CN113604600B - SNP molecular marker for cadmium-resistant character of paspalum vaginatum and application thereof - Google Patents
SNP molecular marker for cadmium-resistant character of paspalum vaginatum and application thereof Download PDFInfo
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Abstract
The invention discloses a Paspalum vaginatum cadmium-resistant SNP molecular marker and application thereof, wherein the nucleotide sequence of the molecular marker is shown as a sequence table SEQ ID NO.1, and SNP loci are as follows: the A/G mutation exists at the 401bp position of the sequence shown in SEQ ID NO. 1. According to the invention, through re-sequencing data of 172 parts of paspalum vaginatum germplasm and by combining phenotype data of the paspalum vaginatum related traits after cadmium stress, whole genome correlation analysis is carried out, molecular markers related to cadmium-resistant traits are positioned and screened, and researches find that the SNP molecular marker Pv.chr04: p41351449 positioned at the 41351449bp position of the No. 04 chromosome has high contribution rate to the cadmium-resistant traits of paspalum vaginatum, plays a key role in the regulation and control of the cadmium-resistant traits of paspalum vaginatum, can be used for site cloning and molecular marker assisted selection, so that a paspalum vaginatum variety with strong cadmium-resistant traits is effectively screened, the breeding process of paspalum vaginatum is accelerated, and the invention is suitable for large-scale popularization and application.
Description
Technical Field
The invention belongs to the technical field of genetic breeding and molecular biology, and particularly relates to a SNP molecular marker for the cadmium-resistant character of paspalum vaginatum and application thereof.
Background
Paspalum vaginatum (Paspalum vaginatum Sw.) also called Paspalum seashore or Hawaii flos is perennial herb of Paspalum paniculatum of Panicum of Gramineae of Monocotyledoneae of angiosperma, is mainly distributed in tropical and subtropical seaside areas, has stolons and rhizomes with developed growth vigor, and has dark green color and uniform and dense blade shape, and is an excellent grass species for greenland lawns and sports ground lawns. The Paspalum vaginatum has certain cadmium resistance, and the root system can enrich a large amount of cadmium and limit the migration of the cadmium to the overground part. . With the development of breeding technology and the intensive research on paspalum vaginatum, paspalum vaginatum is showing more and more extensive application prospect.
Cadmium (Cadmium, cd) is a silvery white, glossy, rare heavy metal, has good toughness and ductility, is a family element in the periodic table with zinc (Zn) and mercury (Hg), and has chemical properties similar to zinc. With the development of industry, cadmium is widely used in the fields of nickel-cadmium batteries, cadmium electroplating, pigment and paint production, atomic reactors and the like, but a large amount of cadmium is also discharged to the environment in the processes of industrial production and waste treatment, so that the problem of soil pollution is increasingly serious. According to the national soil pollution condition survey bulletin, the exceeding rate of cadmium pollution of soil in China is 7.0%, and the top of the localized inorganic pollutants is the most serious soil heavy metal pollutant and is listed as an element for priority control. After the soil is polluted by cadmium, the cadmium-polluted soil is harmful to the yield and quality of crops, is easy to enrich the soil to a human body through a food chain, reduces the beneficial metal efficiency of the human body, damages the liver, causes osteoporosis and cancer pathological changes, and is seriously harmful to the health of the human body, so the cadmium-polluted soil is treated and repaired to become an important environmental problem.
The soil heavy metal pollution remediation technology mainly comprises physical remediation, chemical remediation and biological remediation, and the plant remediation technology in the biological remediation mainly reduces the content of pollutants in soil by utilizing the absorption, accumulation, decomposition, volatilization or fixation of plants on heavy metals, so that the soil heavy metal pollution is reduced. The key for improving the technical efficiency is to screen plant varieties with super-enriched heavy metals.
Genome-wide Association Analysis (GWAS) generally takes a natural population as a research object and Linkage Disequilibrium (LD) as a research basis to search genetic markers or candidate genes associated with a target trait in the population, and GWAS is an effective means for analyzing genetic variation of complex traits at present. GWAS was first proposed by Risch in 1996, after Klein studied age-related macular degeneration with GWAS in 2005, GWAS began to be widely used in the study of human disease. With the development of high throughput sequencing technology, GWAS is widely used in the study of plant traits. Single Nucleotide Polymorphism (SNP) mainly refers to a polymorphism in a DNA sequence caused by a Single Nucleotide base change at the genome level.
Disclosure of Invention
The invention aims to provide an SNP molecular marker of a paspalum vaginatum cadmium-resistant character and application thereof, the invention carries out whole genome correlation analysis by combining 172 parts of heavy sequencing data of paspalum vaginatum germplasm and phenotype data of the paspalum vaginatum related character after cadmium stress, locates and screens out the molecular marker related to the cadmium-resistant character, researches find that the SNP molecular marker Pv.chr04: p41351449 at the 41351449bp position of No. 04 chromosome has high contribution rate to the cadmium-resistant character of the paspalum vaginatum, plays a key role in the regulation and control of the cadmium-resistant character of the paspalum vaginatum, can be used as site-directed cloning and molecular marker-assisted selection, thereby effectively screening and obtaining a paspalum vaginatum variety with strong cadmium-resistant character, accelerating the breeding process of the paspalum vaginatum, and is suitable for large-scale popularization and application.
One of the purposes of the invention is to provide a paspalum cadmium-resistant SNP molecular marker, the nucleotide sequence of the molecular marker is shown in a sequence table SEQ ID NO.1, wherein SNP loci are as follows: the A/G mutation exists at the 401bp position of the sequence shown in SEQ ID NO. 1.
Further, the SNP locus is located at the 41351449bp position of the No. 04 chromosome of Paspalum vaginatum, and the molecular marker is named as Pv.
Further, the method for obtaining the SNP molecular marker comprises the following steps:
a) Collecting 172 parts of paspalum vaginatum which has genetic difference at home and abroad to form an associated population;
b) Extracting total DNA of leaves of each material of the associated population by adopting a CTAB method, and then performing resequencing on the DNA of the population to identify SNP genotype information of the population;
c) Screening a high-quality group SNP data set by carrying out quality filtering on the SNP data;
d) Carrying out cadmium-resistant experimental treatment on 172 parts of materials, and investigating cadmium-resistant phenotypes of different materials;
f) And (3) carrying out whole genome association analysis by combining genotype and cadmium-resistant character phenotype data, and identifying QTL sites which are obviously related to the cadmium-resistant character to obtain the SNP molecular marker related to the cadmium-resistant character of the paspalum vaginatum.
Another object of the present invention is to provide a primer for amplifying the SNP molecular marker.
Further, the nucleotide sequence of the primer is shown in a sequence table SEQ ID NO. 2-3.
The invention also aims to provide the application of the SNP molecular marker or the primer in detecting or identifying the cadmium-resistant character of the paspalum vaginatum.
Further, when the base type of the SNP locus of the Paspalum vaginatum to be detected is AA, the Paspalum vaginatum is a high cadmium-resistant variety; and when the base type of the SNP site of the paspalum vaginatum to be detected is AG, the paspalum vaginatum is a weak cadmium-resistant variety. Namely, the cadmium-resistant property of the Paspalum vaginatum with the base type of AA at the SNP locus is better than that of the Paspalum vaginatum with the base type of AG.
The fourth purpose of the invention is to provide a method for identifying the cadmium resistance of the paspalum vaginatum, which comprises the following steps:
step 1, extracting genome DNA of a paspalum vaginatum material to be detected;
step 2, taking the genomic DNA extracted in the step 1 as a template, performing PCR amplification by using the primers, and then sequencing; or directly carrying out re-sequencing by using the genome DNA so as to determine the genotype of the paspalum vaginatum material to be detected;
step 3, when the genotype of the Paspalum vaginatum material to be detected is AA, the Paspalum vaginatum material is a variety with strong cadmium resistance; and when the genotype of the to-be-detected paspalum vaginatum material is AG, the to-be-detected paspalum vaginatum material is a weak cadmium-resistant variety.
Further, the PCR amplification system in step 2 is 10 μ L, which includes: 50 ng/. Mu.L template DNA 1. Mu.L, 2 XPCR Master Mix 5. Mu.L, 10. Mu. Mol/L forward and reverse primers 0.5. Mu.L each, ddH 2 O3μL。
The invention also aims to provide a detection kit for detecting or identifying the strong and weak cadmium-resistant property of the paspalum vaginatum, wherein the detection kit comprises the primer.
The invention also aims to provide the application of the SNP molecular marker or the primer in the molecular marker assisted breeding of the paspalum vaginatum with strong cadmium resistance.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, through re-sequencing data of 172 parts of paspalum vaginatum germplasm and by combining phenotype data of relevant characters of paspalum vaginatum after cadmium stress, whole genome correlation analysis is carried out, QTL sites influencing the cadmium resistance character in paspalum vaginatum are obtained by first positioning, corresponding SNP molecular markers are developed, and researches show that the SNP molecular marker Pv.chr04: p41351449 at the 41351449bp position of No. 04 chromosome has high contribution rate to the cadmium resistance character of paspalum vaginatum, and the contribution rate is 69.00%, namely the SNP molecular marker plays a key role in regulating and controlling the cadmium resistance character of paspalum vaginatum. The primer designed based on the SNP molecular marker can be used for mapping site cloning and molecular marker assisted selection, so that the paspalum vaginatum variety with strong cadmium resistance is effectively screened and obtained, the detection is convenient and rapid, the method is not influenced by the environment, the breeding efficiency is high, the breeding process of the paspalum vaginatum is accelerated, the defects that the cadmium resistance determination in the traditional breeding method needs specific experimental treatment, the required time is long, the operation is complex, more uncertain factors are caused and the like are overcome, the method has important significance for the treatment and restoration of cadmium pollution, and is suitable for large-scale popularization and application.
Drawings
FIG. 1 is a graph showing the distribution results of the cadmium-resistant trait of Paspalum vaginatum in example 1 of the present invention;
FIG. 2 is a schematic diagram of the cadmium-resistant phenotype corresponding to different genotypes of the Paspalum vaginatum SNP molecular marker Pv.chr04: p41351449 in example 2 of the invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 major QTL site location for the cadmium-tolerant trait of Paspalum vaginatum
(1) Phenotypic determination of cadmium-resistant character of paspalum vaginatum
The Paspalum vaginatum germplasm used in this example was 172 parts from Hainan (47 parts), guangxi (23 parts), guangdong (9 parts), yunnan (7 parts), USA (65 parts), and other countries (21 parts).
Stolons with consistent size and growth are selected from a resource garden, washed clean and planted in seedling growing cups with holes at the bottoms, and the treatment and the Comparison (CK) of each material are repeated for 4 times. And (3) suspending the seedling culture cup with the stolons on a foam board with holes, and covering the foam board on a plastic basin filled with 2L of Hoagland nutrient solution. Pre-culturing for 1 month before a cadmium stress test, pruning once a week and replacing Hoagland nutrient solution; the water lost due to liquid surface evaporation and plant absorption and transpiration is supplemented by tap water every other day; continuously aerating with oxygenation pump.
Uniformly pruning 2 days before the beginning of cadmium stress, and keeping the height of the stubble on the ground to be about 8cm. The phenotype was investigated after 8 days of cadmium stress at a concentration of 1.0mmol/L cadmium, using no cadmium as a control. During this period, four graders visually estimated the percentage of withered and yellow leaf surface to the total leaf surface, with less than 5% indicating that the material had substantially no yellow leaves, 50% indicating half of the withered and yellow, and more than 95% indicating that there were substantially no green leaves and dead. The table values for the 172 parts materials are averaged and the results are shown in table 1.
TABLE 1 mean values of phenotype of 172 materials
Note: RLF8 represents the mean relative withered and yellow ratio of day eight after stress
And (3) drawing a diagram of the withering and yellowing rate distribution result of the associated population according to the data, wherein the result shows that the withering and yellowing rate property expression distribution is in continuous distribution and is biased to a main peak, and the withering and yellowing rate property belongs to quantitative properties and has main effective gene loci as shown in figure 1.
(2) Weight sequencing of paspalum vaginatum population
Sampling in a germplasm resource garden, wherein about 2g of tender leaves of each germplasm are taken, quick-frozen by using liquid nitrogen and sent to a Meiwei metabolism company for sequencing, and the sequencing strategy is Illumina PE150. The sequencing machine-off data (raw data) are subjected to quality control, and the filtering standards are as follows: (1) filtering the linker-containing sequence; (2) Filtering a pair of sequences with the N content exceeding 10% in the single-ended sequence; (3) A pair of sequences with a low mass (Q.ltoreq.5) base number of more than 50% is filtered. And filtering the adaptor sequence, the inaccurate sequence and the low-quality sequence to obtain clean data, and performing the next sequence comparison. After an index file is constructed for the SeaIsle2000 reference genome, the clean data is compared to the reference genome by using BWA software; sequencing the comparison results by using SAMtools software; processing the repeated markers generated in the library preparation process by using Picard Tools software; SNP is further screened and filtered by using GATK software (analyzing group variation information. Using VCFtools software, the command is 'vcfttools-vcf [ vcf _ file ] - -max-missing 0.8-maf 0.05-mac 3-minQ 30-minDP 3-min-alloys 2-max-alloys 2-record-INFO-all-out [ out _ prefix ]'.
(3) Whole genome association analysis
Format conversion is carried out on the vcf file by using plink software; generating a Balding-Nichols genetic relationship matrix by using EMMAX software, wherein the command is' EMMAX-kin-intel64-v-d 10-o [ out _ prefix ] [ tped _ prefix ]; the command for correlation analysis is "emmax-intel64-v-d 10-t [ tped _ prefix ] -p [ pheno _ file ] -k [ kin _ file ] -o [ out _ prefix ]". And (3) obtaining a P value of each site of the paspalum vaginatum according to the correlation analysis result, when the P value is less than 0.5/4833395=1.034E-07, namely the SNP is a remarkable SNP, the SNP with the minimum P value is a peak SNP, grouping materials of different allele types of the peak SNP in a group, and performing variance analysis, wherein the percentage of the ratio of the variance between groups to the total variance is the contribution rate of the peak SNP.
Through analysis, the interval of the main effect QTL site of the cadmium-resistant property of the paspalum vaginatum is limited between the 40437210 th base and the 41652366 th base of the chr04 chromosome of the paspalum vaginatum, the corresponding SNPs are chr04_40437210 (C/T), chr04_41652366 (T/C) and the peak SNP is chr04_41351449 (A/G), and the contribution rate of the QTL to the cadmium-resistant property of the paspalum vaginatum is 69.00 percent (the materials are grouped according to different allele types of the peak SNP, the one-way anova is carried out, and the percentage of the variance between groups divided by the total variance is the contribution rate).
Example 2 development of cadmium-resistant SNP molecular marker for Paspalum vaginatum
Developing an SNP molecular marker according to the identified cadmium resistance QTL and peak SNP of the paspalum vaginatum, extracting 400bp sequences before and after the SNP as characteristic sequences of the SNP molecular marker Pv.chr04: p41351449 (shown as a sequence table SEQ ID NO.1, wherein the 401bp is an SNP locus, and A/G mutation exists), and designing primers for the SNP molecular marker Pv.chr04: p41351449 as follows:
a forward primer: 5 'GGAGTTGTGATAGGTGCAGG-3' (shown in SEQ ID NO. 2);
reverse primer: 5 'ATTCAAGCCGCACAACATGTT-containing 3' (shown in SEQ ID NO. 3).
Through conventional PCR amplification and sequencing detection, the basic groups of the SNP molecular markers are divided into two types, and when the basic group type of Pv.chr04: p41351449 is AA, the average withering and yellowing rate of the material is 10.74 percent, namely the strong cadmium-resistant property; when the base type is AG, the average withering and yellowing rate of the material is 27.97 percent, namely the weak cadmium resistance property. A schematic representation of the cadmium tolerance phenotype for the different genotypes is shown in FIG. 2. The result shows that the cadmium-resistant phenotype corresponding to different genotype types of the SNP molecular marker Pv.chr04: p41351449 has obvious difference. Therefore, the cadmium-resistant property of the seapaspalum to be detected can be quickly and simply evaluated by identifying the type of the SNP molecular marker so as to quickly identify the cadmium-resistant property strength of the material.
Besides the amplification of the sequence by using the primers, the material can be subjected to genome re-sequencing, the SNP molecular marker Pv.chr04: p41351449 disclosed by the invention is subjected to SNP genotyping according to the method in the example 1, and the genotype type of the SNP is determined, so that the cadmium resistance property strength and weakness of the material can be rapidly evaluated.
In conclusion, the SNP molecular marker of the seapaspalum cadmium-resistant character has high contribution rate to the seapaspalum cadmium-resistant character, plays a key role in regulating and controlling the cadmium-resistant character of the seapaspalum, can predict or screen and identify the cadmium-resistant character of the seapaspalum based on the SNP molecular marker, has simple identification method, high selection efficiency and clear selection target, is not influenced by the environment, can be used for site cloning and molecular marker assisted selection, and is suitable for large-scale popularization and application.
Example 3 application of SNP molecular marker Pv.chr04: p41351449 in identifying cadmium-resistant character of paspalum vaginatum
DNA of young leaves of four materials, namely, seaIsle2000, grin _ UPG1743, grin _ UPG145 and FR-4 which are known as high cadmium resistance materials at the early stage are extracted and sent to a sequencing company for genome re-sequencing, the SNP genotypes of the four materials are respectively identified, and the cadmium resistance character identification is carried out on the four materials according to the method of the embodiment 1.
Meanwhile, four early known materials with weak cadmium resistance, namely 17HN-18, 20190131003, 17HN-69 and 17HN-20, are extracted to be sent to a sequencing company for genome re-sequencing, the SNP genotypes of the four materials are respectively identified, and the cadmium resistance traits of the four materials are identified according to the method in the embodiment 1.
The SNP genotypes and cadmium-tolerant traits of the eight materials at the 41351449bp position of the chr04 chromosome (the average value of the relative withering and yellowing rates of the 8 th day under the stress of 1.0mmol/L cadmium concentration) are shown in Table 2.
TABLE 1 Pv.chr04: p41351449 site genotype and cadmium resistance thereof in different cadmium-resistant property materials
Note: RLF8 represents the mean relative yellowing rate at day eight after stress
The results show that for the known materials with strong cadmium resistance, seaIsle2000, FR-4, grin _ UPG1743 and grin _ UPG14, the SNP genotypes at the 41351449bp position (i.e. at the 401bp position of the sequence shown in SEQ ID NO. 1) of the chr04 chromosome are AA, and the phenotypes of the cadmium resistance traits are 5.00%,6.25%,7.50% and 5.00% respectively, which are consistent with the AA genotypes of the SNP molecular marker in the example 2 corresponding to the strong cadmium resistance traits. Similarly, for the known materials with weak cadmium resistance, 17HN-18, 20190131003, 17HN-69 and 17HN-20, the SNP genotypes at the 41351449bp position (i.e., at the 401bp position of the sequence shown in SEQ ID NO. 1) of the chr04 chromosome were all AG, and the phenotypes of the cadmium resistance traits were 38.50%,33.75%,35.00% and 28.75%, respectively, which were consistent with the weak cadmium resistance trait corresponding to the AG genotype of the SNP molecular marker in example 2. The results show that the cadmium resistance of different paspalum vaginatum materials can be effectively identified according to the genotype type of the SNP molecular marker Pv.chr04: p41351449, so that the materials with the high cadmium resistance of the paspalum vaginatum can be screened and specifically retained.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Sequence listing
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<120> SNP molecular marker of cadmium-resistant character of paspalum vaginatum and application thereof
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Claims (10)
1. The SNP molecular marker of the cadimium vulgare cadmium-resistant character is characterized in that the nucleotide sequence of the molecular marker is shown in a sequence table SEQ ID NO.1, wherein SNP loci are as follows: the A/G mutation exists at the 401bp position of the sequence shown in SEQ ID NO. 1.
2. The SNP molecular marker for the cadmium-resistant trait of paspalum vaginatum as claimed in claim 1, wherein the SNP locus is located at the 41351449bp position of chromosome 04 of paspalum vaginatum, and the molecular marker is named as Pv.chr04: p41351449.
3. The SNP molecular marker for the cadmium-resistant trait of paspalum vaginatum according to claim 1, wherein the method for obtaining the SNP molecular marker comprises the following steps:
a) Collecting 172 parts of paspalum vaginatum from home and abroad to form an associated population;
b) Extracting total DNA of leaves of each material of the associated population by adopting a CTAB method, and then performing resequencing on the DNA of the population to identify SNP genotype information of the population;
c) Screening a high-quality group SNP data set by filtering the SNP data quality;
d) Carrying out cadmium-resistant experimental treatment on 172 parts of materials, and investigating cadmium-resistant phenotypes of different materials;
f) And (3) carrying out whole genome association analysis by combining genotype and cadmium-resistant character phenotype data, identifying QTL sites obviously related to the cadmium-resistant character, and obtaining the SNP molecular marker related to the cadmium-resistant character of paspalum vaginatum as claimed in claim 1.
4. A primer for amplifying the SNP molecular marker as set forth in claim 1.
5. The primer according to claim 4, wherein the nucleotide sequence of the primer is shown in SEQ ID NO.2-3 of the sequence table.
6. The application of the SNP molecular marker of claim 1 in detecting or identifying the cadmium-resistant character of paspalum vaginatum.
7. The use as claimed in claim 6, wherein when the base type of the SNP site of paspalum vaginatum to be tested is AA, the paspalum vaginatum is a strong cadmium-resistant variety; and when the base type of the SNP site of the paspalum vaginatum to be detected is AG, the paspalum vaginatum is a weak cadmium-resistant variety.
8. The method for identifying the cadmium resistance of the paspalum vaginatum is characterized by comprising the following steps of:
step 1, extracting genome DNA of a paspalum vaginatum material to be detected;
step 2, taking the genomic DNA extracted in the step 1 as a template, performing PCR amplification by using the primer as claimed in claim 4 or 5, then sequencing, or directly performing re-sequencing by using the genomic DNA to obtain a target fragment containing the sequence shown in SEQ ID NO.1, and determining the base type of the paspalum vaginatum material to be detected at the 401bp position of the sequence shown in SEQ ID NO. 1;
step 3, when the genotype of the Paspalum vaginatum material to be detected is AA, the Paspalum vaginatum material is a variety with strong cadmium resistance; and when the genotype of the to-be-detected paspalum vaginatum material is AG, the to-be-detected paspalum vaginatum material is a weak cadmium-resistant variety.
9. A detection kit for detecting or identifying the cadmium resistance of paspalum vaginatum, which is characterized in that the detection kit comprises the primer as claimed in claim 4 or 5.
10. The application of the SNP molecular marker of claim 1 in molecular marker assisted breeding of the strong cadmium-resistant character of paspalum vaginatum.
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