CN111961746B

CN111961746B - SNP molecular marker related to blight and disease resistance of cotton on land and application thereof

Info

Publication number: CN111961746B
Application number: CN202010927241.6A
Authority: CN
Inventors: 李付广; 王鹏; 杨召恩; 王笑千; 葛晓阳; 靳羽莹; 闫青地; 秦文强; 王晔
Original assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Cotton Research of Chinese Academy of Agricultural Sciences
Priority date: 2020-09-07
Filing date: 2020-09-07
Publication date: 2022-03-15
Anticipated expiration: 2040-09-07
Also published as: CN111961746A

Abstract

The invention discloses SNP molecular markers related to resistance to blight of cotton on land and application thereof. The invention firstly discloses the application of the substance for detecting the polymorphism or genotype of the SNP marker in the cotton genome in identification or auxiliary identification of the resistance of cotton to wilt disease; the SNP marker is located at 2259240 th nucleotide of chromosome D03 of cotton genome, and the nucleotide types are A and C. The invention further discloses a method for identifying or assisting in identifying the resistance of cotton to be detected to the blight. The SNP marker provided by the invention can be used for early prediction of cotton wilt disease resistance, can also be used for cotton genetic background analysis and screening, and cotton wilt resistance molecular marker-assisted selective breeding, and has wide application prospect.

Description

SNP molecular marker related to blight and disease resistance of cotton on land and application thereof

Technical Field

The invention relates to the field of plant breeding, in particular to SNP molecular markers related to resistance to cotton wilt on land and application thereof.

Background

The cotton is the traditional cultivated crop in China and also an important economic crop, and the cotton fiber can be made into fabrics of various specifications and is suitable for making various clothes and fabrics. However, in cotton planting, blight is a common disease. Pathogenic bacteria are soil-borne fungi, namely fusarium oxysporum (wilt specialization type), which mainly damage the vascular bundle and other parts of cotton, so that leaves die or fall off. The molecular mechanism of cotton wilt resistance is still unclear, and the research on the cloning and function of cotton wilt resistance genes is slow. Therefore, the problem to be solved in cotton production is to enhance the resistance of cotton to blight and increase the cotton yield.

With the development of sequencing technology and the completion of cotton genome sequencing, SNP sites related to blight resistance of upland cotton are obtained by combining whole genome association analysis and phenotype, and important reference basis can be provided for molecular marker-assisted selection and molecular breeding improvement of cotton disease resistance.

Disclosure of Invention

The invention aims to solve the technical problem of how to early detect the resistance of cotton to blight.

In order to solve the technical problems, the invention firstly provides the application of a substance for detecting the polymorphism or genotype of an SNP marker in a cotton genome in identification or auxiliary identification of the resistance of cotton to wilt disease;

the SNP marker is located at 2259240 th nucleotide of chromosome D03 of cotton genome, and the nucleotide types are A and C.

The application of the substance for detecting the polymorphism or genotype of the SNP marker in the cotton genome in cotton breeding is also within the protection scope of the invention.

In the application, the cotton breeding is to culture cotton with resistance to blight.

In the application, the SNP marker corresponds to the 448 th site of the GhGLR3.3 gene, namely the 448 th site nucleotide of SEQ ID NO.1, and the nucleotide type is A or C.

In the above application, the detecting polymorphism or genotype of the SNP marker in the cotton genome may specifically be detecting nucleotide species of the SNP marker in the cotton genome. The genotype of the SNP marker is CC (CC genotype for short) or AA (AA genotype for short). Wherein, the CC genotype is the homozygote of the SNP marker C in the cotton genome, and the AA genotype is the homozygote of the SNP marker A in the cotton genome.

In the above application, the substance for detecting the polymorphism or genotype of the SNP marker in the cotton genome is A1) or A2) or A3) below:

A1) a set of primers for amplifying a DNA fragment containing the SNP marker;

A2) PCR reagents containing a 1);

A3) a kit comprising a1) or a 2).

In the application, the primer set consists of a single-stranded DNA molecule shown by SEQ ID NO.2 and a single-stranded DNA molecule shown by SEQ ID NO. 3.

In order to solve the technical problem, the invention further provides a product.

The product provided by the invention is the substance for detecting the polymorphism or genotype of the SNP marker in the cotton genome;

the product has at least one of the following functions:

1) identifying or assisting in identifying the resistance of the cotton to be detected to the blight;

2) breeding cotton with resistance to blight;

3) and (3) breeding cotton with resistance to the wilt.

In order to solve the technical problems, the invention further provides a method for identifying or assisting in identifying the resistance of the cotton to be detected to the blight.

The method for identifying or assisting in identifying the resistance of the cotton to be detected to the blight comprises the following steps: detecting the polymorphism or genotype of the SNP marker in a genome of cotton to be detected, and determining the resistance of the cotton to the blight according to the polymorphism or genotype;

the resistance of the cotton to be detected with the genotype of the SNP marker AA to the blight is larger than that of the cotton to be detected with the genotype of the SNP marker CC;

wherein, the CC genotype is the homozygote of the SNP marker C in the cotton genome, and the AA genotype is the homozygote of the SNP marker A in the cotton genome.

In order to solve the technical problems, the invention further provides a cotton breeding method.

The breeding method of cotton provided by the invention comprises the following steps: detecting the polymorphism or genotype of the SNP marker in the genome of the cotton to be detected, and selecting the cotton with the polymorphism A or the genotype AA of the SNP marker in the genome of the cotton to be detected for breeding.

In the breeding method, the method for detecting the polymorphism or genotype of the SNP marker in the cotton genome to be detected is A) or B) as follows:

A) detecting the polymorphism or genotype of the SNP marker in the cotton genome by sequencing;

B) and carrying out PCR on the cotton genome DNA to be detected by using the substance for detecting the polymorphism or the genotype of the SNP marker to obtain a product, and carrying out genotyping on the product.

Hereinbefore, the cotton is upland cotton.

The SNP marker provided by the invention can be used for early prediction of cotton wilt disease resistance, can also be used for cotton genetic background analysis and screening, and cotton wilt resistance molecular marker-assisted selective breeding, and has wide application prospect.

Drawings

FIG. 1 shows the location of the gene for regulating and controlling the resistance to cotton wilt and the determination of SNP marker sites on land; wherein a is that the SNP marker is positioned on a D03 chromosome by combining the blight phenotype of 419 upland cotton with whole genome association analysis; b is the positioning of the SNP marker on the D03 chromosome within the range of 2250kb to 2270 kb; c is the positioning of the SNP marker at the 2259240 position on the D03 chromosome; d is SNP marker located at 448 th position of GhGLR3.3, and has two nucleotide types of A and C; e is the disease index of the plant after inoculation of blight bacteria when SNP markers are respectively C and A; f is the expression quantity of the gene GhGLR3.3 of the SNP marker in the root after inoculation of fusarium oxysporum when the SNP marker is C and A respectively; g is the expression quantity of GhGLR3.3 gene of disease-resistant cotton variety in different tissues.

FIG. 2 shows the result of VIGS silencing of the GhGLR3.3 gene; wherein a is the disease-resistant phenotype identification results of the VIGS cotton of the treatment group and the empty carrier cotton of the control group; b is the disease index comparison result of the VIGS cotton in the treatment group and the empty carrier cotton in the control group; c is GhGLR3.3 gene expression quantity of the cotton plants of the control group and the treated group; d is the sensitivity test result of the VIGS cotton of the treatment group and the empty carrier cotton cut section of the control group to the infection of verticillium dahliae.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 Regulation and control of mapping of resistance genes to cotton wilt in upland and determination of SNP sites

In the embodiment, 419 parts of upland cotton varieties are inoculated with fusarium wilt bacteria at the seedling stage by collecting and screening 419 parts of upland cotton planting resources, re-sequencing individuals and carrying out SNP analysis, and the phenotype of the fusarium wilt disease at the seedling stage is identified. According to the identified phenotype result, the key SNP locus and the candidate gene thereof are analyzed by combining with the whole genome association, and finally the SNP locus is positioned at the 2259240 th site of chromosome D03 of the cotton genome and the 448 th site of the GhGLR3.3 gene, namely the 448 th nucleotide of SEQ ID NO.1, which is A or C. In SEQ ID NO.1, m is a or c. The specific test steps are as follows:

1. location of cotton wilt resistance gene on land and acquisition of SNP locus

419 different upland cotton varieties are listed, when the upland cotton varieties grow to 3-5 true leaves, all the plants are subjected to root injury treatment and are inoculated with blight bacteria (Li Yunping, Von self-strength, Weifeng, Zhongjinong, Von hongjie, Yuan, Li Shixiang, Shi Yong Qiang, Zhao hong, Guo Qingyuan, Zhu He Qin. the prevention and treatment effect of the new and strong plant growth promoting liquid on cotton wilt disease, plant protection, 2017, 43(6) 187 one 191.), the disease indexes are counted after two weeks, the disease indexes are [ (the number of disease plants at each level multiplied by the corresponding disease level)/(the number of total plants multiplied by the highest disease level) ] × 100, seedlings are divided into 5 levels (0 level, 1 level, 2 level, 3 level and 4 level) according to the symptoms expressed on cotyledon and true leaves, and the grading results are as follows:

based on the statistical results of 419 upland cotton disease indices, a Genome Wide Association analysis (GWAS) was performed with 1,568,133 high quality SNPs using EMMAX (Kang h.m., Sul j.h., Service s.k., Zaitlen n.a., Kong s., Freimer n.b., Sabatti c., eshin e.variety Component Model to Account for Sample Structure in Genome-Wide Association studies, nature Genetics,2010,42(4):348.) to identify disease resistance-related genetically regulated loci. The DI ranged from 0 to 100, indicating that phenotypic differences between different germplasms of the population are suitable for GWAS analysis. A SNP site on chromosome D03 (shown in a in FIG. 1) having a strong association at positions 2250kb to 2270kb on chromosome D03 (shown in b in FIG. 1) was identified by a value of-Log (p) of greater than 10. The site is located at D03-2259240bp (shown as c in FIG. 1) by further alignment analysis, and the gene is named GhGLR3.3. Further alignment analysis located the SNP site at 448bp of the GhGLR3.3 gene, with two nucleotide types A and C (shown in d in FIG. 1). Statistical analysis was performed in combination with the inoculated phenotype, and as a result, it was found that the 448 th nucleotide was C and showed infection (i.e., genotype was CC and indicated by "hap.A") and the 448 th nucleotide was A and showed disease resistance (i.e., genotype was AA and indicated by "hap.B") as shown in e of FIG. 1. After two genotypes of cotton varieties are inoculated with fusarium wilt bacteria, leaves of cotton are taken at 0h, 6h, 12h, 24h and 48h respectively to extract DNA, the expression level of the GhGLR3.3 gene is quantitatively analyzed, and the expression level of the genotype B shows a remarkable rising trend at 24h (shown as f in figure 1). Then, the tissue expression level of the disease-resistant cotton variety is quantitatively analyzed, tissues of roots, stems, leaves, petals, receptacle, sepals, bracts, anthers, filaments, pistils, ovules and fibers are respectively selected to extract DNA, and a quantitative result is shown in a heat map mode as g in figure 1.

According to the statistical result, randomly selecting 5 varieties with high and low disease indexes, extracting RNA of the varieties, carrying out reverse transcription on the varieties to obtain cDNA, carrying out PCR amplification by using a primer F and a primer R by using the cDNA as a template to obtain PCR products, and then carrying out sequencing on the PCR products to obtain the following results according to the sequencing result: if the PCR product can be matched with the nucleotide sequence shown by SEQ ID NO.1 and the nucleotide corresponding to the 448 th site of the SEQ ID NO.1 is C (namely the genotype is CC), the disease index is high, and the variety shows susceptibility; if the PCR product can be matched with the nucleotide sequence shown by SEQ ID NO.1 and the nucleotide corresponding to the 448 th site shown by SEQ ID NO.1 is A (namely, the genotype is AA), the disease index is low, and the variety shows disease resistance. And verifying the result according to the comparison result of the individual re-sequencing to prove that the result is correct.

Wherein the primer sequences are as follows:

and (3) primer F: 5'-TGTAACACAGCCTCAGATGC-3' (SEQ ID NO.2)

And (3) primer R: 5'-AAGTCCTGCGAACAAGTGC-3' (SEQ ID NO.3)

Example 2 verification of resistance to blight and Gene of SNP site

Selecting genes near the SNP locus, carrying out functional verification through a VIGS silent gene, silencing the gene of a plant resisting fusarium wilt, inoculating fusarium wilt, and finding that the plant shows disease susceptibility only when the GhGLR3.3 gene is not expressed. Therefore, the GhGLR3.3 gene is verified to be related to the resistance to the wilt disease. The specific test steps are as follows:

1) construction of VIGS vector

The method takes the genome DNA of the cotton No.2 (Zhang Hua Chong, Zhang Wen mu Wu, Jianguiliang, Qiyang, Si Ning. the main gene + multigene genetic characteristic analysis of the greencotton No.2 anti-greensickness in Zhonghua cotton school report, 2016, 28 (5): 513-GGAATTCTGTAACACAGCCTCAGATGC-3') and VIGS-GLR3.3-832R (5-GGGATCCAAGTCCTGCGAACAAGTGC-3') as primers, and performing PCR amplification to obtain amplification product. After the amplification product is recovered, carrying out enzyme digestion on the product by using EcoRI enzyme and BamHI enzyme respectively, and purifying to obtain a target fragment; simultaneously carrying out double enzyme digestion on pYL-156 vectors (Pang J, Zhu Y, Li Q, Liu J, Tian Y, Liu Y, Wu J: Development of Agrobacterium-mediated gene cloning and performance evaluation of four marker genes in Gossypium barbase. PLoS One 2013,8(9): e73211.) by using the two enzymes, and obtaining a vector fragment after purification; connecting the target fragment with the vector fragment to obtain a connection product; transforming the ligation product to be competent for escherichia coli, extracting plasmids after screening, and transferring positive plasmids into agrobacteriumGV3101, a bacterial solution of the target vector is obtained.

2) Acquisition of VIGS Cotton and identification of disease resistance

Mixing the bacterial liquid of the target vector, the bacterial liquid of pYL-156 empty vector with auxiliary vector pTRV-RNA1(Pang J, Zhu Y, Li Q, Liu J, Tian Y, Liu Y, Wu J: Development of Agrobacterium-mediated gene growing and treatment evaluation of source marker genes in Gossypium bardense PLoS One 2013,8(9): e 73211) respectively, standing for 3h in pairs with equal volume to obtain treated bacterial liquid and control bacterial liquid, injecting cotton cotyledon respectively, culturing in a greenhouse for about 20 days to obtain treated cotton plants and control cotton plants, extracting leaf RNA, measuring expression of GhGLR3.3, using ABI7900 real-time system PCR (Taq PCR) and quantitative PCR reagent for experiment system (Taq kit), and performing quantitative PCR (Taq kit PCR) with PCR (Taq kit) instrument, quantitative primers (sequence shown in table 1) were designed by using His3 in upland cotton as internal reference gene and using primer premier3.0 software, and were synthesized by beijing jinzhi corporation, and each material was subjected to three technical repetitions.

TABLE 1 quantitative primers

The cDNA was diluted 10-fold and then used in a 20uL reaction system as shown in Table 2:

TABLE 2 qRT-PCR reaction System

PCR amplification was performed in three steps, and the reaction procedure is shown in Table 3:

TABLE 3 qRT-PCR reaction procedure

After the reaction is complete, the dissolution curve of each reaction is examined and the Cycle Threshold (CT) is used^2-△△CTThe method is used for calculating the expression level of the gene GhGLR3.3. As a result, as shown in c in fig. 2, it was found that the expression level of ghglrr 3.3 was decreased in the cotton plants of the treated group as compared with the cotton plants of the control group, and VIGS cotton of the treated group whose expression level was decreased and empty vector cotton of the control group whose expression level was normally expressed were obtained. Respectively carrying out root injury inoculation on the VIGS cotton and the empty carrier cotton, carrying out blight germ inoculation for 14 days, and carrying out disease-resistant phenotype identification on the VIGS cotton and the empty carrier cotton, wherein the result is shown as a in figure 2, the VIGS cotton of the treatment group shows disease susceptibility, and the empty carrier cotton of the control group shows disease resistance. The disease index was further determined (see example 1 for details), and the result is shown in b in fig. 2, wherein the disease index of VIGS cotton in the treated group is significantly increased compared to the disease index of the empty carrier cotton in the control group. After 20 days of inoculation, 4cm long stems from the first node were excised from the VIGS cotton of the treated group and the empty vector cotton of the control group, respectively, and the harvested material was sterilized by immersion in 70% ethanol for 20-30 seconds, rinsed 3 times with sterile water, sterilized in 0.1% sodium hypochlorite for 1 minute, rinsed 5 times with sterile water and cut into approximately identical seven segments. Then placing the stem slices cut into seven sections in each plant into the previously prepared PDA culture medium, sealing and then incubating in a constant temperature incubator at 25 ℃, judging the susceptibility of cotton to verticillium dahliae infection according to the number of the stem sections grown by fungi in the cut sections, and as a result, the susceptibility of the VIGS cotton cut sections of the treatment group to verticillium dahliae infection is obviously higher than that of the empty carrier cotton cut sections of the control group as shown by d in figure 2. The results show that the disease resistance of VIGS cotton becomes poor after inoculation, which indicates that the GhGLR3.3 gene is related to the resistance of cotton wilt, and the SNP locus is positioned in the GhGLR3.3 gene.

Example 3 validation of SNP sites associated with resistance to Cotton wilt on land

Culturing 30 cotton varieties in the table 4, culturing 30 cotton varieties, and preparing spore suspension of blight pathogenic bacteria (Li Yunfu, Von Zingiber officinale, Weifeng, Zhongjinglong, Von hongjie, Yuan, Li Shixiang, Shiwarong, Zhaohihong, Guqingyuan, Zhu Heqin, the prevention and treatment effect of new plant growth promoting liquid on cotton blight, plant protection, 2017, 43 (6): 187) when the cotton grows to the first heart stage, respectively injecting 30ml of pathogenic bacteria spore liquid into each cotton plant, and putting into an incubator for continuous culture. The disease index is counted after about two weeks (see example 1 for details). The genomic DNA of 30 different cotton varieties in Table 4 was used as a template, and the primers F and R in example 1 were used as primers to perform PCR amplification and sequencing, and the sequencing result was aligned with the 448 th base of the gene GhGLR3.3.

The disease index and SNP typing results are shown in tables 4 and 5, and when the nucleotide at 448 th position of the gene GhGLR3.3 is A (namely the genotype of the SNP marker is AA), the disease index is lower, namely the disease resistance to cotton fusarium wilt is stronger, and when the nucleotide at 448 th position of the gene GhGLR3.3 is C (namely the genotype of the SNP marker is CC), the disease index is higher, namely the disease resistance to cotton fusarium wilt is stronger, and the disease resistance to cotton fusarium wilt is weaker.

As shown in Table 6, the analysis of variance revealed that there was a significant difference between the disease index of the breed whose SNP marker genotype was AA and the disease index of the breed whose SNP marker genotype was CC.

Table 430 phenotypic identification and SNP typing results of different cotton varieties inoculated with fusarium oxysporum

Note: the biological materials in Table 4 are available to the public from the applicant and are only useful for repeating the relevant experiments of the present invention and are not useful for other purposes.

TABLE 5 differences in resistance of different genotype varieties to blight of Cotton

Genotype(s)	Number of varieties	Index of disease condition
			AA
	15	5.05
			CC	15	55.61

TABLE 6 analysis of variance results

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 embodiments, it will be appreciated that the invention can 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.

SEQUENCE LISTING

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Claims

The application of the SNP marker in identifying or assisting in identifying the resistance of cotton to be detected to the blight; the SNP marker is positioned at the 448 th site of a cotton gene GhGLR3.3, namely the 448 th site nucleotide of SEQ ID NO.1, and the nucleotide type is A or C; the cotton is upland cotton.
2, application of SNP markers in cotton breeding; the cotton breeding is to cultivate cotton with resistance to blight; the SNP marker is positioned at the 448 th site of a cotton gene GhGLR3.3, namely the 448 th site nucleotide of SEQ ID NO.1, and the nucleotide type is A or C; the cotton is upland cotton.
3. A method for identifying or assisting in identifying resistance to blight in cotton to be tested, comprising: detecting polymorphism or genotype of the SNP marker in claim 1 or 2 in a genome of cotton to be detected, and determining the resistance of the cotton to be detected to the blight according to the polymorphism or genotype; the cotton is upland cotton.
4. A method of breeding cotton, comprising: detecting the polymorphism or genotype of the SNP marker in claim 1 or 2 in a cotton genome to be detected, and selecting cotton with the polymorphism A or the genotype AA of the SNP marker in the cotton genome to be detected for breeding; the cotton is upland cotton.