CN102251042A - Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same - Google Patents
Method for quickly detecting purity of seeds of bottle gourd varieties and kit used by same Download PDFInfo
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- CN102251042A CN102251042A CN2011102074561A CN201110207456A CN102251042A CN 102251042 A CN102251042 A CN 102251042A CN 2011102074561 A CN2011102074561 A CN 2011102074561A CN 201110207456 A CN201110207456 A CN 201110207456A CN 102251042 A CN102251042 A CN 102251042A
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Abstract
The invention discloses a method for quickly detecting the purity of the seeds of bottle gourd varieties and a kit used by the same. The method comprises the following steps: (I) partial sequencing of bottle gourd genomic DNA and simple sequence repeat (SSR) primer development and design; (II) calculation of the polymorphism information content (PIC) of SSR primers and screening of combination of diagnostic SSR primers; (III) preparation of standardized DNA fingerprint of the five main cultivated bottle gourd varieties; and (IV) detection of bottle gourd samples to be detected. In the method, because of the use of the 8 pairs of diagnostic SSR primers which are designed independently, the capacity of identifying similar varieties is increased, the efficiency of detection is improved, the detection time is reduced to 4 to 5 hours from 50 to 60 hours which are required by traditional phenotype identification, the detection cost is lowered to about 1/6 of that required by the conventional method, and the accuracy of identification is improved. The method can be promoted and used in seed breeding and seed enterprises.
Description
Technical field
The invention belongs to the crop biological technical field, especially belong to the method and the test kit thereof that utilize diagnostic SSR molecule marker combination rapid detection bottle gourd principal item seed purity.
Background technology
Bottle gourd is the important melon vegetables in summer of China, liked by the people.Scientific research institutions, enterprise that China is engaged in bottle gourd breeding, seed production and operation are numerous, and annual all have new bottle gourd new variety to breed release.The bottle gourd kind kind of using in the current production is numerous; genetic similarity between commercial variety is more and more high; different name of the same race, xenogenesis phenomenon of the same name are serious day by day; seed intellecture property and quality dispute happen occasionally; be necessary to research and develop a kind of energy early stage, annual, fast, accurately identify the method for the bottle gourd kind true and false and purity; serving the needs of bottle gourd breeding, seed operation and production, and protect breeder's legitimum conscientiously.
China's vegetable variety comprises that the bottle gourd seed true and false and purity evaluation rely on the field phenotype to identify mostly at present.But this evaluation required time is grown (generally needing 50-60 days time), and is subjected to the restriction in season, and the phenotype of crop easily changes with the variation of envrionment conditions, particularly for identifying between the less kind of some morphological differencess that difficulty is bigger.
The dna molecular marker technology has been used to differentiate the difference on molecular level between the different varieties at home and abroad.Owing between each kind DNA of same species, have a large amount of polymorphism marks, each kind all has the unique tag that is different from other kind, be the DNA genetic fingerprint that the combination of some DNA fragment specific just is called this kind, the fingerprint fragment of each kind uniqueness constitutes the dna fingerprinting of these species.Compare with traditional identifying,, have and to carry out in any stage of plant strain growth, identify the advantages such as influence that are subjected to genetic expression and envrionment conditions fast and not based on the fingerprint pattern technology of dna level difference according to phenotypic character.
The SSR molecule marker is to utilize a large amount of little satellite tumor-necrosis factor glycoproteins design primer that exists in the eukaryotic gene group, by the placed in-line tumor-necrosis factor glycoproteins of pcr amplification, according to the difference of multiplicity between same species different genotype of little satellite, discloses length polymorphism.SSR is because distribute wide, stability and polymorphic rate height, and simple to operate, good reproducibility, lower etc. to the DNA specification of quality is described as the star of s-generation molecule marker.The SSR molecular marking technique has overcome RAPD stability and poor repeatability, AFLP technical fee costliness, complicated operation, to the purity of DNA and the very high shortcoming of specification of quality of restriction endonuclease.
Can obtain species dna sequence dna information and then design and develop the SSR primer by order-checking wholly or in part the species gene group.Invention is based on the seed purity method for quick of SSR molecular marking technique, will be to standard bottle gourd seed industry, and protection bottle gourd breeder's legitimate rights and interests and peasant's vital interests promote agricultural produce, increasing peasant income significant.
Summary of the invention
The present invention seeks to, identify at present bottle gourd variety seeds purity rely on mostly that phenotype observational technique existing qualification time in field is long, the phenotype that is subjected to seasonal restriction and crop is easily with defectives such as changes in environmental conditions, provide a kind of energy early stage, annual, fast, accurately identify the detection method of the bottle gourd kind true and false and purity; Another object of the present invention provides a kind of test kit of convenient enforcement aforesaid method.
The object of the invention is achieved by the following technical programs.
1, the method for rapid detection bottle gourd variety seeds purity, this method is carried out according to the following steps:
(1) order-checking of the part of bottle gourd genomic dna and the design of SSR primer development:
1) to plant bottle gourd kind " the long melon in Hangzhou " be material with main, and the application sequenator carries out the once sequencing of 1/4 flux to its genomic dna, obtains 150,253 sequencing sequences;
2) operation Newbler sequence assembly program is carried out sequence assembly to institute's calling sequence and is repeated to remove, and obtains 86,561 of break-even dna sequence dnas;
3) move Mreps 2.5SSR Sequence Identification program, contain the sequence section of SSR on the identification of dna sequence, design the PCR primer of crossing over the SSR site simultaneously; Transfer to the synthetic 100 pairs of SSR primers of Shanghai Sani biotech company;
(2) SSR primer polymorphism information content PIC calculates and the screening of diagnostic SSR combination of primers:
1) extract main bottle gourd kind DNA in 44 current productions, with the DNA of these materials of 100 pairs of primers difference of synthetic pcr amplification, the PCR reaction volume is 12.5 microlitres, and annealing temperature is 52 ℃;
2) the amplification situation of every pair of primer of statistics is carried out 0 or 1 assignment to amplified band, and the polymorphism information content that calculates each primer is PIC;
3) carry out comprehensive evaluation from the stability of amplified band, easy resolution degree and three aspects of primer polymorphism PIC index of polymorphic bands, choose LSR011, LSR015, LSR040, LSR045, LSR047, LSR056, LSR063, LSR077 totally 8 pairs of primers as the diagnostic combination of primers, specifically:
(3) preparation of 5 main breed standard DNA of bottle gourd finger printing:
With the listed 8 pairs of diagnostic SSR primers of form increase respectively ' Zhejiang Pu No. 2 ', ' Zhejiang Pu No. 6 ', ' the long melon in Anji ', ' ground, Xiaoshan Pu ' and ' behind the DNA of long melon ' 5, a Hangzhou main breed standard, with amplified production at acrylamide: carry out electrophoretic separation on 8% non-denaturing polyacrylamide gel of methylene diacrylamide=29: 1, silver dyes takes a picture and the record result, obtains the main breed standard finger-print after the Photoshop software processes;
(4) detection of bottle gourd sample to be measured:
Require to differentiate that as testing sample target variety is one of (three) 5 main breeds of step, then only need with this testing sample carry out extracting genome DNA, and carry out same step (two) 1 with the listed 8 pairs of SSR primers of above table) PCR react, gel electrophoresis, the silver of step (three) dye, obtain finger printing after, compare with 5 main breed standard finger-prints, with the true and false of determining testing sample and and then calculate its purity; As require to differentiate that target variety is non-above-mentioned 5 main breeds, then need testing sample and the standard model that requires to differentiate target variety, prepare finger printing respectively and compare, with the true and false of determining the testing sample seed and and then calculate its seed purity as follows:
Seed purity=(detecting gained pure dan number/detection seed sum) * 100%.
2, the test kit of rapid detection bottle gourd variety seeds purity, this test kit comprise box body and 12 eppondorf pipe, 25mM MgCl are housed in 4 respectively therein
210mM dNTP, 10X PCR damping fluid and 5U/ μ L Taq archaeal dna polymerase is characterized in that in all the other 8 eppondorf pipes, be equipped with respectively and contain the diagnostic SSR detection primer LSR011 that forward and reverse concentration is 1mM, LSR015, LSR040, LSR045, LSR047, LSR056, LSR063, LSR077; In addition, also be equipped with in the box body and be printed on the listed bottle gourd of claim 1 step (three) ' Zhejiang Pu No. 2 ', ' Zhejiang Pu No. 6 ', ' G5-3 ', ' G26 ' and ' picture of long melon ' 5, a Hangzhou main breed standard DNA finger printing.
The invention has the beneficial effects as follows:
(1) 8 pairs of diagnostic SSR combination of primers filtering out of application autonomous design have strengthened the ability of differentiating approximate kind.The approximate kind of process increases to compare (Fig. 1) and detect in a large number and facts have proved that this overlaps primer energy service area and divides production to go up common different bottle gourd genotype, can satisfy the needs that main bottle gourd kind in the current production carried out true and false detection.
(2), improved the accuracy of identifying greatly by direct evaluation to the bottle gourd genetic material.The present invention carries out the evaluation of bottle gourd seed purity on dna level, avoided the error that may bring of authentication methods institute indirectly such as phenotypic evaluation, biochemical identification, has easy to operately, and the characteristics of good reproducibility have reliability and authority highly.
(3) efficient and the standardization level that detect have been improved.The present invention will detect with primer and detection reagent and provide so that kit form is supporting, and detected result is reliable and stable, is easy to realize stdn, and only need 4-5 hour detection time, once can detect simultaneously duplicate samples up to a hundred.General seed quality inspection chamber only need possess conventional molecular biology equipment just can carry out actual detected by the schedule of operation that provides.PCR reaction component (MgCl
2, Buffer, dNTP, Taq enzyme) both can provide by this test kit, also can purchase separately by user oneself, easy to use, and detect with low costly, be about the sixth (seeing embodiment 3) of traditional phenotypic evaluation.
Description of drawings
Fig. 1 is the standard DNA finger printing of 5 main breeds of bottle gourd.
1:LSR011 wherein, 2:LSR015,3:LSR040,4:LSR045,5:LSR047,6:LSR056,7:LSR063,8:LSR077; Arrow shows the main difference part of each kind dna fingerprint.
Embodiment
The present invention also is described in further detail in conjunction with the accompanying drawings by following examples, but should be appreciated that the present invention is not placed restrictions on by following content.
Sequenator: German Roche Holding Ag ' 454 ' sequenator.
Embodiment 1:(uses test kit to requiring to differentiate that target variety is the detection of one of main breed testing sample)
Carry out according to the following steps:
(1) DNA extraction: seed to be checked is disseminated in vermiculite is housed: in the seedling culture hole plate of peat=1: 2 matrix, place 25-30 ℃ to grow seedlings, when treating that first pair of true leaf is open and flat, get blade 0.1 gram by individual plant, add the liquid nitrogen grind into powder, extract DNA with conventional CTAB method, standby;
(2) SSR primer amplification and electrophoresis detection: 8 pairs of SSR diagnostic primers that provide with test kit carry out pcr analysis, and reaction volume is 12.5 μ L; Each component of PCR is 10 * buffer, 1.25 μ L, 25mM MgCl
20.75 μ L, 2.5mM dNTPs 1 μ L, Taq enzyme (5U/ μ L) 0.1 μ L, template DNA 10ng adds water to 12.5 μ L; The SSR response procedures is: 94 ℃ of pre-sex change 3min, and 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 50sec, circulate 35 times, and last 72 ℃ are extended 5min; In the enterprising performing PCR amplification of PTC-225 amplification instrument, amplified production is at acrylamide: carry out electrophoretic separation on 8% non-denaturing polyacrylamide gel of methylene diacrylamide=29: 1, carry out silver then and dye colour developing, digital camera photographic recording result obtains the dna fingerprinting of material to be checked;
(3) dna fingerprinting comparison and seed purity are calculated: the bottle gourd ' Zhejiang Pu No. 2 ' that dna fingerprinting and the test kit of material to be checked provided, ' Zhejiang Pu No. 6 ', ' the long melon in Anji ', ' Xiaoshan Pu ' reach that ' long melon ' 5, a Hangzhou main breed standard DNA finger printing is compared; The target variety that for example provides material person to be checked to require to differentiate is ' the long melon in Hangzhou ' of one of 5 main breeds, and the finger printing comparison result both match fully, then this material to be checked is real ' the long melon in Hangzhou ', and and then use formula: seed purity=(detecting gained pure dan number/detection seed sum) * 100% calculates the purity of seed to be checked; Otherwise be pseudo-seed.
Embodiment 2:(uses test kit to requiring to differentiate that target variety is the detection of one of non-main breed testing sample)
Carry out according to the following steps:
(1) DNA extraction: with the standard seed of seed to be detected and requirement discriminating target variety, disseminate respectively in vermiculite is housed: in the seedling culture hole plate of peat=1: 2 matrix, place 25-30 ℃ to grow seedlings, when treating that first pair of true leaf is open and flat, get blade 0.1 gram by individual plant, add the liquid nitrogen grind into powder, extract DNA with the CTAB method, standby;
(2) SSR primer amplification and electrophoresis detection: with embodiment 1;
(3) respectively according to seed to be detected and standard variety seed pcr amplification and electrophoresis detection result, be built into the dna fingerprinting of seed to be detected and standard variety seed;
(4) dna fingerprinting comparison and seed purity are calculated: both dna fingerprintings are compared, the identical pure dan that is of the two banding pattern, and and then use formula: seed purity=(, detect gained pure dan number/detection seed sum) * 100% calculates the purity of seed to be checked; Otherwise be pseudo-seed.
Embodiment 3:(uses the simultaneous test of tradition and the inventive method detection bottle gourd seed purity)
Require to differentiate that target variety is one of 5 main breeds person, then undertaken by embodiment 1 method;
Require to differentiate that target variety is one of non-main breed person, then undertaken by embodiment 2 methods;
Traditional detection method carries out according to the following steps:
1, get kind to be detected and require to differentiate that the standard seed of target variety is seeded in vermiculite respectively: in the 8X8cm nutrition pot of peat=1: 2 matrix, each nutrition pot is broadcast 2 seeds, treats to carry out thinning when cotyledon is open and flat, and each nutrition pot stays 1 strain; Be colonizated in the booth in 1 heart stage of 2 leaves, line-spacing 70cm, spacing in the rows 40cm is more than every kind 100 strains;
2, cultivation technique is carried out daily field management routinely, and begins to carry out characteristic contrast in seedling stage;
3, enter the feature that the after date of bearing fruit examines this standard variety, and the difference of variety characteristic more to be measured one by one and standard variety, to determine the true and false of seed, use following formula after detection is all finished: seed purity=(detecting gained pure dan number/detection seed sum) * 100% calculates the seed purity of each kind to be measured.
The comparing result of table 1 the present invention and traditional technique in measuring bottle gourd seed purity
Claims (2)
1. the method for rapid detection bottle gourd variety seeds purity is characterized in that carrying out according to the following steps:
(1) order-checking of the part of bottle gourd genomic dna and the design of SSR primer development:
1) to plant bottle gourd kind " the long melon in Hangzhou " be material with main, and the application sequenator carries out the once sequencing of 1/4 flux to its genomic dna, obtains 150,253 sequencing sequences;
2) operation Newbler sequence assembly program is carried out sequence assembly to institute's calling sequence and is repeated to remove, and obtains 86,561 of break-even dna sequence dnas;
3) move Mreps 2.5SSR Sequence Identification program, contain the sequence section of SSR on the identification of dna sequence, design the PCR primer of crossing over the SSR site simultaneously; Transfer to the synthetic 100 pairs of SSR primers of Shanghai Sani biotech company;
(2) SSR primer polymorphism information content PIC calculates and the screening of diagnostic SSR combination of primers:
1) extract main bottle gourd kind DNA in 44 current productions, with the DNA of these materials of 100 pairs of primers difference of synthetic pcr amplification, the PCR reaction volume is 12.5 microlitres, and annealing temperature is 52 ℃;
2) the amplification situation of every pair of primer of statistics is carried out 0 or 1 assignment to amplified band, and the polymorphism information content that calculates each primer is PIC;
3) carry out comprehensive evaluation from the stability of amplified band, easy resolution degree and three aspects of primer polymorphism PIC index of polymorphic bands, choose LSR011, LSR015, LSR040, LSR045, LSR047, LSR056, LSR063, LSR077 totally 8 pairs of primers as the diagnostic combination of primers, specifically:
(3) preparation of 5 main breed standard DNA of bottle gourd finger printing:
With the listed 8 pairs of diagnostic SSR primers of form increase respectively ' Zhejiang Pu No. 2 ', ' Zhejiang Pu No. 6 ', ' the long melon in Anji ', ' ground, Xiaoshan Pu ' and ' behind the DNA of long melon ' 5, a Hangzhou main breed standard, with amplified production at acrylamide: carry out electrophoretic separation on 8% non-denaturing polyacrylamide gel of methylene diacrylamide=29: 1, silver dyes takes a picture and the record result, obtains the main breed standard finger-print after the Photoshop software processes;
(4) detection of bottle gourd sample to be measured:
Require to differentiate that as testing sample target variety is one of (three) 5 main breeds of step, then only need with this testing sample carry out extracting genome DNA, and after the PCR reaction, gel electrophoresis, the silver that carry out same step (two) (1) with the listed 8 pairs of SSR primers of above table dyes, obtains finger printing, compare with 5 main breed standard finger-prints, with the true and false of determining testing sample and and then calculate its purity; As require to differentiate that target variety is non-above-mentioned 5 main breeds, then need testing sample and the standard model that requires to differentiate target variety, prepare finger printing respectively and compare, with the true and false of determining the testing sample seed and and then calculate its seed purity as follows:
Seed purity=(detecting gained pure dan number/detection seed sum) * 100%.
2. the test kit of rapid detection bottle gourd variety seeds purity, this test kit comprise box body and 12 eppondorf pipes, 25mM MgCl are housed in 4 respectively therein
210mM dNTP, 10X PCR damping fluid and 5U/ μ L Taq archaeal dna polymerase is characterized in that in all the other 8 eppondorf pipes, be equipped with respectively and contain the diagnostic SSR detection primer LSR011 that forward and reverse concentration is 1mM, LSR015, LSR040, LSR045, LSR047, LSR056, LSR063, LSR077; In addition, also be equipped with in the box body and be printed on the listed bottle gourd of claim 1 step (three) ' Zhejiang Pu No. 2 ', ' Zhejiang Pu No. 6 ', ' the long melon in Anji ', ' ground, Xiaoshan Pu ' and ' picture of long melon ' 5, a Hangzhou main breed standard DNA finger printing.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667487A (en) * | 2013-12-03 | 2014-03-26 | 浙江省农业科学院 | Method for identifying loquat varieties based on EST-SSR (expressed sequence tag-simple sequence repeat) marker |
| CN110923357A (en) * | 2019-12-31 | 2020-03-27 | 浙江省农业科学院 | Bottle gourd core molecular marker set developed based on KASP technology and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701249A (en) * | 2009-11-19 | 2010-05-05 | 浙江省农业科学院 | Molecular marker method for detecting resistance of powdery mildew of bottlegourd and kit thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101701249A (en) * | 2009-11-19 | 2010-05-05 | 浙江省农业科学院 | Molecular marker method for detecting resistance of powdery mildew of bottlegourd and kit thereof |
Non-Patent Citations (2)
| Title |
|---|
| 刘成平: "《瓠瓜EST-SSR、SRAP、ISSR和RAPD反应体系的建立及其纯度鉴定应用的研究》", 《中国优秀硕士学位论文全文数据库农业科技辑（月刊）》 * |
| 王玲平等: "《AFLP分子标记技术在浙蒲2号种子纯度快速鉴定中的应用》", 《浙江农业学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103667487A (en) * | 2013-12-03 | 2014-03-26 | 浙江省农业科学院 | Method for identifying loquat varieties based on EST-SSR (expressed sequence tag-simple sequence repeat) marker |
| CN103667487B (en) * | 2013-12-03 | 2015-10-21 | 浙江省农业科学院 | Based on the method for EST-SSR Marker Identification Loquat Cultivars |
| CN110923357A (en) * | 2019-12-31 | 2020-03-27 | 浙江省农业科学院 | Bottle gourd core molecular marker set developed based on KASP technology and application thereof |
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