CN112349347B - Strawberry functional gene linkage SSR marker development method - Google Patents
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Abstract
The invention discloses a method for developing a strawberry functional gene linkage 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 microsatellite sequences; (3) designing primers at the flanking sequences of the microsatellite sequence; (4) Transcriptome sequencing, splicing and gene annotation of different strawberry varieties; (5) electronic PCR anchors the primer to a specific gene. The SSR marker of the method has the advantages of high polymorphism, co-dominance, good repeatability and the like. 2468 SSR markers and primers thereof are provided for strawberries, and all the markers are related to functional genes, which provides an advantageous tool for strawberry breeding and agricultural trait related gene mining.
Description
Technical Field
The invention relates to a molecular marker screening method, in particular to a method for developing an SSR marker linked with a strawberry functional gene.
Background
Molecular markers are genetic markers based on nucleotide sequence variations of the genetic material of an individual. The molecular marker technology is widely applied to genetic map construction, phylogenetic analysis, population genetic analysis and other aspects. In recent years, the application of the molecular marker method to hybrid identification is also becoming more and more popular. Compared with morphological identification, molecular marker identification is rapid, accurate and good in reproducibility. With the development of molecular technology, more and more molecular marker identification methods are developed, and more than 60 molecular markers are developed, including molecular markers based on traditional Southern hybridization, such as RFLP, SS-CP-RFLP, DGGE-RFLP and the like; PCR-based labels, such as RAPD, AFLP, SSR, ISSR, etc.; genomic sequence-based markers such as SNP, inDel, cSSR and the like. Today, the trend of molecular markers is gradually transitioning from research applications for the RAPD, RFLP, AFLP marker to the SSR, ISSR, SNP marker.
With the development of molecular biology and genetics, DNA molecular markers undergo three stages. The first generation DNA molecular markers mainly comprise RFLP markers and RAPD markers. The two marking methods are mainly used for constructing gene linkage maps, but the sensitivity of RFLP marking to DNA polymorphism detection is not high, and the RAPD marking technology has instability due to random primers and low annealing temperature. The second generation DNA molecular marker is represented by SSR marker. SSR markers have the following three advantages: (1) The whole genome has differential microsatellite sequences, and the number of the designed molecular markers is rich and the polymorphism is high; (2) The SSR molecular marker is a co-dominant marker, so that a plurality of alleles can be identified, and the provided information is high and more accurate; (3) The method is simple, quick, time-saving and labor-saving, free from environmental influence, relatively stable in sequence, good in repeatability and capable of mutually communicating different laboratories to cooperatively develop the primer. The third generation DNA molecular markers are represented by SNP markers. However, SNP markers have high requirements for experimental conditions, and are costly and not universal.
There are about 20 species of strawberry genus (Fragaria), consisting of diploid, tetraploid, hexaploid and octaploid species, of which only one octaploid is used for cultivation, i.e. pineapple strawberry originating from natural hybridization of strawberry and chile strawberry in the american species. Strawberry plants undergo a series of multiple and natural hybridization processes during their evolution. Strawberry (academic name: fragaria x ananasa Duch.) is the most common cultivated hybrid in the genus Fragaria. Also commonly referred to as its fruit. Strawberry is a perennial herb, and is an important horticultural crop. The number of the strawberry cultivars of the excellent variety in China is very large, and the number of the strawberry cultivars is 20000 in the world, but the number of the excellent variety cultivated in a large area is only dozens.
With the continuous development of more and more strawberry SSR primers at home and abroad, the SSR technologies are already used for genetic map construction, variety identification, genetic diversity analysis and the like of strawberry plants.
At present, the development of SSR markers related to strawberry functional genes is not reported yet. The second generation and third generation sequencing technology is vigorously developed, and various genome, transcriptome and functional gene databases are gradually perfected, so that a good foundation is laid for the development of SSR related to strawberry functional genes.
Disclosure of Invention
The invention aims to: the invention aims to provide a method for developing a strawberry functional gene linkage SSR marker with the advantages of high polymorphism, co-dominance, good repeatability and the like.
The technical scheme is as follows: the invention provides a method for developing a strawberry functional gene linkage SSR marker, which comprises the following steps:
(1) Obtaining common data of strawberry genomes;
(2) Performing quality control processing on the data, assembling and splicing the processed sequences, and identifying microsatellite sequences;
(3) Designing primers at the side sequences of the microsatellite sequences;
(4) Transcriptome sequencing, splicing and gene annotation of different strawberry varieties;
(5) Electronic PCR anchors the primers to specific genes.
Further, the method of the step (2) is as follows: converting SRA format data into Fastq format files, adopting parallel cleaning channels to clean the converted files, controlling the data quality including Q20 cleaning and L40 filtering, removing polyT sequences, 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 carrying out SSR site identification on the unigene sequences.
Further, the control data quality comprises: q20 cleaning, wherein the error rate of Phred-Score is more than or equal to 20, namely 1%; l40 filtration: the length is more than or equal to 40bp.
Further, the recognition condition is that the single nucleotide repetition is not less than 10 times, the dinucleotide repetition is not less than 8 times, the trinucleotide repetition is not less than 7 times, the tetranucleotide repetition is not less than 5 times, and the pentanucleotide and hexanucleotide repetition is not less than 4 times; the identification condition of the composite SSR is that the distance between 2 SSRs is not more than 50bp.
Further, the method of the step (3) comprises the following steps: designing primers for the side sequences of the SSR locus, setting the parameters Tm as 58+/-3 ℃, the primer length as 20+/-3 bp, the expected length of the product as 100-450 bp and other parameters as defaults.
Further, the method of the step (4) comprises the following steps: sequencing different strawberry varieties, and filtering the quality and sequencing length of the original sequencing data; subsequently, the treated sequence was subjected to transcriptome de novo assembly to obtain a transcriptome functional gene sequence of strawberry variety tissue, and the gene function was annotated.
Further, the mass is: q20, phred-Score > 20, i.e. error rate of 1%; the sequencing length is as follows: l40 has a length of not less than 40bp.
Further, the method in the step (5) comprises the following steps: the primer pair is anchored to the functional gene sequence by electronic PCR, and the primer pair sequence which cannot be riveted is eliminated.
The beneficial effects are that: the SSR marker has the advantages of high polymorphism, co-dominance, good repeatability and the like, and SSR sites have conservation among intraspecies and even among intraclass. 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 pair;
FIG. 4 is an agarose gel electrophoresis of PCR amplification products of Geno-MK104402 pairs.
Detailed Description
As shown in FIGS. 1-4, in conjunction with the published strawberry genome (https:// www.ncbi.nlm.nih.gov/bioproject/. And then, assembling and splicing the sequences after quality control according to default parameters by using Trinity software, and removing redundant sequences to obtain unigene. SSR site recognition is carried out on the unigene sequence by MISA software (http:// pgrc. Ipk-gaterslben. De/MISA /), wherein the recognition condition is that single nucleotide repetition is not less than 10 times, dinucleotide repetition is not less than 8 times, trinucleotide repetition is not less than 7 times, tetranucleotide repetition is not less than 5 times, pentanucleotide and hexanucleotide repetition is not less than 4 times, and the recognition condition of the composite SSR is that the distance between 2 SSRs is not more than 50bp.
Primer 3.0 software (http:// Primer3.Sourceforge. Net /) is used for designing primers for flanking sequences of the SSR locus, the setting parameter Tm is 58+/-3 ℃, the Primer length is 20+/-3 bp, the expected length of a product is 100-450 bp, and other parameters are defaults.
Ten strawberry varieties, which are commercially common, were then sequenced using the second generation sequencing technique ILLUMINA. Sequencing was performed using an Illumina HiSeq 2000 sequencer. The SolexaQA software package is further utilized to filter the original sequencing data by quality (Q20, error rate of Phred-Score is more than or equal to 20, namely 1 percent) and sequencing length (L40, length is more than or equal to 40 bp). And then, transcriptome de novo assembly is carried out on the cleaned sequence according to default parameters by using Trinity transcriptome assembly software to obtain transcriptome functional gene sequences of tissues such as root, stem, leaf, flower and the like of the ten strawberry varieties, and the gene functions are annotated through KEGG and GO. The primer pair is anchored to the functional gene sequence by electronic PCR, and the primer pair sequence which cannot be riveted is eliminated.
The specific experiment is as follows:
1. experimental materials
1.1 strawberry variety
42 strawberries were selected in total.
1.2SSR primers
A total of 40 pairs of SSR primers.
2. Experimental procedure
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 operation steps are as follows:
1. each sample was mixed with the required CTAB and mercaptoethanol, calculated as 1000ul CTAB extract plus 5ul beta-mercaptoethanol, and the CTAB solution was preheated in a 65℃water bath;
2. placing about 1/3 volume of plant leaves into a 2ml centrifuge tube, adding a steel ball, rapidly freezing in liquid nitrogen, and grinding the leaves by a grinding machine;
3. adding 1ml of the prepared CTAB solution, mixing by vortex, and carrying out water bath at 65 ℃ for 30min (shaking every few minutes during the period);
4. adding 1ml chloroform isoamyl alcohol (CI), mixing, extracting for 10min, centrifuging for 10min at 13000 rpm;
5. absorbing the supernatant, adding equal volume of chloroform isoamyl alcohol (CI), mixing, extracting for 10min, centrifuging for 10min at 13000 rpm;
6. repeating the step 5;
7. absorbing the supernatant (about 500 ul) into a 1.5ml centrifuge tube, adding 2 times of absolute ethyl alcohol, and precipitating at-20 ℃ for more than 2 hours;
centrifuging at 8.13000rpm for 30min;
9. the supernatant was discarded, and 1ml of 75% alcohol was added for washing;
10. repeating the step 8, and discarding the supernatant;
11. drying in a fume hood, adding 50ul ddH 2 O is dissolved;
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 screening SSR primers with good polymorphism by agarose gel electrophoresis.
The method comprises the following steps: experimental conditions:
1) A DNA template: the DNA concentration was diluted to about 50 ng/. Mu.l.
2) SSR primer: and 40 pairs in total.
5) Agarose gel concentration: 2%
By agarose gel electrophoresis of the PCR amplified products, 32 (80%) of 40 pairs of primers were found to be polymorphic in different strawberry varieties.
Claims (7)
1. A method for developing a strawberry functional gene linkage SSR marker is characterized by comprising the following steps: the method comprises the following steps:
(1) Obtaining common data of strawberry genomes;
(2) Performing quality control processing on the data, assembling and splicing the processed sequences, and identifying microsatellite sequences;
(3) Primers were designed flanking the microsatellite sequence: designing a primer of a side sequence of an SSR locus, setting a parameter Tm as 58+/-3 ℃, wherein the primer length is 20+/-3 bp, and the expected length of a product is 100-450 bp; the primer sequences are as follows:
(4) Transcriptome sequencing, splicing and gene annotation of different strawberry varieties;
(5) Electronic PCR anchors the primers to specific genes.
2. The method for developing a strawberry functional gene-linked SSR marker according to claim 1, wherein: the method of the step (2) comprises the following steps: converting SRA format data into Fastq format files, adopting parallel cleaning channels to clean the converted files, controlling the data quality including Q20 cleaning and L40 filtering, removing polyT sequences, 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 carrying out SSR site identification on the unigene sequences.
3. The method for developing a strawberry functional gene-linked SSR marker according to claim 1, wherein: the control data quality includes: q20 cleaning, wherein the error rate of Phred-Score is more than or equal to 20, namely 1%; l40 filtration: the length is more than or equal to 40 and bp.
4. The method for developing a strawberry functional gene-linked SSR marker according to claim 2, wherein: the identification condition is that the single nucleotide repetition is not less than 10 times, the dinucleotide repetition is not less than 8 times, the trinucleotide repetition is not less than 7 times, the tetranucleotide repetition is not less than 5 times, and the pentanucleotide and hexanucleotide repetition is not less than 4 times; the identification condition of the composite SSR is that the distance between 2 SSRs is not more than 50bp.
5. The method for developing a strawberry functional gene-linked SSR marker according to claim 1, wherein: the method of the step (4) is as follows: sequencing different strawberry varieties, and filtering the quality and sequencing length of the original sequencing data; subsequently, the treated sequence was subjected to transcriptome de novo assembly to obtain a transcriptome functional gene sequence of strawberry variety tissue, and the gene function was annotated.
6. The method for developing a strawberry functional gene-linked SSR marker according to claim 5, wherein: the mass is as follows: q20, phred-Score > 20, i.e. error rate of 1%; the sequencing length is as follows: l40, the length is more than or equal to 40 and bp.
7. The method for developing a strawberry functional gene-linked SSR marker according to any one of claims 1 to 6, characterized by: the method of the step (5) is as follows: the primer pair is anchored to the functional gene sequence by electronic PCR, and the primer pair sequence which cannot be riveted is eliminated.
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