CN115838829A

CN115838829A - Upland cotton GhGMP08 gene drought resistance related molecular marker and application thereof

Info

Publication number: CN115838829A
Application number: CN202211580439.7A
Authority: CN
Inventors: 陈琴; 付锦程; 陈全家; 高文举; 孙丰磊; 张虎; 曲延英; 邓晓娟; 谢雨婷
Original assignee: Xinjiang Agricultural University
Current assignee: Xinjiang Agricultural University
Priority date: 2022-12-09
Filing date: 2022-12-09
Publication date: 2023-03-24

Abstract

The invention discloses a drought resistance related molecular marker of upland cotton GhGMP08 gene and application thereof. The invention provides application of the following single nucleotide polymorphisms of SNP sites in a cotton genome or substances for detecting the following single nucleotide polymorphisms of SNP sites in the cotton genome in identification or auxiliary identification of the drought resistance of cotton to be detected; the physical position of the SNP locus in a cotton reference genome TM-1_ZJU _V2.1 is A07:8291850; the nucleotide at the SNP site is C or T. The KASP marker developed by the invention can be used for selecting the foreground and the background in the seedling stage, thus reducing the field planting scale of breeding groups, shortening the breeding period and accelerating the breeding process.

Description

Upland cotton GhGMP08 gene drought resistance related molecular marker and application thereof

Technical Field

The invention relates to the field of plant assisted breeding, in particular to a upland cotton GhGMP08 gene drought resistance related molecular marker and application thereof.

Background

GDP-mannose pyrophosphorylase (GMP) is a key enzyme for synthesizing GDP-mannose in plants, provides a precursor for synthesizing GDP-mannose and plays an important role in synthesizing plant polysaccharide. Despite the myriad structures of plant polysaccharides, the synthetic pathways remain essentially consistent. The biosynthetic pathway from sucrose to GDP-mannose and GDP-fucose is also clear. First, sucrose is catalyzed by sucrose synthase (SUS) to produce fructose, which is then converted by Hexokinase (HK) to produce fructose-6-phosphate. The fructose-6-phosphate is catalyzed by mannose-6-phosphate isomerase (MPI) to generate mannose-6-phosphate, which is then converted into mannose-1-phosphate by mannose-phosphate mutase (PMM), and finally into GDP-mannose under the action of GDP-mannose pyrophosphorylase (GMPP). The GMP gene is also the rate-limiting enzyme of the L-galactose pathway, the key pathway of plant L-ascorbic acid (AsA) biosynthesis, and ascorbic acid (AsA) plays an important role in the redox homeostasis of plants and animals, mainly by scavenging reactive oxygen species, and is involved in the growth of various plants and can resist plant biotic and abiotic stresses. The GDP-mannose pyrophosphorylase of rice can regulate the content of AsA in the rice body, participate in salt stress and drought stress response of the rice and influence the root system growth of plants (Wangyun. GDP-mannose pyrophosphorylase genes OsVTC1-1 and OsVTC1-3 function research in ascorbic acid synthesis and stress response of rice [ D ]. Chinese agricultural academy of sciences, 2016.), the expression level of AgGMP gene in celery is obviously increased under abiotic stress, the AsA content of AgGMP gene transferred Arabidopsis thaliana is higher than that of wild plants, and the AgGMP gene regulates the celery AsA accumulation and also participates in drought stress response (Liu Y H, wang H, liu J X, et al. AgGMP encoding GDP-D-mannose pyrophosphorylase gene expressed by the wild plants, and the AgGMP gene regulates the AspGA accumulation and also participate in drought stress response [ J ]. 20210, 20212976 ].

Cotton is an important economic crop in the world, and the yield and the fiber quality of cotton are greatly influenced by drought and water shortage. The cotton planting is mainly distributed in three cotton areas, namely a yellow river valley, a Yangtze river valley and the northwest inland, the Xinjiang cotton area is the largest cotton production base in China and belongs to arid and semiarid areas, and the sustainable development of the Xinjiang cotton is seriously influenced by the shortage of water resources.

The functional marker is a novel molecular marker developed according to polymorphic motifs which cause phenotypic character variation in functional genes. There are two types of functional molecular markers, direct type functional molecular marker (DFM) and indirect type functional molecular marker (IFM). Wherein indirect functional markers are established based on statistical evidence obtained from association analysis, and direct functional markers are determined by comparing phenotypic differences and sequence polymorphisms between pairs of near-isogenic lines. Compared with random DNA markers and target gene markers, the functional markers have more advantages in application: more efficiently fixing alleles in a population; help to control the balanced selection and screening of alleles in natural populations and breeding populations; it is helpful to combine allelic FM affecting the same or different traits and construct linked FM haplotypes in breeding. In addition, the development of functional markers requires the following basic conditions: the separation and cloning of genes with definite functions are the premise and the basis of the development of functional markers; according to the needs of determining the allelic motif information of the genes, whether excellent allelic variation can be found and identified for the target functional gene is an important factor influencing the development of the functional marker; the determination of functional motifs within genes that cause phenotypic variation is a key culprit in the development of functional markers.

The breeding years of cotton under traditional breeding are long, and the expected result cannot be obtained even if manpower and material resources are too high. The functional molecular marker brings a new direction, is developed based on functional Single Nucleotide Polymorphism (SNP) sites in functional gene motifs, is co-separated from genes, can be directly applied to gene detection once being developed, is suitable for both natural groups and artificial groups, and can detect target genes and target traits in time. In addition, the functional marker is from the inside of the gene and directly reflects the target character expression, so that when backcross is used for excellent character transformation, the functional marker can better avoid linkage drag, reduce the introduction of useless genes of a donor into a receptor and realize the excellent combination of the target characters. The combination of functional markers and molecular marker assisted breeding provides effective means and ways for promoting breeding speed, aggregating excellent functional genes and improving target characters. The breeding and popularization of the drought-resistant cotton variety are one of effective ways for promoting the continuous and stable development of cotton production, genes related to drought resistance are searched, specific molecular markers are developed, and a foundation can be laid for the auxiliary breeding of the drought-resistant molecular markers of the cotton.

Disclosure of Invention

The invention aims to solve the technical problem of developing a drought-resistance related molecular marker which can closely relate to drought-resistance related character indexes of cotton breeding materials in the molecular-assisted selective breeding process of cotton to be applied to rapid identification and breeding of cotton drought-resistance excellent materials, lay a foundation for rapidly identifying and screening large-scale cotton drought-resistance groups and make a contribution to subsequent cotton molecular breeding.

In order to solve the technical problems, the invention provides a drought resistance related molecular marker based on upland cotton GhGMP08 gene and application thereof.

In a first aspect, the invention claims application of the following single nucleotide polymorphisms of SNP sites in a cotton genome or substances for detecting the following single nucleotide polymorphisms of SNP sites in the cotton genome in identification or auxiliary identification of the drought resistance of cotton to be detected.

The physical position of the SNP locus in a cotton reference genome TM-1_ZJU _V2.1 is A07:8291850; the reference genomic sequence TM-1. ZJU. V2.1 is available from the cotton COTTONGEN database (https:// www.cottongen. Org/data/download/genome _ tetraploid/AD 1); the nucleotide at the SNP site is C or T. Note: the physical location on the TM-1_ZJU _V2.1 reference genome is the physical location of the sense strand of the GhGMP08 gene. The SNP site is A or G at the 22 nd position of the DNA fragment shown in SEQ ID No.4 in the cotton genome corresponding to the antisense strand.

Further, the substance for detecting a single nucleotide polymorphism of said SNP site in the cotton genome is a KASP primer described in the second aspect hereinafter or a reagent or kit described in the third aspect hereinafter.

In a second aspect, the invention claims KASP primers for identifying or assisting in identifying drought resistance in cotton.

The KASP primer for identifying or assisting in identifying the drought resistance of cotton, which is claimed by the invention, consists of a primer 1, a primer 2 and a primer 3; the primer 1 is single-stranded DNA which is provided with a tag sequence A and 22 th to 43 th sites of SEQ ID No.1 in sequence from 5 'end to 3' end; the primer 2 is single-stranded DNA which is provided with a tag sequence B and 22 th to 42 th sites of SEQ ID No.2 in sequence from 5 'end to 3' end; the primer 3 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No.3 in a sequence table.

Further, the nucleotide sequence of the tag sequence A is 1 st to 21 st positions of SEQ ID No. 1; the nucleotide sequence of the tag sequence B is 1 st to 21 st of SEQ ID No. 2.

In a specific embodiment of the invention, the primer 1 is a single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 1; the primer 2 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 2.

In a third aspect, the invention claims a reagent or a kit for identifying or assisting in identifying cotton drought resistance.

The reagent or kit for identifying or assisting in identifying cotton drought resistance, which is claimed by the invention, contains the KASP primer described in the second aspect.

Further, the reagent or the kit also contains a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B. The nucleotide sequence of the fluorescent probe A is consistent with that of the tag sequence A, and the 5' end is connected with a fluorescent group A; the nucleotide sequence of the quenching probe A is reverse complementary to the nucleotide sequence of the label sequence A, and a quenching group is connected to the 3' terminal. The nucleotide sequence of the fluorescent probe B is consistent with that of the label sequence B, and the 5' end is connected with a fluorescent group B; the nucleotide sequence of the quenching probe B is reversely complementary with the nucleotide sequence of the label sequence B, and the 3' terminal is connected with a quenching group.

In a specific embodiment of the invention, the fluorophore a is VIC; the fluorophore B is FAM; the quenching group is BHQ.

In a fourth aspect, the invention claims the use of a KASP primer as described in the second aspect above or a reagent or kit as described in the third aspect above in any one of:

(A1) Identifying or assisting in identifying the drought resistance of cotton;

(A2) Identifying or assisting in identifying yield traits and/or apparent morphology and/or photosynthesis of cotton under drought stress;

(A3) Comparing the drought resistance of the cotton to be tested;

(A4) Comparing the yield traits and/or apparent morphology and/or photosynthesis of cotton to be tested under drought stress;

(A5) Selecting single plants or strains or varieties of cotton with relatively strong drought resistance;

(A6) Breeding a single cotton plant or a strain or a variety with relatively high yield and/or relatively high plant height and/or relatively more fruit branches and/or relatively more effective fruit branches and/or relatively stronger photosynthesis under drought stress;

(A7) Breeding a single plant or strain or variety of cotton with relatively weak drought resistance;

(A8) Breeding a cotton single plant or strain or line or variety with relatively low yield and/or relatively low plant height and/or relatively low fruit branch number, less and/or relatively low effective fruit branch number and/or relatively weak photosynthesis under drought stress;

(A9) And (5) cotton breeding.

In a fifth aspect, the invention claims any of the following methods:

the method I comprises the following steps: a method for comparing drought resistance of cotton to be tested comprises the following steps: detecting nucleotides at the following SNP sites in a genome of cotton to be detected, determining the genotype of the cotton to be detected, and determining the drought resistance of the cotton to be detected according to the genotype of the cotton to be detected as follows: the drought resistance of the to-be-detected cotton with the T: T genotype is stronger than or candidate stronger than that of the to-be-detected cotton with the C: T genotype.

The method can also be used for comparing the yield traits and/or the apparent morphology and/or the photosynthesis of cotton to be tested under drought stress. The yield of the cotton to be detected with the T: T genotype under drought stress is higher than or is more than candidate of the cotton to be detected with the C: T genotype, the plant of the cotton to be detected with the T: T genotype under drought stress is higher than or is more than candidate of the cotton to be detected with the C: T genotype, the fruit number of the cotton to be detected with the T: T genotype under drought stress is more than or is more than candidate of the cotton to be detected with the C: T genotype, the effective fruit number of the cotton to be detected with the T: T genotype under drought stress is more than or is more than candidate of the cotton to be detected with the C: T genotype, and the photosynthesis of the cotton to be detected with the T: T genotype under drought stress is stronger than or is more candidate of the cotton to be detected with the C: T genotype.

Method II: a method for breeding a single plant or strain or line or variety of cotton with relatively strong drought resistance comprises the following steps: detecting the nucleotides at the following SNP loci in the genome of the cotton to be detected, determining the genotype of the cotton to be detected, selecting the cotton to be detected with the SNP loci of T: T genotype in the genome as a parent to carry out breeding, selecting the cotton with the SNP loci of T: T genotype in the genome in each generation of breeding, and finally obtaining the single plant or strain or variety of the cotton with relatively strong drought resistance.

The method can also be used for breeding a single cotton plant or a strain or a variety with relatively high yield and/or relatively high plant height and/or relatively more fruit branches and/or relatively more effective fruit branches and/or relatively stronger photosynthesis under drought stress. The single cotton plant or strain or variety with relatively strong drought resistance is finally obtained, namely the single cotton plant or strain or variety with relatively high yield and/or relatively high plant height and/or relatively large fruit branch number and/or relatively large effective fruit branch number and/or relatively strong photosynthesis under drought stress.

Method III: a method for breeding cotton single plant or strain or line or variety with relatively weak drought resistance comprises the following steps: detecting nucleotides at the following SNP loci in a genome of cotton to be detected, determining the genotype of the cotton to be detected, selecting the cotton to be detected with the SNP loci of C: T genotype in the genome as a parent to carry out breeding, selecting the cotton with the SNP loci of C: T genotype in the genome in each generation of breeding, and finally obtaining the cotton single plant or strain or variety with relatively weak drought resistance.

The method can also be used for breeding cotton single plants or lines or varieties with relatively low yield and/or relatively low plant height and/or relatively few fruit branches and/or relatively few effective fruit branches and/or relatively weak photosynthesis under drought stress. The finally obtained cotton single plant or strain or variety with relatively weak drought resistance is the cotton single plant or strain or variety with relatively low yield and/or relatively low plant height and/or relatively few fruit branches and/or relatively few effective fruit branches and/or relatively weak photosynthesis under drought stress.

The physical position of the SNP locus in a cotton reference genome TM-1_ZJU _V2.1 is A07:8291850; the nucleotide at the SNP site is C or T. Note: the physical location on the TM-1_ZJU _V2.1 reference genome is the physical location of the sense strand of the GhGMP08 gene. The SNP site is A or G at the 22 nd position of the DNA fragment shown in SEQ ID No.4 in the cotton genome corresponding to the antisense strand.

The T genotype is at physical location A07 in the cotton reference genome TM-1_ZJU _V2.1: 8291850 nucleotides are homozygous for T; the T genotype is homozygous for A at the nucleotide at position 22 of the DNA fragment shown in SEQ ID No.4 in the cotton genome corresponding to the antisense strand.

The C: T genotype is at physical location A07 in the cotton reference genome TM-1_ZJU _V2.1: 8291850 nucleotides are heterozygous for C and T; the nucleotide at the 22 nd position of the DNA segment shown as SEQ ID No.4 in the cotton genome of the genotype C: T corresponds to the antisense strand, and the nucleotide is a hybrid of G and A.

In addition, there are theoretically C: C genotypes in addition to the T: T genotype, the C: T genotype (none of the 186 natural materials validated in this invention, and unknown genotypes for some materials). The C: C genotype is the physical location A07 in the cotton reference genome TM-1 _ZJU2.1: 8291850 nucleotides are homozygous for C; the C genotype is homozygous for G at the nucleotide at position 22 of the DNA fragment shown in SEQ ID No.4 in the cotton genome corresponding to the antisense strand.

In the above method, the "detecting nucleotides at the following SNP sites in the genome of the cotton to be tested and determining the genotype of the cotton to be tested" may be performed according to a method comprising the following steps: performing PCR amplification on the genomic DNA of the cotton to be detected by using the reagent or the kit in the third aspect, performing fluorescence signal scanning on the amplified product, and then determining the genotype of the SNP locus in the genome of the cotton to be detected according to the following steps:

if the fluorescence signal of the amplification product of the cotton to be detected is the signal of the fluorophore A, the SNP locus of the cotton to be detected is the T: T genotype;

if the fluorescence signal of the amplification product of the cotton to be detected is the signal of the fluorophore A and the fluorophore B, the SNP locus of the cotton to be detected is the C: T genotype;

if the fluorescence signal of the amplification product of the cotton to be detected is the signal of the fluorophore B, the SNP locus of the cotton to be detected is the C: C genotype;

and if the fluorescence signal of the amplification product of the cotton to be detected does not show the signal of the fluorophore A and the signal of the fluorophore B, determining that the SNP locus of the cotton to be detected is an unknown genotype.

In the present invention, said fluorescent Probe A, said fluorescent Probe B, said quenching Probe A and said quenching Probe B are present in KASP HiGeno 2x Probe Mix, wherein said KASP HiGeno 2x Probe Mix is a product of Kagaku Biotech Co., ltd, beijing (product No. AQP-001S).

In the above aspects, the cotton is upland cotton.

In a specific embodiment of the invention, the cotton is selected from the 186 parts materials shown in table 3.

In the above aspects, the drought resistance is mainly reflected in changes in yield traits, apparent morphology and/or photosynthesis under drought stress. Further, the yield traits are embodied by the following indexes: bell Number (BN), effective Bell Number (EBN), single Bell Seeds Weight (SBSW), and/or Single Bell Weight (SBW); the apparent morphology is represented by the following indexes: plant Height (PH), fruit Branch Number (FBN) and/or Effective Fruit Branch Number (EFBN); the photosynthesis is embodied by the following indexes: porosity (Gs), transpiration rate (Tr), and/or Vapor Pressure Deficit (VPD).

The invention has the beneficial effects that:

(1) The KASP marker phenotype selection efficiency developed by the invention is basically consistent with that of field identification, and the method can be used for quickly and accurately detecting the drought resistance in different germplasm resources of cotton.

(2) The molecular marker KASP developed by the invention can be applied to commercial molecular breeding with high flux, and complicated procedures such as enzyme digestion, electrophoresis and sequencing are not needed in the detection process, so that the pollution of aerosol and the use of toxic substances such as EB (electron beam) are reduced, the base is directly detected, the accuracy is not influenced by the length of an amplified fragment, and the method is simple, convenient, rapid, accurate and high in automation degree, the gene breeding efficiency is greatly improved, and the cost is reduced.

(3) The KASP marker developed by the invention can be used for selecting the foreground and the background in the seedling stage, thus reducing the field planting scale of breeding groups, shortening the breeding period and accelerating the breeding process.

Drawings

FIG. 1 is a diagram showing the results of genotyping.

FIG. 2 is a diagram of 186 parts of resource material drought resistance clusters. A is a graph of the result of cluster analysis based on the CDC value, and B is a graph of the result of cluster analysis based on the D value.

FIG. 3 is a boxplot of the drought resistance related trait indexes DC value, CDC value and D value combination genotypes of 186 parts of upland cotton resource materials. A-Q are box line graphs drawn by combining drought resistance coefficient (DC) values respectively calculated by 17 individual indexes of a Plant Height (PH), a first fruit branch node Height (HNFFB), a Fruit Branch Number (FBN), an Effective Fruit Branch Number (EFBN), a Boll Number (BN), an Effective Boll Number (EBN), a boll seed cotton weight (SBSW), a boll lint cotton weight (SBLW), a boll weight (SBW), clothes fraction (LP), an intercellular carbon dioxide concentration (Ci), a net photosynthetic rate (Pn), a gas pore conductance (Gs), a transpiration rate (Tr), a water Vapor Pressure Deficiency (VPD), water Utilization Efficiency (WUE) and a chlorophyll relative value (SPAD) with genotype data, and R and S are box line graphs drawn by combining the comprehensive drought resistance coefficient (CDC) values and the drought resistance metric (D) of the materials with the genotype data.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 development of molecular markers for identifying drought resistance of Cotton and design of KASP primers

The gene used for developing the molecular marker is the Gossypium hirsutum GhGMP08 gene, and the annotation information of the gene is mannose-1-phosphate guanyltransferase (mannose-1-phosphate guanyltransferase) or GDP-mannose pyrophosphorylase (GDP-mannose pyrophosphorylase). The source background is a gene in a drought resistance related candidate interval for carrying out BSA-seq simplified genome sequencing and positioning by a drought resistance related RIL group based on a drought resistance metric value D, a reference genome is TM-1 \/ZJU \/V2.1, the gene ID is GH _ A07G0705, the physical position information is A07:8,288,401-8,293,141 (sense strand), the CDS sequence fragment size is 1,248bp, the DNA full-length sequence fragment size is 4741bp, the protein sequence length is 415aa, and related information can be queried in a cotton cottonFGD database (https:// cottonfgd.net/profiles/gene/GH _ A07G0705 /).

The SNP information and the non-synonymous gene information in the positioning candidate interval are analyzed and found, and the SNP information and the non-synonymous gene information are positioned at a genome physical position A07:8291850 One SNP mutation is present (at the sense strand): C/T, this site is at base 820 in the CDS sequence of the gene, and its mutation causes amino acid change: the mutation of the Cat/Tat from histidine to tyrosine is the only nonsynonymous mutation site in the gene. The SNP site is named GhGMP08-820SNP according to the position of the SNP site in the CDS sequence of the GhGMP08 gene.

Designing KASP marker primer sequence according to the antisense strand of the SNP locus (GhGMP 08-820 SNP), as follows:

an upstream primer F1:5' -GAAGGTCGGAGTCAACGGATTTGAACCGAAATTGTGCAAGAATA-3' (SEQ ID No.1, the specific fluorescent tag sequence VIC is underlined);

an upstream primer F2:5' -GAAGGTGACCAAGTTCATGCTGAACCGAAATTGTGCAAGATG-3' (SEQ ID No.2, the underlined part is the specific fluorescent tag sequence FAM);

a downstream primer R:5' AGAAAGTCCTTAAAGTGTTCTGGC-.

The last base pair at the 3' end of the two upstream primers should be the SNP site (GhGMP 08-820 SNP). The GhGMP08-820SNP corresponds to position 22 of SEQ ID No.4 in the cotton genome and is A or G (indicated by R in SEQ ID No. 4).

The upstream primer F1 was used to amplify the physical position A07 of the cotton reference genome TM-1_ZJU _V2.1: 8291850 In the case where the nucleotide at (sense strand) is T, the forward primer F2 is used to amplify the physical position A07 of the cotton reference genome TM-1 \/ZJU \/V2.1: 8291850 (sense strand) nucleotide is C; the downstream primer R is a universal primer.

Amplifying the single-stranded DNA molecule shown in SEQ ID No.1 and the single-stranded DNA molecule shown in SEQ ID No.3 to obtain the DNA sequence of the cotton reference genome TM-1_ZJU_V2.1 at the physical position A07:8291850 (sense strand) a fragment in which the genotype of the nucleotide is T: T homozygous (i.e., the base at position 22 of SEQ ID No.4 on the cotton genome is A: A homozygous). The sequence of the theoretical amplification product (without the specific fluorescent tag sequence) is shown in SEQ ID No.4 (A at position 22).

The single-stranded DNA molecule shown in SEQ ID No.2 and the single-stranded DNA molecule shown in SEQ ID No.3 are used for amplifying the physical position A07 of the cotton reference genome TM-1_ZJU_V2.1: 8291850 (sense strand) a fragment in which the genotype of the nucleotide is C: C homozygous (i.e., G: G homozygous at the 22 nd base of SEQ ID No.4 on the cotton genome). The sequence of the theoretical amplification product (without the specific fluorescent tag sequence) is shown in positions 2-49 of SEQ ID No.4 (G at position 22).

Amplifying the physical position A07 of the cotton reference genome TM-1_ZJU_V2.1 by the single-stranded DNA molecule shown in SEQ ID No.1, the single-stranded DNA molecule shown in SEQ ID No.2 and the single-stranded DNA molecule shown in SEQ ID No. 3: 8291850 (sense strand) is a fragment of a C: T hybrid (i.e., G: A hybrid at base 22 of SEQ ID No.4 on the cotton genome). The theoretical amplification product (without specific fluorescent tag sequence) has two DNA fragments, namely DNA fragment 1 shown in SEQ ID No.4 (A at position 22) and DNA fragment 2 shown in positions 2-49 of SEQ ID No.4 (G at position 22).

Example 2 establishment and application of method for detecting SNP genotype with KASP primer

1. DNA sample preparation

DNA extraction: genomic DNA was extracted from cotton leaves by the CTAB method.

Determination of DNA concentration: several DNA working solutions were randomly drawn and the concentration was measured using a NanoDrop2000 instrument.

And (3) DNA integrity identification: agarose gel electrophoresis, 1.5%,120V,40min. The main belt is qualified.

SNP Primer Mix (4X) preparation: the primer dry powder was diluted to 100mM and the three sequences were sequenced according to F1: f2: r: ddH ₂ O = 24.

DNA dilution: the DNA stock was diluted to 20 ng/. Mu.l in bulk.

2. KASP reaction

The Xinjiang Edison Biotechnology Ltd is entrusted with KSAP detection of GhGMP08-820SNP locus by LGC high-flux gene typing detection platform.

The KASP detection PCR amplification system is shown in Table 1.

TABLE 1 KASP detection PCR amplification System

Name (R)	384 well plate (4 μ L system)
		KASP HiGeno 2×Probe Mix	2μL
SNP Primer Mix(4x)	1μL
		DNA sample	2μL

Note: KASP HiGeno 2 XProbe Mix is a product of Biotechnology Ltd of Jia Cheng, beijing (product number AQP-001S). The KASP HiGeno 2 XProbe Mix contains fluorescent Probe A, fluorescent Probe B, quenching Probe A, quenching Probe B, ROX dye, high fidelity Taq enzyme, dNTP, mg ²⁺ And the like. The nucleotide sequence of the fluorescent probe A is 5' and-GAAGGTCGGAGTCAACGGATT-3 ', and the 5' end is connected with a VIC fluorescent group. The nucleotide sequence of the fluorescent probe B is 5' GAAGGTGACCAAGTTCATGCT-3', and the 5' end is connected with a FAM fluorescent group. The nucleotide sequence of the quenching probe A is 5' AATCCGTTGACTCCGACCTTC-3', and the 3' end is connected with a quenching group BHQ. The nucleotide sequence of the quenching probe B is 5. The KASP detection PCR reaction program is shown in table 2.

TABLE 2 KASP detection PCR reaction program

3. Analysis of results

Fluorescence value reading: after the PCR amplification cycle is finished, reading the fluorescence value by using a fluorescent quantitative PCR instrument in the environment of lower than 40 ℃. In this method, SNP site detection uses the fluorophores FAM and VIC to distinguish between two isogenic sites. A passive reference dye ROX (passive reference dye ROX) was used to correct for the difference in signal from well to well due to reaction volume error. The relevant excitation and emission wavelengths are shown in table 3 below. The reading software is an Omega device of LGC.

TABLE 3 excitation and emission wavelengths of fluorophores

Fluorescent group	Exciting light (nm)	Light emission (nm)
			FAM	485	520
VIC	535	556
			ROX	575	610

Note: if the fluorescence scanning instrument uses HEX fluorophores as detection signals, no modification of the settings is required, since the excitation and emission light values of VIC and HEX are very similar.

The data from the fluorescence readings were analyzed using the LGC genotyping software (Kluster Caller). And correcting the VIC and FAM values of each reaction well through the value of the specific well reference dye (ROX), and standardizing the data fluorescence values to obtain the relative fluorescence values corresponding to the VIC and FAM of each PCR reaction well. Clustering the samples according to the relative fluorescence value, and further determining the genotype of the GhGMP08-820SNP site in the cotton genome to be detected (namely whether the base at the 22 nd position of SEQ ID No.4 on the cotton genome is T or C) according to the sample cluster and the fluorescence type: if the fluorescence signal data of the amplification product of the cotton to be detected is close to a Y axis (VIC signal) through the analysis of a genotyping software KlustERCaller, the genotype of the GhGMP08-820SNP site in the genome of the cotton to be detected is T: T homozygote (namely, the 22 nd base of SEQ ID No.4 on the genome of the cotton is A: A homozygote); if the fluorescence signal data of the amplification product of the cotton to be detected is close to an X axis (FAM signal) through the analysis of Klustercaller of the genotyping software, the genotype of the GhGMP08-820SNP site in the genome of the cotton to be detected is C: C homozygote (namely the 22 nd base of SEQ ID No.4 on the genome of the cotton is G: G homozygote); if the fluorescence signal data of the amplification product of the cotton to be detected is positioned between the X axis and the Y axis (simultaneously, VIC and FAM signals) through the Klustercaller analysis of the genotyping software, the genotype of the GhGMP08-820SNP site in the genome of the cotton to be detected is a C: T heterozygote (namely, the base at the 22 nd position of SEQ ID No.4 on the genome of the cotton is a G: A heterozygote).

The genotyping results are shown in FIG. 1. Genotype T: T is marked orange (cross), genotype C: T is marked blue (circle), and genotype unknown is marked green (square).

4. Mark typing data analysis

The total 186 parts of upland cotton resource materials for testing are collected and provided by crop genetic improvement and germplasm innovation key laboratories of agricultural college of Xinjiang university.

On the one hand, the genotype of the GhRF09-110SNP site of each cotton material to be tested is detected according to the method. On the other hand, the field resistance identification is carried out by adopting a conventional method. The method comprises the following specific steps:

186 parts of resource material (see Table 4, recorded in "Yan Cheng Chuan, great waves, chen Qin, pao jin Cheng, wang Ting Wei, chen quan, quyanying, upland cotton boll stage drought resistance index screening and evaluation [ J]China agricultural science and technology guide, 2022,24 (07): 46-57 "article, publicly available from the applicant, and only available for duplication of experiments not otherwise used in the invention) drought resistance identification related tests were conducted in Sankyo county 144 group 3, xinjiang agricultural university Cotton Breeding base (43 ° 20 '-45' N,84 ° 45 '-86' E) in 2021. The test set up 2 treatments of drought stress and normal control, each 2 replicates, with a 2m protective row between drought stress and normal control. The length of each cell line is 2m, the 14 membranes in 1 area, the 3 membranes in 1 line and 1 part of the material of 1 membrane are planted, and drip irrigation and watering are carried out under the membranes. The test material was sown at 26 days 4 months, emerged at 5 days 5 months and topped at 7 days 7 months. The stress treatment is started in 7 months and 4 days, and a water meter is arranged on a main pipeline in an arid region to record the water control amount before the stress. The management method during cultivation is the same as that in the field. After drought stress lasts for 15 days, soil layers of 0-20 cm, 20-40 cm and 40-60 cm are sampled and weighed by a 5-point sampling method, the soil layers are placed in an oven to be dried to constant weight and weighed, and the water content is measured. The results show that the water content change of 0-20 cm soil layer after drought stress is maximum, the water content is reduced by 8.257 percentage points, the water content of 3 soil layers is reduced by 7.921 percentage points on average, and the total water control of 2 drought stress treatments is 566m during the stress period ³ And drought stress conditions are achieved.

Selecting 3 cotton leaves, and measuring photosynthetic indexes including transpiration rate (Tr), intercellular carbon dioxide concentration (Ci), net photosynthetic rate (Pn), stomatal conductance (Gs), vapor Pressure Deficiency (VPD) and Water Use Efficiency (WUE) by using a photosynthesizer (CIRAS-3), wherein 5 cotton leaves are continuously measured and the average value is 1 repeat.

And 7, 20 days, measuring a chlorophyll relative value (SPAD value) by using a SPAD instrument (SYS-SPAD-502 Plus, seasa, japan), selecting 3 leaves of cotton, measuring the upper part, the middle part and the lower part of the 3 leaves of the cotton to obtain an average value, continuously measuring 5 strains, and recording the average value as 1 repeat. 9, 17 days, determining agronomic traits of drought stress and normal control, including Plant Height (PH), first fruit branch node Height (Height at the node of the first fruit branch, HNFFB), fruit Branch Number (FBN), effective Fruit Branch Number (EFBN), bell Number (BN), effective Bell Number (EBN); on 25 days 9, 20 bolls of cotton were mixed, and the Lint (LP), single Boll Weight (SBW), single boll seed cotton weight (SBSW) and Single boll lint cotton weight (SBLW) were measured, with reference to cotton germplasm description specification and data standard (duandrin, zhou loyal. Cotton germplasm description specification and data standard [ M ]. Beijing: chinese agriculture press, 2005.

Statistical analysis was performed on the data using EXCEL 2010 software and SPSS 25.0 software. The test totally has 17 individual indexes, and the drought resistance evaluation method comprises a drought resistance coefficient (DC), a drought resistance index (DI), a comprehensive drought resistance coefficient (CDC value) and a drought resistance measurement value (D), and the calculation formula is calculated by referring to the calculation methods of Sunfeng Lei (Sunfeng Lei, quyanying, chen quanjiao, and the like, cotton drought resistance related index comprehensive evaluation and gray correlation analysis [ J ]. Agricultural research in arid regions, 2019,37 (1): 233-239), and the like:

drought resistance coefficient:

drought resistance index:

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comprehensive drought resistance coefficient:

membership functions:

drought resistance measurement:

in each of the above calculation formulas, X _d 、X _w The measured values of the indexes of the materials under drought stress and normal control are respectively,

average of this index under drought stress, DI _i _min 、DI _i _max The minimum value and the maximum value of drought resistance coefficients of all characters are obtained; r is _i The contribution rate of the ith comprehensive index.

The larger the comprehensive drought resistance coefficient CDC value and the drought resistance measurement value D value are, the stronger the drought resistance (tolerance) is, the clustering results of the two are approximately the same, and the clustering chart of the comprehensive drought resistance coefficient CDC value and the drought resistance measurement value D value of 186 parts of upland cotton resource materials is shown in FIG. 2. The drought resistance value D is classified into 5 types as shown in B in figure 2, the type I is strong drought resistance type (D is more than or equal to 0.54 and less than or equal to 0.61), and the total amount of the materials is 13 parts, wherein 5 parts (36.5%) of genotype CT and 8 parts (61.5%) of genotype TT are provided. The II type is drought resistant type (D is more than or equal to 0.46 and less than 0.54), and 47 parts of materials are used, wherein 39 parts of genotype CT (83.0%) and 6 parts of genotype TT (12.8%) are used, and 2 parts of unknown genotypes of the materials are used. The class III is medium drought resistant type (D is more than or equal to 0.42 and less than 0.46), and 40 parts of materials are used, wherein 35 parts of genotype CT (87.5%) and 4 parts of genotype TT (10%) are used, and 1 part of unknown genotype of the materials is used. The IV group is sensitive drought type (D is more than or equal to 0.38 and less than 0.42), and 46 parts of materials are used, wherein 40 parts of genotype CT (87.0%) and 5 parts of genotype TT (10.9%) are contained, and 1 part of unknown genotype of the materials is contained. Class V is extremely sensitive drought type (D is more than or equal to 0.27 and less than 0.38), and 40 parts of material are used, wherein 38 parts of genotype CT (95.0%) and 2 parts of genotype TT (5.0%) are included.

Among 186 parts of the material, 157 parts of the material have the genotype CT,25 parts of the material have the genotype TT and 4 parts of the material have unknown genotype. The drought-resistant genotype TT contained in the first three grades accounts for 18 parts of 72.0 percent of the total number of the drought-resistant genotype TT.

The drought resistance coefficient DC value, the comprehensive drought resistance coefficient CDC value, the drought resistance measurement value D value and the genotype information of the GhGMP08 non-synonymous mutation site of 186 parts of upland cotton resource material 17 are shown in Table 4. The DC values for the 17 personality specifications for 186 parts of upland cotton resource material are shown in table 5.

TABLE 4 CDC value and corresponding grade of 186 parts upland cotton resource material 17 personality index, D value and corresponding grade, and genotype information of GhGMP08-820SNP locus

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Note: -represents an unknown genotype.

TABLE 5 DC value of 17 personality index of 186 upland cotton resource material

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5. Genotype-phenotype association analysis

And (4) carrying out correlation analysis on the drought resistance identification result based on 186 parts of upland cotton resource materials obtained in the step (4) and the genotype result of the upland cotton resource materials on the GhGMP08 gene so as to achieve the purpose of correlating the genotype with one or more character indexes and search the relation between the genotype and the drought resistance.

The results of a two-tailed T-test between phenotype and genotype using the software GraphPad Prism 9 are shown in FIG. 3. ns represents no significant difference; * Representing that P is less than 0.05, which shows that the character indexes of different genotypes are obviously different on the whole; * P <0.01, indicating that the trait indexes between different genotypes are very significantly different on the whole; * P <0.001, indicating that the trait indices between different genotypes differ very significantly overall.

The result shows that the comprehensive drought resistance coefficient CDC value and the drought resistance measurement value D are very obviously different, which shows that the genotype change of the gene is strongly related to the drought resistance of the resource material, and the TT genotype material is more drought resistant compared with the CT genotype material on the whole. The 17 measured individual character indexes have significant difference of Gs and Tr, six indexes of PH, FBN, BN, SBSW, SBW and VPD and significant difference of EFBN and EBN, which shows that the genotype of the gene is related to a plurality of character indexes, different genotypes can influence the differential expression of a plurality of characters through a certain regulation mechanism, and the drought resistance of plants is increased by influencing the plurality of characters.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Claims

1. The application of the following SNP site single nucleotide polymorphism in cotton genome or the substance for detecting the following SNP site single nucleotide polymorphism in cotton genome in identification or auxiliary identification of the cotton drought resistance to be detected;

the physical position of the SNP locus in a cotton reference genome TM-1_ZJU _V2.1 is A07:8291850; the nucleotide at the SNP site is C or T.

2. Use according to claim 1, characterized in that: the substance for detecting single nucleotide polymorphism of SNP site in cotton genome is KASP primer as defined in any one of claims 3-5 or reagent or kit as defined in claim 6 or 7.

3. KASP primer for identifying or assisting in identifying cotton drought resistance is characterized in that: the KASP primer consists of a primer 1, a primer 2 and a primer 3; the primer 1 is single-stranded DNA which is provided with a tag sequence A and 22 th to 43 th sites of SEQ ID No.1 in sequence from 5 'end to 3' end; the primer 2 is single-stranded DNA which is provided with a tag sequence B and 22 th to 42 th sites of SEQ ID No.2 in sequence from 5 'end to 3' end; the primer 3 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No.3 in a sequence table.

4. The KASP primer of claim 3, wherein: the nucleotide sequence of the tag sequence A is 1 st-21 st of SEQ ID No. 1; the nucleotide sequence of the tag sequence B is 1 st to 21 st of SEQ ID No. 2.

5. A KASP primer according to claim 3 or 4, wherein: the primer 1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 1; the primer 2 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 2.

6. A reagent or a kit for identifying or assisting in identifying cotton drought resistance is characterized in that: the reagent or kit contains KASP primers as claimed in any one of claims 3-5.

7. The reagent or kit of claim 6, wherein: the reagent or the kit also contains a fluorescent probe A, a fluorescent probe B, a quenching probe A and a quenching probe B;

the nucleotide sequence of the fluorescent probe A is consistent with that of the tag sequence A, and the 5' end is connected with a fluorescent group A; the nucleotide sequence of the quenching probe A is reversely complementary with the nucleotide sequence of the tag sequence A, and a quenching group is connected to the 3' terminal;

the nucleotide sequence of the fluorescent probe B is consistent with that of the label sequence B, and the 5' end is connected with a fluorescent group B; the nucleotide sequence of the quenching probe B is reversely complementary with the nucleotide sequence of the label sequence B, and the 3' terminal is connected with a quenching group;

further, the fluorophore A is VIC; the fluorophore B is FAM; the quenching group is BHQ.

8. Use of a KASP primer according to any one of claims 3 to 5 or a reagent or kit according to claim 6 or 7 in any one of:

(A1) Identifying or assisting in identifying the drought resistance of cotton;

(A3) Comparing the drought resistance of the cotton to be tested;

(A5) Breeding a single cotton plant or strain or variety with relatively strong drought resistance;

(A9) And (5) cotton breeding.

9. Any one of the following methods:

the method I comprises the following steps: a method for comparing drought resistance of cotton to be tested comprises the following steps: detecting nucleotides at the following SNP sites in a genome of cotton to be detected, determining the genotype of the cotton to be detected, and determining the drought resistance of the cotton to be detected according to the genotype of the cotton to be detected as follows: the drought resistance of the cotton to be detected with the T-gene type is stronger than or candidate stronger than that of the cotton to be detected with the C-T gene type;

method II: a method for breeding a single plant or strain or line or variety of cotton with relatively strong drought resistance comprises the following steps: detecting nucleotides at the following SNP loci in a genome of cotton to be detected, determining the genotype of the cotton to be detected, selecting the cotton to be detected with the SNP loci of T: T genotype in the genome as a parent to carry out breeding, selecting the cotton with the SNP loci of T: T genotype in the genome in each generation of breeding, and finally obtaining a single plant or a strain or a variety of the cotton with relatively strong drought resistance;

method III: a method for breeding cotton single plant or strain or line or variety with relatively weak drought resistance comprises the following steps: detecting nucleotides at the following SNP loci in a genome of cotton to be detected, determining the genotype of the cotton to be detected, selecting the cotton to be detected with the SNP loci of C: T genotype in the genome as a parent to carry out breeding, selecting the cotton with the SNP loci of C: T genotype in the genome in each generation of breeding, and finally obtaining a single plant or a strain or a variety of the cotton with relatively weak drought resistance;

the physical position of the SNP locus in a cotton reference genome TM-1_ZJU _V2.1 is A07:8291850; the nucleotide at the SNP site is C or T;

the T genotype is at physical location A07 in the cotton reference genome TM-1_ZJU _V2.1: the nucleotide at 8291850 is homozygous for T;

the C: T genotype is at physical location A07 in the cotton reference genome TM-1_ZJU _V2.1: 8291850 nucleotides are heterozygotes of C and T.

10. The method of claim 9, wherein: the method for detecting the nucleotides at the following SNP sites in the genome of the cotton to be detected and determining the genotype of the cotton to be detected is carried out according to the following steps: performing PCR amplification on the genomic DNA of the cotton to be detected by using the reagent or the kit as claimed in claim 7, performing fluorescence signal scanning on the amplified product, and then determining the genotype of the SNP site in the genome of the cotton to be detected according to the following steps:

and if the fluorescence signal of the amplification product of the cotton to be detected is the signal of the fluorophore A and the fluorophore B, the SNP locus of the cotton to be detected is the C: T genotype.