay was first performed. Recombinant primers pSP1-LUC F / R were designed using Kpn I and Hind III restriction sites. The GbSP1 / GhSP1 promoter sequence was ligated into the pGreenII0800-LUC (mini35S) vector, replacing the mini35S promoter to initiate downstream LUC gene expression, generating the target gene promoter::LUC reporter gene vector. The original mini35S promoter carried by this vector is the minimal promoter of CAMV35S driving LUC reporter gene expression, using pGreenII0800-LUC as the vector backbone (Wang et al., 2025). (Wang L, Wang G, Wang F, Chen Z, Wang X, et al. (2025) Transcription factor GbTCP20 confers plant wilt resistance by regulating ACC biosynthesis pathway and lignin deposition. Plant Sci 359: 112663.) The expression of reporter genes was observed after 3 days of treatment with water or Verticillium wilt pathogen strain V991. The GbSP1 promoter showed higher activity in regulating LUC than the GhSP1 promoter, and this difference was even more pronounced after induction with Verticillium wilt pathogen. Figure 7 a). The GATCt motif is a specific binding element for GATA transcription factors. Therefore, based on the Hai7124 and TM-1 genomes and Verticillium wilt induced expression transcriptome data, we identified three GATA transcription factors that were significantly upregulated at all time points after induction. Their expression patterns were similar to GbSP1, and based on their homology with Arabidopsis thaliana, we named them GATA8 / 9 / 10 ( Figure 7 b). Binding element prediction was performed using the PlantPAN 4.0 promoter analysis website, and the results showed that they all had strong binding ability to GATCt. Figure 7 c). Recombinant primers pHis F / R were designed using EcoRI and SacI restriction sites. The GbSP1 / GhSP1 promoter sequence, carrying approximately 100 bp of promoter differential regions, was ligated into the yeast one-hybrid vector pHis2. GATA8 / 9 / 10 was ligated into the pGADT7 vector (Wang et al., 2024). Yeast one-hybrid experiments demonstrated that GATA10 specifically binds to the GhSP1 promoter sequence in TM-1 to regulate gene expression, but cannot bind to the GbSP1 promoter sequence in Hai7124. Figure 7d). Furthermore, using electrophoretic mobility shift assays (EMSA), recombinant primers GhGATA10-GST F / R were designed with BamHI and EcoRI restriction sites to construct a GST-GhGATA10 prokaryotic expression vector fused with a GST expression sequence tag. The GST-GhGATA10 protein was expressed and purified in vitro. A biotin-labeled GbSP1 / GhSP1 promoter sequence containing 55 bp carrying GATCt was used as a probe. The purified protein was incubated with the probe, and electrophoretic mobility shift analysis showed that the recombinant GST-GhGATA10 protein could bind to the GhSP1 promoter sequence probe containing GATCt, while the unlabeled competitive probe inhibited the binding; it did not bind to the GbSP1 promoter sequence probe. Figure 7 e).

[0102] Furthermore, GhGATA10 was constructed into a plant expression vector driven by the 35S promoter and used as an effector. This vector, along with the GbSP1 / GhSP1 promoter::LUC reporter gene vector, was co-infected with tobacco leaves via Agrobacterium-mediated transformation, and the expression of the LUC gene was observed. In upland cotton, GhGATA10 significantly inhibited the expression of the GhSP1 promoter::LUC reporter gene, while in sea island cotton, the expression of the GbSP1 promoter::LUC reporter gene showed no significant change. Figure 7 f). Furthermore, using a dual-luciferase reporter system with the REN gene as an internal control, co-transformation of the GhGATA10 effector and the GbSP1 / GhSP1 promoter::LUC reporter gene vector yielded the same results: GhGATA10 significantly inhibited the expression of the GhSP1 promoter::LUC reporter gene. Figure 7 g). The primers used in the study are listed in Table 5.

[0103] Table 5: Primers used for amplification

[0104]

[0105] Based on the above results, we identified a gene, GhSP1, encoding a small peptide 1 (SP1), using the important marine-terrestrial infiltration system IL64. GbThis gene originates from an introgression of a chromosomal segment from Hai7124. Sequence analysis of the promoter regions between marine and terrestrial materials revealed a 5 bp insertion 591 bp upstream of the ATG in TM-1. Using TM-1 and Hai7124 as reference systems, the TM-1 type promoter sequence was named haplotype 1, and the Hai7124 type promoter sequence was named haplotype 2. Haplotype 2 showed a significant advantage over haplotype 1 in improving Verticillium wilt resistance. Silencing GbSP1 in Hai7124 significantly weakened the disease resistance of cotton plants. Heterologous expression of GhSP1 and GbSP1 (GhSP1...) was also observed. Gb The resistance of Arabidopsis thaliana plants to Verticillium wilt was significantly improved by GhSP1, derived from sea island cotton. Gb Molecular markers and the SP1 gene play an important role and have promising applications in improving cotton resistance to Verticillium wilt and in breeding high-quality new cotton varieties.

[0106] sequence list

[0107] SEQ ID NO.1 (1271 bp genome sequence upstream of the promoter of the GhSP1 gene in haplotype 1 upland cotton TM-1) 1271

[0109] Gossypium hirsutum

[0110] TAGAATTTAAAAATAAGAAGGGTTAAATTTATTGAACTATTTAGAATTAAGATCCAAATTGATACAATATGTAAATATTGAAGACTAAATAGAACTACACTCATTCTATTTGACATTAATTAAAATAGGGTTTTTTATTATTTAAGTAGATGTAGTCTTATATTATTCAGTTTTTAACATTAGTAAATTTCTCTTCTCTTACTCATAATTTTTTTCCGAAAAGGTTTCCATATAAAAATATGTGTGTTTTATTTTTCCTTTTTCTTATTGTTGTCGTTTTATTACCATTATCGACGTCTATTATAACAATTATATCTATAAATTATTTAAAATATAAAT TGAATGCTAAATTTTAAATAAGAAATAAAATTCAATCAAAACAAAAATAGAGTGACAAAATTCACTATTAATTTAAATTATAATGTAAATTTATTTTTAATAATTAAGTACAAACACTGAAGCATAATATAGTGATAAATTTACACTTTAATTCTATGTTTAAGCCATTGAAATATCATTTACATGCGATTCTAAATTTGAAATCGACACAATGCAATTGATCTTTTAAATATAAAAGTGGAATTCGATTTAGCAAAATTTTATTTGATATTATTTTAATTTCCCATAAGTATGAAAAATATTGTCTTCAGAATCGATTAGATTTATTAGATTATACT GATCTGAAATGAAGAGCTGAACGGATGAATTATTTTTAGCTATTTTTAAATTTTTTATTCAATAAAATAAAAAGAATATTAAATTAAAAATTACTAATCTAATTGATCCGACCAGAAGGAAAAAAGAAATCCAAACACCTGAACTGTGGCCCACGTATCAAAGTGAAAGCTCAGCAGATTTTATCACCCCACGTCCTCGACAAATTTAGCTGGCTAACAGGGAATTCTTACCGGTAATACAAGAAAAACCTATAGAAAAAAAGAGTGCCCAAGGCACACACTACAATCCTGTATTCCTCTCCGTCAAGTCTAAAAAAGTTGAAGCAGAGAAGGGTCCAATATTAAAATGGATTAAGTTGAAGAGTCAAATGAAAGGTAATGTGGGTGTCCACAAACCGTAATCCAGAAGGCATGGAAATGGAAAATTGCTACTCTAACTAAGTTTTCACATCACAACTTTGAAAGCAAACCCAACTCACCTATAAATTCTAACATTGCACCATTCATACAACAAACAACCTTTCTATTCTCAATTTCCCCATCTCTCTCGTCTCTGGGTCTCGTAAGTGGGTGAATAGTGTTGTAATTAGTGCCA

[0111] SEQ ID NO.2 (CDS sequence of GhSP1 in Gossypium hirsutum TM-1) 201

[0113] Gossypium hirsutum

[0114] ATGGCTCGGATTGGGACCTCTGCAGCTCACATTGTGTTGGCAATATTTGCTGTGGCCATGTTTGTTGTGTCCGGGACCATGGCACAGGATATTGCTCCTTCTCCTGCAATGGCTACCGGAGCAGGCTCTGCTTTGCCGGTTTCCGCTGTCTTCTTATGCTCTTCCATGTTGGTCTCTTTAATTGCTCTCTTGGTGCATTGA

[0115] SEQ ID NO.3 (Amino acid sequence of GhSP1 in Gossypium hirsutum TM-1) 66

[0117] Gossypium hirsutum

[0118] MARIGTSAAHIVLAIFAVAMFVVSGTMAQDIAPSPAMATGAGSALPVSAVFLCSSMLVSLIALLVH

[0119] SEQ ID NO.4 (GbSP1 gene in haplotype 2 sea island cotton sea 7124 (GhSP1 in IL64)) Gb (1267 bp genome sequence upstream of the promoter) 1267

[0121] Gossypium barbadense

[0122]

[0123] SEQ ID NO.5 (GbSP1 in sea island cotton sea 7124 (GhSP1 in IL64)) Gb CDS sequence) 201

[0125] Gossypium barbadense

[0126] ATGGCTCGGATTGGGACCTCTGCAGCTCACATTGTGTTGGCAATATTTTCTGTGGCCATGTTTGTTGTGTCCGGGACCATGGCACAGGATATTGCTCCTTCTCCTGCAATGGCTACCGGAGCAGGCTCTGCTTTGCCGGTTTCCGCTGTCTTCTTATGCTCTTCCATGTTGGTCTCTTTAATTGCTCTCTTGGTGCATTGA

[0127] SEQ ID NO.6 (GbSP1 in sea island cotton sea 7124 (GhSP1 in IL64)) Gb (amino acid sequence) 66

[0129] Gossypium barbadense

[0130] MARIGTSAAHIVLAIFSVAMFVVSGTMAQDIAPSPAMATGAGSALPVSAVFLCSSMLVSLIALLVH

[0131] SEQ ID NO.7 (Forward primer sequence of molecular marker for GhSP1 InDel site) twenty three

[0133] ATGTGTGTTTTATTTTTCCTTTT

[0134] SEQ ID NO.8 (Reverse primer sequence of molecular marker for GhSP1 InDel site) twenty three

[0136] ATCCGTTCAGCTCTTCATTTCAG.

Claims

1. The application of InDel molecular markers related to cotton resistance to Verticillium wilt, or substances for detecting said InDel molecular markers, characterized in that, The InDel molecular marker is an insertion of a 5 bp nucleotide fragment GATCT at -591 bp in the promoter sequence of the cotton small peptide protein gene GhSP1, as shown in SEQ ID NO.

1. The application is at least one of the following (a1)-(a5): (a1) Screening or assisting in the screening of cotton germplasm with different resistance to Verticillium wilt, or preparing products for screening or assisting in the screening of cotton germplasm with different resistance to Verticillium wilt; (a2) To identify or assist in the identification of cotton resistance to Verticillium wilt, or to prepare products for the identification or assistance in the identification of cotton resistance to Verticillium wilt; (a3) Identify the promoter haplotype of cotton small peptide protein gene GhSP1, or prepare a product for identifying the promoter haplotype of cotton small peptide protein gene SP1; (a4) Improve cotton’s resistance to Verticillium wilt, or prepare products for improving cotton’s resistance to Verticillium wilt; (a5) Breeding of cotton with high resistance to Verticillium wilt, or preparing products for breeding of cotton with high resistance to Verticillium wilt.

2. The application according to claim 1, characterized in that, The promoter region of the cotton peptide protein gene GhSP1 has two haplotypes. The genomic nucleotide sequence of haplotype 1 is shown in SEQ ID NO.1, and the genomic nucleotide sequence of haplotype 2 is shown in SEQ ID NO.

4. Compared with haplotype 2, haplotype 1 has a 5 bp nucleotide fragment GATCT inserted at -591 bp in the promoter sequence. Compared with the cotton peptide protein gene GhSP1 corresponding to haplotype 1, the cotton peptide protein gene GhSP1 corresponding to haplotype 2 has... Gb The expression level in plant roots, the expression level induced by Verticillium wilt, and the disease resistance were all superior to haplotype 1.

3. A product characterized in that, The product contains the substance for detecting InDel molecular markers as described in claim 1, and is at least one of the following (b1)-(b5): (b1) Products for screening or assisting in screening cotton germplasm with different Verticillium wilt resistance; (b2) Products used to identify or assist in the identification of cotton resistance to Verticillium wilt; (b3) Products for identifying the promoter haplotype of the cotton small peptide protein gene GhSP1; (b4) Products used to improve cotton's resistance to Verticillium wilt; (b5) Products used in the breeding of cotton with high resistance to Verticillium wilt.

4. A method, characterized in that, The method is at least one of the following (c1)-(c5): (c1) Methods for screening or assisting in the screening of cotton germplasm with high resistance to Verticillium wilt; (c2) Methods for identifying or assisting in the identification of cotton Verticillium wilt resistance; (c3) A method for identifying the promoter haplotype of the cotton small peptide protein gene GhSP1; (c4) Methods to improve cotton's resistance to Verticillium wilt; (c5) Methods for breeding cotton with high resistance to Verticillium wilt; This method involves using a substance that detects InDel molecular markers to determine whether the promoter region of the cotton small peptide protein gene GhSP1 contains the InDel molecular markers as described above, thereby identifying the haplotype of the GhSP1 promoter. The GhSP1 promoter region contains two haplotypes. Haplotype 1 has a 5 bp nucleotide fragment GATCT insertion at -591 bp in the promoter sequence, unlike haplotype 2. Haplotype 2 exhibits superior resistance to Verticillium wilt. Cotton germplasm carrying haplotype 2 is selected as cotton germplasm with high resistance to Verticillium wilt for cotton breeding to improve cotton resistance to the disease.

5. The application according to claim 1, the product according to claim 3, and the method according to claim 4, characterized in that, The substances that are detected by the InDel molecular label are (d1) or (d2) or (d3) or (d4) as follows: (d1) Contains in vitro nucleic acid amplification primers as shown in SEQ ID NO.7 and SEQ ID NO.8 that specifically amplify the InDel molecular marker; (d2) In vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primers described in (d1); (d3) A kit containing the in vitro nucleic acid amplification primers described in (d1) or the in vitro nucleic acid amplification reagents described in (d2); (d4) A detection instrument containing the in vitro nucleic acid amplification primers described in (d1), the in vitro nucleic acid amplification reagents described in (d2), or the kit described in (d3).

6. The application according to claim 1 or 2, the product according to claim 3, and the method according to claim 4, characterized in that, The trait of resistance to Verticillium wilt in cotton is at least one of the disease phenotype and the incidence rate.

7. The small peptide protein gene GhSP with the nucleotide sequence shown in SEQ ID NO.2 or the small peptide protein gene GhSP1 with the nucleotide sequence shown in SEQ ID NO.

5. Gb Promote the small peptide protein gene GhSP1 or GhSP1 Gb The expressed substance or the enhancement of the small peptide protein gene GhSP1 or GhSP1 Gb The application of substances encoding protein activity or abundance in the following e1) or e2) : el) Improve cotton resistance to Verticillium wilt or cultivate new germplasm with improved resistance to Verticillium wilt; e2) Improve the disease resistance of target plants.

8. The application according to claim 7, characterized in that, Promotes the small peptide gene GhSP1 or GhSP1 Gb The expressed substance or the small peptide protein gene GhSP1 or GhSP1 Gb The substance encoding the activity or abundance of a protein is a biological material, and the biological material is any one of the following (fl) to (f6): f1) Contains the small peptide gene GhSP1 or GhSP1 Gb Expression box; f2) Contains the small peptide gene GhSP1 or GhSP1 Gb The recombinant vector, or the recombinant vector containing the expression cassette described in f1); f3) Contains the small peptide gene GhSP1 or GhSP1 Gb Recombinant microorganisms, or recombinant microorganisms containing the expression cassette described in f1), or recombinant microorganisms containing the recombinant vector described in f2); f4) Contains the small peptide gene GhSP1 or GhSP1 Gb The transgenic plant cell line, or the transgenic plant cell line containing the expression cassette described in f1), or the transgenic plant cell line containing the recombinant vector described in f2); f5) Contains the small peptide gene GhSP1 or GhSP1 Gb The transgenic plant tissue, or the transgenic plant tissue containing the expression cassette described in f1), or the transgenic plant tissue containing the recombinant vector described in f2); f6) Contains the small peptide gene GhSP1 or GhSP1 Gb The transgenic plant organ, or the transgenic plant organ containing the expression cassette described in f1), or the transgenic plant organ containing the recombinant vector described in f2).

9. The application according to claim 7 or 8, characterized in that, The amino acid sequence of the protein encoded by the small peptide gene GhSP1 is shown in SEQ ID NO.

3. Gb The amino acid sequence encoding the protein is shown in SEQ ID NO.

6.

10. A method for improving resistance to Verticillium wilt in cotton, characterized in that: With small peptide protein gene GhSP1 or GhSP1 Gb The target gene is the small peptide protein gene GhSP1 with the nucleotide sequence shown in SEQ ID NO.2 or the small peptide protein gene GhSP1 with the nucleotide sequence shown in SEQ ID NO.5, which is overexpressed in cotton through genetic transformation. Gb To improve cotton's resistance to Verticillium wilt.

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