CN117757974A - Combined drought resistance related molecular marker of upland cotton GhGMP family and application thereof - Google Patents
Combined drought resistance related molecular marker of upland cotton GhGMP family and application thereof Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract
The invention discloses a combined drought resistance related molecular marker of a upland cotton GhGMP family and application thereof. The invention provides application of a single nucleotide polymorphism of two SNP locus combinations in a cotton genome or a substance for detecting the single nucleotide polymorphism of two SNP locus combinations in the cotton genome in identification or auxiliary identification of drought resistance of cotton to be detected; the two SNP locus combinations are GhGMP08-820SNP and GhGMP30-874SNP respectively, and the physical positions of the two loci in the cotton reference genome TM-1_ZJU_V2.1 are A07 respectively: 8291850 (C/T) and D07:52577953 (A/G). The invention develops a combined drought-resistance related molecular marker which can be closely related to drought-resistance related character indexes of cotton breeding materials, is applied to rapid identification and breeding of cotton drought-resistance excellent materials, lays a foundation for rapid identification and screening of large-scale cotton drought-resistance populations, and contributes to subsequent cotton molecular breeding.
Description
Technical Field
The invention relates to the field of plant molecular marker assisted breeding, in particular to a combined drought resistance related molecular marker of a upland cotton GhGMP family and application thereof.
Background
GDP-mannose pyrophosphorylase (GDP-mannose Pyrophosphorylase, GMP) is the first key enzyme in the plant's ascorbic acid (AsA) biosynthetic pathway, and ascorbic acid (AsA) plays an important role in redox homeostasis in plants and animals, mainly by scavenging active oxygen, and is involved in the growth of a variety of plants and is resistant to plant biotic and abiotic stresses. In tomato and potato, increasing GMP enzyme activity can increase AsA content, pigment content and net photosynthetic rate of leaf are increased significantly, and plant senescence is delayed (Christelle C, M GG, R FA, jan B, jens K, rolene B: manipulation of L-ascorbic acid biosynthesis pathways in Solanum lycopersicum: eleeved GDP-mannose pyrophosphorylase activity enhances L-ascorbate levels in red fri. Planta 2012,235 (3). Lin Lingling: GDP-D-mannose pyrophosphorylase gene (GMPase) functional analysis in potato. Shuoshi. Shandong agricultural university; 2009.). In rice, the content of AsA in the rice can be regulated and controlled, and the rice is involved in salt stress and drought stress response of the rice, so that root growth of plants is affected (Wang Yayun: functional research on synthesis of rice ascorbic acid and stress response of GDP-mannose pyrophosphorylase genes OsVTC1-1 and OsVTC 1-3. Shuoshi. National academy of agricultural science; 2016.); in Arabidopsis, overexpression of the rice OsVTC1-1 gene can restore the synthesis of AsA, enhance the drought resistance of Arabidopsis (Ritesh K, ananda M, kumar SK, vishal S, subhasis S, kumar SS, ashwani P, lata S-PS: functional screening of cDNA library from a salt tolerant rice genotype Pokkaliidentifies mannose-1-phosphate guanyl transferase gene (OsMPG 1) as a key member of salinity stress response.plant molecular biology2012,79 (6)); in celery, the expression level of AgGMP gene under abiotic stress is remarkably increased, and the regulation of celery AsA accumulation is also involved in drought stress response (Liu Y H, wang H, liu J X, et al AgGMP encoding GDP-D-mannose pyrophosphorylase from celery enhanced the accumulation of ascorbic acid and resistance to drought stress in Arabidopsis [ J ]. PeerJ,2022, 10:e12976.). GMP genes play an important role in the growth and development of plants and play an important role in abiotic stress.
Cotton is an important economic crop in the world, and drought and water shortage can have a great influence on cotton yield and fiber quality. The functional marker is a novel molecular marker developed according to polymorphic motifs within functional genes that cause phenotypic variation. Under traditional breeding, cotton breeding is long in year limit, and the cost of manpower and material resources is too high to obtain expected results. The functional molecular marker brings new directions, is developed based on functional Single Nucleotide Polymorphism (SNP) loci in functional gene motifs, is co-separated from genes, can be directly applied to gene detection after development, is applicable to natural populations and artificial populations, and can timely detect target genes and target traits. In addition, as the functional marker is derived from the interior of the gene and directly reflects the expression of the target trait, when the backcross is utilized to carry out the transformation of the excellent trait, the functional marker can better avoid linkage encumbrance, reduce the introduction of the non-beneficial gene of the donor into the receptor and realize the excellent assembly of the target trait. The combination of the functional markers and the molecular marker assisted breeding provides effective means and ways for promoting breeding speed, polymerizing excellent functional genes and improving target characters. The breeding and popularizing of drought-resistant cotton varieties are one of effective ways for promoting the continuous and stable development of cotton production, searching genes related to drought resistance, developing specific molecular markers, and laying a foundation for auxiliary breeding of cotton drought-resistant molecular markers.
Disclosure of Invention
The invention aims to provide a combined drought resistance related molecular marker of a upland cotton GhGMP family and application thereof.
In a first aspect, the invention claims the use of a single nucleotide polymorphism of the following two SNP site combinations in the cotton genome or a substance for detecting single nucleotide polymorphism of the following two SNP site combinations in the cotton genome for the identification or assisted identification of drought resistance of cotton to be tested;
the two SNP locus combinations are respectively marked as GhGMP08-820SNP and GhGMP30-874SNP;
the physical position of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1 is A07:8291850 (sense strand), the nucleotides of which are C or T. The GhGMP08-820SNP is A or G at the 22 nd position of the DNA fragment shown in SEQ ID No.4 in cotton genome corresponding to the antisense strand.
The physical position of the GhGMP30-874SNP in the cotton reference genome TM-1_ZJU_V2.1 is D07:52577953 (sense strand), the nucleotide of which is A or G. The GhGMP30-874SNP is A or G at the 22 nd position of the DNA fragment shown in SEQ ID No.8 in the cotton genome.
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).
Further, the substance for detecting a single nucleotide polymorphism of the combination of the two SNP loci in the cotton genome is the set of KASP primers as described in the second aspect hereinafter or the reagent or kit as described in the third aspect hereinafter.
In a second aspect, the invention claims a kit of KASP primers for identifying or aiding in the identification of drought resistance in cotton.
The set of KASP primers claimed in the present invention consists of KASP primers for detecting GhGMP08-820SNP and KASP primers for detecting GhGMP30-874 SNP;
the physical position of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1 is A07:8291850, the nucleotide thereof is C or T;
the physical position of the GhGMP30-874SNP in the cotton reference genome TM-1_ZJU_V2.1 is D07:52577953, the nucleotide of which is A or G.
Wherein, the KASP primer for detecting the GhGMP08-820SNP consists of a primer A1, a primer A2 and a primer A3; the primer A1 is single-stranded DNA with a fluorescent tag sequence M and 22-43 positions of SEQ ID No.1 from the 5 'end to the 3' end; the primer A2 is single-stranded DNA with a fluorescent tag sequence N and 22-42 bits of SEQ ID No.2 from the 5 'end to the 3' end; the primer A3 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No.3 in a sequence table;
The KASP primer for detecting the GhGMP30-874SNP consists of a primer B1, a primer B2 and a primer B3; the primer B1 is single-stranded DNA with a fluorescent tag sequence M ' and 22-43 bits of SEQ ID No.5 from the 5' end to the 3' end; the primer B2 is single-stranded DNA with a fluorescent tag sequence N ' and 22-42 bits of SEQ ID No.6 from the 5' end to the 3' end; the primer B3 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No.7 in a sequence table.
Further, the fluorescent tag sequence M is a fluorescent tag sequence VIC, and the nucleotide sequence of the fluorescent tag sequence M is the 1 st-21 st position of SEQ ID No. 1; the fluorescent tag sequence N is a fluorescent tag sequence FAM, and the nucleotide sequence of the fluorescent tag sequence N is 1 st-21 st position of SEQ ID No. 2.
Further, the fluorescent tag sequence M 'is a fluorescent tag sequence VIC, and the nucleotide sequence of the fluorescent tag sequence M' is the 1 st-21 st position of SEQ ID No. 5; the fluorescent tag sequence N 'is a fluorescent tag sequence FAM, and the nucleotide sequence of the fluorescent tag sequence N' is the 1 st-21 st position of SEQ ID No. 6.
Further, the primer A1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 1; the primer A2 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 2.
Further, the primer B1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 5; the primer B2 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 6.
In a third aspect, the invention claims a reagent or kit for identifying or aiding in the identification of drought resistance in cotton.
The invention claims the kit or kit containing the second aspect of the kit of KASP primer.
In a fourth aspect, the invention claims the kit of KASP primers as described in the second aspect above or the reagent or kit as described in the third aspect above for use in any of the following:
(A1) Identifying or assisting in identifying drought resistance of cotton;
(A2) Identifying or assisting in identifying the yield traits and/or apparent morphology of cotton under drought stress;
(A3) Comparing drought resistance of cotton to be tested;
(A4) Comparing the yield characteristics and/or apparent morphology of cotton to be tested under drought stress;
(A5) Selecting cotton single plants or lines or strains or varieties with relatively strong drought resistance;
(A6) Breeding cotton single plants or lines or varieties with relatively high yield under drought stress;
(A7) Selecting a single plant or strain or variety of cotton having a relatively high effective number of bolls (efficiency bollnumber, EBN) and/or a relatively high weight of single bolls (single boll seeds weight, SBSW) and/or a relatively high weight of single bolls (Single boll weight, SBW) and/or a relatively high number of Bolls (BN) under drought stress;
(A8) Selecting a single plant or strain or variety of cotton with relatively high effective number of branches (efficiency fruit branch number, EFBN) and/or relatively high number of fruit branches (fruit branch number, FBN) under drought stress;
(A9) Screening out cotton single plants with relatively weak drought resistance;
(A10) Screening and removing cotton single plants with relatively low yield under drought stress;
(A11) Screening cotton individuals with relatively few effective bolls (efficiency bollnumber, EBN) and/or relatively light single boll seed cotton weights (single boll seeds weight, SBSW) and/or relatively light single boll weights (Single boll weight, SBW) and/or relatively few Boll Numbers (BN) under drought stress;
(A12) Cotton individuals with relatively few effective shoots (efficiency fruit branch number, EFBN) and/or relatively few fruit shoots (fruit branch number, FBN) under drought stress were selected for removal.
In a fifth aspect, the invention claims any of the following methods:
method I: a method for comparing drought resistance of cotton to be tested comprises the following steps: detecting single nucleotide polymorphism of the following two SNP locus combinations in the genome of the cotton to be detected, determining the haplotype of the cotton to be detected, and determining the drought resistance of the cotton to be detected according to the haplotype of the cotton to be detected as follows: the drought resistance of the cotton to be tested of the Hap4 (TT/GG) haplotype is stronger or candidate stronger than that of the cotton to be tested of the Hap1 (CT/GG) haplotype and the Hap3 (CT/AA) haplotype.
The method can also be used for comparing the yield characteristics and/or apparent morphology of cotton to be tested under drought stress. The yield of the cotton to be tested of the Hap4 (TT/GG) haplotype under drought stress is higher than or the cotton to be tested of which the candidates are higher than that of the Hap1 (CT/GG) haplotype and the Hap3 (CT/AA) haplotype; specifically, the cotton under test of Hap4 (TT/GG) haplotype has a relatively higher effective number of bolls (efficiency bollnumber, EBN) and/or a relatively higher weight of single bolls (single boll seeds weight, SBSW) and/or a relatively higher weight of single bolls (Single boll weight, SBW) and/or a relatively higher number of Bolls (BN) under drought stress than the cotton under test of Hap1 (CT/GG) haplotype and Hap3 (CT/AA) haplotype. The number of effective shoots (efficiency fruit branch number, EFBN) and/or number of fruit shoots (fruit branch number, FBN) of the test cotton of Hap4 (TT/GG) haplotype is higher or candidate higher than the test cotton of Hap1 (CT/GG) haplotype and Hap3 (CT/AA) haplotype under drought stress.
Method II: a method for breeding cotton single plant or strain or variety with relatively strong drought resistance comprises the following steps: detecting single nucleotide polymorphism of the following two SNP locus combinations in a genome of cotton to be detected, determining haplotype of the cotton to be detected, selecting the cotton to be detected, of which the two SNP locus combinations are Hap4 (TT/GG) haplotype, as a parent for breeding, and selecting the cotton of which the two SNP locus combinations are Hap4 (TT/GG) haplotype in each generation of breeding, thereby finally obtaining cotton single plants or strains or varieties with relatively strong drought resistance.
The method can also be used for breeding cotton single plants or lines or varieties with relatively high yield and/or relatively high effective number of bolls (efficiency bollnumber, EBN) and/or relatively heavy single boll seed cotton weight (single boll seeds weight, SBSW) and/or relatively heavy single boll weight (Single boll weight, SBW) and/or relatively high number of Bolls (BN) and/or relatively high effective number of branches (efficiency fruit branch number, EFBN) and/or relatively high number of fruit branches (fruit branch number, FBN) under drought stress. The cotton single plant or strain or variety with relatively strong drought resistance is obtained by the method that the yield is relatively high and/or the effective number of bolls (efficiency bollnumber, EBN) is relatively high and/or the weight of single boll seed cotton (single boll seeds weight, SBSW) is relatively heavy and/or the weight of single boll seed cotton (Single boll weight, SBW) is relatively heavy and/or the number of Boll Numbers (BN) is relatively high and/or the effective number of branches (efficiency fruit branch number, EFBN) is relatively high and/or the number of fruit branches (fruit branch number, FBN) is relatively high.
Method III: a method for screening out cotton single plants with relatively weak drought resistance comprises the following steps: detecting single nucleotide polymorphism of the following two SNP locus combinations in a genome of a cotton single plant to be detected, determining the haplotype of the cotton to be detected, and eliminating the cotton single plant to be detected, of which the two SNP locus combinations in the genome are Hap1 (CT/GG) haplotype and/or Hap3 (CT/AA).
The method may also be used to screen cotton individuals that reject relatively low yields and/or relatively low effective number of bolls (efficiency bollnumber, EBN) and/or relatively light weight of single bolls (single boll seeds weight, SBSW) and/or relatively light weight of single bolls (Single boll weight, SBW) and/or relatively low number of Bolls (BN) and/or relatively low effective number of shoots (efficiency fruit branch number, EFBN) and/or relatively low number of fruit shoots (fruit branch number, FBN) under drought stress. The cotton individual with relatively weak drought resistance finally removed is the cotton individual with relatively low yield and/or relatively low effective number of bolls (efficiency bollnumber, EBN) and/or relatively light weight of single boll seed cotton (single boll seeds weight, SBSW) and/or relatively light weight of single boll seed (Single boll weight, SBW) and/or relatively low number of Bolls (BN) and/or relatively low effective number of branches (efficiency fruit branch number, EFBN) and/or relatively low number of fruit branches (fruit branch number, FBN) under drought stress.
In each of the above methods, the two SNP site combinations were designated as GhGMP08-820SNP and GhGMP30-874SNP, respectively.
The physical position of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1 is A07:8291850, the nucleotide is C or T.
The physical position of the GhGMP30-874SNP in the cotton reference genome TM-1_ZJU_V2.1 is D07:52577953, the nucleotide of which is A or G.
The Hap4 (TT/GG) haplotype refers to the physical position A07 of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1: the nucleotide at 8291850 is homozygous for T, and the GhGMP30-874SNP is at physical position D07 in cotton reference genome TM-1_zju_v2.1: the nucleotide at 52577953 is homozygous for G.
The Hap1 (CT/GG) haplotype refers to the physical position a07 of the GhGMP08-820SNP in cotton reference genome TM-1_zju_v2.1: the nucleotides at 8291850 are heterozygous for C and T, and the physical position D07 of the GhGMP30-874SNP in cotton reference genome TM-1_zju_v2.1: the nucleotide at 52577953 is homozygous for G.
The Hap3 (CT/AA) haplotype refers to the physical position a07 of the GhGMP08-820SNP in cotton reference genome TM-1_zju_v2.1: the nucleotides at 8291850 are heterozygous for C and T, and the physical position D07 of the GhGMP30-874SNP in cotton reference genome TM-1_zju_v2.1: the nucleotide at 52577953 is homozygous for a.
Further, the "detecting single nucleotide polymorphism of the following two SNP locus combinations in the genome of the cotton to be detected" may be performed according to a method comprising the steps of: PCR amplification is carried out on the GhGMP08-820SNP and the GhGMP30-874SNP respectively by taking genomic DNA of the cotton to be detected as a template and using the set of KASP primers described in the second aspect or the reagent or the kit described in the third aspect, fluorescent signal scanning is carried out on the two amplified products respectively, and then single nucleotide polymorphism of the combination of the two SNP loci in the genome of the cotton to be detected is determined as follows:
Aiming at the GhGMP08-820SNP, if the fluorescent signal of the amplified product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence M, the nucleotide at the GhGMP08-820SNP of the cotton to be detected is homozygous for T; if the fluorescent signal of the amplified product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence N, the nucleotide at the GhGMP08-820SNP of the cotton to be detected is homozygote of C; if the fluorescent signal of the amplified product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence M and the signal corresponding to the fluorescent tag sequence N (namely a double signal), the nucleotides at the GhGMP08-820SNP of the cotton to be detected are heterozygous of C and T;
aiming at the GhGMP30-874SNP, if the fluorescent signal of the amplification product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence M', the nucleotide at the GhGMP30-874SNP of the cotton to be detected is homozygous for G; if the fluorescent signal of the amplified product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence N', the nucleotide at the SNP of GhGMP30-874 of the cotton to be detected is homozygous A; if the fluorescent signal of the amplified product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence M 'and the signal corresponding to the fluorescent tag sequence N' (i.e. a double signal), the nucleotides at the GhGMP08-820SNP of the cotton to be detected are heterozygous for A and G.
In a sixth aspect, the invention claims the use of a set of KASP primers as described in the second aspect above or a reagent or kit as described in the third aspect above or a method as described in the fifth aspect above in cotton breeding.
In the above aspects, the cotton is upland cotton. Such as upland cotton with haplotypes of the Hap1 (CT/GG) haplotype, the Hap3 (CT/AA) haplotype or the Hap4 (TT/GG) haplotype.
In a specific embodiment of the present invention, the cotton is specifically 186 parts cotton material as shown in table 4. Among the 186 cotton material, there were also Hap2 (CT/GA) haplotype and Hap5 (TT/AA) haplotype in addition to the Hap1 (CT/GG) haplotype, the Hap3 (CT/AA) haplotype and the Hap4 (TT/GG) haplotype, the drought resistance of the cotton material of these two haplotypes was biased toward the intermediate type. The Hap2 (CT/GA) haplotype refers to the physical position a07 of the GhGMP08-820SNP in cotton reference genome TM-1_zju_v2.1: the nucleotides at 8291850 are heterozygous for C and T, and the physical position D07 of the GhGMP30-874SNP in cotton reference genome TM-1_zju_v2.1: the nucleotide at 52577953 is a hybrid of G and a. The Hap5 (TT/AA) haplotype refers to the physical position A07 of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1: the nucleotide at 8291850 is homozygous for T, and the GhGMP30-874SNP is at physical position D07 in cotton reference genome TM-1_zju_v2.1: the nucleotide at 52577953 is homozygous for a.
In the aspects, the drought resistance is mainly represented by yield traits and/or apparent morphology under drought stress. Further, the yield trait is represented by the following indicators: effective number of bolls (efficiency bollnumber, EBN), single boll seed cotton weight (single boll seeds weight, SBSW), single boll weight (Single boll weight, SBW), number of Bolls (BN); the apparent morphology is represented by the following index: effective branch number (efficiency fruit branch number, EFBN), fruit branch number (fruit branch number, FBN).
Experiments prove that the invention develops the combined drought-resistance related molecular marker which can be closely related to the drought-resistance related character indexes of cotton breeding materials, is applied to the rapid identification and breeding of cotton drought-resistance excellent materials, lays a foundation for rapid identification and screening of large-scale cotton drought-resistance populations, and makes a contribution to subsequent cotton molecular breeding.
Drawings
FIG. 1 is a genotyping map of GhGMP08-820SNP site (A) and GhGMP30-874SNP site (B).
FIG. 2 is a cluster map of drought resistance for 186 parts of resource materials. A is a clustering analysis result graph based on CDC values, and B is a clustering analysis result graph based on D values.
FIG. 3 is a box plot of 186 parts upland cotton resource material drought resistance related property index CDC value and D value combined with different combination haplotypes. A and B are box line diagrams drawn by combining comprehensive drought resistance coefficient (CDC value) and drought resistance metric value (D) of each material with different combination haplotype data.
FIG. 4 is a box plot of 186 parts of upland cotton resource material drought resistance related property index DC value combined with main combination haplotype. a-Q is a box plot drawn by combining drought resistance coefficient (DC) values calculated respectively by 17 personality indexes of Plant Height (PH), first fruit branch position Height (HNFFB), fruit Branch Number (FBN), effective branch number (EFBN), bell Number (BN), effective Bell Number (EBN), single bell seed cotton weight (SBSW), single Bell Lint Weight (SBLW), single Bell Weight (SBW), clothing component (LP), intercellular carbon dioxide concentration (Ci), net photosynthetic rate (Pn), pore conductance (Gs), transpiration rate (Tr), water Vapor Pressure Deficiency (VPD), water Utilization Efficiency (WUE), chlorophyll relative value (SPAD), and main combination haplotype data.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1 development of molecular markers for identifying drought resistance in cotton and design of KASP primers
The genes used for developing the molecular markers are upland cotton GhGMP08 and GhGMP30 genes, and the gene annotation information is mannose-1-guanosine phosphate transferase (mannase-1-phosphate guanylyltransferase) and GDP mannose pyrophosphorylase (GDP-mannose pyrophosphorylase). The reference genome is TM-1_ZJU_2.1.
The gene ID of GhGMP08 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 the related information can be inquired in a cotton cottonFGD database (https:// cottonfgd.net/profiles/trans-script/GH_ A07G0705.1 /). Based on SNP information obtained by BSA-seq simplified genome sequencing of drought resistance-related RIL populations based on drought resistance metric D values, analysis of non-synonymous gene information found that there was one SNP mutation at genomic physical position A07__8291850 (sense strand): C/T, this site is at base 820 in CDS sequence of the gene, its mutation causes amino acid change: cat/Tat, in which histidine is mutated into tyrosine, is the only non-synonymous mutation site in the gene, and is named GhGMP08-820SNP.
The gene ID of GhGMP30 is GH_D07G2142, the physical position information is D07:52303026-52305696 (sense strand), the CDS sequence fragment size is 1,563bp, the DNA full-length sequence fragment size is 2671bp, the protein sequence length is 520aa, and the related information can be inquired in a cotton cottonFGD database (https:// cottonfgd.net/profiles/trans-script/GH_ D07G2142.1 /). Based on SNP information obtained by BSA-seq simplified genome sequencing of drought resistance related RIL populations based on drought resistance metric D values, analysis of non-synonymous gene information shows that one SNP mutation exists at the physical position D07_52577953: + of the genome: a/G, this site being at base 874 in the CDS sequence of the gene, the mutation of which causes an amino acid change: aag/Gag, a mutation from lysine to glutamic acid, is the only non-synonymous mutation site in this gene and is designated GhGMP30-874SNP.
1. KASP primer design for detecting GhGMP08-820SNP
Designing KASP mark primer sequence according to antisense strand of GhGMP08-820SNP, as follows:
GhGMP08-820SNP-F1：5’-GAAGGTCGGAGTCAACGGATTTGAACCGAAATTGTGCAAGATA-3' (SEQ ID No.1, underlined is the specific fluorescent tag sequence VIC);
GhGMP08-820SNP-F2：5’-GAAGGTGACCAAGTTCATGCTGAACCGAAATTGTGCAAGATG-3' (SEQ ID No.2, underlined is the specific fluorescent tag sequence FAM);
GhGMP08-820SNP-R：5’-AGAAAGTCCTTAAAGTGTTCTGGC-3’(SEQ ID No.3)。
The amplification product sequence of the GhGMP08-820SNP marker primer is as follows (the 22 nd site is a mutation site):
TGAACCGAAATTGTGCAAGATRTAGGCCAGAACACTTTAAGGACTTTCT(SEQ ID No.4)。
the last base at the 3' -end of the two upstream primers corresponds to 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 (represented by R in SEQ ID No. 4).
The upstream primer F1 (i.e., ghGMP08-820 SNP-F1) is used for amplifying the case that the nucleotide at the physical position A07:8291850 (sense strand) of the cotton reference genome TM-1_ZJU_V2.1 is T, and the upstream primer F2 (i.e., ghGMP08-820 SNP-F2) is used for amplifying the case that the nucleotide at the physical position A07:8291850 (sense strand) of the cotton reference genome TM-1_ZJU_V2.1 is C; the downstream primer R (namely GhGMP08-820 SNP-R) is a universal primer.
The single-stranded DNA molecule shown in SEQ ID No.1 and the single-stranded DNA molecule shown in SEQ ID No.3 amplify physical position A07 of cotton reference genome TM-1_ZJU_V2.1: 8291850 A fragment of the (sense) nucleotide having a genotype of T: T homozygote (i.e., a homozygote of A: A at the 22 nd base of SEQ ID No.4 on cotton genome). The sequence of the theoretical amplification product (without specific fluorescent tag sequence) is shown as 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 amplify physical position A07 of cotton reference genome TM-1_ZJU_V2.1: 8291850 A fragment of the (sense) nucleotide having a genotype of C: C homozygote (i.e., G: G homozygote at the 22 nd base of SEQ ID No.4 on the cotton genome). The sequence of the theoretical amplification product (without specific fluorescent tag sequence) is shown in positions 2-49 of SEQ ID No.4 (G at position 22).
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 amplify physical position A07 of cotton reference genome TM-1_ZJU_V2.1: 8291850 A fragment of the (sense) nucleotide having a genotype of C: T heterozygous (i.e., a heterozygous G: A base at position 22 of SEQ ID No.4 on the cotton genome). The theoretical amplification product (without specific fluorescent tag sequence) has two DNA fragments, namely a DNA fragment 1 shown in SEQ ID No.4 (A at 22 nd position) and a DNA fragment 2 shown in 2 nd to 49 nd positions of SEQ ID No.4 (G at 22 nd position).
2. KASP primer design for detecting GhGMP30-874SNP
The KASP-labeled primer sequence was designed based on the sense strand of the GhGMP30-874SNP as follows:
GhGMP30-874SNP-F1：5’-GAAGGTCGGAGTCAACGGATTCTTGATGATGAGAGGGCAAAAG-3' (SEQ ID No.5, underlined is the specific fluorescent tag sequence VIC);
GhGMP30-874SNP-F2：5’-GAAGGTGACCAAGTTCATGCTCTTGATGATGAGAGGGCAAAAA-3' (SEQ ID No.6, underlined is the specific fluorescent tag sequence FAM);
GhGMP30-874SNP-R：5’-CCATACTAGCAATGTAAGGCATC-3’(SEQ ID No.7)。
the amplification product sequence of the GhGMP30-874SNP marker primer is as follows (the 22 nd site is a mutation site):
CTTGATGATGAGAGGGCAAAARAGATGCCTTACATTGCTAGTATGG(SEQ ID No.8)。
the last base at the 3' -end of the two upstream primers corresponds to the SNP site (GhGMP 30-874 SNP). The GhGMP30-874SNP corresponds to position 22 of SEQ ID No.8 in the cotton genome, and is G or A (represented by R in SEQ ID No. 8).
The upstream primer F1 (i.e., ghGMP30-874 SNP-F1) is used to amplify the case where the nucleotide at the physical position D07_52577953 (sense strand) of the cotton reference genome TM-1_ZJU_V2.1 is G, and the upstream primer F2 (GhGMP 30-874 SNP-F2) is used to amplify the case where the nucleotide at the physical position D07_52577953 (sense strand) of the cotton reference genome TM-1_ZJU_V2.1 is A; the downstream primer R (namely GhGMP30-874 SNP-R) is a universal primer.
The single-stranded DNA molecule shown in SEQ ID No.1 and the single-stranded DNA molecule shown in SEQ ID No.3 amplify the physical position D07 of the cotton reference genome TM-1_ZJU_V2.1: 52577953 A fragment of the (sense) nucleotide having the genotype G: G homozygote (i.e., the base at position 22 of SEQ ID No.8 on the cotton genome is G: G homozygote). The sequence of the theoretical amplification product (without specific fluorescent tag sequence) is shown in SEQ ID No.8 (G 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 amplify the physical position D07 of the cotton reference genome TM-1_ZJU_V2.1: 52577953 A fragment with the genotype A: A homozygote (i.e., A: A homozygote at the 22 nd base of SEQ ID No.8 on cotton genome) is the nucleotide at (sense strand). The sequence of the theoretical amplification product (without specific fluorescent tag sequence) is shown in SEQ ID No.8 (A at position 22).
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 amplify the physical position D07 of the cotton reference genome TM-1_ZJU_V2.1: 52577953 A fragment with the genotype G: A heterozygous (i.e., the base at position 22 of SEQ ID No.8 on the cotton genome is G: A heterozygous) of the nucleotide at (sense strand). The theoretical amplification products (without specific fluorescent tag sequences) are two, namely a DNA fragment 1 shown in SEQ ID No.8 (G at position 22) and a DNA fragment 2 shown in SEQ ID No.8 (G at position 22).
Example 2 establishment and use of method for detecting SNP genotype Using KASP primer
1. DNA sample preparation
DNA extraction: genomic DNA was extracted from cotton leaves using CTAB method.
Determination of DNA concentration: several DNA working solutions were randomly extracted and concentration was measured using a NanoDrop2000 instrument.
DNA integrity identification: agarose gel electrophoresis, 1.5%,120V,40min. And the main belt is qualified.
SNP Primer Mix (4×) formulation: the primer dry powder was diluted to 100mM and the three sequences were then mixed according to F1: f2: r: ddH 2 O=24:24:48:100.
DNA dilution: the DNA stock was diluted uniformly to 20 ng/. Mu.l.
2. KASP reaction
The GhGMP08-820SNP and the KSAP detection of the GhGMP30-874SNP sites were carried out by the Izosen Biotechnology Co.Ltd.
The KASP detection PCR amplification system is shown in Table 1.
TABLE 1 PCR amplification System for KASP detection
| Name of the name | 384 well plate (4 mu L system) |
| KASP HiGeno 2×Probe Mix | 2μL |
| SNP Primer Mix(4x) | 1μL |
| DNA sample | 2μL |
Note that: KASP HiGeno 2X Probe Mix is a product of Beijing Jiacheng Biotechnology Co., ltd. (product number AQP-001S). KASP HiGeno 2X Probe Mix contains fluorescent Probe A, fluorescent Probe B, quenching Probe A, quenching Probe B, ROX dye, high-fidelity Taq enzyme, dNTP, mg 2+ Etc. The nucleotide sequence of the fluorescent probe A is 5'-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'-AGCATGAACTTGGTCACCTTC-3', and the 3' -end is connected with a quenching group BHQ. The KASP detection PCR reaction procedure is shown in Table 2.
TABLE 2 KASP detection PCR reaction procedure
3. Analysis of results
Fluorescence value reading: after the PCR amplification cycle is completed, the fluorescent value is read by a fluorescent quantitative PCR instrument in an environment lower than 40 ℃. In this method, SNP site detection uses fluorophores FAM and VIC to distinguish between two isogenic sites. The passive reference dye ROX (passive reference dye ROX) was used to correct for signal differences from well to well due to reaction volume errors. The associated excitation and emission wavelengths are shown in table 3 below. The read software was Omega device of the LGC.
TABLE 3 excitation light and emission light wavelength of fluorophores
| Fluorescent group | Excitation light (nm) | Emission light (nm) |
| FAM | 485 | 520 |
| VIC | 535 | 556 |
| ROX | 575 | 610 |
Note that: if the fluorescent scanner uses HEX fluorophores as detection signals, no modification of the settings is required, as the excitation light and emission light values of VIC and HEX are very similar.
The resultant data from the fluorescent value reading was analyzed using the genotyping software (Kmaster Caller) for LGC. The VIC and FAM values of each reaction well are corrected by the specific Kong Can specific dye (ROX) value, and the data fluorescence values are subjected to standardized treatment to obtain the corresponding relative fluorescence values of the VIC and FAM of each PCR reaction well. Clustering samples according to relative fluorescence values, and further determining genotypes of GhGMP08-820SNP and GhGMP30-874SNP loci in a cotton genome to be detected according to the sample clusters and fluorescence types (namely whether the base at the 22 nd position of SEQ ID No.4 on the cotton genome is A or G and whether the base at the 22 nd position of SEQ ID No.8 is G or A):
aiming at the GhGMP08-820SNP locus, if the fluorescence signal data of the amplified product of the cotton to be detected is analyzed by a genotyping software KlumterCaller to approach to the Y axis (VIC signal), the genotype of the GhGMP08-820SNP locus in the genome of the cotton to be detected is T:T homozygosity (namely, the 22 nd base of SEQ ID No.4 on the genome of the cotton is A:A homozygosity); if the fluorescence signal data of the amplified product of the cotton to be detected is analyzed by a genotyping software KlumterCaller to be close to the X axis (FAM signal), the genotype of the GhGMP08-820SNP locus in the genome of the cotton to be detected is C:C homozygosity (namely, the base at the 22 nd position of SEQ ID No.4 on the genome of the cotton is G:G homozygosity); if the fluorescence signal data of the amplified product of the cotton to be detected is located in the middle of the X axis and the Y axis (with VIC and FAM signals at the same time) through the genotyping software KiusterCaller analysis, the genotype of the GhGMP08-820SNP locus in the genome of the cotton to be detected is T:C heterozygous (namely, the 22 nd base of SEQ ID No.4 on the genome of the cotton is A:G heterozygous).
Aiming at the GhGMP30-874SNP locus, if the fluorescence signal data of the amplified product of the cotton to be detected is analyzed by a genotyping software KlumterCaller to approach to the Y axis (VIC signal), the genotype of the GhGMP30-874SNP locus in the genome of the cotton to be detected is G:G homozygosity (namely, the 22 nd base of SEQ ID No.8 on the genome of the cotton is G:G homozygosity); if the fluorescence signal data of the amplified product of the cotton to be detected is analyzed by a genotyping software KlumterCaller to be close to the X axis (FAM signal), the genotype of the SNP locus GhGMP30-874 in the genome of the cotton to be detected is A:A homozygote (namely, the base at the 22 nd position of SEQ ID No.8 on the genome of the cotton is A:A homozygote); if the fluorescence signal data of the amplified product of the cotton to be detected is located in the middle of the X axis and the Y axis (with VIC and FAM signals at the same time) through the genotyping software KiusterCaller analysis, the genotype of the SNP locus GhGMP30-874 in the genome of the cotton to be detected is A: G heterozygous (namely, the base at the 22 nd position of SEQ ID No.8 on the genome of the cotton is A: G heterozygous).
The genotyping results are shown in FIG. 1. A is a genotyping result diagram of GhGMP08-820SNP locus, which is marked orange (crossed) and is marked with genotype T, which is marked with genotype C, which is marked with blue (circular), which is marked with green (square), which is marked with genotype unknown, and which is marked with red (cross) and is marked with template-free negative control (No Template Control, NTC for short, other reagent additions are the same, namely the DNA template is replaced by water). B is a genotyping result diagram of the GhGMP30-874SNP locus, the genotype G is marked orange (crossed), the genotype G is marked blue (circular), the genotype G is marked green (square), the genotype A is marked A, the genotype is unknown, and the genotype is marked red (cross), and the genotype is marked purple (square) and is marked as a control NTC.
4. Marking type data analysis
186 parts of tested upland cotton resource materials are used, and the conventional planting varieties, part of foreign introduced materials and laboratory self-raising materials of all large cotton areas including a yellow river basin cotton area, a northwest inner upland cotton area, a Yangtze river basin cotton area, a northeast ultra-early maturing cotton area and the like are collected and provided by a key laboratory of crop genetic improvement and germplasm innovation of Xinjiang agricultural university college.
In one aspect, genotypes of each of the cotton tested GhGMP08-820SNP and GhGMP30-874SNP sites were detected as described above. On the other hand, the field resistance identification is performed by a conventional method. The method comprises the following steps:
186 parts of resource material (see Table 4, described in "Chengchuan, zeng Qingtao, chen Qin, fujin Cheroke, wang Tingwei, chen Quangu, qu Yanying. Screening and evaluation of drought resistance index of upland cotton in boll period [ J ]]Chinese agricultural science and technology guide 2022,24 (07): 46-57", available to the public from the applicant and used only for repeated experiments of the invention but not for him), was tested in relation to drought resistance identification at 144 in the county of Shawan, 3 in 2021Is carried out in connection with cotton breeding bases (43 DEG 20 '-45 DEG 20' N,84 DEG 45 '-86 DEG 40' E) of Xinjiang agricultural university. The test sets 2 treatments of drought stress and normal control, 2 replicates each, and 2m protection rows between drought stress and normal control. 2m is grown in each cell, 14 films are grown in 1 region, 1 film is grown in 3 rows, 1 film is planted in 1 part of material, and drip irrigation under the film is adopted for watering. The test materials were sown on 26 days of 4 months, seedlings were emerged on 5 days of 5 months, and topped on 7 days of 7 months. The stress treatment is started at 7 months and 4 days, and a water meter is installed on a main pipeline of an arid region to record the water control amount before the stress. The management method during cultivation is the same as that of the field. And after drought stress for 15d, sampling and weighing soil layers of 0-20, 20-40 and 40-60 cm by adopting a 5-point sampling method, putting the soil layers into an oven, baking the soil layers to constant weight, and measuring the water content. The results show that the water content of the soil layer with the thickness of 0-20 cm after drought stress is changed maximally, 8.257 percentage points are reduced, the water content of the soil layer with the thickness of 3 soil layers is reduced by 7.921 percentage points on average, and the total water control of 2 drought stress treatments during the stress period is 566m 3 Drought stress conditions are reached.
Cotton was selected and 3 leaves were inverted, and photosynthesis metrics were measured using a photosynthesis apparatus (hansa, cisas-3, england), including transpiration rate (transpiration rate, tr), intercellular carbon dioxide concentration (intercellular carbon dioxide concentration, ci), net photosynthetic rate (photosynthetic net, pn), stomatal conductance (gas, gs), water vapor pressure deficiency (vapor pressure deficit, VPD), and water use efficiency (water use efficiency, WUE), and 3 plants were continuously measured for each material and averaged for 1 repeat.
For 7 months and 20 days, chlorophyll relative value (SPAD) was measured with SPAD instrument (SYS-SPAD-502 Plus, japan), cotton was selected to be 3 leaves, average was measured on 3 leaves, middle and lower, 3 plants were continuously measured, and the average was recorded as 1 repeat. Determining agronomic traits of drought stress and normal controls, including Plant Height (PH), first fruit branch position height (Height at the node of the first fruit branch, HNFFB), fruit branch number (fruit branch number, FBN), effective branch number (efficiency fruit branch number, EFBN), bell Number (BN), effective bell number (efficiency bollnumber, EBN), at day 17, 9 months; cotton 20 bolls were harvested at 25 months, the coat weight (Single boll weight, SBW), the seed cotton weight (single boll seeds weight, SBSW) and the lint weight (single boll lint weight, SBLW) were measured, and the measurement method was referred to "cotton germplasm resource description Specification and data Standard" (Du Xiongming, zhou Zhongli. Cotton germplasm resource description Specification and data Standard [ M ]. Beijing: china agricultural Press, 2005:1-89.).
Statistical analysis of the data was performed using EXCEL 2010 software and SPSS25.0 software. The method for evaluating drought resistance by 17 personality indexes in total comprises the steps of calculating drought resistance coefficients (DC), drought resistance indexes (DI), comprehensive drought resistance coefficients (CDC value) and drought resistance measurement values (D) of related character indexes, wherein a calculation formula is calculated by referring to a calculation method of Sun Fenglei (Sun Fenglei, qu Yanying, chen Quangu, and the like; comprehensive evaluation of cotton drought resistance related indexes and gray correlation analysis [ J ]. Agricultural research in arid regions, 2019,37 (1): 233-239.):
drought resistance coefficient:
drought resistance index:
comprehensive drought resistance coefficient:
membership function:
drought resistance metric value:
in the above calculation formulas, X d 、X w The measured values of various indexes of various materials under drought stress and normal control are respectively obtained,to average of the index under drought stress, DI i min 、DI i max The minimum value and the maximum value of drought resistance indexes of all characters are obtained; r is (r) i And contributing rate for the ith comprehensive index.
After genotype detection of GhGGMP 08-820SNP and GhGGMP 30-874SNP loci was performed on 186 parts of upland cotton materials, it was found that 5 kinds of combined haplotypes, which are Hap1 (CT/GG), hap2 (CT/GA), hap3 (CT/AA), hap4 (TT/GG) and Hap5 (TT/AA), were formed by using GhGGMP 08-820SNP and GhGGMP 30-874SNP markers in the materials. Wherein "/" is preceded by the genotype of the GhGMP08-820SNP locus and "/" is followed by the genotype of the GhGMP30-874SNP locus.
And (3) measuring and analyzing 17 individual indexes of 186 parts of upland cotton resource materials by using the test design and analysis method. Calculating a drought resistance coefficient DC value of an average value after normal treatment and drought stress treatment, calculating a comprehensive drought resistance coefficient CDC value and a drought resistance measurement value D value, classifying drought resistance of 186 parts of upland cotton resource materials through cluster analysis, and displaying that the classification of the comprehensive drought resistance coefficient CDC value and the drought resistance measurement value D value is approximately the same. Finally, determining the final drought resistance grade according to the drought resistance measurement value D value: class I is strong drought-resistant (D is more than or equal to 0.54 and less than or equal to 0.61), and 13 materials are provided; class II is drought-resistant (D is more than or equal to 0.46 and less than 0.54), and 47 materials are provided; class III is medium drought resistant (D is more than or equal to 0.42 and less than 0.46), and 40 materials are provided; class IV is sensitive drought type (D is more than or equal to 0.38 and less than 0.42), and 46 materials are contained; class V is extremely sensitive drought type (D is more than or equal to 0.27 and less than or equal to 0.38), and 40 materials are provided.
Materials with Hap4 (TT/GG) haplotypes total 11, with materials in class i and class ii (strong drought and drought resistant) accounting for about 81.8% (9/11), materials in class iv (drought sensitive) accounting for about 18.2% (2/11), and materials in class v (extreme drought sensitive) accounting for 0% (0/11).
Materials with Hap5 (TT/AA) haplotypes total 12, with material ratios of about 58.3% (7/12) for class i, class ii and class iii (strong drought, drought and medium drought) and about 41.7% (5/12) for class iv (drought sensitive) and class v (extreme drought).
The total of 41 materials with Hap1 (CT/GG) haplotypes, wherein the material ratio of the materials in the I type, the II type and the III type (strong drought resistance type, drought resistance type and medium drought resistance type) is about 63.4 percent (26/41), and the material ratio of the materials in the IV type (drought sensitive type) and the V type (extremely drought sensitive type) is about 36.6 percent (15/41).
The total of 2 materials with Hap2 (CT/GA) haplotypes are respectively positioned in class II (drought-resistant type) and class IV (drought-sensitive type).
Materials with Hap3 (CT/AA) haplotypes total 102, with material ratios of about 45.1% for class i, class ii and class iii (strong drought, drought and medium drought) (46/102) and about 54.9% for class iv (drought sensitive) and class v (extreme drought) (56/102).
A cluster diagram of 186 parts of resource materials for drought resistance is shown in FIG. 2. Fig. 2 a shows a graph of the result of the clustering analysis based on the CDC value, and fig. 2B shows a graph of the result of the clustering analysis based on the D value. Color represents the magnitude of a value, from blue to red representing the magnitude from small to large. The results show that: the clustering analysis results of the CDC value and the D value are approximately the same, drought resistance grades of 186 parts of resource materials are evenly divided into I-V types, and meanwhile, the range of the CDC value and the D value is larger, which shows that the genetic background of 186 parts of resource materials is rich, and drought resistance comparison is representative.
The comprehensive drought resistance coefficient CDC value, drought resistance metric value D value, grading condition according to the D value and genotype information of GhGMP08-820SNP and GhGMP30-874SNP loci calculated by 186 parts of upland cotton resource materials based on the drought resistance coefficient DC value of 17 personality indexes are shown in table 4. Table 5 is the DC value of 17 personality indicators for 186 parts upland cotton resource material.
TABLE 4 CDC value, D value and corresponding grade of 186 parts upland cotton resource material and genotypes of GhGMP08-820SNP, ghGMP30-874SNP, and combination haplotypes
Note that: the "class" in the table corresponds to the preceding classes I to V. "-" indicates undetected.
Table 5, 186 parts DC value of 17 personality index for upland cotton resource material
5. Genotype and phenotype association analysis
And (3) carrying out association analysis on the drought resistance identification result based on 186 parts of upland cotton resource materials obtained in the step (4) and the combined haplotype result of the drought resistance identification result at GhGMP08-820SNP locus and GhGMP30-874SNP locus so as to achieve the aim of associating the combined haplotype with single or multiple character indexes and explore the association of the combined haplotype and drought resistance.
And (3) carrying out double-tail T test on the CDC value, the D value and each haplotype of the comprehensive drought resistance coefficient by using software GraphPad Prism 9, wherein the result is shown in figure 3. ns represents no significant difference; * Representing P <0.05, indicating that the character indexes among different haplotypes are obviously different as a whole; * Representing P <0.01, indicating that the character indexes among different haplotypes are very significant differences overall; * P <0.001 is represented, indicating that the trait indices between different haplotypes are very significantly different overall.
The results show that the main three combined haplotypes: the comprehensive drought resistance coefficient CDC value and drought resistance measurement value D value of Hap1 (CT/GG), hap3 (CT/AA) and Hap4 (TT/GG) are extremely obviously different, and the comparison of Hap2 (CT/GA) and Hap5 (TT/AA) is biased to the middle haplotype, so that the change of three combined haplotypes of Hap1 (CT/GG), hap3 (CT/AA) and Hap4 (TT/GG) has stronger correlation strength with the overall drought resistance strength of the resource material.
The results of the significance correlation analysis were performed for three combined haplotypes, hap1 (CT/GG), hap3 (CT/AA) and Hap4 (TT/GG), with 17 personality indicators, as shown in FIG. 4. The results show that the appearance of Hap4 (TT/GG) represents better drought resistance in most of the trait indexes, while the appearance of Hap1 (CT/GG) and Hap3 (CT/AA) represents poorer drought resistance, and that Hap3 (CT/AA) and Hap4 (TT/GG) have stronger significance differences in most of the trait indexes, and Hap1 (CT/GG) and Hap4 (TT/GG) have significance differences but are relatively weaker. Among the 17 individual character indexes, the measured 4 individual character indexes such as EFBN, EBN, SSW, SBW and the like show very significant differences, the measured 2 individual character indexes such as FBN1 and the like show very significant differences, and the indexes can directly show the apparent morphology and yield information of cotton. The other character indexes have no significant difference on the whole, but all show the drought resistance difference trend on the whole. These results indicate that the combined haplotype of GhGMP08-820SNP and GhGMP30-874SNP loci are significantly associated with a plurality of property indexes related to drought resistance of cotton, and that different combined haplotypes represent the drought resistance to a certain extent as a whole.
In conclusion, different combined haplotypes of the GhGMP08-820SNP locus and the GhGMP30-874SNP locus can influence the differential expression of a plurality of property indexes of cotton through a certain drought stress regulation mechanism, compared with the material of the Hap4 (TT/GG) haplotype, the material of the Hap4 (TT/GG) haplotype is drought-resistant on the whole, and meanwhile, the effect of increasing the drought resistance of plants is achieved through the influence of the materials on a plurality of properties, so that the GhGMP08-820SNP locus and the GhGMP30-874SNP locus are taken as a combined molecular marker, have important significance in drought resistance identification and screening of cotton resource materials, and lay a good foundation for auxiliary selective breeding of cotton molecular markers.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present 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 respect to specific embodiments, 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.
Claims (10)
1. The application of single nucleotide polymorphism of the following two SNP locus combinations in a cotton genome or substances for detecting the single nucleotide polymorphism of the following two SNP locus combinations in the cotton genome in the identification or auxiliary identification of drought resistance of cotton to be detected;
the two SNP locus combinations are respectively marked as GhGMP08-820SNP and GhGMP30-874SNP;
the physical position of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1 is A07:8291850, the nucleotide thereof is C or T;
the physical position of the GhGMP30-874SNP in the cotton reference genome TM-1_ZJU_V2.1 is D07:52577953, the nucleotide of which is A or G.
2. The use according to claim 1, characterized in that: the substance for detecting single nucleotide polymorphism of the combination of the two SNP loci in cotton genome is the kit KASP primer set as set forth in any one of claims 3 to 5 or the reagent or kit as set forth in claim 6 or 7.
3. A set of KASP primers for identifying or aiding in the identification of drought resistance in cotton, characterized in that: the set of KASP primers consists of KASP primers for detecting GhGMP08-820SNP and KASP primers for detecting GhGMP30-874SNP;
the physical position of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1 is A07:8291850, the nucleotide thereof is C or T;
The physical position of the GhGMP30-874SNP in the cotton reference genome TM-1_ZJU_V2.1 is D07:52577953, the nucleotide thereof is A or G;
wherein, the KASP primer for detecting the GhGMP08-820SNP consists of a primer A1, a primer A2 and a primer A3; the primer A1 is single-stranded DNA with a fluorescent tag sequence M and 22-43 positions of SEQ ID No.1 from the 5 'end to the 3' end; the primer A2 is single-stranded DNA with a fluorescent tag sequence N and 22-42 positions of SEQ ID No.2 from the 5 'end to the 3' end; the primer A3 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No.3 in a sequence table;
the KASP primer for detecting the GhGMP30-874SNP consists of a primer B1, a primer B2 and a primer B3; the primer B1 is single-stranded DNA with a fluorescent tag sequence M ' and 22-43 bits of SEQ ID No.5 from the 5' end to the 3' end; the primer B2 is single-stranded DNA with a fluorescent tag sequence N ' and 22-42 bits of SEQ ID No.6 from the 5' end to the 3' end; the primer B3 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No.7 in a sequence table.
4. A kit of KASP primers according to claim 3, wherein: the fluorescent tag sequence M is a fluorescent tag sequence VIC, and the nucleotide sequence of the fluorescent tag sequence M is the 1 st-21 st position of SEQ ID No. 1; the fluorescent tag sequence N is a fluorescent tag sequence FAM, and the nucleotide sequence of the fluorescent tag sequence N is the 1 st-21 st position of SEQ ID No. 2; and/or
The fluorescent tag sequence M 'is a fluorescent tag sequence VIC, and the nucleotide sequence of the fluorescent tag sequence M' is the 1 st-21 st position of SEQ ID No. 5; the fluorescent tag sequence N 'is a fluorescent tag sequence FAM, and the nucleotide sequence of the fluorescent tag sequence N' is the 1 st-21 st position of SEQ ID No. 6.
5. The kit of KASP primers according to claim 3 or 4, wherein: the primer A1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 1; the primer A2 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 2; and/or
The primer B1 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 5; the primer B2 is single-stranded DNA with a nucleotide sequence shown as SEQ ID No. 6.
6. A reagent or kit for identifying or assisting in identifying drought resistance of cotton, characterized in that: the reagent or kit contains the kit of KASP primers as described in any one of claims 3-5.
7. Use of a kit of KASP primers according to any one of claims 3 to 5 or a reagent or kit according to claim 6 in any one of the following:
(A1) Identifying or assisting in identifying drought resistance of cotton;
(A2) Identifying or assisting in identifying the yield traits and/or apparent morphology of cotton under drought stress;
(A3) Comparing drought resistance of cotton to be tested;
(A4) Comparing the yield characteristics and/or apparent morphology of cotton to be tested under drought stress;
(A5) Selecting cotton single plants or lines or strains or varieties with relatively strong drought resistance;
(A6) Breeding cotton single plants or lines or varieties with relatively high yield under drought stress;
(A7) Selecting single cotton plants or lines or strains or varieties with relatively more effective bolls and/or relatively heavier single boll seed cotton weights and/or relatively heavier single bolls and/or relatively more bolls under drought stress;
(A8) Selecting single cotton plants or strains or varieties with relatively more effective branches and/or relatively more fruit branches under drought stress;
(A9) Screening out cotton single plants with relatively weak drought resistance;
(A10) Screening and removing cotton single plants with relatively low yield under drought stress;
(A11) Screening and removing cotton single plants with relatively fewer effective bolls and/or relatively lighter single boll seed cotton weights and/or relatively lighter single bolls and/or relatively fewer bolls under drought stress;
(A12) Screening and removing cotton single plants with relatively fewer effective branches and/or relatively fewer fruit branches under drought stress.
8. The method comprises the following steps:
method I: a method for comparing drought resistance of cotton to be tested comprises the following steps: detecting single nucleotide polymorphism of the following two SNP locus combinations in the genome of the cotton to be detected, determining the haplotype of the cotton to be detected, and determining the drought resistance of the cotton to be detected according to the haplotype of the cotton to be detected as follows: drought resistance of the cotton to be tested of the Hap4 (TT/GG) haplotype is stronger or candidate is stronger than drought resistance of the cotton to be tested of the Hap1 (CT/GG) haplotype and the Hap3 (CT/AA) haplotype;
Method II: a method for breeding cotton single plant or strain or variety with relatively strong drought resistance comprises the following steps: detecting single nucleotide polymorphism of the following two SNP locus combinations in a genome of cotton to be detected, determining haplotype of the cotton to be detected, selecting the cotton to be detected, of which the two SNP locus combinations are Hap4 (TT/GG) haplotype, as a parent for breeding, and selecting the cotton of which the two SNP locus combinations are Hap4 (TT/GG) haplotype in each generation of breeding, so as to finally obtain cotton single plant or strain or variety with relatively strong drought resistance;
method III: a method for screening out cotton single plants with relatively weak drought resistance comprises the following steps: detecting single nucleotide polymorphism of the following two SNP locus combinations in a genome of a cotton single plant to be detected, determining the haplotype of the cotton to be detected, and eliminating the cotton single plant to be detected, of which the two SNP locus combinations in the genome are Hap1 (CT/GG) haplotype and Hap3 (CT/AA);
the two SNP locus combinations are respectively marked as GhGMP08-820SNP and GhGMP30-874SNP;
the physical position of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1 is A07:8291850, the nucleotide thereof is C or T;
The physical position of the GhGMP30-874SNP in the cotton reference genome TM-1_ZJU_V2.1 is D07:52577953, the nucleotide thereof is A or G;
the Hap4 (TT/GG) haplotype refers to the physical position A07 of the GhGMP08-820SNP in the cotton reference genome TM-1_ZJU_V2.1: the nucleotide at 8291850 is homozygous for T, and the GhGMP30-874SNP is at physical position D07 in cotton reference genome TM-1_zju_v2.1: the nucleotide at 52577953 is homozygous for G;
the Hap1 (CT/GG) haplotype refers to the physical position a07 of the GhGMP08-820SNP in cotton reference genome TM-1_zju_v2.1: the nucleotides at 8291850 are heterozygous for C and T, and the physical position D07 of the GhGMP30-874SNP in cotton reference genome TM-1_zju_v2.1: the nucleotide at 52577953 is homozygous for G;
the Hap3 (CT/AA) haplotype refers to the physical position a07 of the GhGMP08-820SNP in cotton reference genome TM-1_zju_v2.1: the nucleotides at 8291850 are heterozygous for C and T, and the physical position D07 of the GhGMP30-874SNP in cotton reference genome TM-1_zju_v2.1: the nucleotide at 52577953 is homozygous for a.
9. The method according to claim 8, wherein: the method for detecting the single nucleotide polymorphism of the following two SNP locus combinations in the genome of the cotton to be detected comprises the following steps: PCR amplification is carried out on the GhGMP08-820SNP and the GhGMP30-874SNP respectively by taking genomic DNA of the cotton to be detected as a template and using the set KASP primer as set in any one of claims 3-5 or the reagent or kit as set in claim 6, fluorescent signal scanning is carried out on two amplified products respectively, and then single nucleotide polymorphism of the combination of the two SNP loci in the genome of the cotton to be detected is determined as follows:
Aiming at the GhGMP08-820SNP, if the fluorescent signal of the amplified product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence M, the nucleotide at the GhGMP08-820SNP of the cotton to be detected is homozygous for T; if the fluorescent signal of the amplified product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence M and the signal corresponding to the fluorescent tag sequence N, the nucleotides at the GhGMP08-820SNP of the cotton to be detected are heterozygous of C and T;
aiming at the GhGMP30-874SNP, if the fluorescent signal of the amplification product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence M', the nucleotide at the GhGMP30-874SNP of the cotton to be detected is homozygous for G; if the fluorescent signal of the amplified product of the cotton to be detected is the signal corresponding to the fluorescent tag sequence N', the nucleotide at the SNP of GhGMP30-874 of the cotton to be detected is homozygous A.
10. Use of a set of KASP primers according to any one of claims 3 to 5 or a reagent or kit according to claim 6 or a method according to claim 8 or 9 in cotton breeding.
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