CN112349347B - Strawberry functional gene linkage SSR marker development method - Google Patents
Strawberry functional gene linkage SSR marker development method Download PDFInfo
- Publication number
- CN112349347B CN112349347B CN202011028667.4A CN202011028667A CN112349347B CN 112349347 B CN112349347 B CN 112349347B CN 202011028667 A CN202011028667 A CN 202011028667A CN 112349347 B CN112349347 B CN 112349347B
- Authority
- CN
- China
- Prior art keywords
- geno
- strawberry
- protein
- sequences
- isoform
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 235000011363 Fragaria x ananassa Nutrition 0.000 title claims abstract description 44
- 235000016623 Fragaria vesca Nutrition 0.000 title claims abstract description 43
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 39
- 238000000034 method Methods 0.000 title claims abstract description 31
- 239000003550 marker Substances 0.000 title claims abstract description 19
- 238000011161 development Methods 0.000 title description 7
- 244000307700 Fragaria vesca Species 0.000 title 1
- 241000220223 Fragaria Species 0.000 claims abstract description 50
- 238000012163 sequencing technique Methods 0.000 claims abstract description 15
- 108091092878 Microsatellite Proteins 0.000 claims abstract description 7
- 238000003908 quality control method Methods 0.000 claims abstract description 6
- 238000012545 processing Methods 0.000 claims abstract description 5
- 238000004140 cleaning Methods 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 239000002131 composite material Substances 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 102000001708 Protein Isoforms Human genes 0.000 claims 12
- 108010029485 Protein Isoforms Proteins 0.000 claims 12
- 101710159648 Uncharacterized protein Proteins 0.000 claims 6
- 101100170084 Arabidopsis thaliana At4g01020 gene Proteins 0.000 claims 2
- 101000800606 Arabidopsis thaliana Xyloglucan O-acetyltransferase 1 Proteins 0.000 claims 2
- 101710093804 ELMO domain-containing protein C Proteins 0.000 claims 2
- 108010016918 Histone-Lysine N-Methyltransferase Proteins 0.000 claims 2
- 101710134436 Putative uncharacterized protein Proteins 0.000 claims 2
- 102100027172 Transposon Hsmar1 transposase Human genes 0.000 claims 2
- 101150094830 setd3 gene Proteins 0.000 claims 2
- 238000011144 upstream manufacturing Methods 0.000 claims 2
- 101710161990 1-aminocyclopropane-1-carboxylate oxidase homolog Proteins 0.000 claims 1
- 108010028700 Amine Oxidase (Copper-Containing) Proteins 0.000 claims 1
- 108010092778 Autophagy-Related Protein 7 Proteins 0.000 claims 1
- 229930192334 Auxin Natural products 0.000 claims 1
- 101710181320 Chaperone protein DnaJ 1 Proteins 0.000 claims 1
- 101710179123 Cleavage stimulation factor subunit 77 Proteins 0.000 claims 1
- 102000008954 Copper amine oxidases Human genes 0.000 claims 1
- 101710174377 DEAD-box ATP-dependent RNA helicase 22 Proteins 0.000 claims 1
- 101710125629 Dihydroxy-acid dehydratase, chloroplastic Proteins 0.000 claims 1
- 102100040620 Dynein regulatory complex subunit 6 Human genes 0.000 claims 1
- 101710095704 Dynein regulatory complex subunit 6 Proteins 0.000 claims 1
- 101710126430 F-box/LRR-repeat protein 13 Proteins 0.000 claims 1
- 108010093223 Folylpolyglutamate synthetase Proteins 0.000 claims 1
- 101000689807 Gallus gallus Replication termination factor 2 Proteins 0.000 claims 1
- 101710120918 Histone-lysine N-methyltransferase ATXR4 Proteins 0.000 claims 1
- 101710169245 Kinesin-related protein 11 Proteins 0.000 claims 1
- 101710192752 Nuclear pore complex protein NUP50A Proteins 0.000 claims 1
- 101710134325 Nudix hydrolase 23, chloroplastic Proteins 0.000 claims 1
- 108090000854 Oxidoreductases Proteins 0.000 claims 1
- 102000004316 Oxidoreductases Human genes 0.000 claims 1
- 102100039654 Phosphoribosylglycinamide formyltransferase Human genes 0.000 claims 1
- 108010064209 Phosphoribosylglycinamide formyltransferase Proteins 0.000 claims 1
- 101710156996 Probable LRR receptor-like serine/threonine-protein kinase At1g07650 Proteins 0.000 claims 1
- 101710107061 Protein ROOT PRIMORDIUM DEFECTIVE 1 Proteins 0.000 claims 1
- 101710102240 Protein TPLATE Proteins 0.000 claims 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims 1
- 102000040945 Transcription factor Human genes 0.000 claims 1
- 108091023040 Transcription factor Proteins 0.000 claims 1
- 102100022979 Ubiquitin-like modifier-activating enzyme ATG7 Human genes 0.000 claims 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims 1
- 239000002363 auxin Substances 0.000 claims 1
- 102000023732 binding proteins Human genes 0.000 claims 1
- 108091008324 binding proteins Proteins 0.000 claims 1
- 102000030722 folylpolyglutamate synthetase Human genes 0.000 claims 1
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims 1
- 101710204818 mRNA-decapping enzyme-like protein Proteins 0.000 claims 1
- 108010034343 phosphoribosylamine-glycine ligase Proteins 0.000 claims 1
- 210000001519 tissue Anatomy 0.000 claims 1
- 239000011701 zinc Substances 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 claims 1
- 235000021012 strawberries Nutrition 0.000 abstract description 3
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000005065 mining Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 11
- 239000003147 molecular marker Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 6
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 6
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- BKHZIBWEHPHYAI-UHFFFAOYSA-N chloroform;3-methylbutan-1-ol Chemical compound ClC(Cl)Cl.CC(C)CCO BKHZIBWEHPHYAI-UHFFFAOYSA-N 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 208000005652 acute fatty liver of pregnancy Diseases 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 240000009088 Fragaria x ananassa Species 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- 208000035199 Tetraploidy Diseases 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000003517 fume Substances 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 238000007671 third-generation sequencing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/30—Detection of binding sites or motifs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Immunology (AREA)
- Botany (AREA)
- Bioinformatics & Computational Biology (AREA)
- Evolutionary Biology (AREA)
- Medical Informatics (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Theoretical Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011028667.4A CN112349347B (en) | 2020-09-25 | 2020-09-25 | Strawberry functional gene linkage SSR marker development method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011028667.4A CN112349347B (en) | 2020-09-25 | 2020-09-25 | Strawberry functional gene linkage SSR marker development method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112349347A CN112349347A (en) | 2021-02-09 |
| CN112349347B true CN112349347B (en) | 2023-11-10 |
Family
ID=74360522
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011028667.4A Active CN112349347B (en) | 2020-09-25 | 2020-09-25 | Strawberry functional gene linkage SSR marker development method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112349347B (en) |
Families Citing this family (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 |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684481A (en) * | 2009-03-30 | 2010-03-31 | 电子科技大学 | Method for preparing salvia miltiorrhiza EST-SSR molecular mark, specific primer and application thereof |
| CN105713984A (en) * | 2016-04-21 | 2016-06-29 | 中国科学院西北高原生物研究所 | Lancea tibetica microsatellite molecular markers |
| CN105969767A (en) * | 2016-07-18 | 2016-09-28 | 黄冈师范学院 | SSR molecular marker primer based on transcriptome data of azalea as well as screening method and application of SSR molecular marker primer |
| CN106636342A (en) * | 2016-10-27 | 2017-05-10 | 四川省农业科学院经济作物育种栽培研究所 | EST-SSR marker primer group developed on basis of sequence of transcriptome of ligusticum wallichii, and acquisition method and application of EST-SSR marker primer group |
| CN106754886A (en) * | 2017-01-19 | 2017-05-31 | 北京林业大学 | Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers |
| CN106987648A (en) * | 2017-06-01 | 2017-07-28 | 中国农业大学 | A kind of high-throughout plant organ development correlation SSR molecular marker method |
| CN108660239A (en) * | 2018-04-09 | 2018-10-16 | 中国林业科学研究院森林生态环境与保护研究所 | The specific SSR primers and its application of Malus sieversii and cultivation apple |
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6566479B2 (en) * | 2015-03-18 | 2019-08-28 | 国立研究開発法人農業・食品産業技術総合研究機構 | Four-seasonal markers related to strawberry genus plants and their use |
-
2020
- 2020-09-25 CN CN202011028667.4A patent/CN112349347B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101684481A (en) * | 2009-03-30 | 2010-03-31 | 电子科技大学 | Method for preparing salvia miltiorrhiza EST-SSR molecular mark, specific primer and application thereof |
| CN105713984A (en) * | 2016-04-21 | 2016-06-29 | 中国科学院西北高原生物研究所 | Lancea tibetica microsatellite molecular markers |
| CN105969767A (en) * | 2016-07-18 | 2016-09-28 | 黄冈师范学院 | SSR molecular marker primer based on transcriptome data of azalea as well as screening method and application of SSR molecular marker primer |
| CN106636342A (en) * | 2016-10-27 | 2017-05-10 | 四川省农业科学院经济作物育种栽培研究所 | EST-SSR marker primer group developed on basis of sequence of transcriptome of ligusticum wallichii, and acquisition method and application of EST-SSR marker primer group |
| CN106754886A (en) * | 2017-01-19 | 2017-05-31 | 北京林业大学 | Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers |
| CN106987648A (en) * | 2017-06-01 | 2017-07-28 | 中国农业大学 | A kind of high-throughout plant organ development correlation SSR molecular marker method |
| CN108660239A (en) * | 2018-04-09 | 2018-10-16 | 中国林业科学研究院森林生态环境与保护研究所 | The specific SSR primers and its application of Malus sieversii and cultivation apple |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112349347A (en) | 2021-02-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754886B (en) | The method for obtaining black fruit fructus lycii SSR primer is sequenced based on transcription | |
| CN110144418B (en) | Common camellia oleifera SSR molecular marker primer, marking method and application | |
| CN114231654B (en) | PARMS molecular marker associated with thousand grain weight of rape and application thereof | |
| CN106987648B (en) | High-flux SSR molecular marking method related to plant organ development | |
| CN106916897B (en) | Molecular marker for identifying purity of pumpkin hybrid seeds 'Yinhui No. three' of Indian pumpkin and application of molecular marker | |
| CN113637789B (en) | Wheat stripe rust resistance gene YrTD121 linked KASP molecular marker, primer, kit and application | |
| CN111893209B (en) | Indel site detection marker related to thousand grain weight of wheat and application thereof | |
| CN110894542A (en) | Primer for identifying types of GS5 gene and GLW7 gene of rice and application of primer | |
| CN108588255B (en) | Indel marker development for distinguishing five pepper cultivars and application thereof | |
| KR20180077873A (en) | SNP markers for selection of marker-assisted backcross in watermelon | |
| CN113637787B (en) | DNA fragment related to quality of single oil tea fruit and application thereof | |
| CN112349347B (en) | Strawberry functional gene linkage SSR marker development method | |
| CN110106281B (en) | Hexaploid I.trifida genome specific SNP molecular marker primer and application | |
| CN116515858A (en) | Peanut early leaf spot resistance major gene AhESR 1 and application of molecular marker thereof | |
| CN114736979B (en) | Molecular marker closely linked with watermelon complete leaf shape gene ClLL and application thereof | |
| CN112553360B (en) | Sophora alopecuroides identification related SNP marker and application thereof | |
| CN117230240B (en) | InDel locus related to soybean seed oil content, molecular marker, primer and application thereof | |
| CN117821650B (en) | Taro whole genome SNP-Panel and application thereof | |
| CN116479164B (en) | SNP locus, molecular marker, amplification primer and application of SNP locus and molecular marker related to soybean hundred-grain weight and size | |
| CN114606341B (en) | dCAPS molecular marker of aegilops sieboldii based on genome resequencing SNP and application | |
| CN115820907B (en) | Application of primer pair in identification of melon leaf shape | |
| CN117265176B (en) | SNP locus related to soybean kernel oil content, molecular marker and application | |
| CN116622877B (en) | SNP molecular marker related to lotus rhizome internode shape and application thereof | |
| CN116024368B (en) | Molecular marker closely linked with soybean plant high-efficiency gene locus and application thereof | |
| CN117904127A (en) | Gene, SNP molecular marker and primer set related to macadimia nut germplasm resource identification and identification method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |