Indel molecular marker related to drought resistance in upland cotton, method and application

Abstract

The present invention relates to the field of biotechnology, and in particular to an Indel molecular marker related to drought resistance of upland cotton, as well as a method and application thereof. The Indel molecular marker is located at bases 1083-1100 of the nucleotide sequence shown in SEQ ID NO: 1. In drought-resistant cotton germplasm, the genotype of the Indel molecular marker is CAGCGAAAAACTAAGTGT, and in drought-sensitive cotton germplasm, the genotype of the Indel molecular marker is C. The Indel molecular marker provided by the present invention is closely linked to the drought-resistant phenotype of upland cotton, can be used to efficiently and accurately identify or assist in identifying the drought resistance of tested cotton, effectively distinguish between drought-resistant and drought-sensitive cotton germplasm, and provide an effective means and approach for molecular marker-assisted selection of drought-resistant cotton and cultivation of new cotton germplasm resources.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an Indel molecular marker related to drought resistance of upland cotton, a method and an application thereof. Background Art

[0002] Cotton, one of the world's major cash crops, is widely cultivated in over 30 countries and plays an indispensable role in numerous fields, including textiles and medicine. However, its growth is highly susceptible to extreme environmental conditions, particularly drought. Cotton is highly sensitive to water deficits during the seedling, flowering, and boll development stages. Drought stress not only hinders normal cotton growth and development but also leads to a sharp drop in yield and a significant decline in fiber quality, dealing a heavy blow to the cotton industry. Therefore, cultivating drought-resistant cotton varieties and mitigating the impact of drought are key tasks for the sustainable development of the cotton industry.

[0003] Traditional cotton breeding relies primarily on direct observation and screening of phenotypic traits. However, due to the complex and changing natural environment, external factors such as light, temperature, and soil fertility significantly influence cotton phenotype, making it difficult to accurately determine the relationship between phenotype and genotype. This significantly limits the efficiency of selecting superior varieties and makes it difficult to meet the current cotton industry's urgent need for drought-resistant varieties.

[0004] With the rapid development of molecular biology techniques, molecular marker-assisted selection (MAS) breeding has emerged and is gradually gaining prominence. Compared to traditional breeding methods, this technology breaks through the dual constraints of time and location, enabling precise and rapid identification of target traits at the genetic level, significantly improving the accuracy and timeliness of breeding. It has become the preferred strategy for identifying drought-resistant cotton varieties, and its core position in cotton breeding is becoming increasingly prominent. In the construction of MAS breeding systems, the development and regulation of molecular markers closely linked to genes associated with cotton drought resistance is the cornerstone of efficient breeding.

[0005] Insertion-deletion markers (InDels), an important class of molecular markers, are based on sequence differences at the same genomic site between closely related species or individuals of the same species, i.e., the insertion or deletion of one or more bases, resulting in length polymorphism. InDel markers are widely distributed throughout the genome, have considerable density, are numerous, and exhibit good polymorphism and high stability, making them widely applicable to multiple species. In practical applications, primers designed using InDel polymorphic molecular markers for PCR amplification can be used to efficiently and quickly test large quantities of samples based on the size differences of the amplified products, accurately determining their genotypes and effectively distinguishing the sample phenotypes, which is highly consistent with the needs of molecular marker-assisted selection breeding.

[0006] At present, although molecular marker research on cotton has achieved certain results, it mainly focuses on the screening of molecular markers related to traits such as yield, fiber quality and disease resistance. In terms of abiotic stress resistance, especially drought resistance, there is a lack of research on related molecular markers. There are few effective molecular markers closely linked to drought resistance genes. This bottleneck has seriously hindered the breeding process of using molecular markers to assist in the screening of new drought-resistant varieties. In view of this, in-depth exploration of key genes closely related to drought resistance and the subsequent development of highly specific Indel molecular markers have both great scientific significance and broad application value for building a complete molecular marker-assisted selection breeding system, promoting the rapid and accurate identification of drought-resistant cotton varieties and achieving genetic improvement. Summary of the Invention

[0007] In light of this, the present invention proposes an indel molecular marker, method, and application for drought resistance in upland cotton. This indel molecular marker is tightly linked to the drought resistance phenotype of upland cotton and can be used to efficiently and accurately identify or assist in the identification of drought resistance in tested cotton species, effectively distinguishing between drought-resistant and drought-sensitive cotton germplasm. This provides an effective means and approach for molecular marker-assisted selection of drought-resistant cotton materials and the cultivation of new cotton germplasm resources.

[0008] The technical solution of the present invention is achieved as follows:

[0009] In a first aspect, the present invention provides an Indel molecular marker related to drought resistance of upland cotton, wherein the Indel molecular marker comprises at least one DNA molecule in SEQ ID NOs: 8-11.

[0010] In a second aspect, the present invention provides detection primers for detecting the Indel molecular marker, wherein the detection primers include: a primer pair of SEQ ID NO: 4-5 or SEQ ID NO: 6-7.

[0011] In a third aspect, the present invention provides a reagent / kit for detecting the Indel molecular marker, wherein the components of the reagent / kit include at least one of the following: (1) PCR primers for amplifying the Indel molecular marker; and (2) the detection primers (primer pair SEQ ID NO: 4-5 or SEQ ID NO: 6-7).

[0012] In a fourth aspect, the present invention provides applications of the Indel molecular marker, the detection primer, or the reagent / kit, wherein the application includes at least one of the following applications:

[0013] (1) Identify or assist in identifying the drought resistance of cotton;

[0014] (2) Compare the drought resistance of the tested cotton;

[0015] (3) Breeding drought-resistant cotton plants, strains, lines, or varieties;

[0016] (4) Cotton breeding;

[0017] (5) Prepare detection products related to cotton drought resistance.

[0018] In a fifth aspect, the present invention provides a method for detecting the Indel molecular marker, the method comprising the following steps:

[0019] S1 performs PCR amplification on the genomic DNA of the cotton to be tested;

[0020] S2 determines the drought resistance of the cotton to be tested by DNA sequencing or gel electrophoresis results of the PCR amplification products.

[0021] The beneficial effects of the present invention include at least the following:

[0022] Through genome-wide association analysis, the present invention discovered an indel variant in the upstream regulatory region of the GhCYCU4-1 gene, which is tightly linked to a drought resistance phenotype. Furthermore, the CIDT values ​​of the drought resistance phenotypes corresponding to the different genotypes at the indel variant site differed significantly, indicating a significant correlation and high consistency between the genotypes and the drought resistance phenotype.

[0023] The present invention provides an Indel molecular marker associated with cotton drought resistance and an accurate and efficient detection method. Using the genomic DNA of a cotton sample as a template, specific primers are used for targeted amplification, and the Indel molecular marker genotype is accurately determined based on the sequence information of the amplified product. This method allows for efficient and accurate identification of the drought resistance of the cotton sample, or assists in the identification of drought-resistant cotton germplasm, and allows differentiation between drought-resistant and drought-sensitive cotton germplasm. This invention provides a practical means and approach for molecular marker-assisted selection and breeding of drought-resistant cotton materials, as well as for the cultivation of novel cotton germplasm resources, and has important practical significance and application value.

[0024] the term

[0025] Indel (Insertion-Deletion) mutations refer to insertions or deletions that occur in genomic sequences. This refers to the addition or deletion of a base sequence compared to a reference genome. In some embodiments of the present invention, indel molecular markers are based on insertion / deletion (ID) mutations in DNA sequences; they are polymorphic markers resulting from the insertion or deletion of a DNA sequence at a specific location in the genome. These insertions or deletions can range in length from a single base pair to thousands of base pairs.

[0026] In some specific embodiments of the present invention, the Indel molecular marker includes a molecular marker developed based on an Indel mutation in the upstream regulatory region of the GhCYCU4-1 gene. The specific location of the Indel mutation in the upstream regulatory region of the GhCYCU4-1 gene is as follows:

[0027] The indel variant is located at position 76,507,400 of chromosome A12 in the Ghirsutum_genome_HAU_v1.0 reference genome of upland cotton TM-1. Compared to the reference genome, the indel variant CAGCGAAAAACTAAGTGT→C occurs. The Ghirsutum_genome_HAU_v1.0 reference genome of upland cotton TM-1 is publicly available from the CottonFGD database at https: / / cottonfgd.net / about / download.html.

[0028] The Indel molecular marker includes: base sequences from positions 1083 to 1100 in SEQ ID NO: 1.

[0029] Furthermore, in drought-resistant cotton germplasm, the Indel molecular marker includes SEQ ID NO: 3. In drought-sensitive cotton germplasm, the sequence of the Indel molecular marker is missing from positions 2 to 18 compared with the sequence shown in SEQ ID NO: 3.

[0030] In some specific embodiments of the present invention, the detection primers for the Indel molecular marker include: primer pair F1 and R1 (SEQ ID NO: 4-5), or primer pair F2 and R2 (SEQ ID NO: 6-7).

[0031] In some specific embodiments of the present invention, a PCR reaction is performed using genomic DNA of a sample to be tested as a template and primers for detecting the indel molecular marker; the genotype of the indel molecular marker in the PCR amplification product is detected. The drought resistance characteristics of the tested cotton germplasm are then determined based on the detection results, wherein the determination comprises the following steps:

[0032] When the primers are F1 and R1 (SEQ ID NOs: 4-5), if the PCR amplification product can specifically amplify a band and the 346 bp band pattern as shown in SEQ ID NO: 8 is present, it can be determined that the cotton germplasm to be tested has drought resistance and belongs to drought-resistant cotton germplasm; conversely, if the PCR amplification product cannot specifically amplify a band, or the amplified band pattern does not include the 346 bp band pattern, for example, the PCR amplification product has a 329 bp band pattern as shown in SEQ ID NO: 10, then the cotton germplasm to be tested is drought-sensitive and belongs to drought-sensitive cotton germplasm.

[0033] When the primers are F2 and R2 (SEQ ID NOs: 6-7), if the PCR amplification product can specifically amplify a band, and there is a 349 bp band pattern as shown in SEQ ID NO: 9, it can be determined that the cotton germplasm to be tested has drought resistance and belongs to a drought-resistant cotton germplasm; if the PCR amplification product has a 332 bp band pattern as shown in SEQ ID NO: 11, the cotton germplasm to be tested is drought-sensitive and belongs to a drought-sensitive cotton germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 This is the correlation between the variant sites at the GhCYCU4-1 locus and the drought resistance phenotype in upland cotton in Example 1 of the present invention; wherein Figure a is a Manhattan plot and linkage disequilibrium block diagram between the variant sites at different physical locations and the drought resistance phenotype, and Figure b is the distribution position of the Indel variant at the GhCYCU4-1 locus;

[0036] Figure 2 This is a CIDT statistical graph corresponding to different haplotypes of Indel mutations at the GhCYCU4-1 locus in a natural population of upland cotton in Example 1 of the present invention;

[0037] Figure 3The genotypes and sequencing peaks of the Indel mutations at the GhCYCU4-1 locus in drought-resistant and drought-sensitive cotton germplasms of Example 2 of the present invention are shown; wherein, Figure a is the drought-sensitive cotton germplasm Junmian 1, and Figure b is the drought-resistant cotton germplasm Wankangmian 9;

[0038] Figure 4 Figures 2 show the results of amplifying the Indel variant at the GhCYCU4-1 locus using different primers in Example 2 of the present invention; Figure a is a sequencing peak graph of the product amplified using the primers set forth in SEQ ID NOs: 6-7, and Figure b is an electrophoresis graph of the product amplified using the primers set forth in SEQ ID NOs: 4-5;

[0039] Figure 5 This is an electrophoresis diagram of the product of amplifying a natural cotton population using the primers shown in SEQ ID NO: 4-5 in Example 3 of the present invention; wherein, Figure a is a drought-sensitive cotton material, and Figure b is a drought-resistant cotton material. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the embodiments described are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0041] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor). The methods for obtaining various biological materials described in the examples are only to provide an experimental method for obtaining them to achieve the specific disclosed purposes and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological material that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples. The genes, proteins or fragments thereof involved in the present invention can be naturally purified products, or chemically synthesized products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants, etc.).

[0042] Table 1

[0043]

[0044]

[0045]

[0046] Table 2

[0047]

[0048]

[0049] Example 1 Candidate gene association analysis based on natural variation of GhCYCU4-1 gene

[0050] (1) Comprehensive drought resistance and population phenotypic analysis of cotton

[0051] The present invention uses the Comprehensive Index of Drought Tolerance (CIDT) to evaluate the comprehensive drought resistance of cotton. The calculation formula is as follows:

[0052] CIDT=DRC(LP+LW+YPH+FSBN) / (GP+SW+FL+FS+FE+PH+FFSH+FFSBN+EBN), where DRC is the ratio of the phenotypic value of drought treatment to that of the control. Each trait corresponds to a separate DRC value. The population means of lint percentage (LP), lint index (LW), seed cotton yield per hectare (YPH), and fruiting branch number (FSBN) increased significantly under water restriction (p<0.001), while the population means of growth period (GP), seed index (SW), fiber length (FL), fiber strength (FS), fiber elongation (FE), plant height (PH), height at the first node of fruiting branch (FFSH), position at the first node of fruiting branch (FFSBN), and effective boll number per plant (EBN) decreased significantly under water restriction (p<0.001).

[0053] (II) Construction of natural variation map of upland cotton and genome-wide association

[0054] The present invention utilizes publicly available resequencing data from 517 upland cotton accessions (raw resequencing data: see the SRA database in NCBI, project number PRJNA556955) to construct a natural variation map for upland cotton. Drought resistance phenotypes were assessed using CIDT from the 517 accessions. Genome-wide association analysis, combined with the resequencing data, identified drought quantitative trait loci (QTLs) associated with CIDT. Differentially expressed genes within the QTLs were identified, and functional annotation of the associated genes was performed. The candidate gene GhCYCU4-1 (Ghir_A12G010520 in the Cotton FGD database) was identified. GhCYCU4-1 plays a role in mediating BR regulation of cell division and thus controlling leaf uprightness. Multiple variants within the GhCYCU4-1 locus (including the sequence upstream of the GhCYCU4-1 gene start codon) were found to be significantly associated with CIDT. Among them, the insertion / deletion of 17bp (see positions 2-18 of SEQ ID NO: 3) located in the upstream regulatory region of the candidate gene GhCYCU4-1 (918bp from the start codon ATG) was significantly correlated with drought-resistance-related phenotypes. At the same time, verification of the Indel molecular marker revealed that the drought-sensitive material did indeed have a 17bp base deletion, while the drought-resistant material did not have this variation.

[0055] (III) Candidate gene association analysis based on natural variation of the GhCYCU4-1 gene

[0056] 1. Obtain the variation information between 76505993 bp and 76512080 bp on chromosome A12 in the natural variation map, that is, the variation information of the GhCYCU4-1 locus (including the 2000 bp before the start codon of the GhCYCU4-1 gene) (as shown in Table 3).

[0057] There are 36 mutations in the GhCYCU4-1 locus, of which 14 are in the upstream regulatory region, including 7 Indel mutations and 7 SNP mutations; 9 are in the upstream regulatory region, including 1 Indel mutation and 8 SNP mutations; 3 SNP mutations are in the 5'UTR region of the gene; 6 SNP mutations and 3 Indel mutations are in the 3'UTR region of the gene; there is also a synonymous mutation.

[0058] The reference genome of upland cotton TM-1, Ghirsutum_genome_HAU_v1.0, has an Indel mutation of CAGCGAAAAACTAAGTGT→C at position 76507400 of chromosome A12 compared to the reference genome. The reference genome version of upland cotton TM-1, Ghirsutum_genome_HAU_v1.0, can be obtained from the CottonFGD database at:

[0059] https: / / cottonfgd.net / about / download.html.

[0060] Table 3

[0061]

[0062]

[0063] Note: The meanings of the categories in the table are as follows: Type: variant type; Pos: physical position in the genome; Ref: reference genotype; Alt: variant genotype; DisttoATG: physical distance from the start codon, negative values ​​indicate upstream of the start codon.

[0064] 2. The cotton materials were grouped according to different genotypes based on the variation information. For example, the SNP variation located at the 76506930bp position of chromosome A12 of the reference genome had two haplotypes, A and T, and its allele genotypes were AA, TT, and AT. CIDT was used as the drought resistance phenotypic value for association analysis, and the grouped t-test of drought resistance-related phenotypic values ​​was calculated. The p-value reflected the correlation between the variation and the phenotype after the t-test, with a significance threshold of less than 0.05.

[0065] Figure 1 The correlation between variants at different physical locations and CIDT phenotypic values ​​is shown; Figure 1 Figure (a) shows, from top to bottom, the Manhattan plot and linkage disequilibrium block (LD Block) plots between variants at different physical locations and drought resistance phenotypes. In the Manhattan plot, the horizontal axis represents the physical position of the variant on the reference genome, and the vertical axis represents -log(p), where p represents the t-test result. In the LD Block plot, the upper bar shows the relative physical position of the variant on the reference genome, and the lower red squares represent the LD values ​​for different variants. Darker squares indicate higher LD values. Typically, a group of highly linked genetic variants form a linkage disequilibrium block, and variants within these blocks are often inherited together. The results showed that 28 variants were significantly associated with drought resistance-related phenotypic values, including one indel variant.

[0066] Figure 1 Figure b shows the distribution of the aforementioned Indel mutations at the GhCYCU4-1 locus, located at position 76507400bp on chromosome A06 of the upland cotton TM-1 reference genome. AGCGAAAAACTAAGTGT(SEQ ID NO: 3) → C deletion variant, the underlined portion is the deleted sequence (length 17 bp).

[0067] (IV) Development and validation of functional molecular markers for indel mutations

[0068] Because regulatory regions play a crucial role in gene expression, and linkage disequilibrium analysis also showed a significant correlation between indel variants in the GhCYCU4-1 gene regulatory region and drought resistance phenotypes, the present invention selected these indel variants for development of functional molecular markers. 517 natural cotton populations were grouped according to haplotypes corresponding to SEQ ID NO: 3 and C. The CIDT values ​​corresponding to plants with different haplotypes were calculated and used as phenotypic values ​​for a grouped t-test. Because heterozygotes of these two haplotypes are extremely rare in natural samples, heterozygous genotypes were not included in the analysis.

[0069] Figure 2 The phenotypic data for 517 natural cotton populations, grouped by haplotypes harboring the indel variant at position 76507400bp on chromosome A12, are shown. The horizontal axis represents haplotype, and the vertical axis represents CIDT. The figure shows that the different haplotypes of the indel variant are highly correlated with the drought resistance phenotype of cotton (p=5.8e-05). The CIDT values ​​of the different haplotypes differ significantly. Cotton accessions carrying haplotype SEQ ID NO: 3 (18bp) have significantly higher CIDT values ​​than those carrying haplotype C, indicating that the presence of this deletion impairs cotton's tolerance to drought.

[0070] In summary, this example develops a drought-resistant molecular marker based on the Indel variation SEQ ID NO: 3→C in the upstream regulatory region of the GhCYCU4-1 gene of upland cotton. The Indel molecular marker is located at the 76507400bp position of chromosome A12 of the upland cotton TM-1 "Ghirsutum_genome_HAU_v1.0" reference genome, based on the position of the haplotype SEQ ID NO: 3. The genotype in the drought-resistant cotton germplasm is SEQ ID NO: 3, and the genotype in the drought-sensitive cotton germplasm is C.

[0071] As shown in Table 1, SEQ ID NO: 1 is a nucleotide sequence containing the Indel variation. The sequence segment is excerpted from the regulatory sequence 2000 bp upstream of the GhCYCU4-1 gene. Haplotype SEQ ID NO: 3 is located at bases 1083-1100 in SEQ ID NO: 1 (indicated by shading and bold). The nucleotide sequence of haplotype "C" is obtained by replacing bases 1083-1100 in SEQ ID NO: 3 with "C".

[0072] Example 2 Molecular markers, detection primers and detection methods developed based on Indel mutations in the upstream regulatory region of the GhCYCU4-1 gene in upland cotton

[0073] 1. Molecular marker authenticity verification

[0074] Based on the material information and corresponding genotype information in the variation map of Example 1, one extreme drought-resistant material was selected, each with the genotypes of SEQ ID NO: 3 and C at the 76507400 bp position. In this example, molecular marker amplification was performed using natural populations Wankangmian 9 and Junmian 1. Wankangmian 9 is a typical drought-resistant variety with a genotype of haplotype SEQ ID NO: 3, and Junmian 1 is a drought-sensitive germplasm with a genotype of haplotype C. The main steps are as follows:

[0075] (1) Primer design:

[0076] According to the upstream regulatory sequence of GhCYCU4-1 and the marker position in SEQ ID NO: 1, PCR primers were designed, the upstream primer and the downstream primer were F2 and R2, respectively (see Table 1).

[0077] (2) Genomic DNA extraction:

[0078] The CTAB method was used to extract genomic DNA from Wankangmian 9 and Junmian 1. Specifically, 100 mg of fresh cotton leaves were added to 200 μL of extraction buffer, ground, and then 800 μL of CTAB lysis buffer was added. The samples were lysed in a 65°C water bath for 30 minutes. 800 μL of chloroform was added to the lysed samples, and the samples were inverted and mixed for 5 minutes to remove impurities such as pigments. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was transferred and mixed with an equal volume of -20°C pre-cooled isopropanol. The samples were placed at -20°C for precipitation for 2 hours, centrifuged at 12,000 rpm for 1 minute, and the supernatant was discarded. The pellet was rinsed with 1 mL of 75% ethanol for a total of two 10-minute rinses. The supernatant was discarded after centrifugation and the pellet was left to dry at room temperature. The DNA was dissolved in ddH2O. The extraction buffer consists of 0.35 M glucose, 0.1 M Tris-HCl, 5 mmol / L Na2EDTA, 2% (w / v) PVP K-30, and 0.1% (w / v) DIECA, pH 7.5. 0.2% β-mercaptoethanol was added before use. The CTAB lysis buffer consists of 0.1 M Tris-HCl, 1.4 M NaCl, 0.02 M Na2EDTA, 2% CTAB, 2% (w / v) PVP K-30, and 0.1% (w / v) DIECA, pH 8.0.

[0079] (3) PCR amplification:

[0080] 75-100 ng of genomic DNA was used as a template for PCR amplification. The PCR amplification reaction system (20 μL) included: 1 μL of genomic DNA, 2.0 μL of 10× buffer, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 0.4 μL of dNTP mix, 0.2 μL of Taq DNA polymerase, and 16 μL of sterile water. In the reaction system, the concentrations of upstream and downstream primers were 1-2 mM, and the concentration of genomic DNA was 3.75-5 ng / μL.

[0081] The PCR amplification reaction procedure included: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 51°C for 30 s, and extension at 72°C for 30 s, for 30 cycles; and extension at 72°C for 5 min.

[0082] (4) Result detection:

[0083] After PCR amplification of F2 and R2 using primers, the PCR amplification products were sent to the company for Sanger sequencing to confirm the authenticity of the marked Indel in the sequence.

[0084] The results are as follows Figure 3 As shown, Figures a and b are the genotype information and sequencing peak diagrams of Junmian 1 and Wankangmian 9 respectively. Figure 3 It can be seen that the genotype of Wankangmian 9 at the physical position of 76507400bp is SEQ ID NO: 3 (drought-resistant genotype); the genotype of Junmian1 at the physical position of 76507400bp is C (drought-sensitive genotype), thereby confirming the existence of Indel variation and can be used as a drought-resistance molecular marker to distinguish drought-resistant and drought-sensitive cotton germplasm.

[0085] 2. PCR detection primer sequence design

[0086] After confirming the authenticity of the marker, the marker primer sequence was designed. The upstream primer was located upstream of the mutation site, and the downstream primer contained the Indel mutation at the physical position of 76507400bp (CAGC GAAAAACTAAGTGT) is underlined in the partial sequence, and the target sequence amplification size is 346bp. The detection principle is: cotton materials containing the Indel molecular marker can amplify the target sequence, while cotton materials that do not contain the Indel molecular marker have no specific binding with the downstream primers, and their genomes basically do not amplify or cannot amplify a band of the target size. Therefore, the presence or absence of PCR amplification products and their band size can be used to conveniently determine whether the Indel molecular marker is contained, and then identify the cotton germplasm genotype. The nucleotide sequences (5'-3') of the PCR amplification primers F1 and R1 of the target sequence (SEQ ID NO: 8) are shown in Table 1;

[0087] Five cotton accessions of each genotype were selected for molecular marker amplification using the same PCR amplification system and procedure as described above. The drought-sensitive accessions were Xinluzao 9, Xinluzao 32, Xinluzhong 4, Zhongmiansuo 13, and Jinmian 24, while the drought-resistant accessions were Jihan 3, Xuzhou 142, Xuzhou 1514, Shanmian 401, and Xiaomian 1. PCR amplification and sequencing were performed simultaneously using the upstream and downstream primers indicated by F2 and R2 to confirm that the PCR primer amplification results were consistent with the sequencing results.

[0088] The results are as follows Figure 4 Figure a shows the sequencing results of the product amplified by PCR using F2 and R2, and Figure b shows the electrophoresis results of the product amplified by PCR using F1 and R1. The results of Figures a and b match, indicating that the PCR detection primers of the present invention are effective. The presence of the PCR amplification product can be used to determine whether the indel molecular marker is present, thereby identifying or assisting in the identification of cotton drought resistance.

[0089] The judgment rules for using detection primers to identify drought resistance of cotton germplasm are as follows:

[0090] When the detection primers are F1 and R1 (SEQ ID NOs: 4-5), if the PCR amplification product can specifically amplify a band and the 346 bp band pattern as shown in SEQ ID NO: 8 is present, it can be determined that the cotton germplasm to be tested has drought resistance and belongs to drought-resistant cotton germplasm; conversely, if the PCR amplification product cannot specifically amplify a band, or the amplified band pattern does not include the 346 bp band pattern, for example, the PCR amplification product has a 329 bp band pattern as shown in SEQ ID NO: 10, then the cotton germplasm to be tested is drought-sensitive and belongs to drought-sensitive cotton germplasm.

[0091] When the detection primers are F2 and R2 (SEQ ID NOs: 6-7), if the PCR amplification product can specifically amplify a band, and there is a 349bp band pattern as shown in SEQ ID NO: 9, it can be determined that the cotton germplasm to be tested has drought resistance and belongs to a drought-resistant cotton germplasm; if the PCRPCR amplification product has a 332bp band pattern as shown in SEQ ID NO: 11, the cotton germplasm to be tested is drought-sensitive and belongs to a drought-sensitive cotton germplasm.

[0092] Example 3 Application of the Indel Molecular Markers, Detection Primers, and Detection Methods Provided by the Present Invention in Natural Populations

[0093] In this example, the detection primers identified in Example 2 were used to select 28 drought-resistant and drought-sensitive accessions based on CIDT phenotypic data from natural populations. Genomic DNA was extracted. PCR amplification was performed using F1 and R1 as upstream and downstream primers, respectively. The PCR amplification system and procedure were the same as in Example 2.

[0094] The electrophoresis diagram of PCR amplification products is as follows Figure 5 Figure a shows drought-sensitive accessions, and Figure b shows drought-resistant accessions. * indicates accessions with unclear or nonspecific amplification. Results showed that among the drought-sensitive accessions, six amplified bands were detected, while 22 failed to amplify clear specific bands. Among the drought-resistant accessions, one failed to amplify clear specific bands, while 27 amplified the target bands. A chi-square test revealed a significant difference in amplification efficiency between drought-resistant and drought-sensitive accessions, with a p-value of 1.17E-08, confirming a significant correlation between the indel variant genotype and drought-resistant phenotypic traits in natural populations.

[0095] In summary, the Indel mutations provided by the present invention and the detection primers developed based on the Indel mutations can be used to detect drought resistance in cotton. The detection accuracy is high, and the genotype and phenotype are highly consistent, providing a convenient method for high-throughput screening of drought-resistant cotton varieties.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An application of an Indel molecular marker related to drought resistance in upland cotton, characterized in that: The Indel molecular marker includes at least one DNA molecule selected from SEQ ID NOs: 8, 9, and 11; if the Indel molecular marker SEQ ID NO: 8 or SEQ ID NO: 9 is detected in the upland cotton genome to be tested, the upland cotton germplasm to be tested is determined to have drought resistance; if the Indel molecular marker SEQ ID NO: 11 is detected in the upland cotton genome to be tested, the upland cotton germplasm to be tested is determined to be drought-sensitive; and the application includes at least one of the following applications: (1) Identify or assist in identifying the drought resistance of upland cotton; (2) Compare the drought resistance of the tested upland cotton; (3) Breeding drought-resistant upland cotton plants, lines, strains or varieties; (4) Prepare testing products related to drought resistance of upland cotton.

2. A method for detecting drought resistance of upland cotton, characterized in that: The method comprises the following steps: S1 PCR amplifies the target region containing the Indel molecular marker in the genomic DNA of the cotton to be tested; S2 Determine the drought resistance of the cotton to be tested by DNA sequencing or gel electrophoresis of the PCR amplification product; The Indel molecular marker includes at least one DNA molecule of SEQ ID NO: 8, 9 and 11; If the Indel molecular marker SEQ ID NO: 8 or SEQ ID NO: 9 is detected in the upland cotton genome to be tested, the upland cotton germplasm to be tested is determined to have drought resistance; if the Indel molecular marker or SEQ ID NO: 11 is detected in the upland cotton genome to be tested, the upland cotton germplasm to be tested is determined to be drought-sensitive.

3. The method according to claim 2, characterized in that In the step S1, The PCR amplification reaction system, in 20 μL, includes: 1 μL of genomic DNA, 2.0 μL of 10× buffer, 0.2 μL of upstream primer, 0.2 μL of downstream primer, 0.4 μL of dNTP mix, 0.2 μL of Taq DNA polymerase, and 16 μL of sterile water; in the reaction system, the concentrations of the upstream primer and the downstream primer are 1-2 mM, and the concentration of the genomic DNA is 3.75-5 ng / μL.

4. The method according to claim 2 or 3, characterized in that In step S1: the primers for PCR amplification include primers with nucleotide sequences as shown in SEQ ID NO: 4-5; In step S2: judging by the sequencing results of the PCR amplification products, if the PCR amplification products can specifically amplify a band and the 346bp band pattern as shown in SEQ ID NO: 8 is present, it can be determined that the upland cotton germplasm to be tested has drought resistance; if the PCR amplification products cannot specifically amplify a band, or the amplified band pattern does not include the 346bp band pattern, the upland cotton germplasm to be tested is drought-sensitive.

5. The method according to claim 2 or 3, characterized in that In step S1: the primers for PCR amplification include primers with nucleotide sequences as shown in SEQ ID NOs: 6-7; In step S2: judging by sequencing the PCR amplification product, if the PCR amplification product can specifically amplify a band and there is a 349bp band pattern as shown in SEQ ID NO: 9, it can be determined that the upland cotton germplasm to be tested has drought resistance; if the PCR amplification product has a 332bp band pattern as shown in SEQ ID NO: 11, the upland cotton germplasm to be tested is drought-sensitive.

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