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

Abstract

The invention relates to the technical field of biology, in particular to an Indel molecular marker related to upland cotton drought resistance and a method and application thereof. The Indel molecular marker is located at the 1083rd to 1100th basic groups of a nucleotide sequence as shown in SEQ ID NO: 1, the genotype of the Indel molecular marker is CAGCGAAAAACATAAGTGT in a drought-resistant cotton germplasm, and the genotype of the Indel molecular marker is C in a sensitive drought cotton germplasm. The Indel molecular marker provided by the invention is closely linked with the drought resistance phenotype of upland cotton, can be used for efficiently and accurately identifying or assisting in identifying the drought resistance of to-be-detected cotton and effectively distinguishing drought-resistant and sensitive drought-resistant cotton germplasm, and provides an effective means and way 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] As one of the world's major cash crops, cotton is widely planted in more than 30 countries and plays an indispensable role in many fields such as textiles and medicine. However, its growth is easily restricted by extreme environmental conditions, among which drought is particularly prominent. Cotton is highly sensitive to water deficit during the seedling stage, flowering stage and boll development stage. Drought stress not only hinders the normal growth and development of cotton, but also causes a sharp drop in yield and a significant decline in fiber quality, bringing a heavy blow to the cotton industry. Therefore, cultivating cotton varieties with strong drought resistance and reducing drought damage have become key tasks for the sustainable development of the cotton industry.

[0003] The traditional cotton breeding model mainly relies on direct observation and screening of phenotypic traits. However, due to the complex and changeable natural environment, external factors such as light, temperature, and soil fertility have a huge impact on the cotton phenotype, making it difficult to accurately determine the relationship between phenotype and genotype, greatly limiting the efficiency of breeding excellent varieties and making it difficult to meet the urgent needs of the current cotton industry for drought-resistant varieties.

[0004] With the vigorous development of molecular biology technology, molecular marker-assisted selection breeding has emerged and gradually emerged. Compared with traditional breeding methods, this technology breaks through the dual constraints of time and region, can accurately and quickly identify target traits at the genetic level, and significantly improves the accuracy and timeliness of breeding. It has become the priority strategy for identifying drought-resistant cotton varieties at this stage, and its core position in the field of cotton breeding is becoming increasingly prominent. In the construction of molecular marker-assisted selection breeding system, the development and regulation of molecular markers that are closely linked to genes related to cotton drought resistance is the cornerstone for achieving efficient breeding.

[0005] Insertion-deletion markers (InDel) are an important type of molecular markers. They are based on sequence differences at the same site in the genome between closely related species or different individuals of the same species, that is, the presence of one or more base insertions or deletions, thus presenting length polymorphism. InDel markers are widely distributed in the genome, have considerable density, are numerous, have good polymorphism and high stability, and can be widely applied to multiple species. In practical applications, primers are designed using InDel polymorphic molecular markers for PCR amplification. Based on the size differences of the amplified products, large quantities of samples can be tested efficiently and quickly, their genotypes can be accurately determined, and then the sample phenotypes can be effectively distinguished, 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, related molecular marker research is quite scarce. 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 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 view of this, the present invention proposes an Indel molecular marker, method and application related to drought resistance of upland cotton. The Indel molecular marker is closely linked to the drought resistance phenotype of upland cotton, and can be used to efficiently and accurately identify or assist in identifying the drought resistance of the cotton to be tested, effectively distinguish drought-resistant and drought-sensitive cotton germplasm, and provide an effective means and approach for the selection of cotton drought-resistant materials and the cultivation of new cotton germplasm resources through molecular marker assistance.

[0008] The technical solution of the present invention is achieved in this way:

[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 NO: 8-11.

[0010] In a second aspect, the present invention provides detection primers for detecting the Indel molecular marker, and 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, the components of the reagent / kit include at least one of the following: (1) PCR primers for amplifying the Indel molecular marker; (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 an application 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 plants;

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

[0016] (4) Cotton breeding;

[0017] (5) Prepare testing 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] The present invention has discovered an Indel variation in the upstream regulatory region of the GhCYCU4-1 gene through genome-wide association analysis, and the variation is closely linked to the drought resistance phenotype. At the same time, the CIDT values ​​of the drought resistance phenotype corresponding to different genotypes at the Indel variation site are extremely different, and the genotype is significantly correlated with the drought resistance phenotype and has high consistency.

[0023] The present invention provides an Indel molecular marker related to drought resistance of cotton, and provides an accurate and efficient detection method: using the genomic DNA of the cotton sample to be tested as a template, using specific primers for targeted amplification, and accurately obtaining the Indel molecular marker genotype through the sequence information of the amplified product; thereby efficiently and accurately identifying, or assisting in identifying the drought resistance of the cotton to be tested, and distinguishing drought-resistant cotton germplasm from drought-sensitive cotton germplasm. The present invention provides a practical means and approach for molecular marker-assisted selection and breeding of cotton drought-resistant materials, as well as the cultivation of new cotton germplasm resources, and has important practical significance and application value.

[0024] the term

[0025] Indel (Insertion-Deletion) mutation refers to an insertion or deletion mutation that occurs in a genome sequence. That is, a segment of bases is added or subtracted in a DNA sequence compared to a reference genome. In some specific embodiments of the present invention, the Indel molecular marker is a molecular marker based on insertion / deletion (Insertion / Deletion) mutations in a DNA sequence; it refers to a polymorphic marker generated by the insertion or deletion of a segment of a DNA sequence at a specific position in the genome. The length of these inserted or deleted sequences can range from 1 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 the Indel variation in the upstream regulatory region of the GhCYCU4-1 gene. The specific location of the Indel variation in the upstream regulatory region of the GhCYCU4-1 gene is as follows:

[0027] The Indel mutation is located at the 76507400bp position of chromosome A12 of the upland cotton TM-1 reference genome "Ghirsutum_genome_HAU_v1.0", and compared with the reference genome, an Indel mutation of CAGCGAAAAACTAAGTGT→C occurs. The upland cotton TM-1 reference genome version Ghirsutum_genome_HAU_v1.0 can be openly obtained from the CottonFGD database at https: / / cottonfgd.net / about / download.html.

[0028] The Indel molecular marker includes: the base sequence from position 1083 to position 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 the 2nd to the 18th position 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 markers 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, the genomic DNA of the sample to be tested is used as a template, and the detection primers of the Indel molecular marker are used to perform a PCR reaction; the genotype of the Indel molecular marker in the PCR amplification product is detected. The drought resistance characteristics of the cotton germplasm to be tested are then determined based on the detection results, wherein the determination includes the following steps:

[0032] When the primers are F1 and R1 (SEQ ID NO: 4-5), if the PCR amplification product can specifically amplify a band, and there is a 346bp band pattern as shown in SEQ ID NO: 8, 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 contain the 346bp band pattern, for example: the PCR amplification product is a 329bp 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 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 drought-resistant cotton germplasm; if the PCR 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 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 creative work.

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

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

[0037] Figure 3The genotypes and sequencing peaks of the Indel variation at the GhCYCU4-1 locus of drought-resistant and drought-sensitive cotton germplasms in 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 The results of amplifying the Indel variation at the GhCYCU4-1 locus using different primers in Example 2 of the present invention are shown; wherein, Figure a is a sequencing peak diagram of the product amplified using the primers shown in SEQ ID NOs: 6-7, and Figure b is an electrophoresis diagram of the product amplified using the primers shown in SEQ ID NOs: 4-5;

[0039] Figure 5 This is the electrophoresis diagram of the product of amplifying the 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] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in 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 various ways of obtaining biological materials described in the examples are only to provide an experimental way of obtaining in order to achieve the specific disclosed purpose, and should not be a limitation on the source of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials 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 from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants, etc.) using recombinant technology.

[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] (I) Comprehensive drought resistance and population phenotype analysis of cotton

[0051] The present invention uses the Comprehensive Index of Drought Tolerance (CIDT) to evaluate the comprehensive drought resistance of cotton, and 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 drought treatment phenotypic value to control phenotypic value. Each trait corresponds to a respective DRC value. The population average values ​​of lint percentage (LP), lint index (LW), seed cotton yield per hectare (YPH), and fruiting branch number (FSBN) increased significantly under water restriction conditions (p<0.001), while the population average values ​​of growth period (GP), seed index (SW), fiber length (FL), fiber strength (FS), fiber elongation (FE), plant height (PH), height of first node of fruiting branch (FFSH), first node position of fruiting branch (FFSBN), and number of effective bolls per plant (EBN) decreased significantly under water restriction conditions (p<0.001).

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

[0054] The present invention uses 517 publicly published resequencing data of upland cotton (resequencing raw data: see the SRA database in NCBI, project number PRJNA556955) to construct a natural variation map of upland cotton. The drought resistance phenotype of 517 materials is evaluated by CIDT, and a whole genome association analysis is performed in combination with the resequencing data to identify the drought quantitative trait locus (QTL) segment associated with CIDT, screen out differentially expressed genes in the QTL segment, and further perform functional annotation on the related genes to obtain the candidate gene GhCYCU4-1 (Cotton FGD database gene number Ghir_A12G010520), GhCYCU4-1 mediates BR to regulate cell division and thus plays a role in controlling leaf uprightness, and it is found that multiple variations on the GhCYCU4-1 locus (including the upstream sequence of the GhCYCU4-1 gene start codon) are 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 away from the start codon ATG) was significantly correlated with the drought resistance-related phenotypes. At the same time, the Indel molecular marker was verified, and it was found that the drought-sensitive material did 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 GhCYCU4-1 gene

[0056] 1. Obtain the variation information between 76505993 bp and 76512080 bp of 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; and 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 76507400bp on chromosome A12 compared with 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 grouped t-tests of drought resistance-related phenotypic values ​​were calculated. The p-value reflected the correlation between the variation and the phenotype after the t-test, with a significant threshold of less than 0.05.

[0065] Figure 1 The correlation between variants at different physical locations and CIDT phenotype values ​​is shown; Figure 1 As shown in Figure a, from top to bottom are the Manhattan plot and linkage disequilibrium block (LD Block) between variant sites at different physical locations and drought resistance phenotypes; in the Manhattan plot, the horizontal axis is the physical position of the variant site on the reference genome, the vertical axis is -log(p), and p is the t test result; in the LD block plot, the upper bar shows the relative physical position of the variant site in the reference genome, and the lower red square represents the LD value of different variant sites. The darker the square, the higher the LD value. Usually, a group of highly linked gene variants form a linkage disequilibrium block, and the variants in these blocks are usually inherited together. The results showed that 28 variants were significantly correlated with drought resistance-related phenotypic values, including an Indel variant.

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

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

[0068] Since the regulatory region plays an important role in the expression change of the gene, and linkage disequilibrium analysis also shows that there is a significant correlation between the Indel variation in the regulatory region of the GhCYCU4-1 gene and the drought resistance phenotype, the present invention selects the Indel variation to develop a functional molecular marker. 517 natural cotton populations were grouped according to the two haplotypes of SEQ ID NO: 3 and C, and the CIDT corresponding to the plants of different haplotypes was counted and used as the phenotypic value for a group t test; since the heterozygotes of these two haplotypes accounted for a very small proportion in the natural samples, the heterozygous genotype was not included in the analysis.

[0069] Figure 2 The phenotypic data statistics of 517 cotton natural populations grouped according to different haplotypes of Indel mutation at position 76507400bp on chromosome A12 are shown, wherein the abscissa represents haplotype and the ordinate represents CIDT. It can be seen from the figure that different haplotypes of the Indel mutation are highly correlated with the drought resistance phenotype of cotton (p=5.8e-05), and the CIDT values ​​of different haplotypes are extremely significantly different. The CIDT value of cotton germplasm carrying haplotype SEQ ID NO: 3 (18bp) is significantly higher than that of cotton germplasm carrying haplotype C, indicating that the presence of the above-mentioned deletion mutation will weaken the tolerance of cotton to drought environment.

[0070] In summary, this embodiment develops a drought-resistant molecular marker for 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 reference genome of upland cotton TM-1 "Ghirsutum_genome_HAU_v1.0" 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 segment sequence is excerpted from the regulatory sequence of 2000 bp upstream of the GhCYCU4-1 gene, haplotype SEQ ID NO: 3 is located at bases 1083-1100 in SEQ ID NO: 1 (shown in bold shade), and the nucleotide sequence of haplotype "C" is obtained by replacing bases 1083-1100 of 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] According to the material information and corresponding genotype information in the variation map of Example 1, one copy of each drought-resistant extreme material with genotypes of SEQ ID NO: 3 and C at the 76507400bp position was selected. In this example, natural populations Wankangmian 9 and Junmian 1 were used for molecular marker amplification. 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 and marker position of GhCYCU4-1 in SEQ ID NO: 1, PCR primers were designed, and the upstream primer and downstream primer were F2 and R2, respectively (see Table 1).

[0077] (2) Genomic DNA extraction:

[0078] The CTAB method was used to extract the genomic DNA of Wankangmian 9 and Junmian 1 materials. Specifically, 100 mg of fresh cotton leaves were added to 200 μL of extraction buffer, ground, added to 800 μL of CTAB lysis solution, lysed in a 65°C water bath for 30 minutes, and 800 μL of chloroform was added to the lysed sample. The sample was turned over and mixed for 5 minutes to remove impurities such as pigments. After centrifugation at 12000 rpm for 10 minutes, the supernatant was transferred and mixed with an equal volume of -20°C pre-cooled isopropanol, placed at -20°C for precipitation for 2 hours, centrifuged at 12000 rpm for 1 minute, and the supernatant was discarded. The precipitate was rinsed with 1 mL of 75% ethanol, rinsed twice for a total of 10 minutes each time, centrifuged and the supernatant was discarded, and the precipitate was placed at room temperature to dry naturally, and washed with ddH 2 O to dissolve DNA. The extraction buffer composition is: 0.35M glucose, 0.1M Tris-HCl, 5mmol / LNa 2 EDTA, 2% (W / V) PVP K-30 and 0.1% (W / V) DIECA, pH 7.5, add 0.2% β-mercaptoethanol before use. CTAB lysis buffer composition: 0.1M Tris-HCl, 1.4MNaCl, 0.02MNa 2EDTA, 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 reaction system for PCR amplification was 20 μL, including: 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 dNTPmix, 0.2 μL of TaqDNA polymerase and 16 μL of sterile water; wherein, in the reaction system, the concentrations of upstream primer and downstream primer were 1-2 mM, and the concentration of genomic DNA was 3.75-5 ng / μL.

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

[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 (CAGC) at the physical position of 76507400bp. GAAAAACTAAGTGT) is underlined in the partial sequence, and the target sequence amplification size is 346bp. The detection principle is: the cotton material containing the Indel molecular marker can amplify the target sequence, while the cotton material without the Indel molecular marker has no specific binding with the downstream primer, and its genome basically does not amplify or cannot amplify the band of the target size. Therefore, the presence or absence of the PCR amplification product and its 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 copies of each of the two genotype cotton materials were selected for molecular marker amplification, and the PCR amplification system and amplification procedure were the same as above. The drought-sensitive materials were Xinluzao 9, Xinluzao 32, Xinluzhong 4, Zhongmiansuo 13 and Jinmian24, and the drought-resistant materials were Jihan 3, Xuzhou 142, Xuzhou 1514, Shanmian 401 and Xiaomian 1. At the same time, the upstream primers and downstream primers shown in F2 and R2 were used for PCR amplification and sequencing to confirm that the PCR detection primer amplification results were consistent with the sequencing results.

[0088] The results are as follows Figure 4 As shown, Figure a is the sequencing result of the product amplified by PCR using F2 and R2, and Figure b is the electrophoresis result of the product amplified by PCR using F1 and R1. The results of Figure a and Figure b are consistent, indicating that the PCR detection primers of the embodiment of the present invention are applicable, and whether the PCR amplification product contains the Indel molecular marker can be judged by the presence or absence of the PCR amplification product, thereby identifying or assisting in identifying the drought resistance of cotton.

[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 NO: 4-5), if the PCR amplification product can specifically amplify a band, and there is a 346bp band pattern as shown in SEQ ID NO: 8, 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 contain the 346bp band pattern, for example: the PCR amplification product is a 329bp 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 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 drought-sensitive cotton germplasm.

[0092] Example 3 Application of Indel molecular markers, detection primers and detection methods provided by the present invention in natural populations

[0093] In this example, the detection primers confirmed in Example 2 were used, and according to the CIDT phenotype data of the natural population, 28 drought-resistant materials and 28 drought-sensitive materials were selected to extract genomic DNA. F1 and R1 were used as upstream and downstream primers for PCR amplification. The PCR amplification system and amplification procedure were the same as in Example 2.

[0094] The electrophoresis of PCR amplification products is shown in Figure 5 , where Figure a is drought-sensitive material, Figure b is drought-resistant material, and the * mark in the figure indicates that the amplification is not obvious or non-specific. The results showed that 6 materials in the drought-sensitive material could amplify the bands, and 22 materials did not amplify the obvious specific bands; 1 material in the drought-resistant material did not amplify the obvious specific bands, and 27 materials amplified the target bands. The chi-square test found that the amplification efficiency between drought-resistant materials and drought-sensitive materials was significantly different, with a p value of 1.17E-08, confirming that the genotype of Indel variation in natural populations is significantly correlated with the drought resistance phenotypic traits of cotton.

[0095] In summary, the Indel variation provided by the present invention and the detection primers developed based on the Indel variation can be used to detect cotton drought resistance, and can distinguish cotton drought-resistant and sensitive germplasm based on the electrophoretic band distribution of the amplified product, with high accuracy, and high consistency between the genotype and the phenotype, 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 protection scope of the present invention.

Claims

1. An Indel molecular marker related to drought resistance of upland cotton, characterized in that: The Indel molecular marker comprises at least one DNA molecule in SEQ ID NO: 8-11.

2. The Indel molecular marker according to claim 1, characterized in that: The Indel molecular markers include: In the drought-resistant cotton germplasm, the genotype of the Indel mutation site is SEQ ID NO: 3; In the drought-sensitive cotton germplasm, the genotype of the Indel mutation site is C; The Indel mutation site is located at bases 1083-1100 of the sequence shown in SEQ ID NO:

1.

3. A detection primer for detecting the Indel molecular marker according to claim 1, characterized in that: Primer pairs SEQ ID NO: 4-5 or SEQ ID NO: 6-7 are included.

4. A reagent / kit for detecting the Indel molecular marker according to claim 1, characterized in that: The components of the reagent / kit include at least one of the following: (1) a PCR primer for amplifying the Indel molecular marker of claim 1; (2) a detection primer of claim 3.

5. The reagent / kit according to claim 4, characterized in that: The components of the reagent / kit also include: Taq DNA polymerase, dNTPs and buffer reagent.

6. Use of the Indel molecular marker according to any one of claims 1 to 2, the detection primer according to claim 3, or the reagent / kit according to any one of claims 4 to 5, characterized in that: The application includes at least one of the following applications: (1) Identify or assist in identifying the drought resistance of cotton; (2) Compare the drought resistance of the tested cotton plants; (3) Breeding drought-resistant cotton plants, strains, lines or varieties; (4) Cotton breeding; (5) Prepare testing products related to cotton drought resistance.

7. A method for detecting the Indel molecular marker according to claim 1, characterized in that: The method comprises the following steps: S1 performs PCR amplification on the genomic DNA of the cotton to be tested; S2 determines the drought resistance of the cotton to be tested by DNA sequencing or gel electrophoresis results of the PCR amplification products.

8. The method according to claim 7, characterized in that In the step S1, The reaction system for PCR amplification, 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 TaqDNA 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.

9. The method according to claim 7 or 8, characterized in that: In the step S1: the primers for PCR amplification include SEQ ID NO: 4-5; In the step S2: judging by sequencing the PCR amplification product, if the PCR amplification product can specifically amplify a band and there is a 346 bp band pattern as shown in SEQ ID NO: 8, it can be determined that the cotton germplasm to be tested has drought resistance; If the PCR amplification product cannot specifically amplify a band, or the amplified band pattern does not include the 346 bp band pattern, the cotton germplasm to be tested is drought-sensitive.

10. The method according to claim 7 or 8, characterized in that: In the step S1: the primers for PCR amplification include SEQ ID NO: 6-7; In the step S2: judging by sequencing the PCR amplification product, 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; 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.

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