CN113801952A - SNP molecular marker for enriching cadmium content character of paspalum vaginatum root system and application thereof - Google Patents
SNP molecular marker for enriching cadmium content character of paspalum vaginatum root system and application thereof Download PDFInfo
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Abstract
The invention discloses an SNP molecular marker for enriching the character of cadmium content in a paspalum vaginatum root system and application thereof, wherein a nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and SNP loci are as follows: the G/A mutation is present at the 401bp position of the sequence shown in SEQ ID NO. 1. According to the invention, 86 parts of paspalum vaginatum germplasm is subjected to re-sequencing, the phenotype data of enriched cadmium content of the paspalum vaginatum root system after cadmium stress treatment is combined to perform whole genome correlation analysis, and molecular markers related to the character of enriched cadmium content of the paspalum vaginatum root system are positioned and screened out, researches show that the contribution rate of the SNP molecular marker Pv.chr02: p30702908 at the 30702908bp position of the No. 02 chromosome on the character of enriched cadmium content of the paspalum vaginatum root system is high, and the method can be used for auxiliary mapping by using locus cloning and molecular markers, quickly predicting or identifying the height of the enriched cadmium content of the root system, effectively screening the paspalum vaginatum variety capable of efficiently enriching, not only accelerates the breeding process of paspalum vaginatum, but also has important significance on prevention and control of soil cadmium pollution, and 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 an SNP molecular marker for enriching a cadmium content character of a paspalum vaginatum root system and application thereof.
Background
Cadmium (Cadmium, Cd) is a silvery white and glossy rare heavy metal, and with industrial development, Cadmium is widely applied to 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 industrial production and waste treatment processes, so that the problem of soil pollution is increasingly serious. According to research, the exceeding rate of cadmium pollution of soil in China reaches 7.0%, and the lead of the resident 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 pollution is not only harmful to the yield and quality of crops, but also easily enriches the cadmium pollution to human bodies through food chains, so that the beneficial metal efficiency of the human bodies is reduced, the liver is damaged, osteoporosis, cancer pathological changes and serious harm to the health of the human bodies are caused, and the cadmium pollution treatment and restoration become important environmental problems.
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. How to screen plant varieties with super-enriched heavy metals is the key for improving the technical efficiency.
Paspalum vaginatum Sw, also called Paspalum vaginatum or Hawaii, is an perennial grass plant of Paspalum in Panicum of Gramineae of Monocotyledoneae of angiosperma, is mainly distributed in tropical and subtropical seashore areas, has creeping stems and rhizomes with developed growth, and has a leaf line shape or a needle shape, and the formed lawn is uniform and compact, has dark green color and is an excellent grass seed of greenland lawns and sports field lawns. Paspalum vaginatum has certain cadmium resistance, and the root system can be enriched with cadmium and limit the migration of cadmium to the overground part. Therefore, the screened paspalum vaginatum which can effectively enrich cadmium has important significance for soil heavy metal pollution remediation and cadmium pollution protection.
Genome-wide Association Study (GWAS) usually takes natural populations as research objects, and takes Linkage Disequilibrium (LD) as a research basis to search genetic markers or candidate genes associated with target traits in the populations, and GWAS is currently an effective means for analyzing genetic variation of complex traits. 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 DNA sequence polymorphism caused by Single Nucleotide base change on genome level.
Disclosure of Invention
The invention aims to provide an SNP molecular marker for enriching the cadmium content character of a paspalum vaginatum root system and application thereof, the invention carries out re-sequencing on 86 parts of paspalum vaginatum germplasm, carries out whole genome correlation analysis by combining with phenotype data of the enriched cadmium content of the paspalum vaginatum root system after cadmium stress treatment, locates and screens out molecular markers related to the character of the enriched cadmium content of the root system thereof, researches find that the SNP molecular marker Pv.chr02: p30702908 positioned at the 30702908bp position of the No. 02 chromosome has high contribution rate to the character of the enriched cadmium content of the paspalum vaginatum root system, plays a key role in regulating and controlling the character of the enriched cadmium content of the paspalum vaginatum root system, can be used for site-specific cloning and molecular marker-assisted selection, thereby being capable of rapidly predicting or identifying the height of the enriched cadmium content of the root system thereof in the whole growth period of the paspalum vaginatum, effectively screens and obtains the high-efficiency enriched paspalum vaginatum breed variety of the paspalum vaginatum, not only accelerates the breeding process, and has important significance for preventing and treating the heavy metal pollution of the soil, and is suitable for large-scale popularization and application.
One of the purposes of the invention is to provide an SNP molecular marker for enriching the character of cadmium content in a paspalum vaginatum root system, wherein the molecular marker is Pv.chr02: p30702908, the nucleotide sequence of the SNP molecular marker is shown as SEQ ID NO.1, and SNP loci are as follows: the G/A mutation is present at the 401bp position of the sequence shown in SEQ ID NO. 1.
Furthermore, the contribution rate of the molecular marker to the character of enriching the cadmium content in the root system is 89.20%.
Further, the method for obtaining the SNP molecular marker comprises the following steps:
a) collecting 86 parts of paspalum vaginatum from home and abroad with hereditary difference to form a related group;
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 stress experiment treatment on 86 parts of materials, and investigating the phenotype of cadmium content enrichment of root systems of different materials;
e) and (3) carrying out whole genome association analysis by combining genotype and phenotypic data of the root system cadmium content enrichment character, and identifying QTL (quantitative trait loci) sites which are obviously related to the root system cadmium content enrichment character to obtain the SNP molecular marker related to the root system cadmium content enrichment 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 predicting or identifying the character of the enriched cadmium content of the paspalum vaginatum root system.
Further, when the base type of the SNP locus of the paspalum vaginatum to be detected is GG, the paspalum vaginatum is a material with a root system rich in high cadmium content; when the base type of the SNP locus of the paspalum vaginatum to be detected is GA, the paspalum vaginatum is a material with a root system enriched with medium cadmium content; and when the base type of the SNP locus of the paspalum vaginatum to be detected is AA, the paspalum vaginatum is a material with a root system rich in cadmium and low in content.
The fourth purpose of the invention is to provide a method for predicting the high and low content of enriched cadmium in the root system of 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, and performing PCR amplification and sequencing by adopting the primers; 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 seapaspalum to be detected is GG, enriching a material with high cadmium content for a root system; when the genotype of the paspalum vaginatum to be detected is GA, the paspalum vaginatum is a material with a root system enriched with medium cadmium content; and when the genotype of the to-be-detected paspalum vaginatum is AA, the material with the root system enriched with low cadmium content is obtained.
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, ddH2O 3μL。
The invention also aims to provide a detection kit for predicting or identifying the high content and the low content of the enriched cadmium in the paspalum vaginatum root system, wherein the detection kit comprises the primer.
The sixth purpose of the invention is to provide the application of the SNP molecular marker or the primer in the molecular marker assisted breeding of the phoenix dactylicaba root system for efficiently enriching the cadmium property.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, 86 parts of paspalum vaginatum germplasm is subjected to re-sequencing, and the whole genome correlation analysis is carried out by combining with the phenotypic data of cadmium content enriched in the root system of paspalum vaginatum after cadmium stress, QTL loci related to the high and low cadmium content enriched in the root system of paspalum vaginatum are obtained by first positioning, and corresponding SNP molecular markers are developed, wherein the contribution rate of the SNP molecular marker Pv.chr02: p30702908 positioned at the 30702908bp position of the No. 02 chromosome to the cadmium content enriched in the root system of paspalum vaginatum is the highest and is up to 89.20%, and the result shows that the SNP molecular marker plays a key role in regulating and controlling the content of cadmium which can be enriched in the root system of paspalum vaginatum. The primer designed based on the SNP molecular marker can be used for site cloning and molecular marker assisted selection, and can rapidly identify the content of the enriched cadmium in the root system of the paspalum vaginatum in the whole growth period, so that the paspalum vaginatum variety capable of efficiently enriching the cadmium is effectively screened, the breeding process of the paspalum vaginatum is accelerated, the soil cadmium pollution is efficiently repaired, the soil heavy metal pollution is reduced, the breeding efficiency is high, the environment influence is avoided, the defects of complex operation, time consumption and many uncertain factors in the root system cadmium content determination in the traditional breeding method are effectively overcome, the method has important significance for cadmium pollution prevention and treatment, and is suitable for large-scale popularization and application.
Drawings
FIG. 1 is a schematic diagram showing the distribution results of the cadmium content-enriched character of the root system of Paspalum vaginatum in example 1 of the present invention;
FIG. 2 is a schematic diagram of the phenotype of the cadmium-rich trait of the root system corresponding to different genotypes of the Paspalum vaginatum root system SNP molecular marker Pv.chr02: p30702908 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 enrichment of cadmium content traits in Carex baccans root systems
(1) Phenotypic determination of enriched cadmium content character of paspalum vaginatum root system
The total of 86 parts of Paspalum vaginatum germplasm used in this example. 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 overground stubbles about 8 cm. And (3) increasing the cadmium concentration to 1.5mmol/L to stress for 13 days after the cadmium concentration is stressed for 8 days without adding cadmium as a control, and investigating the phenotype of the root system with high and low enriched cadmium content.
Dividing the paspalum vaginatum into stem leaves and root parts, collecting samples, washing the samples clean by deionized water and removing surface moisture. Deactivating enzyme at 105 deg.C for 30min, and oven drying at 65 deg.C to constant weight. After grinding, 0.20g of the powder is weighed and placed in a polytetrafluoroethylene microwave digestion tank, 4mL of concentrated nitric acid and 2mL of deionized water are added, the tank is covered and sealed, and digestion treatment is carried out according to a preset program. And transferring the digested solution to a centrifuge tube, fixing the volume to 25mL by using deionized water, shaking up, measuring the reading value by using an atomic absorption spectrometer, and calculating the cadmium content of the root system.
And drawing a schematic diagram of the distribution result of the cadmium content-enriched character of the root system of the associated population according to the data, wherein the result is shown in fig. 1, and the result shows that the character of the cadmium content-enriched root system is continuously distributed and is biased to one main peak, which indicates that the character of the cadmium content-enriched root system belongs to quantitative characters and has main effective gene sites.
(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 PE 150. The sequencing machine-off data (raw data) are subjected to quality control, and the filtering standard is as follows: (1) filtering the sequence containing the linker; (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, comparing clean data 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; using EMMAX software to generate a Balding-Nichols genetic relationship matrix with a command of' EMMAX-kin-intel64-v-d 10-o [ out _ prefix ] [ tped _ prefix ]; the command for correlation analysis is "emmax-intel 64-v-d 10-t [ tped _ prefix ] -p [ pheno _ file ] -k [ kin _ file ] -o [ out _ prefix ]". And obtaining a P value of each site of the paspalum vaginatum by the correlation analysis result, when the SNP of which the P value is less than 1.0E-06 is the obvious SNP, the SNP of which the P value is the minimum is the peak SNP, grouping the materials by different allele types of the peak SNP in the group, carrying out variance analysis, and obtaining the percentage of the ratio of the variance between the groups to the total variance, namely the contribution rate of the peak SNP.
Through analysis, the interval of the main effect QTL site for enriching the cadmium content character of the paspalum vaginatum root system is limited between the 30653812 th base and the 30779422 th base of the chr02 chromosome of the paspalum vaginatum, the corresponding SNP is chr02_30653812(C/T), chr02_30779422(T/C), the peak SNP is chr02_30702908(G/A), the contribution rate of the QTL to the cadmium content character of the paspalum vaginatum root system is 89.20 percent (the materials are grouped according to different allele types of the peak SNP, the analysis of single-factor variance is carried out, and the percentage of the variance between groups divided by the total variance is the contribution rate).
Example 2 development of SNP molecular marker for gathering character of cadmium content in paspalum vaginatum root system
Developing an SNP molecular marker according to the identified enriched cadmium content QTL and peak value SNP of the paspalum vaginatum root system, extracting 400bp sequences before and after the SNP as characteristic sequences of the SNP molecular marker Pv.chr02: p30702908 (shown as a sequence table SEQ ID NO.1, wherein the 401bp is an SNP locus, and G/A mutation exists), and designing primers for the SNP molecular marker Pv.chr02: p30702908 as follows:
a forward primer: 5'-ATCTCCTGCGAGTCTGGTGT-3' (shown in SEQ ID NO. 2);
reverse primer: 5'-GCCAGGGTAAGCGTGAATGT-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 three types, and when the basic group type Pv.chr02: p30702908 is GG, the average cadmium content of the root system of the material is 30332.48mg/kg, namely the material is a variety with high enriched cadmium content of the root system; when the basic group type is GA, the average cadmium content of the root system of the material is 24299.50mg/kg, namely the root system is a variety with medium enriched cadmium content; when the base type is AA, the average cadmium content of the root system of the material is 19130.28mg/kg, namely the variety with the root system enriched with low cadmium content.
A schematic representation of the cadmium-rich phenotype of the root system for different genotypes is shown in FIG. 2. The result shows that the phenotype of the root system enriched cadmium content character corresponding to different genotype types of the SNP molecular marker Pv.chr02: p30702908 has obvious difference. Therefore, the cadmium content of the root system of the paspalum vaginatum to be detected can be quickly and simply evaluated by identifying the type of the SNP molecular marker, so that the paspalum vaginatum material capable of efficiently enriching the cadmium can be quickly screened and obtained.
Besides the amplification sequence of the primer, the material can be subjected to genome re-sequencing, the SNP molecular marker Pv.chr02: p30702908 disclosed by the invention is subjected to SNP genotyping according to the method in the embodiment 1, and the genotype type of the SNP is determined so as to quickly predict the content of cadmium which can be enriched in the root system of the material.
In conclusion, the SNP molecular marker Pv.chr02: p30702908 has the highest contribution rate to the character of enriching the cadmium content in the roots of the paspalum vaginatum, namely has a key regulation and control function on the high and low content of the enriched cadmium in the roots of the paspalum vaginatum, can predict or screen and identify the character of enriching the cadmium content in the roots of the paspalum vaginatum based on the SNP molecular marker, has the advantages of simple identification method, high selection efficiency, clear selection target and no influence of 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.chr02: p30702908 in trait identification of enriched cadmium content in paspalum vaginatum root system
Extracting DNA of young leaf blades of the earlier known materials 18HN-53, 19HN-01 and 17HN-37 with low cadmium-enriched root systems, sending the extracted materials to a sequencing company for genome re-sequencing, respectively identifying SNP genotypes of the three materials, and identifying the cadmium-enriched root system characters of the three materials according to the method in the embodiment 1.
Meanwhile, the early known materials 18HN-67, 17HN-23 and 17HN-13 with the root systems enriched with medium cadmium content are extracted to be sent to a sequencing company for genome re-sequencing, the SNP genotypes of the three materials are respectively identified, and the characters of the root systems enriched with cadmium content of the three materials are identified according to the method in the embodiment 1.
Meanwhile, extracting DNA of young and tender leaves of the materials 18HN-90 and 18HN-72 with high cadmium content in the known root systems at the early stage, sending the extracted DNA to a sequencing company for genome re-sequencing, respectively identifying SNP genotypes of the two materials, and identifying the cadmium content enriched character of the root systems of the two materials according to the method in the embodiment 1.
The SNP genotypes of the eight materials at the 30702908bp position of the chr02 chromosome and the character of the root system enriched with cadmium (mean value of cadmium content of the root system at day 13 under cadmium stress) are shown in Table 1.
TABLE 1 enrichment of cadmium content in genotype and root system at different Pv.chr02: p30702908 loci of different materials
Note: RDW represents the mean value of the enriched cadmium content of the root system at the 13 th day after cadmium stress
The result shows that for the known materials 18HN-53, 19HN-01 and 17HN-37 with low root system enriched cadmium content, the SNP genotypes at the 30702908bp position of the chr02 chromosome (namely, the 401bp position of the sequence shown in SEQ ID NO. 1) are all AA, the phenotypes of the traits with the root system enriched cadmium content are 19050.83mg/kg, 19135.00mg/kg and 19205.00mg/kg respectively, and the low-property corresponding to the AA genotype of the SNP molecular marker in the embodiment 2 and the root system enriched cadmium content are consistent; for known 18HN-67, 17HN-23 and 17HN-13 with common cadmium content enriched root systems, the SNP genotypes of the 30702908bp positions of the chr02 chromosome are all AG, the phenotypes of the traits of enriched cadmium content of the root systems are 24334.18mg/kg, 24342.50mg/kg and 26046.75mg/kg respectively, and the traits are consistent with the traits such as enriched cadmium content of the root systems corresponding to the AG genotype of the SNP molecular marker in the embodiment 2; similarly, 18HN-90 and 18HN-72 with high cadmium content enriched in known root systems have GG genotypes at the 30702908bp position of the chr02 chromosome, and the phenotypes of the traits with high cadmium content enriched in root systems are 36750.00mg/kg and 43208.25mg/kg, respectively, which are consistent with the traits with high cadmium content enriched in root systems corresponding to the GG genotypes of the SNP molecular markers in example 2. The results show that the method can effectively identify the content of cadmium enriched in the root systems of different paspalum vaginatum materials according to the genotype type of the SNP molecular marker Pv.chr02: p30702908, and can screen and specifically reserve the materials which can efficiently enrich cadmium, namely, the materials of which the genotypes are AG or GG, so that the remediation efficiency of the cadmium pollution of the soil is improved, and the method has extremely high application value.
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
<110> university of Hainan
<120> SNP molecular marker for enriching cadmium content character of paspalum vaginatum root system and application thereof
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<170> SIPOSequenceListing 1.0
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<211> 801
<212> DNA
<213> Paspalum vaginatum Sw.)
<400> 1
gctcacatgg aaaggtgtgg agagcaagag aagtcctatc ttagagtgtt tcactccttt 60
cttagtagtt aggatagtcc taggtgttag agttgagttg agctttgcct cagaatctcc 120
ggagttatct ttgagtttgt ggtaaagcat tgtatatttc tactcaagtt atcttcaata 180
taatttagtt atattttact ctaagctcta tctcctgcga gtctggtgta cctttctttg 240
catacttgct aggcttagat tgtgtttaac tttgttattc aaatcttgtt attcttgccg 300
gtgctataac tcaaccctag actctgacgc tagcctctag ctagatcccc tcctctgatc 360
aaggaagatc tatcagttgt tcaagcaaaa catttatagt gtgccatcct ctccacgcag 420
gtttgcttgg tagccccact cggtgacagt gtgtgatgtg cctgtgtagt ccaggcccat 480
cattcgggag gcctgtaggt aatagctaga ctctctttcg ggaccatcta ccccttgata 540
cccttgagac tctagaattt cgctcgacgc aagaacctga cttgtttatt ggaatatctt 600
ggttagacac ggataatctt tgtagccaaa tagaagtaga gttggttgac tttatcgcac 660
tcgttgtcct cccaggcaac tacacctttc tactgatttc tttgataatc gactgtctct 720
caaccataca tctattacat tcacgcttac cctggcttaa ttactagtcg gtacaattta 780
tccaagttta cagtttattc g 801
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<213> Artificial Sequence (Artificial Sequence)
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atctcctgcg agtctggtgt 20
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<213> Artificial Sequence (Artificial Sequence)
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Claims (10)
1. The SNP molecular marker for enriching the cadmium content character of the paspalum vaginatum root system is characterized in that the molecular marker is Pv.chr02: p30702908, the nucleotide sequence of the SNP molecular marker is shown as a sequence table SEQ ID NO.1, wherein SNP loci are as follows: the G/A mutation is present at the 401bp position of the sequence shown in SEQ ID NO. 1.
2. The SNP molecular marker for the trait of the enrichment of the cadmium content in the paspalum vaginatum root system according to claim 1, wherein the contribution rate of the molecular marker to the trait of the enrichment of the cadmium content in the root system is 89.20%.
3. The SNP molecular marker for enriching the cadmium content character of the paspalum vaginatum root system according to claim 1, wherein the method for obtaining the SNP molecular marker comprises the following steps:
a) collecting 86 parts of paspalum vaginatum from home and abroad with hereditary difference to form a related group;
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 stress experiment treatment on 86 parts of materials, and investigating the phenotype of cadmium content enrichment of root systems of different materials;
e) carrying out whole genome association analysis by combining genotype and phenotypic data of the root system cadmium content enrichment character, identifying QTL sites which are obviously related to the root system cadmium content enrichment character, and obtaining the SNP molecular marker related to the root system cadmium content enrichment character of the 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 as SEQ ID NO.2-3 of the sequence Listing.
6. Use of the SNP molecular marker according to claim 1 or the primer according to any one of claims 4 to 5 for predicting or identifying the cadmium content-rich trait of the root system of paspalum vaginatum.
7. The application of claim 6, wherein when the base type of the SNP site of the paspalum vaginatum to be detected is GG, the paspalum vaginatum is a material with a root system rich in high cadmium content; when the base type of the SNP locus of the paspalum vaginatum to be detected is GA, the paspalum vaginatum is a material with a root system enriched with medium cadmium content; and when the base type of the SNP locus of the paspalum vaginatum to be detected is AA, the paspalum vaginatum is a material with a root system rich in cadmium and low in content.
8. A method for predicting the enriched cadmium content of a paspalum vaginatum root system is characterized by comprising 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, and performing PCR amplification and sequencing by using the primer according to claim 4 or 5; 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 seapaspalum to be detected is GG, enriching a material with high cadmium content for a root system; when the genotype of the paspalum vaginatum to be detected is GA, the paspalum vaginatum is a material with a root system enriched with medium cadmium content; and when the genotype of the to-be-detected paspalum vaginatum is AA, the material with the root system enriched with low cadmium content is obtained.
9. A detection kit for predicting or identifying the enriched cadmium content in the paspalum vaginatum root system, which is characterized in that the detection kit comprises the primer as claimed in claim 4 or 5.
10. The use of the SNP molecular marker according to claim 1 or the primer according to any one of claims 4 to 5 in molecular marker assisted breeding for the efficient enrichment of cadmium traits in the root system of paspalum vaginatum.
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| CN114540537A (en) * | 2022-04-01 | 2022-05-27 | 海南大学 | SNP molecular marker related to melaleuca sinica resistant lentigo disease state and application thereof |
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| CN114540537A (en) * | 2022-04-01 | 2022-05-27 | 海南大学 | SNP molecular marker related to melaleuca sinica resistant lentigo disease state and application thereof |
| CN115141263A (en) * | 2022-06-29 | 2022-10-04 | 海南大学 | PvHMA5 protein, coding gene, expression vector and application thereof |
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