CN107217101A - Differentiate and really weigh the detection method of identification suitable for variety of crops molecular identity - Google Patents
Differentiate and really weigh the detection method of identification suitable for variety of crops molecular identity Download PDFInfo
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Abstract
The invention discloses a kind of detection method for differentiating suitable for variety of crops molecular identity and really weighing identification.A variety of methods and techniques are comprehensively utilized, carrying out comprehensive, multi-angle to marker site assesses, it is determined that applicable core and extension Sites Combination, under the premise of detection accuracy is met, prominent detection efficiency.(1) core Sites Combination：Efficiently to distinguish different cultivars as target, the features such as possessing high stability, high kind separating capacity, compatible with multiple platforms, number tens to hundreds of.(2) Sites Combination is extended：Using genetic similarty between accurate evaluation kind as target, possess be uniformly distributed, compatible chip platform the features such as, number is thousands of to tens of thousands of.The use in (3) two groups of sites：In cultivar identification, first detected using core bit point, such as can not effectively distinguish to be measured and control sample, then continue to detect using extension site.The detection method of the present invention is built suitable for variety of crops standard DNA finger print data, and molecular identity differentiates and really power identification etc..
Description
Technical field
The invention belongs to biology field, specifically, it is related to a kind of variety of crops molecular identity that is suitable to and differentiates
With the detection method of true power identification.
Background technology
State is using agriculture as this, and agriculture is to plant as elder generation.Breeding and the fast development for planting industry, the variety of crops such as corn, paddy rice, wheat
Quantity is sharply increased, and is all brought challenges to Variety identification and seed management.It is counterfeit set, how hash, a product are several, fastens one person's story upon another person
Outstanding problem, these problems seriously govern the modern development for planting industry.Solve these problems be at all to variety of crops,
Breeding material etc. effectively identify, monitor and protect.
Traditional field trapping test by cycle length, cost height, ambient influnence, be difficult to be formed the limitation such as normal data,
The demand of variety of crops identification can not be met.In the urgent need to utilizing advanced molecular engineering, to each kind one clearly, have
Effect, molecular identity card easy to identify.It is to implement cultivar identification, monitoring to carry out identification to variety of crops from DNA molecular level
With the basis of protection, be ensure market fair competition in the urgent need to.DNA molecular identification technology is applied to by many developed countries
In field crop and garden crop cultivar identification, international Seed Inspection association (ISTA), UPOV
(UPOV), international organization's recommendation DNA identification technologies such as international seed alliance (ISF) build auxiliary with cultivar identification for varieties systematics
Aid method.
Crops DNA molecular identification technology development experience three important stages, the first generation mark of the nineties in last century
Note technology such as RFLP (RFLP) and RAPD (DNArandom amplified polymorphic DNA), the second generation that the beginning of this century rises
Labelling technique SSR (simple repeated sequence), third generation labelling technique SNP (mononucleotide polymorphic) and INDEL in recent years
(insertion and deletion is polymorphic).Wherein SSR, SNP, INDEL are due to being that codominant inheritance, chromosome mapping understand, can realize high pass
Amount automation is in crops Molecular Identification field by system research or popularization and application.
Based on SSR molecular marker：China has promulgated the professional standard of corn variety identification in 2007 first, by the end of
There are within 2015 16 crops to promulgate SSR fingerprint identification standards；China's staple crops kind is carried out based on above-mentioned standard
Standard DNA fingerprint construction work, wherein corn, paddy rice have completed more than 20,000 parts and 5,000 multiple sample SSR fingerprints are built；And
The staple crops such as corn, paddy rice SSR Molecular Identifications standard and fingerprint database are widely used in variety certification, registration, product
Plant power protection, market monitorings, judicial expertise etc..External variety of crops DNA fingerprint is built mainly to be carried out under UPOV frameworks
Plant variety right protects the database work of sample, as France sets up 1537 parts of corn variety SSR fingerprints, and Germany sets up 480 parts of Europe
Continent wheat breed SSR fingerprints etc..
Based on SNP marker：With the continuous progress of modern molecular biology, the completion of multiple kinds of crops genome sequencing
And the rise of high flux DNA typing technology etc., it is that the research and development of the New molecular marker identification technology such as SNP bring opportunity, it is main
It is embodied in the following aspects.First, based on genomics Research foundation, the staple crops such as corn have developed largely
Excellent SNP, IDNEL, SSR site, and carry out dependent evaluation checking.Second, since 2010, each crop high density parting
Chip is issued successively, maizeSNP50K, maizeSNP3072, maizeSNP600K, 6H90K in such as corn, in paddy rice
660K and 90K in Rice 6K, 50K, 90K, wheat etc..3rd, the highdensity chip gene typing method in site is such as
Illumina Infinium, Affymetrix Axiom, the high-throughout typing method LGC KASP of sample, Array Tape etc.
It is applied in crops Molecular Identification technical research.
Although variety of crops Molecular Identification have 20 years of researches experiences, abundant Research foundation, variety managements,
Extensive use in market monitorings, judicial expertise, but existing standard of perfection or technology are relatively independent, can only be in a class or a few classes
Played a role in detection demand, without a set of comprehensive and systematic detection method.
The content of the invention
In order to change the present situation of variety of crops Molecular Detection, comprehensively solve above mentioned problem, present invention foundation molecular genetic
, genomics correlation theory, binding molecule detection practice, it is proposed that one kind is suitable for the discriminating of variety of crops molecular identity and really
Weigh the system detecting method of identification.
The design of the present invention is as follows：Genomics, genetic statistics and high flux genotyping technique are comprehensively utilized,
Full-length genome horizontal alignment point carries out comprehensive, multi-angle deep assessment checking, differentiates according to kind identity and really power identification
Etc. the requirement of different testing goals, it is determined that applicable core and extension Sites Combination, under the premise of detection accuracy is met, protrude inspection
Efficiency is surveyed, it is workable, with practical application value.(1) core Sites Combination：It is excellent during DNA molecular marker method identification of species
The one group of site first selected, efficiently to distinguish different cultivars as target, possesses high stability, high kind separating capacity, compatibility many
Platform, take into account and be uniformly distributed and many laboratory proofing results, number of loci is tens to hundreds of magnitudes, can distinguish a kind of work
The kind material of thing more than 90%, for improved variety (seed of single cross, self-mating system, DH pure lines etc.) standard fingerprint structure, kind
Identity discriminating etc..(2) Sites Combination is extended：It is available outside core Sites Combination during DNA molecular marker method identification of species
One group of site, using hereditary similarity degree between accurate evaluation kind as target, it is desirable to which site is uniformly distributed, compatible middle-high density core
Piece, loci polymorphism is taken into account, thousands of to tens of thousands of magnitudes of number of loci can distinguish a kind of kind material of crop more than 99%
Material, identification etc. is weighed for approximate kind, derivation kind really.The use of (3) two groups of Sites Combinations：When carrying out cultivar identification,
Detected first by core Sites Combination, such as can not effectively distinguish testing sample and control sample, then using extension site
Proceed detection, based on the relation between genetic similarty parameter decision testing sample and control sample.
In order to realize the object of the invention, what the present invention was provided is suitable to the discriminating of variety of crops molecular identity and really power identification
Detection method, comprises the following steps：
S1, core DNA molecular labeling Sites Combination acquisition；
S2, the acquisition for extending DNA molecular marker Sites Combination；
S3, two groups of DNA molecular marker Sites Combinations use.
In step S1, the core DNA molecular labeling includes efficiently distinguishing different cultivars, possesses high stability, Gao Pin
Kind of separating capacity, compatible with multiple platforms, take into account and be uniformly distributed and many laboratory proofing results, number of loci is tens to hundreds of amounts
Level, can distinguish a kind of kind material of crop more than 90%, available for improved variety (seed of single cross, self-mating system, DH pure lines
Deng) standard fingerprint build, kind identity differentiate etc. molecular labeling.
In step S2, the extension DNA molecular marker refers to outside step S1 core DNA molecular labeling Sites Combination
Available molecular labeling, can hereditary similarity degree between accurate evaluation kind, site is uniformly distributed, compatible middle-high density core
Piece, loci polymorphism is taken into account, thousands of to tens of thousands of magnitudes of number of loci can distinguish a kind of kind material of crop more than 99%
Material, available for approximate kind, derives from the molecular labeling that kind really weighs identification etc..
In step S3, when carrying out cultivar identification, the core DNA molecular labeling Sites Combination first by step S1 is carried out
Detection, such as can not effectively distinguish testing sample and control sample, then using step S2 extension DNA molecular marker Sites Combination after
It is continuous to be detected, based on the relation between genetic similarty parameter decision testing sample and control sample.
Variety of crops identifies the determination in core site and extension site：
The selection of DNA molecular marker type：Codominance, rich polymorphism, experimental repeatability is good, and data are easy to integrate altogether
Enjoy, site on genome distribution situation, it is known that corresponding typing method mature and reliable.Based on mentioned above principle, recommend SSR, SNP,
The DNA molecular marker type that the types such as INDEL are identified as variety of crops.
Core site and the determination in extension site：(1) site is extended：From the excellent marker site pond of the crops such as corn
In, with reference to the analysis of biological information means such as haplotype block, linkage disequilibrium, obtain hereditary equally distributed non-redundant candidate site
Set.Polymorphic type sample (cenospecies, self-mating system/routine are planted) is chosen, using chip, contour throughput platform progress candidate bit is sequenced
Point experimental evaluation, is mainly analyzed from data pick-up rate, genotyping effect, triplet feature.Finally, statistics is utilized
Method determines to be suitable to the extension bits point set that breeding material is really weighed, formation middle-high density authentication chip etc..Number is thousands of to several
Ten thousand.(2) core site：The demand differentiated based on improved variety identity, using platforms such as superchip, sequencings, to full-page proof
This (based on known kind) carries out test assessment, candidate's core site that screening-gene type parting effect is good, polymorphism is high.Knot
Many laboratories, multi-platform individual authentication result are closed, the site set that a set of compatible with multiple platforms, stability are high, polymorphism is high is determined.
Optimal Sites Combination (can distinguish more than 90% known kind) is screened based on Analysis of Genetic Algorithms, and carries out random sample test,
Sites Combination is optimized and revised, consequently recommended a set of high accuracy, high stability, high kind separating capacity, compatible with multiple platforms are taken into account
Equally distributed core Sites Combination.Number of loci is tens to hundreds of.
The technology path that middle peasant's Identifying Crop Cultivars core of the present invention and extension Sites Combination are determined is shown in Fig. 1.
The step of variety of crops is identified：
Whether the 1st, identify testing sample A is control sample B：Detected first with core bit point, if core site
Combination difference number of sites, which reaches, can be determined that sample A is different from sample B, can termination detection experiment；If using core site group
Conjunction not can determine that sample A is different from sample B, then continue to carry out detection experiment using extension Sites Combination, using genetic similarty
Mode judges sample A and sample B relation, determines that both relations belong to different cultivars, query and derive from kind also according to threshold value
It is to derive from kind.
2nd, it is which known kind belonged to identify unknown testing sample C：Sample C fingerprint is set up first with core site
Data, C fingerprint is compared two-by-two with known breed standard fingerprint database, if sample C with it is known in database
Kind more can be determined that as difference then the sample may be with specific new varieties two-by-two；If sample C with it is a certain
Individual or multiple kinds can not be determined as difference, then testing sample C is continued with into extension bits point with serial near sample is examined
Test is tested, and is analyzed using genetic similarty, it is determined that with sample C genetic background similarity highest kinds, and determine be matter
Doubt and derive from or derive from kind.
Middle peasant's Identifying Crop Cultivars technology path of the present invention is shown in Fig. 2.
The detection method that the present invention is provided can be applied in following any field.(1) variety of crops standard DNA
Finger print data is built；(2) variety of crops molecular identity differentiates；(3) variety of crops really weighs identification etc..This method can be in farming
The fields such as thing variety certification, registration, protection, market monitorings, judicial expertise are applied, and are varieties distribution and to set up fair competition
Market environment technical support and guarantee are provided.
Above-mentioned core DNA molecular labeling site and combinations thereof and/or extension DNA molecular marker site and combinations thereof can be prepared
Into chip, crops Molecular Identification/detection is carried out using constructed chip platform.
The present invention also provides the core, extension Sites Combination in variety of crops standard DNA fingerprint database structure
Application.
The present invention also provides the core, extension Sites Combination in variety of crops molecular identity differentiates or kind is really weighed
Application.
The present invention differentiates according to variety of crops identity and really the different testing goals such as power identification require to determine core and expansion
The method for opening up Sites Combination, cultivar identification technology path is carried out using core and extension bits point.(1) solved with a small amount of core site
Standard fingerprint storehouse builds the discriminating problem with most kinds, realizes reliable and stable, convenient and efficient, is particularly greatly lowered
The problems such as cost；The approximate Variety identification of genetic affinity relatively near, similarity height etc. is solved with superchip or multidigit point data, with
And breeding material is the problems such as really weigh.(2) core Sites Combination is determined：It is mainly used in improved variety standard fingerprint structure, kind body
Part discriminating etc., efficiently to distinguish different cultivars as target, proposes its site screening principles and requirements, process, site in the present invention
Number range.(3) extension Sites Combination is determined：It is mainly used in approximate kind, derivation kind, breeding material and weighs identification etc. really,
Using hereditary similarity degree between accurate evaluation kind as target, its site screening principles and requirements, process, site are proposed in the present invention
Number range.(4) applied with a small amount of core site and extension site：Based on variety of crops present situation and detection practical experience, carry
Go out the two step system detecting methods for recycling extension bits point to be detected from core site first.
The present invention at least has following advantages and beneficial effect：
In view of the problem of variety of crops Molecular Identification is present at present, the present invention provides a kind of suitable for variety of crops molecule
A kind of system detecting method that identity differentiates and really power is identified, comprehensive farming article can be applied to compared with existing authentication method
Plant in the demands such as identity differentiates, breeding material is really weighed, approximate or derivation cultivar identification, expanded the application of Molecular Detection；
Core and the flexible Application in extension site, on the basis of raising efficiency reduces cost, breach the limitation of number of loci, realize
In the cultivar identification of genomic level；The present invention differentiates for variety of crops molecular identity, breeding material is really weighed and germplasm
Stock assessment provides new idea and method, and this method can be in Crop breed audit, registration, protection, market monitorings, the administration of justice
Applied in identification, be that varieties distribution and the market environment for setting up fair competition provide technical support and guarantee.
Brief description of the drawings
Fig. 1 is that variety of crops of the present invention identifies that core and extension bits point assess determination Technology Roadmap.
Fig. 2 is variety of crops identification technology route map of the present invention.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual (Sambrook J＆Russell DW,
Molecular Cloning:A Laboratory Manual, 2001), or according to the condition of manufacturer's specification suggestion.
The acquisition of the core and extension SNP marker Sites Combination of the corn of embodiment 1 (diplont) cultivar identification
1st, the determination of Sites Combination is extended：
(1) the excellent SNP site pond of corn：The domestic and international representativeness corn inbred line sample of selection (9 class germplasm groups are covered,
And the torrid zone, the glutinous, local varieties of sweet tea etc.) full-length genome deep sequencing (10 ×) is carried out, excavated from high-quality heavy sequencing data,
SNP site is positioned, the SNP site of single copy, two condition is determined based on Bioinformatic methods；With reference to the SNP in the chip announced
SNP site in site or haplotype collection of illustrative plates, forms the more comprehensive excellent SNP site pond of corn (number of loci is more than 20,000,000).
(2) it is suitable to the candidate locus set that corn variety is identified：Above-mentioned excellent SNP site information submits two, U.S. chip
Production company Affymetrix, Illumina, design team is based on probe design and the requirement of biochemical reaction to SNP site
It is estimated.According to feedback information is assessed, the site for not meeting chip design requirement is deleted, point value of evaluation is deleted and is less than 0.6
Site, remaining site further carries out analysis and evaluation based on the data that self-mating system resurveys sequence.Assessing parameter is, MAF (Minor
Allele Frequency) value is more than or equal to 0.2, and heterozygosis rate is less than 0.05, site both sides or at least 60bp sequences in side have
Conservative (sequence of 60bp length has taken into account Affymetrix and the large chips of Illumina two design the requirement of probe length).Through
Site after above-mentioned assessment, according to equally distributed principle (being analyzed with reference to physical location and haplotype block), it is chain not
The methods such as equilibrium analysis, obtain the candidate locus set of equally distributed nonredundancy on genome (number of loci is more than 2,000,000).
(3) it is estimated using screening chip：Above-mentioned candidate locus set is submitted Affymetric companies to be estimated and set
Meter, and form screening chip.Choose about 200 assessment samples, including the self-mating systems of different genetic backgrounds, triplet (parents and
F1 cenospecies), breeding material etc., carry out test assessment.Assessing parameter is, if be PHR (poly high resolution)
The site of class, data pick-up rate is more than or equal to 95%, MAF values and is more than or equal to 0.2, and triplet sample meets Mendelian inheritance, three
Genotype energy automatic distinguishing is planted, site heterozygosis rate is less than 0.05, deletes undesirable site, obtains the SNP of high-effect high-quality
Site.
(4) extension Sites Combination is determined：The above-mentioned site assessed through screening chip, it is further excellent according to principle is uniformly distributed
Change, submit Affymetrix companies to be estimated design, customized a high density SNP chip (number of loci about 90,000).It is based on
The customized chip, enlarged sample quantity, the self-mating system and Inbred Lines material for choosing acquisition corn variety power protection is commented
Estimate, sample size about 2000 sets up the finger print data of these self-mating systems.It is to be classified as the site of PHR classes to assess parameter,
Data pick-up rate is more than or equal to 97%, MAF values and is more than or equal to 0.2, and site heterozygosis rate is less than 0.05, deletes undesirable position
Point.Further optimize using statistical method, according to equally distributed principle, obtain and be suitable to the extension site that corn variety is identified
Combine (number of loci about 50,000).
2nd, the determination of core Sites Combination：
(1) candidate's core site is obtained based on large sample amount, big data analysis：Corn hybridization has been authorized or authorized in selection
Kind, sample size about 3000, using above-mentioned customized chip, sets up corn hybrid seed finger print data, and to extension bits therein
Point is estimated.Assessing parameter is, if be the site of PHR classes, and data pick-up rate is more than 99%, MAF values and is more than or equal to 0.4,
Genotype data can automatic parting direction, physics heredity is uniformly distributed.Undesirable site is deleted, a set of candidate's core bit is obtained
Point, number about 1000.
(2) multi-platform, many laboratories independently carry out site checking：Above-mentioned candidate's core site, chip, KASP,
Multiple platforms such as TaqMan are verified, while submitting to other three laboratories carries out individual authentication, often take turns verification experimental verification sample
This quantity about 200.With reference to the feedback result of multi-platform, many laboratory proofings, quality minute is carried out to candidate's core bit point on
Class, obtains the site set that a set of compatible with multiple platforms, stability are high, polymorphism is high (number is hundreds of).
(3) it is based on the optimal Sites Combination of Analysis of Genetic Algorithms：From the superchip finger print data of about 5000 samples
The data in above-mentioned hundreds of site are extracted, optimization analysis is combined based on genetic algorithm, a set of high-resolution SNP are filtered out
Point, can distinguish more than 90% sample, and consider to be uniformly distributed principle, determine a set of optimal SNP site combination (number of loci
Within 100).
(4) determination of core Sites Combination：Above-mentioned optimal Sites Combination, randomly selects 1000 corn samples and is surveyed
Examination, adjusts optimal Sites Combination, comprehensive according to the development of kind SNP finger print data situations known to corn, with due regard to following kind
Close and recommend a set of high accuracy differentiated suitable for corn identity, high stability, high kind separating capacity, the core of compatible with multiple platforms
Sites Combination (number of loci is tens).
The core of the wheat of embodiment 2 (hexaploid) cultivar identification and the acquisition of extension SNP site combination
1st, the determination of Sites Combination is extended：
(1) Distinctive Wheat SNP site pond：Based on the physical map information in wheat full-length genome hereditary variation site, comprehensively
Parse distribution of the SNP site in subgenome between subgenome, number of copies, homology and polymorphism level, screening tool
There is discriminating homologue ability, and there is well-conserved specific mark in cultigen, set up hexaploid wheat A sub-
The excellent marker site pond of group, B subgroups, the special SNP of D subgroup chromosomes.
(2) it is suitable to the candidate locus set that wheat breed is identified：Above-mentioned excellent SNP site information submits two, U.S. chip
Production company Affymetrix, Illumina are estimated.Chip design evaluation and based on biological information assess with embodiment 1 in
The corn principle of correspondence it is identical.Through the site after above-mentioned assessment, based on being uniformly distributed, linkage disequilibrium value method, with reference to
With SNP marker polymorphism level, section base composition and design feature in subgroup between subgroup, three relatively uniform point of subgroups are obtained
The candidate locus set of cloth.
(3) determination of Sites Combination is estimated and extended using screening chip：The assessment of screening chip and extension site
The determination method of combination is identical with the corn principle of correspondence in embodiment 1.
2nd, the determination of core Sites Combination：
(1) candidate's core site is obtained based on large sample amount, big data analysis：Wheat descriptive literature has been authorized or authorized in selection
Kind, sample size about 1000 sets up its finger print data, and extension bits point therein is estimated using customized chip.
Assessing parameter is, if be the site of PHR classes, and data pick-up rate is more than 97%, MAF values and is more than or equal to 0.2, genotype data energy
Enough automatic parting directions, physics heredity is uniformly distributed.
(2) multi-platform, many laboratories independently carry out site checking：Above-mentioned candidate's core site, in multiple platforms such as chip
Verified, while submitting to other three laboratories carries out individual authentication, often take turns verification experimental verification sample size about 100.Knot
Close feedback result, obtain the site set that a set of compatible with multiple platforms, stability are high, polymorphism is high (number is hundreds of).
(3) it is based on the optimal Sites Combination of Analysis of Genetic Algorithms：From the superchip finger print data of about 1000 samples
The data in above-mentioned hundreds of site are extracted, optimization analysis is combined based on genetic algorithm, a set of high-resolution SNP are filtered out
Point, can distinguish more than 90% sample, and consider to be uniformly distributed principle, determine a set of optimal SNP site combination (number of loci
Within 100).
(4) determination of core Sites Combination：Above-mentioned optimal Sites Combination, randomly selects 200 wheat samples and is tested,
Adjust Sites Combination scheme, recommend it is a set of suitable for wheat identity differentiate high accuracy, high stability, high kind separating capacity,
The core Sites Combination of compatible with multiple platforms (number of loci is tens).
The structure of the maize dna fingerprint database of embodiment 3
(1) Reference cultivars：From corn hybrid seed capital section 968 two parent capital 724 and capital 92 as Reference cultivars, its
The homozygosity of sample is detected that homozygosity reaches more than 99.9% using Sites Combination is extended.
(2) prepared by corn variety DNA：Each sample mixes the greenery of 30 individual plants, liquid nitrogen grinding, and DNA extracts specific step
It is rapid to perform (Wang Fengge etc., 2014) according to maize dna Molecular Identification standard.Distinguished with ultraviolet specrophotometer and agarose electrophoresis
DNA quality is extracted in detection, and agarose electrophoresis shows that DNA bands are single, do not degraded；UV spectrophotometer measuring
A260/280 is between 1.8-2.0；A260/230 is more than 1.8.Dilution DNA formation working solution, concentration is 20ng/ μ L.
(3) corn variety fingerprint construction platform is selected：Flux based on each detection platform sample and site, with reference to actual feelings
Condition selects suitable platform to carry out fingerprint structure.Standard for Maize sample number is more than 5000 in the present invention, and number of loci is
Tens, so being adapted to selection sample high flux, the simple KASP platforms of experiment process.
(4) PCR is expanded：Enter performing PCR reaction from 1536 hole elisa Plates, water-bath PCR instrument.Reaction system is 1 μ L, including
1.5 μ L STb genes (drying), 0.5 μ L KASP ROX standard reaction mix (Kbiosciences, Herts UK),
0.014 μ L primer mixtures, 0.5 μ L deionized waters.Response procedures are 94 DEG C of 15min；Take touchdown PCR mode, 61 DEG C of landing
To 55 DEG C, circulate 10 times, each 0.6 DEG C of circulation reduction；94 DEG C of 20sec, 58 DEG C of 1min, are circulated 30 times.
(5) fingerprint data collection：Amplified production is carried out using BMG Pherastar (LGC, Middlesex, UK) instrument
Fluorescence signal is scanned, and obtains initial data.Initial data imports Kraken softwares (LGC, Middlesex, UK) progress analysis and obtained
Obtain the finger print data in each each site of corn variety.Finger print data record format be A/B, homozygous genotype be recorded as AA or
BB, heterozygous genotypes are recorded as AB, and A and B are defined using Reference cultivars simultaneously.
The application of the core of embodiment 4 and the combination of extension SNP site in corn variety identification
(1) whether identify corn sample (code name is D) to be measured is known control corn variety capital section 968 (code name is E).
DNA is extracted and quality determination：Extract corn sample to be measured (code name is D) and (code name of control corn sample capital section 968
For E) genomic DNA, step (2) in specific method be the same as Example 3.
Detect experiment process：First with core site, detection experiment is carried out based on KASP platforms.PCR is expanded and fingerprint
Step (4) and (5) in collection, specific method be the same as Example 3.
Data analysis is with judging：Sample D and sample E fingerprint, one by one site be compared, difference number of loci, which reaches, to be sentenced
Determine D different from E, it is not control corn variety capital section 968 to judge corn sample D to be measured.
(2) whether identify corn sample (code name is F) to be measured is known control corn variety capital section 968 (code name is E).
DNA is extracted and quality determination：Extract corn sample to be measured (code name is F) and (code name of control corn sample capital section 968
For E) genomic DNA, step (2) in specific method be the same as Example 3.
Detect experiment process：First with core site, detection experiment is carried out based on KASP platforms.PCR is expanded and fingerprint
Step (4) and (5) in collection, specific method be the same as Example 3.
First time finger print data is analyzed with comparing：Sample F and sample E fingerprint, one by one site be compared, difference site
Number not can determine that F is different from E, continue to carry out detection experiment using extension Sites Combination.
Extend the experiment of site combine detection：It is suitable based on each detection platform sample and the selection of site flux combination actual conditions
Platform tested.Corn extends number of loci about 50,000 in the present invention, and test sample number is 2, so selection site
High flux chip platform.Two samples of F and E are utilized respectively 150ngDNA, and two sample DNAs are divided together with other test samples
It is fitted on 1 96 cell formula chip board.SNP chip base is carried out based on Affymetrix companies GeneTitan chip technologies platform
Because of blood grouping.The Axiom 2.0Assay manual Workflow guides that operating process is announced according to Affymetrix companies
Carry out.
Fingerprint data collection：Initial data CEL formatted files are obtained after chip scanning and imported into Axiom
Analysis Suite softwares, are set using Best Practices and are automatically analyzed, export F and E finger print data.
Second of finger print data analysis is with comparing：Finger print data based on sample F and sample E, is joined using genetic similarty
Number judges that corn sample F to be measured, with compareing the relation between corn variety capital section 968, determines that sample F is and capital section according to threshold value
968 be to belong to different cultivars, query and derive from kind, derive from kind relation.
(3) it is to belong to which in known kind corn variety to identify corn sample (code name is G) to be measured.
DNA is extracted and quality determination：The genomic DNA of corn sample to be measured (code name is G) is extracted, specific method is with implementation
Step (2) in example 3.
Detect experiment process：First with core site, sample G finger print data is set up based on KASP platforms.PCR is expanded
And fingerprint collecting, step (4) and (5) in specific method be the same as Example 3.
First time, data analysis was with comparing：Sample G fingerprint is compared two-by-two with known breed standard finger print data
Analysis, can be determined that as difference if sample G is compared two-by-two with the known kind in database, and sample G may be to have
There are specific new varieties.If sample G can not be determined as difference with some or multiple kinds, by testing sample G with being
Row near sample continues with extension bits point and detected.
Extend the experiment of site combine detection：Tested based on Affymetrix chip technology platforms.G and the approximate sample of series
Originally 150ngDNA is utilized respectively, is assigned to together with other test samples on 1 96 cell formula chip board.It is based on
Affymetrix companies GeneTitan chip technologies platform carries out SNP chip genotypic assays.Operating process foundation
The Axiom 2.0Assay manual Workflow guides that Affymetrix companies announce are carried out.
Fingerprint data collection：Initial data CEL formatted files are obtained after chip scanning and imported into Axiom
Analysis Suite softwares, are set using Best Practices and are automatically analyzed, the finger of export G and the approximate sample of series
Line data.
Second of finger print data analysis is with comparing：Finger print data based on sample G and the approximate sample of series, using hereditary phase
Analyzed like degree parameter, it is determined that with testing sample G genetic background similarity highest kinds, and sample G is determined according to threshold value
Belong to query between the kind to derive from kind or derive from kind relation.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be modified or improved, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
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Claims (9)
1. differentiate and really weigh the detection method of identification suitable for variety of crops molecular identity, it is characterised in that comprise the following steps：
S1, core DNA molecular labeling Sites Combination acquisition；
S2, the acquisition for extending DNA molecular marker Sites Combination；
S3, two groups of DNA molecular marker Sites Combinations use；
In step S1, the core DNA molecular labeling includes efficiently distinguishing different cultivars, possesses high stability, high variety plot
Point ability, compatible with multiple platforms, take into account and be uniformly distributed and many laboratory proofing results, number of loci is tens to hundreds of magnitudes,
A kind of kind material of crop more than 90% can be distinguished, is built available for improved variety standard fingerprint, kind identity differentiates
Molecular labeling；
In step S2, the extension DNA molecular marker refers to optional outside step S1 core DNA molecular labeling Sites Combination
Molecular labeling, can hereditary similarity degree between accurate evaluation kind, site is uniformly distributed, compatible middle-high density chip, simultaneous
Loci polymorphism is turned round and look at, thousands of to tens of thousands of magnitudes of number of loci can distinguish a kind of kind material of crop more than 99%, can use
In approximate kind, derive from the molecular labeling that kind really weighs identification；
In step S3, when carrying out cultivar identification, the core DNA molecular labeling Sites Combination first by step S1 is examined
Survey, such as can not effectively distinguish testing sample and control sample, then continued using step S2 extension DNA molecular marker Sites Combination
Detected, based on the relation between genetic similarty parameter decision testing sample and control sample.
2. according to the method described in claim 1, it is characterised in that the DNA molecular marker is marked including SSR, SNP, INDEL
Note.
3. according to the method described in claim 1, it is characterised in that in step S1 from the excellent marker site pond of crops,
With reference to haplotype block, linkage disequilibrium analysis of biological information means, hereditary equally distributed non-redundant candidate site set is obtained；
Polymorphic type sample is chosen, candidate locus experimental evaluation is carried out using chip, sequencing high flux platform, from data pick-up rate, gene
Type parting effect, triplet feature are analyzed；Finally, determine to be suitable to the extension bits that breeding material is really weighed using statistical method
Point set, forms middle-high density authentication chip, and number is thousands of to tens of thousands of.
4. according to the method described in claim 1, it is characterised in that superchip, microarray dataset are utilized in step S2, with
Know and test assessment, candidate's core site that screening-gene type parting effect is good, polymorphism is high are carried out to large sample based on kind；Knot
Many laboratories, multi-platform individual authentication result are closed, the site set that a set of compatible with multiple platforms, stability are high, polymorphism is high is determined；
The optimal Sites Combination of more than 90% known kind can be distinguished by being filtered out based on Analysis of Genetic Algorithms, and carry out random sample survey
Examination, optimizes and revises Sites Combination, finally determines a set of high accuracy, high stability, high kind separating capacity, compatible with multiple platforms,
Equally distributed core Sites Combination is taken into account, number of loci is tens to hundreds of.
5. the method according to claim any one of 1-4, it is characterised in that carried out in step S3 first with core bit point
Detection, if core Sites Combination difference number of sites, which reaches, can be determined that sample A is different from sample B, termination detection experiment；If
It not can determine that sample A is different from sample B using core Sites Combination, then continue to carry out detection experiment using extension Sites Combination,
Sample A and sample B relation are judged using genetic similarty mode, according to threshold value determine both relation belong to different cultivars,
Query and derive from kind or derivation kind.
6. core DNA molecular labeling site obtained according to any one of claim 1-5 methods described and combinations thereof.
7. extension DNA molecular marker site obtained according to any one of claim 1-5 methods described and combinations thereof.
8. DNA molecular mark is extended described in core DNA molecular labeling site and combinations thereof described in claim 6 and/or claim 7
Remember application of the site and combinations thereof in variety of crops standard DNA fingerprint database structure.
9. DNA molecular mark is extended described in core DNA molecular labeling site and combinations thereof described in claim 6 and/or claim 7
Remember that site and combinations thereof differentiates or the really application in power identification in variety of crops molecular identity.
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