CN112349347A - Development method of strawberry functional gene linked SSR marker - Google Patents
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Abstract
The invention discloses a method for developing a strawberry functional gene linked SSR marker, which comprises the following steps: (1) obtaining strawberry genome public data; (2) performing quality control processing on the data, assembling and splicing the processed sequences, and identifying a microsatellite sequence; (3) designing primers on flanking sequences of the microsatellite sequence; (4) transcriptome sequencing, splicing and gene annotation of different strawberry varieties; (5) electronic PCR anchors primers to specific genes. The SSR marker of the method has the advantages of high polymorphism, codominance, good repeatability and the like. 2468 SSR markers and primers thereof are provided for the strawberries, and the markers are all related to functional genes, so that an advantageous tool is provided for strawberry breeding and gene mining related to agricultural traits.
Description
Technical Field
The invention relates to a molecular marker screening method, in particular to a development method of a strawberry functional gene linked SSR marker.
Background
Molecular markers are genetic markers based on nucleotide sequence variations of the genetic material of an individual. Molecular marker technology is widely applied to the aspects of genetic map construction, phylogenetic analysis, population genetic analysis and the like. In recent years, the application of molecular marker methods to hybrid identification has also begun to become increasingly popular. Compared with morphological identification, the molecular marker identification is rapid, accurate and good in reproducibility. With the development of molecular technology, more and more molecular marker identification methods are used, and more than 60 molecular markers are developed, including molecular markers based on traditional Southern hybridization, such as RFLP, SS-CP-RFLP, DGGE-RFLP, etc.; PCR-based markers such as RAPD, AFLP, SSR, ISSR, etc.; genomic sequence-based markers such as SNPs, InDel, cSSR, etc. At present, the development trend of molecular markers is gradually transiting from research and application of RAPD, RFLP and AFLP markers to research and application of SSR, ISSR and SNP markers.
With the development of molecular biology and genetics, DNA molecular markers go through three stages. The first generation of DNA molecular markers mainly include RFLP markers and RAPD markers. The two marking methods are mainly used for constructing gene linkage maps, but the RFLP marking has low sensitivity on DNA polymorphism detection, and the RAPD marking technology has instability due to random primers and low annealing temperature. Second generation DNA molecular markers are represented by SSR markers. SSR markers have three advantages: (1) the whole genome has different microsatellite sequences, so that the designed molecular markers have rich quantity and high polymorphism; (2) the SSR molecular marker is a codominant marker, can mark a plurality of alleles, and has high and more accurate provided information; (3) the method is simple, rapid, time-saving and labor-saving, is not influenced by environment, has relatively stable sequence and good repeatability, and different laboratories can mutually exchange and cooperate to develop primers. The third generation of DNA molecular markers are represented by SNP markers. However, SNP markers have high requirements on experimental conditions, high cost and no universality.
There are about 20 species of the strawberry genus (Fragaria) consisting of diploid, tetraploid, hexaploid and octaploid species, of which only one octaploid is used for cultivation, namely pineapple strawberries originating from the natural cross between the american famid and chile strawberries. Strawberry plants undergo a series of polyploidization and natural crossing processes during their evolution. Strawberry (academic name: Fragaria x ananassa Duch.) is the most common hybrid cultivated in the strawberry genus. Also commonly referred to as the fruit. Strawberries are perennial herbaceous plants and are important horticultural crops. The excellent varieties of strawberries in China are many, and the number of the excellent varieties is 20000 in all over the world, but the excellent varieties cultivated in a large area are only dozens.
With the development of SSR primers of more and more strawberry plants at home and abroad, the SSR techniques have been used for genetic map construction, variety identification, genetic diversity analysis and the like of the strawberry plants.
At present, the development of SSR markers related to strawberry functional genes is not reported. However, the second generation and third generation sequencing technology is developed vigorously, and various genomes, transcriptomes and functional gene databases are improved day by day, which lay a good foundation for the development of SSR related to strawberry functional genes.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a method for developing a strawberry functional gene linked SSR marker with the advantages of high polymorphism, codominance, good repeatability and the like.
The technical scheme is as follows: the invention provides a method for developing a strawberry functional gene linked SSR marker, which comprises the following steps:
(1) obtaining strawberry genome public data;
(2) performing quality control processing on the data, assembling and splicing the processed sequences, and identifying a microsatellite sequence;
(3) designing primers on flanking sequences of the microsatellite sequence;
(4) transcriptome sequencing, splicing and gene annotation of different strawberry varieties;
(5) electronic PCR anchors primers to specific genes.
Further, the method of the step (2) is as follows: converting SRA format data into a Fastq format file, cleaning the converted file by adopting a parallel cleaning channel, controlling the data quality, including Q20 cleaning and L40 filtering, removing polyT, polyA sequences and carrier sequences at 5 and 3 ends, assembling and splicing the sequences after quality control processing, removing redundant sequences to obtain unigene, and identifying SSR sites of the unigene sequences.
Further, the controlling the data quality comprises: q20 cleaning, wherein Phred-Score is more than or equal to 20, namely the error rate is 1%; l40 filtration: the length is more than or equal to 40 bp.
Further, the recognition conditions are that the mononucleotide repeat is not less than 10 times, the dinucleotide repeat is not less than 8 times, the trinucleotide repeat is not less than 7 times, the tetranucleotide repeat is not less than 5 times, and the pentanucleotide and the hexanucleotide repeat is not less than 4 times; the recognition condition of the composite SSR is that the distance between 2 SSRs does not exceed 50 bp.
Further, the method of step (3) is: designing primers for flanking sequences of SSR sites, setting the Tm as 58 +/-3 ℃, setting the length of the primers as 20 +/-3 bp, setting the expected length of products as 100-450 bp, and setting other parameters as defaults.
Further, the method in the step (4) is as follows: sequencing different strawberry varieties, and filtering the quality and sequencing length of original sequencing data; subsequently, the processed sequences were subjected to transcriptome de novo assembly to obtain transcriptome functional gene sequences of strawberry variety tissues, and the gene functions were annotated.
Further, the mass is: q20, Phred-Score is more than or equal to 20, namely the error rate is 1 percent; the sequencing length was: l40, length ≥ 40 bp.
Further, the method of step (5) is: and (3) anchoring the primer pair to a functional gene sequence through electronic PCR, and removing primer pair sequences which cannot be riveted.
Has the advantages that: the SSR marker disclosed by the invention has the advantages of high polymorphism, codominance, good repeatability and the like, and SSR sites are conserved among species, even family species. The invention provides 2468 SSR markers and primers thereof for strawberries, and the markers are all related to functional genes, which provides a favorable tool for strawberry breeding and agricultural trait related gene mining.
Drawings
FIG. 1 is a flow chart of the method of the present invention;
FIG. 2 is an agarose gel electrophoresis of PCR amplification products of Geno-MK10224 pairs;
FIG. 3 is an agarose gel electrophoresis of PCR amplification products of Geno-MK10263 pairs;
FIG. 4 is an agarose gel electrophoresis image of the PCR amplification product of Geno-MK104402 pair.
Detailed Description
As shown in FIGS. 1-4, combining the published strawberry genome (https:// www.ncbi.nlm.nih.gov/bioproject/. And then, assembling and splicing the sequence after quality control by using Trinity software according to default parameters, and removing redundant sequences to obtain unigene. The identification conditions of the SSR locus of the unigene sequence are that the repetition of mononucleotide is not less than 10 times, the repetition of dinucleotide is not less than 8 times, the repetition of trinucleotide is not less than 7 times, the repetition of tetranucleotide is not less than 5 times, the repetition of pentanucleotide and hexanucleotide is not less than 4 times, and the distance between 2 SSRs is not more than 50 bp.
Primer 3.0 software (http:// Primer3. sourceforce. net /) is used for designing primers for the flanking sequences of the SSR loci, the set parameter Tm is 58 ℃ plus or minus 3 ℃, the Primer length is 20 plus or minus 3bp, the expected product length is 100-450 bp, and other parameters are defaults.
Ten commercially common strawberry varieties were then sequenced using the second generation sequencing technique ILLUMINA. Sequencing was performed using an Illumina HiSeq 2000 sequencer. Further using SolexaQA software package to filter the quality (Q20, error rate of Phred-Score is more than or equal to 20, namely 1%) and sequencing length (L40, length is more than or equal to 40bp) of the original sequencing data. Subsequently, the Trinity transcriptome assembly software is used for carrying out transcriptome de novo assembly on the cleaned sequence according to default parameters to obtain the transcriptome functional gene sequences of the tissues of the root, the stem, the leaf, the flower and the like of the ten strawberry varieties, and the gene functions are annotated by KEGG and GO. And (3) anchoring the primer pair to a functional gene sequence through electronic PCR, and removing primer pair sequences which cannot be riveted.
The specific experiment is as follows:
1. experimental Material
1.1 strawberry variety
A total of 42 strawberries were selected.
1.2SSR primers
There are 40 pairs of SSR primers.
2. Procedure of experiment
2.1 extraction of strawberry genomic DNA
The DNA of the 42 strawberry leaves was extracted as a PCR template.
The method comprises the following steps: CTAB extraction method
The method comprises the following operation steps:
1. uniformly mixing the required CTAB and mercaptoethanol according to 1000ul CTAB extracting solution and 5ul beta-mercaptoethanol of each sample, and preheating CTAB solution in a water bath at 65 ℃;
2. placing plant leaves with about 1/3 volume into a 2ml centrifuge tube, adding a steel ball, quickly freezing in liquid nitrogen, and grinding the leaves by a sample grinder;
3. adding 1ml of prepared CTAB solution, mixing uniformly by vortex, and carrying out water bath at 65 ℃ for 30min (shaking every few minutes during the process);
4. adding 1ml chloroform isoamyl alcohol (CI), reversing and mixing, extracting for 10min, centrifuging at 13000rpm for 10 min;
5. sucking supernatant, adding equal volume of chloroform isoamyl alcohol (CI), reversing, mixing, extracting for 10min, and centrifuging at 13000rpm for 10 min;
6. repeating the step 5;
7. sucking supernatant (about 500ul) into a 1.5ml centrifuge tube, adding 2 times of anhydrous ethanol, and precipitating at-20 deg.C for more than 2 h;
8.13000rpm for 30 min;
9. discarding the supernatant, adding 1ml of 75% alcohol for washing;
10. repeating the step 8, and discarding the supernatant;
11. blow-drying in fume hood, adding 50ul ddH2Dissolving O;
12. measuring the DNA concentration;
2.2 screening of polymorphic SSR primers
And carrying out PCR amplification on 42 strawberry DNA templates by using the 40 pairs of SSR primers, and then carrying out agarose gel electrophoresis to screen out SSR primers with better polymorphism.
Attached: the experimental conditions are as follows:
1) DNA template: the DNA concentration was diluted to about 50 ng/. mu.l.
2) SSR primers: and 40 pairs in total.
5) Agarose gel concentration: 2 percent of
32 of the 40 primers (80%) were found to be polymorphic in different strawberry varieties by agarose gel electrophoresis of the PCR amplification products.
Claims (8)
1. A method for developing a strawberry functional gene linked SSR marker is characterized in that: the method comprises the following steps:
(1) obtaining strawberry genome public data;
(2) performing quality control processing on the data, assembling and splicing the processed sequences, and identifying a microsatellite sequence;
(3) designing primers on flanking sequences of the microsatellite sequence;
(4) transcriptome sequencing, splicing and gene annotation of different strawberry varieties;
(5) electronic PCR anchors primers to specific genes.
2. The method for developing a strawberry functional gene-linked SSR marker according to claim 1, wherein the SSR marker comprises: the method of the step (2) comprises the following steps: converting SRA format data into a Fastq format file, cleaning the converted file by adopting a parallel cleaning channel, controlling the data quality, including Q20 cleaning and L40 filtering, removing polyT, polyA sequences and carrier sequences at 5 and 3 ends, assembling and splicing the sequences after quality control processing, removing redundant sequences to obtain unigene, and identifying SSR sites of the unigene sequences.
3. The method for developing a strawberry functional gene-linked SSR marker according to claim 1, wherein the SSR marker comprises: the controlling the data quality comprises: q20 cleaning, wherein Phred-Score is more than or equal to 20, namely the error rate is 1%; l40 filtration: the length is more than or equal to 40 bp.
4. A method for developing a strawberry functional gene-linked SSR marker according to claim 2, characterized in that: the recognition conditions are that mononucleotide repeat is not less than 10 times, dinucleotide repeat is not less than 8 times, trinucleotide repeat is not less than 7 times, tetranucleotide repeat is not less than 5 times, pentanucleotide and hexanucleotide repeat is not less than 4 times; the recognition condition of the composite SSR is that the distance between 2 SSRs does not exceed 50 bp.
5. A method for developing a strawberry functional gene-linked SSR marker according to claim 1 or 2, wherein: the method in the step (3) comprises the following steps: designing primers for flanking sequences of SSR sites, setting the Tm as 58 +/-3 ℃, setting the length of the primers as 20 +/-3 bp, setting the expected length of products as 100-450 bp, and setting other parameters as defaults.
6. A method for developing a strawberry functional gene-linked SSR marker according to any one of claims 1 to 5, wherein: the method in the step (4) comprises the following steps: sequencing different strawberry varieties, and filtering the quality and sequencing length of original sequencing data; subsequently, the processed sequences were subjected to transcriptome de novo assembly to obtain transcriptome functional gene sequences of strawberry variety tissues, and the gene functions were annotated.
7. A method for developing a strawberry functional gene-linked SSR marker according to claim 6, characterized in that: the mass is as follows: q20, Phred-Score is more than or equal to 20, namely the error rate is 1 percent; the sequencing length was: l40, length ≥ 40 bp.
8. A method for developing a strawberry functional gene-linked SSR marker according to any one of claims 1 to 7, wherein: the method in the step (5) comprises the following steps: and (3) anchoring the primer pair to a functional gene sequence through electronic PCR, and removing primer pair sequences which cannot be riveted.
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| CN108998553A (en) * | 2018-08-14 | 2018-12-14 | 西北大学 | The method and primer of a kind of quick screening polymorphic micro-satellite site target primer |
| CN110343767A (en) * | 2019-06-25 | 2019-10-18 | 天津市水产研究所 | Litopenaeus vannamei microsatellite molecular marker specific primer and its application in analysis of genetic diversity |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115108855A (en) * | 2022-07-01 | 2022-09-27 | 江苏农林职业技术学院 | Method for preparing fertilizer by using household kitchen waste |
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