CN104561315A - SNP (Single Nucleotide Polymorphism) detection method of rice high-resistant starch content mutant gene sbe3-rs - Google Patents
SNP (Single Nucleotide Polymorphism) detection method of rice high-resistant starch content mutant gene sbe3-rs Download PDFInfo
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Abstract
The invention discloses an SNP (Single Nucleotide Polymorphism) detection method of a rice high-resistant starch content mutant gene sbe3-rs. The SNP detection method disclosed by the invention comprises the following steps of: designing four gene specific primers by adopting a four-primer amplification refractory mutation system PCR (Polymerase Chain Reaction), namely an ARMS-PCR method, according to the fact that the sbe3-rs gene has single nucleotide polymorphism, adding into the same PCR reaction system to amplify different rice DNAs, and rapidly and accurately detecting whether a rice planting resource or a breeding population thereof contains the mutant gene sbe3-rs according to the characteristic patterns of the PCR thereof. The SNP detection method disclosed by the invention has the characteristics of being high in sensitivity and specificity; the selection efficiency of the high-resistant starch mutant gene sbe3-rs can be increased; and the breeding process of high-resistant starch rice varieties can be accelerated.
Description
Technical field
The invention belongs to agriculture field of molecular biotechnology, relate to the SNP detection method of a kind of paddy rice high resistant starch content mutator gene sbe3-rs particularly, for qualification and the breed breeding of paddy rice high-resistance starch mutator gene sbe3-rs rice pest insects.
Background technology
Along with improving constantly of China's expanding economy and living standards of the people, there is great variety in the dietary structure of people.China resident is staple food traditionally with cereal, but the consumption of cereal constantly declines now, and meat and egg etc. are higher fatty acid constantly to rise with intake that is high protein food, causes human diseases to be composed and very big change also occurs.Because diet formula is uneven and overnutrition causes " ciril disease " to occur in China and be the trend expanded year by year.Diabetes, obesity, hypertension, hyperlipidemia, cardiovascular and cerebrovascular disease and coronary heart disease etc. have become the principal disease of harm our people health.Especially increase the most remarkable in these diseases with onset diabetes rate.
Diabetes have become the chronic disease of the third-largest serious threat human health after tumour and cardiovascular pathological changes.One section of article display that on September 4th, 2013, " JAMA " was issued, according to the epidemiological investigation data display of China in 2010, the morbidity of diabetes mellitus in China has turned over nearly twice at nearly 10 years, diabetes mellitus in China incidence reaches 11.6%, reach alert level, be significantly higher than world average level 6.4%.Diabetes mellitus in China high risk population is also expanding, and about 1.5 hundred million, there is ten hundreds of patients to die from diabetes and complication thereof every year.The diabetes present situation of China is very severe, and China has become the fastest-rising area of global range diabetes, and age of onset is more tending towards rejuvenation.Future 50 years, diabetes will be China's the most serious public health problems.
Paddy rice (Oryza sativa L.) is as one of Three major grain crops, Starch content of rice is the highest, for one of the most important carbohydrate of the mankind and energy derive, nutritive ingredient in improvement rice is conducive to reducing human body to the absorption of carbohydrate, thus may reduce the sickness rate of " ciril disease ".Improving Resistant starch (Resistant Starch, RS) content in rice is one of a kind of very effective and feasible way.Resistant starch refers to " in the small intestine of healthy individuals unabsorbable starch or starch decomposition products " (European Resistant starch association EURESTA defines).RS occupies very consequence in adjustment diet formula, by the rice genetic resource of screening and identification height RS content, improvement rice varieties, the dietary structure improving people is one of sick most economical effective means sent out of the ciril disease such as treatment and prevent diabetes.
Chinese patent 201210266649.9 located the major gene sbe3-rs controlling paddy rice RS content, this mutator gene has the base mutation of T → C at the 105th place corresponding to rice fecula q enzyme SBE3 gene the 16th exon, and develop a CAPS functional label molecule marker for this site, for the high-yield rice new variety of being cultivated high RS content by Molecular Marker Assisted Selection Technology (Marker-assisted selection, MAS) or transgenic approach provide theory and technology to instruct.
But, step is cut with restriction enzyme SpeI enzyme because CAPS molecule marker relates to after first PCR reacts, complex steps is there is so use, the series of malpractice such as somewhat expensive, therefore, the methods of genotyping setting up a kind of new PCR-based technology identifies that paddy rice high-resistance starch mutant gene sbe3-rs genotype has become technical barrier urgently to be resolved hurrily in current Resistant starch Breeding Application fast and accurately.
Summary of the invention
The object of the present invention is to provide the SNP detection method of a kind of paddy rice high resistant starch content mutator gene sbe3-rs, the method of tetra-primer ARMS-PCR PCR and ARMS-PCR is adopted to detect the different genotype of paddy rice resistance mutant amylase gene sbe3-rs quickly and accurately, its highly sensitive, high specificity, for high-resistance starch rice pest insects qualification and molecular marker assisted selection breeding in cultivate high-resistance starch new rice variety.
In order to achieve the above object, the present invention adopts following technical scheme:
A SNP detection method of paddy rice high resistant starch content mutator gene sbe3-rs, is the molecule marking method of tetra-primer ARMS-PCR PCR and ARMS-PCR, comprises:
1) ARMS design of primers
There is at the 105th place of the 16th exon according to sbe3-rs gene the mononucleotide base mutation of T → C, design four gene-specific primers, be respectively: forward outer primer, reverse outer primer, forward inner primer and reverse inner primer, wherein, 3 ' end of forward inner primer and reverse inner primer terminates in mutational site to be detected, arranges a base mispairing in 3 ' end the 3rd increase reciprocal of forward inner primer and reverse inner primer;
2) ARMS-PCR amplification
By step 1) in four gene-specific primers add same PCR reaction system, and pcr amplification is carried out to the DNA of rice plant, row agarose gel electrophoresis of going forward side by side;
3) according to electrophoresis result determination genotype
If there is 572bp, 218bp two characteristic bands are not containing sbe3-rs genotype homozygote and low resistant starch content plant; If there is 572bp and 397bp two band to be high-resistance starch mutant gene sbe3-rs homozygote; If there is 572bp simultaneously, 218bp and 397bp tri-band is then the heterozygote of high-resistance starch mutant gene sbe3-rs.
Further, forward inner primer is non-sbe3-rs amplified allele inner primer, introduce the exchange of A-C in its 3 ' end the 3rd bit mismatch reciprocal, reverse inner primer is mutant sbe3-rs genotype inner primer, introduces the exchange of C-A in its 3 ' end the 3rd bit mismatch reciprocal.
Particularly, the sequence of described specificity ARMS primer is:
Forward outer primer sbe3-rs-O-F1:5 '-TGTGATGTGCTGGATTTGGT-3 ';
Reverse outer primer sbe3-rs-O-R1:5 '-GCTGTGGTTTTCATACCGTTC-3 ';
Forward inner primer sbe3-rs-I-F2:5 '-TGAAAGTCATGATCAAGCCCT-3 ';
Reverse inner primer sbe3-rs-I-R2:5 '-GCAATAGTTTTGTCACCAAATG-3 '.
Further again, during ARMS-PCR amplification, in PCR reaction system, the consumption of DNA profiling is 50 ~ 100ng, and the final concentration of forward outer primer and reverse outer primer is respectively 0.2 ~ 0.6 μM, preferably 0.4 ~ 0.6 μM; The final concentration of forward inner primer and reverse inner primer is respectively 1 ~ 5 times of forward outer primer or reverse outer primer, preferably 3 ~ 5 times.
Further, the cumulative volume of described PCR reaction system is 10 ~ 50 μ L; Wherein, DNA profiling, 50 ~ 100ng; 10 × Buffer, 1 ~ 5 μ L; Mg
2+final concentration is 1.5 ~ 2.0mM; DNTP final concentration is 0.2 ~ 0.25mM; The final concentration of forward outer primer and reverse outer primer is respectively 0.2 ~ 0.6 μM; The final concentration of forward inner primer and reverse inner primer is respectively 1 ~ 5 times of forward outer primer or reverse outer primer; Taq polysaccharase consumption is 0.5U ~ 5U, surplus ddH
2o supplies.
Preferably, the cumulative volume of described PCR reaction system is 25 μ L, wherein, and DNA profiling, 50 ~ 100ng; 10 × Buffer, 2.5 μ L; Mg
2+final concentration is 1.5mM; DNTP final concentration is 0.25mM; The final concentration of forward outer primer and reverse outer primer is respectively 0.2 μM; The final concentration of forward inner primer and reverse inner primer is respectively 5 times of forward outer primer or reverse outer primer, Taq polysaccharase 2.5U, surplus ddH
2o supplies.
Further, during ARMS-PCR amplification, the condition of circulating reaction is: 94 DEG C of denaturation 5min; 94 DEG C of 30s; 56 ~ 58 DEG C of 30s; 72 DEG C of 40 ~ 60s; 35 circulations; Then 72 DEG C extend 10min.
After utilizing the genomic DNA of the SNP detection method amplifying rice kind of paddy rice resistant starch content mutator gene sbe3-rs of the present invention, all occurred in each DNA sample by the band of the 572bp of forward outer primer sbe3-rs-O-F1 and reverse outer primer sbe3-rs-O-R1 amplification, can it not only play the effect (detecting DNA effectively be increased) of positive control, also effectively can reduce the amplification of non-specific PCR products and the formation of primer dimer simultaneously.
When the rice varieties of amplification is sbe3-rs homozygous genotype, except the contrast band amplifying 572bp, also amplify the band of 397bp, this 397bp band is increased by forward outer primer sbe3-rs-O-F1 and reverse inner primer sbe3-rs-I-R2 and produces, its specific amplification be the 105th Nucleotide C allelotrope site of SBE3 the 16th exon.
When the rice varieties of amplification is non-sbe3-rs genotype, contrast except band except 572bp can be amplified, the band of 218bp can also be amplified, this 218bp band is increased by reverse outer primer sbe3-rs-O-R1 and forward inner primer sbe3-rs-I-F2 and produces, its specific amplification be the 105th of SBE3 the 16th exon the be Nucleotide T allelotrope site.
The rice varieties of amplification can amplify three band when being sbe3-rs gene hybridizing type, comprises 572bp, 397bp and 218bp.
Beneficial effect:
The four primer ARMS-PCR molecule marking methods for the identification of paddy rice high-resistance starch mutator gene sbe3-rs different genotype provided by the invention, have the following advantages:
1) molecule marker provided by the invention be based on mutator gene mutational site design molecule marker, be functional indicia, with Resistant starch proterties be divided into completely from, directly can reflect the phenotype of plant, do not exist cause due to exchange mistake qualification.
2) molecule marking method of the present invention can realize the Rapid identification of antagonism mutant amylase gene sbe3-rs rice pest insects.
3) molecule marking method provided by the invention can be effective to the assistant breeding of the high resistant starch content rice varieties that high-resistance starch gene causes.The four primer ARMS-PCR molecule markers utilizing gene sbe3-rs mutational site to design carry out DNA detection to plant, the individual plant of high-resistance starch mutant gene sbe3-rs homozygous genotype can be gone out at seedling stage assay, eliminate other individual plant, so not only save breeding cost, and substantially increase the efficiency of selection of high-resistance starch rice varieties.
4) compared with the CAPS molecule marker of existing qualification mutator gene sbe3-rs, molecule marking method provided by the invention adopts the method for four primer One_step PCR amplifications to identify sbe3-rs different genotype, effectively can not only distinguish isozygotying and heterozygous genotypes of sbe3-rs, and owing to not relating to the use of restriction enzyme, therefore there is not complex operation, the drawbacks such as somewhat expensive, more efficiently, fast.
Accompanying drawing explanation
Fig. 1 is the strategy being designed for the four primer ARMS-PCR systems detecting paddy rice high-resistance starch mutator gene sbe3-rs different genotype in embodiments of the invention;
Wherein, black background base represents single nucleotide mutation base position, and gray background represents four different primers sequence correspondence positions.
Fig. 2 is that four primer ARMS-PCR molecule marking methods in embodiments of the invention are to the genotypic detected result of different rice varieties sbe3-rs;
Wherein, M:DNA marker, DL2000; 1 ~ 2: not containing close positive 23 two repetitions of sbe3-rs genotypic rice kind; Two repetitions of 3 ~ 4:sbe3-rs gene pure type paddy rice hypoglycemic rice; 5 ~ 6:sbe3-rs heterozygous rice plant, hypoglycemic rice No. 1 and close positive 23 hybridizes F
1for two repetitions of individual plant.
Fig. 3 be in embodiments of the invention four primer ARMS-PCR molecule markers to hypoglycemic rice No. 1/close positive 23F
2the genotypic detected result of colony sbe3-rs;
Wherein, M:DNA marker DL2000; 1: close positive 23; 2: hypoglycemic rice No. 1; 3 ~ 17 is F
2individual plant, wherein 3,4,5,11,14,16,17 is mutator gene sbe3-rs heterozygous individual plant; 7 is the homozygous individual plant of sbe3-rs; 6,8,9,10,12,13,15 is not containing the individual plant of mutator gene sbe3-rs.
Embodiment
Below in conjunction with specific embodiment and accompanying drawing, technical scheme of the present invention is described in further detail.It is important to note that these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.The various apparent correction made the present invention and changing also is included in scope of the present invention.
The SNP of embodiment paddy rice high resistant starch content mutator gene sbe3-rs detects
1. experiment material
High-resistance starch rice varieties: hypoglycemic rice No. 1 (yang et al.A putative gene sbe3-rsfor resistant starch mutanted from SBE3 for starch branching enzyme in rice (Oryza sativa L.), Plos One, 2012,0043026);
General kind: close positive 23;
Sbe3-rs genotype segregating population: the F of hypoglycemic rice No. 1/close positive 23 hybridization
2for 15 strains of random choose in 178 individual plants.
2. the exploitation of molecule marker
There is according to the 105th place of rice fecula q enzyme SBE3 gene the 16th exon the base mutation of T → C, design ARMS-PCR primer, see Fig. 1, black background base represents single nucleotide mutation base position, gray background represents four different primers sequence correspondence positions, and design primer sequence is as follows:
Forward outer primer sbe3-rs-O-F1:5 '-TGTGATGTGCTGGATTTGGT-3 ';
Reverse outer primer sbe3-rs-O-R1:5 '-GCTGTGGTTTTCATACCGTTC-3 ';
Forward inner primer sbe3-rs-I-F2:5 '-TGAAAGTCATGATCAAGCCCT-3 ';
Reverse inner primer sbe3-rs-I-R2:5 '-GCAATAGTTTTGTCACCAAATG-3 '.
3. the checking of molecule marker
1) extraction of oryza sativa genomic dna
Paddy rice miniprep dna extraction method, the report of Primary Reference McCouch etc. (1988), method is summarized as follows:
A) clip one small pieces blade 4 ~ 5cm, adds 700 μ L 1.5 × CTAB (containing 1.5%CTAB, 75mM Tris-HCl, 15mM EDTA, 1.05M NaCl), fully grinds;
B) homogenate is proceeded to the centrifuge tube of 1.5mL, after 56 DEG C of water-bath 20min, be cooled to room temperature;
C) chloroform of 700 μ L is added: primary isoamyl alcohol (v:v, 24:1), shakes up;
D) at full throttle (13200rpm) centrifugal 10min;
E) supernatant liquor is proceeded to new centrifuge tube, and add 100% alcohol of the precooling of two volumes, after static 20min, collected by centrifugation DNA;
F) remove supernatant, add 70% alcohol 600-800 μ L, jog centrifuge tube, 12000rpm is centrifugal;
G) remove supernatant, air-dry DNA, add 50 ~ 100 μ L distilled waters and dissolve, detect in ultraviolet spectrophotometer.Dilution DNA, prepares a set of DNA working solution, and its concentration is 50 ~ 100ng/ μ about L, and 4 DEG C of Refrigerator stores are for subsequent use.
2) amplification of molecule marker and electrophoresis detection
Above-mentioned four molecule marker primers sbe3-rs-O-F1, sbe3-rs-O-R1, sbe3-rs-I-F2 and sbe3-rs-I-R2 are added same PCR reaction system, and the DNA of Rice Germplasm Resources or breeding population plant is increased;
25 μ LPCR reaction systems comprise: DNA profiling, 50 ~ 100ng/ μ L, 1 μ L (representing that 1 μ L concentration is the DNA profiling of 50 ~ 100ng/ μ L, lower same); 10 × Buffer, 2.5 μ L; MgCl
2,25mM (reagent concentration, lower same), 1.5 μ L; DNTP, 2.5mM, 2.5 μ L; Forward outer primer sbe3-rs-O-F1,10 μMs, 0.5 μ L; Reverse outer primer sbe3-rs-O-R1,10 μMs, 0.5 μ L; Forward inner primer sbe3-rs-I-F2,10 μMs, 2.5 μ L; Reverse inner primer sbe3-rs-I-R2,10 μMs, 2.5 μ L; Taq polysaccharase, 5U/ μ L, 0.5 μ L, surplus ddH
2o supplies.
The reaction conditions of PCR circulation is: 94 DEG C of denaturation 5min; 94 DEG C of 30s; 57 DEG C of 30s; 72 DEG C of 40s; 35 circulations; Then 72 DEG C extend 10min.
Amplified production is added sample-loading buffer (10 × loading buffer) termination reaction, get 10 μ LPCR products, on the agarose of 1.2%, gel electrophoresis is separated pcr amplification product, and imaging record.
4. interpretation of result
To hypoglycemic rice No. 1, close positive 23 and hybridization F
1carry out four primer ARMS-PCR for individual plant to increase, amplified production is through the band of 1.2% agarose gel electrophoresis detection display three types.
See Fig. 2, wherein, all occurred in each DNA sample by the band of the 572bp of outer primer sbe3-rs-O-F1 and sbe3-rs-O-R1 amplification, can it not only play the effect (detecting DNA effectively be increased) of positive control, also effectively can reduce the amplification of non-specific PCR products and the formation of primer dimer simultaneously.
Close positive 23 contrast except band except amplifying 572bp, the band of 218bp can also be amplified, this band is increased by forward inner primer sbe3-rs-I-F2 and reverse outer primer sbe3-rs-O-R1 and produces, its specific amplification be the 105th of SBE3 the 16th exon for Nucleotide T allelotrope site (non-sbe3-rs genotype, Fig. 2: 1 ~ 2); Hypoglycemic rice No. 1 is containing sbe3-rs homozygous genotype, except the contrast band amplifying 572bp, also amplify the band of 397bp, this band is increased by forward outer primer sbe3-rs-O-F1 and reverse inner primer sbe3-rs-I-R2 and produces, its specific amplification be the 105th Nucleotide C allelotrope site (sbe3-rs genotype, see Fig. 2: 3 ~ 4) of SBE3 the 16th exon; Hypoglycemic rice No. 1 and close positive 23 hybridizes F
1three band can be amplified for individual plant, comprise 572bp, 397bp and 218bp (sbe3-rs gene hybridizing type, Fig. 2: 5 ~ 6).
In order to verify that four primer ARMS-PCR molecule markers are to the effect of high-resistance starch mutator gene sbe3-rs genotype detection further, to 15 strain hypoglycemic rice No. 1 and close positive 23 F of hybridizing
2individual plant carries out DNA cloning detection, 15 individual plants selected are cut with the CAPS marker enzyme developed before to identify (reference yang et al.A putative gene sbe3-rs for resistant starchmutanted from SBE3for starch branching enzyme in rice (Oryza sativa L.), Plos One2012,0043026), concrete detected result is see Fig. 3, and its detected result and CAPS enzyme trimscript remember that qualification result is completely the same.
Therefore, four primer ARMS-PCR molecule marking methods of the present invention effectively can distinguish three kinds of different genotype of paddy rice high-resistance starch mutator gene sbe3-rs, improve the efficiency of selection to its gene, accelerate the seed selection process of high-resistance starch rice varieties.
Claims (9)
1. a SNP detection method of paddy rice high resistant starch content mutator gene sbe3-rs, it is the molecule marking method of tetra-primer ARMS-PCR PCR and ARMS-PCR, comprises the steps:
1) ARMS design of primers
According to the mononucleotide base mutation that sbe3-rs gene has T → C at the 105th place of the 16th exon, design four gene-specific primers, be respectively: forward outer primer, oppositely outer primer, forward inner primer and reverse inner primer; Wherein, 3 ' end of forward inner primer and reverse inner primer terminates in mutational site to be detected, arranges base mispairing at 3rd reciprocal of the 3 ' end of forward inner primer and reverse inner primer;
2) ARMS-PCR amplification
By step 1) in four gene-specific primers add same PCR reaction system, and pcr amplification is carried out to the DNA of rice plant, carries out agarose gel electrophoresis;
3) according to electrophoresis result determination genotype
If there are 572bp and 218bp two characteristic bands, be not containing sbe3-rs genotype homozygote and low resistant starch content plant; If there is 572bp and 397bp two band, be high-resistance starch mutant gene sbe3-rs homozygote; If there is 572bp, 218bp and 397bp tri-band simultaneously, then it is the heterozygote of high-resistance starch mutant gene sbe3-rs.
2. SNP detection method according to claim 1, is characterized in that, described forward inner primer is non-sbe3-rs amplified allele inner primer, introduces the exchange of A-C in its 3 ' end the 3rd bit mismatch reciprocal; Reverse inner primer is mutant sbe3-rs genotype inner primer, introduces the exchange of C-A in its 3 ' end the 3rd bit mismatch reciprocal.
3. SNP detection method according to claim 1, it is characterized in that, in described ARMS-PCR amplification step, in PCR reaction system, the consumption of DNA profiling is 50 ~ 100ng, the final concentration of forward outer primer and reverse outer primer is respectively 0.2 ~ 0.6 μM, and the final concentration of forward inner primer and reverse inner primer is respectively 1 ~ 5 times of forward outer primer or reverse outer primer.
4. SNP detection method according to claim 1, it is characterized in that, in described ARMS-PCR amplification step, in PCR reaction system, the consumption of DNA profiling is 50 ~ 100ng, the final concentration of forward outer primer and reverse outer primer is respectively 0.4 ~ 0.6 μM, and the final concentration of forward inner primer and reverse inner primer is respectively 3 ~ 5 times of forward outer primer or reverse outer primer.
5. SNP detection method according to claim 1, is characterized in that, the cumulative volume of described PCR reaction system is 10 ~ 50 μ L; Wherein, DNA profiling, 50 ~ 100ng; 10 × Buffer, 1 ~ 5 μ L; Mg
2+final concentration is 1.5mM ~ 2.0mM; DNTP final concentration is 0.2 ~ 0.25mM; The final concentration of forward outer primer and reverse outer primer is respectively 0.2 ~ 0.6 μM; The final concentration of forward inner primer and reverse inner primer is respectively 1 ~ 5 times of forward outer primer or reverse outer primer; Taq polysaccharase consumption is 0.5U ~ 5U, surplus ddH
2o supplies.
6. SNP detection method according to claim 1, is characterized in that, the cumulative volume of described PCR reaction system is 25 μ L, wherein, and DNA profiling, 50 ~ 100ng; 10 × Buffer, 2.5 μ L; Mg
2+final concentration is 1.5mM; DNTP final concentration is 0.25mM; The final concentration of forward outer primer and reverse outer primer is respectively 0.2 μM; The final concentration of forward inner primer and reverse inner primer is respectively 5 times of forward outer primer or reverse outer primer, Taq polysaccharase 2.5U, surplus ddH
2o supplies.
7. the SNP detection method according to any one of claim 1 ~ 6, is characterized in that, the sequence of four described gene specific ARMS primers is:
Forward outer primer sbe3-rs-O-F1:5 '-TGTGATGTGCTGGATTTGGT-3 ';
Reverse outer primer sbe3-rs-O-R1:5 '-GCTGTGGTTTTCATACCGTTC-3 ';
Forward inner primer sbe3-rs-I-F2:5 '-TGAAAGTCATGATCAAGCCCT-3 ';
Reverse inner primer sbe3-rs-I-R2:5 '-GCAATAGTTTTGTCACCAAATG-3 '.
8. the SNP detection method according to any one of claim 1 ~ 6, is characterized in that, when carrying out described ARMS-PCR amplification, the condition of circulating reaction is: 94 DEG C of denaturation 5min; 94 DEG C, 30s; 56 ~ 58 DEG C, 30s; 72 DEG C, 40 ~ 60s; 35 circulations; Then 72 DEG C extend 10min.
9. SNP detection method according to claim 7, is characterized in that, when carrying out described ARMS-PCR amplification, the condition of circulating reaction is: 94 DEG C of denaturation 5min; 94 DEG C, 30s; 56 ~ 58 DEG C, 30s; 72 DEG C, 40 ~ 60s; 35 circulations; Then 72 DEG C extend 10min.
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| CN109448789A (en) * | 2018-09-17 | 2019-03-08 | 上海派森诺生物科技股份有限公司 | A kind of automated analysis method of the special SNP site of population based on perl language |
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