CN105550537A - Method for identifying rice DNA identities and application thereof - Google Patents

Abstract

The invention provides a method for identifying rice DNA identities, comprising following steps: obtaining standard gene fingerprint data of rice through detecting the genotypes of the genetic diversity molecular markers of the whole genome of the rice, identifying the DNA identities of the rice, or further, identifying the DNA identities of the rice through feature gene fingerprint data. According to the method provided by the invention, the DNA identities of the rice can be identified rapidly and accurately; and the method is applicable for new rice product identification, seed supervision and management or variety right protection.

Description

The method of qualification paddy DNA identity and application thereof

Technical field

The application belongs to seed quality detection field and field of molecular breeding.Particularly, the application relates to method and the application thereof of qualification paddy DNA identity.

Background technology

For a long time, the subject matter existed in China's rice seed industry has following several respects: one is that breeding material is complicated, genetic background is unintelligible, Main Agronomic Characters and excellent proterties lack effective functional label or linked marker, the kind homogeneity that authorization is promoted is serious, and breeding needs commercialization integrated technology to raise the efficiency; Two is that China's rice paddy seed market exists " many, random, assorted " phenomenon in varying degrees, and seed quality detects and market monitorings lacks modern science and technology support; Three is breeding material relative closures of each breeding team, lacks the evaluation of breeding intermediate materials and transaction platform, makes a lot of breeding material be confined to indivedual team Inner eycle and uses, fully extensively can not exchange, play the potential value of breeding material.

In order to set up modern market environment of planting industry fair competition; strengthen seed management and supervision and management; the intellecture property of protection breeder, safeguard seed industry enterprises just rights and interests and ensure the legitimate rights and interests of peasant, need to set up a set ofly to improve reliably, rice varieties identity authentication system fast and accurately.

China's New variety protection depends on the identity that DUS (specificity (Distinctness), consistance (Uniformity) and stability (Stability)) test carrys out plant identification kind.But the DUS phenotype test cycle is long, and along with identifying that place meeting different from the time is variant, be not suitable for building kind identity database.

DNA molecular marker is the important core technology of carrying out Hybrid breeding in commercial system, is the important means accurately and fast, efficiently identifying the variety of crops such as paddy rice, corn authenticity and purity.

The seed quality examination criteria of current employing utilizes SSR marker to carry out kind genetic fingerprinting, such as People's Republic of China's agricultural industry criteria NY/T1433-2014 " rice varieties authenticate technology code SSR marker method ", wherein utilizes 48 SSR marker to identify rice varieties.The application of SSR marker, relied on the seed quality of variable rate technology and seed morphology proterties to detect relative to the past completely, reliability and or be all significantly increased in accuracy.But, because SSR marker number is few, the genetic background of breeding material can not be reflected comprehensively, can not distinguish well for Sister Lines kind, intravarietal variation and deck kind for height.

Summary of the invention

On the one hand, this application provides the method for qualification paddy DNA identity, it comprises and utilizes paddy rice standard gene finger print data to carry out DNA identity authentication to paddy rice; Or utilize paddy rice standard gene finger print data and characterizing gene finger print data to carry out DNA identity authentication to paddy rice.

Particularly, the method of the qualification paddy DNA identity that the application provides, it comprises by detecting the genotype being distributed in one group of genetic diversity molecular labeling of paddy rice full-length genome, obtains the standard gene finger print data of paddy rice, identifies the DNA identity of described paddy rice thus.

In a particular embodiment, the method for the qualification paddy DNA identity that the application provides, after the standard gene finger print data obtaining paddy rice, utilizes genome similarity to identify the DNA identity of described paddy rice.

In another embodiment, the method of the qualification paddy DNA identity that the application provides comprises the following steps: (1) detects the genotype of the one group of genetic diversity molecular labeling being distributed in paddy rice full-length genome respectively, obtains the standard gene finger print data of at least two kinds of paddy rice; (2) full-length genome of described paddy rice is divided into multiple section; (3) the standard gene finger print data of obtained at least two kinds of paddy rice is compared by divided section, determine whether the genotype of the genetic diversity molecular labeling in each section there are differences respectively; And the differential gene group number of sections that the genotype of (4) statistics genetic diversity molecular labeling there are differences, be calculated as follows genome similarity, thus identify the DNA identity of described paddy rice:

GI＝(1-x/n)×100％

Wherein GI is genome similarity, and x is differential gene group number of sections, and n is the total number of sections of full-length genome.

In one embodiment, the number of wherein detected genetic diversity molecular labeling is 3000 ~ 6000, preferably 4000 ~ 5000.

In another embodiment, with 750kb ~ 1.0Mb, the preferably physical distance of about 1.0Mb, the full-length genome of described paddy rice is divided 300 ~ 400 sections, preferably 350 ~ 400 sections, more preferably 360 ~ 380 sections, the number of genetic diversity molecular labeling that wherein average each section comprises is at least 5, preferably 5 ~ 20, more preferably 10 ~ 15.

In one embodiment, in the step (1) of said method, the genotype of described genetic diversity molecular labeling is detected by SNP chip.In another embodiment, the genotype of described genetic diversity molecular labeling is detected by paddy rice full-length genome breeding chip Rice6K.

In one embodiment, in the step (4) of said method, if genome similarity > 95%, then identify that described paddy rice is no significant difference; Or, if genome similarity≤95%, then identify that described paddy rice is for there being notable difference.

In yet another embodiment, if when described paddy rice is accredited as no significant difference, said method also comprises the characteristic molecular mark by detecting one or more groups reflection rice varieties feature, obtains the characterizing gene finger print data of paddy rice, identifies the DNA identity of paddy rice thus further.

In optional embodiment, the characteristic molecular that uses in said method mark comprise in gene function molecular labeling, gene haplotype molecular labeling, constant gene segment C recombinant molecule mark, parent's specific genetic molecular labeling or other marks one or more.Further optionally, the characteristic molecular mark used in said method is selected from the genetic diversity molecular labeling in the Different regions that detects from described standard gene finger print data.In another optional embodiment, detected the genotype of described characteristic molecular mark by unit point or multidigit point detection platform.Further optionally, described unit point or multidigit point detection platform are TaqMan/KASPSNP mark or OpenArray chip.

On the other hand, the method of the qualification paddy DNA identity that the application provides, specifically comprise the following steps: (1) uses paddy rice full-length genome breeding chip Rice6K, detect 3000 ~ 6000 that are distributed in paddy rice full-length genome respectively, the preferably genotype of 4000 ~ 5000 genetic diversity molecular labelings, obtains the standard gene finger print data of at least two kinds of paddy rice; (2) with 750kb ~ 1.0Mb, the preferably physical distance of about 1.0Mb, the full-length genome of described paddy rice is divided into 300 ~ 400 sections, preferably 350 ~ 400 sections, more preferably 360 ~ 380 sections, the number of genetic diversity molecular labeling that wherein average each section comprises is at least 5, preferably 5 ~ 20, more preferably 10 ~ 15; (3) the standard gene finger print data of obtained at least two kinds of paddy rice is compared by divided section, if have the genotype of at least 1 genetic diversity molecular labeling to detect in the section wherein divided there are differences, then determine that this section is differential gene group section; And (4) statistical discrepancy genomic segment number, be calculated as follows genome similarity, thus identify the DNA identity of described paddy rice:

GI＝(1-x/n)×100％

Wherein GI is genome similarity, and x is differential gene group number of sections, and n is the total number of sections of full-length genome; Wherein, if genome similarity > 95%, then described paddy rice is accredited as no significant difference; Or if genome similarity≤95%, then described paddy rice has been accredited as notable difference.

Another aspect, the method of the qualification paddy DNA identity that the application provides, specifically comprise the following steps: (1) uses paddy rice full-length genome breeding chip Rice6K, detect 3000 ~ 6000 that are distributed in paddy rice full-length genome respectively, the preferably genotype of 4000 ~ 5000 genetic diversity molecular labelings, obtains the standard gene finger print data of at least two kinds of paddy rice; (2) with 750kb ~ 1.0Mb, the preferably physical distance of about 1.0Mb, the full-length genome of described paddy rice is divided into 300 ~ 400 sections, preferably 350 ~ 400 sections, more preferably 360 ~ 380 sections, the number of genetic diversity molecular labeling that wherein average each section comprises is at least 5, preferably 5 ~ 20, more preferably 10 ~ 15; (3) the standard gene finger print data of obtained at least two kinds of paddy rice is compared by divided section, if have the genotype of at least 1 genetic diversity molecular labeling to detect in the section wherein divided there are differences, then determine that this section is differential gene group section; And (4) statistical discrepancy genomic segment number, be calculated as follows genome similarity, thus identify the DNA identity of described paddy rice:

GI＝(1-x/n)×100％

Wherein GI is genome similarity, and x is differential gene group number of sections, and n is the total number of sections of full-length genome; Wherein, if genome similarity > 95%, then described paddy rice is accredited as no significant difference; Or if genome similarity≤95%, then described paddy rice has been accredited as notable difference; (5) if when described paddy rice is accredited as no significant difference, by detecting the genotype of the characteristic molecular mark of one or more groups reflection rice varieties feature, obtaining the characterizing gene finger print data of paddy rice, identifying the DNA identity of paddy rice thus further.

In optional embodiment, the characteristic molecular that uses in said method mark comprise in gene function molecular labeling, gene haplotype molecular labeling, constant gene segment C recombinant molecule mark, parent's specific genetic molecular labeling or other marks one or more.Further optionally, the characteristic molecular mark used in said method is selected from the genetic diversity molecular labeling in the differential gene group section that detects from described standard gene finger print data.

In another optional embodiment, detected the genotype of described characteristic molecular mark by unit point or multidigit point detection platform.Further optionally, described unit point or multidigit point detection platform are TaqMan/KASPSNP mark or OpenArray chip.

In the method for the qualification paddy DNA identity provided in the application, the brood body of paddy rice is detected.Such as, at least 20 seeds, preferably the compound sample of 20 seeds or single seed detect, or at least 20 strain blades, and preferably the compound sample of 20 strain blades or single-strain blade detect.

In addition, present invention also provides and the method for qualification paddy DNA identity described above also be can be used for, in the fields such as new rice variety identification, rice paddy seed supervision and management or rice varieties right-safeguarding, comprising the supervision etc. in new rice variety identity authentication, Rice Germplasm Resources evaluation, rice paddy seed market.

Therefore, the method for the qualification paddy DNA identity that the application provides, its advantage is at least one of following several respects:

(1) generally, if difference reference numerals is certain, the more concentrated interracial difference of indicia distribution is less; If some sections are variant, as long as number of labels and representativeness are enough, the undulatory property by selected specific markers and detection platform is less.The replacement of the application's utilization variance genomic segment ratio is existing judges kind degree differentiation with difference number of labels or ratio, the statistical error that the differential gene phenotypic marker so both can having eliminated concentrations causes, solves again the use problem of certain segment mark selectivity and detection platform.

(2) although paddy rice functional genome research obtains remarkable progress, a large amount of Main Agronomic Characters functional gene is cloned, but also cannot explain function and the mutual relationship of paddy rice full genome gene at present, genotype and phenotype also cannot accomplish complete correspondence.Only can not satisfy the demands with a set of standard gene fingerprint.The paddy rice standard gene fingerprint that the application provides further and the combination of characterizing gene fingerprint solve this difficult problem.

Particularly, in the method for the qualification paddy DNA identity that the application provides, the genetic fingerprints for kind identity authentication comprise the breed standard genetic fingerprints and the varietal characteristic genetic fingerprints data utilizing varietal characteristic Markers for Detection that utilize and be uniformly distributed in the detection of genomic genetic diversity mark.So both solve the qualification of different cultivars generality, solve again the specificity identification of similar varieties.For New variety protection and variety certification, both can obtain the standard gene fingerprint of kind, the characterizing gene fingerprint that this kind is different from approximate kind can also be obtained.Therefore, when seed supervision and management and right-safeguarding, usually there is certain understanding to the essential characteristic of this kind, only need just to be identified kind by characterizing gene finger print data.

(3) method of qualification paddy DNA identity that the application provides has the reliability of testing result and easy operability.Breed standard genetic fingerprints are applicable to the genetic fingerprints database setting up all kinds, can realize digitizing and robotization.Varietal characteristic genetic fingerprints Data Detection cost is lower, is applicable to the detection of daily scale.Both combinations add the reliability of testing result.

(4) method of qualification paddy DNA identity that provides of the application; be applicable to the directions such as New variety protection, variety certification, seed supervision and management and kind right-safeguarding, run through germplasm resource evaluation, breeding material really weigh and new varieties identity authentication etc. wholely to educate, numerous, push through journey.

Accompanying drawing explanation

Fig. 1 is the suitableeest number of labels utilizing DNA differential identification between 195 parts of Mini core collection resource chip data analysis kinds.Transverse axis is SNP marker number, and the longitudinal axis is the difference average of sample value and standard value, and lines are Trendline.

Fig. 2 utilizes 137 parts of representative kinds order sequenced data of resurveying to evaluate the effect of different DNA identity identifying method.The dark-grey color dot of upper triangular is with about 5000SNP for data set, the genetic distance before calculating between two by section difference; Middle circular light grey point for about 5000SNP for data set, calculate the genetic distance between two between kind by difference SNP number; Below Dark grey Diamond spot be with all SNP of gained that check order for data set, calculate the genetic distance between two between kind by SNP number difference; Straight line and formula are the result of linear analogue.

Fig. 3 calculates LD average die-away curve for utilizing 5000 parts of rice varieties SNP marker.Transverse axis is physical distance on chromosome, and the longitudinal axis is R ²value, grey lines are simulation curve.

Fig. 4 identifies rice varieties " imperial round-grained rice 31 " and " no loadtransformer " DNA identity comparison diagram for utilizing distinct methods.Wherein A identifies for utilizing conventional genetic similarity, and black lines represents two breed difference SNP marker positions; B identifies for utilizing the application's genome similarity, and black region represents two breed difference sections.In figure, square frame represents paddy rice 12 chromosomes successively, and ordinate numeral is the physical location on rice genome.

Fig. 5 is rice varieties " 93-11 " and " peace selects No. 6 " seed phenotypes difference comparison chart.A is for there being awns kind " 93-11 ", and B is awnless variety " peace selects No. 6 ".

Fig. 6 is rice varieties " 93-11 " and " peace selects No. 6 " standard gene fingerprint disparity map.Black region represents " 9311 " and " peace selects No. 6 " Different regions; Square frame represents paddy rice 12 chromosomes successively, and ordinate numeral is the physical location on rice genome.

Embodiment

There is provided to give a definition with method in order to define the application better and instruct those of ordinary skill in the art in the application's practice.Unless otherwise mentioned, term is understood according to the common usage of person of ordinary skill in the relevant.All patent documentations, scientific paper, industry standard and other public publications etc. quoted herein, full content entirety is wherein incorporated to herein as a reference.

" genetic polymorphism molecular labeling " as used herein, refer to can heredity and the detectable DNA fragment specific that can reflect certain species diversity in genome between bion or population.In the method for the application, preferably use SNP marker.

" standard gene finger print data " as used herein, refer to utilize filter out fully can reflect genome mutation between kind (Rice Genetic diversity) and be uniformly distributed in one group of full-length genome representative molecular labeling, detect the genotype of these molecular labelings in the full-length genome of rice varieties, obtain the standard gene finger print data of this rice varieties thus, it can represent the overall permanence of this rice varieties.

The standard gene finger print data of these different rice varieties can be built into rice varieties standard gene fingerprint database.Such as, the structure of rice varieties standard gene fingerprint database comprises the following steps:

(1) rice genetic diversity mark preliminary screening.Utilize the rice varieties of public database resurvey order sequenced data or voluntarily collect rice varieties carry out resurveying sequence, utilize these data screenings one group can reflect the multifarious group echo being distributed in full-length genome of Rice Genetic.Rice genome size is about 400Mb, if average every Mb10 mark, approximately needs 4000 marks, recommends 3000-6000 mark, preferred 4000-5000 mark during practical operation.

(2) rice genetic diversity mark is determined.The genetic diversity mark screened needs through test to determine mark quality and representativeness, determines the mark that one group of high-quality mark builds as standard gene fingerprint after test.Because number of labels is more, during integrated testability, select the high flux such as chip or order-checking detection platform.Because order-checking has randomness, recommendation marks metastable SNP chip detection platform.

(3) acquisition of rice varieties standard gene fingerprint.Utilize selected detection platform to detect to obtain the genotype of genetic diversity mark to rice varieties, markd genotype combination constitute the standard gene fingerprint of this kind.

(4) structure of rice varieties standard gene fingerprint database.The standard gene finger print data of rice varieties is stored and builds database.By data importing to MySQL/Oracle or other types relevant database.This database has inquiry, than reciprocity basic function.

" characterizing gene finger print data " as used herein, refer to one or more groups characteristic molecular mark that can reflect variety characteristic utilizing and filter out, detect the genotype of these molecular labelings in specific rice varieties, obtain the characterizing gene finger print data of this rice varieties thus.Varietal characteristic genetic fingerprints are supplementing, only for specific kind and specific demand of standard gene fingerprint.

Building varietal characteristic genetic fingerprints key is to obtain varietal characteristic molecular labeling, and it has following two sources:

(1) based on the feature section in standard fingerprint.When utilizing standard gene fingerprint to carry out detecting and judging, if find that the genome similarity of testing sample and known kind is between 95 ~ 100%, but be evident as different cultivars by phenotypic evaluation (as DUS test), development features can mark in the Different regions of standard gene fingerprint, or the polymorphism mark in direct selection differences section marks as characteristic molecular.

(2) provided by breeding man, kind holder or the mechanism through qualification certification.In order to kind to be measured and known approximate kind are distinguished; or in order to protect certain certain species better; characteristic molecular can be provided to mark by breeding man or agency, but the characterizing gene fingerprint that the characteristic molecular provided mark obtains can not be identical with other known approximate kinds.

A few class mark as varietal characteristic molecular labeling, but can be not limited to following a few class below:

(1) gene function molecular labeling

Gene function mark derive from functional gene inside directly cause phenotypic character to make a variation Genetic polymorphism sequence, it and target gene be divided into from.In fact, paddy rice full-length genome breeding chip Rice6K has included gene function mark, as Plant height gene Sd-1, grain length gene GS3, the wide gene GW5 of grain, scent gene fgr etc.

Equally also can mark for individual gene function labeling development unit point detection platform, as KASP mark.Such as, according to bibliographical information, temperature sensitive genic male sterile gene tms5 is the main effect sterile gene in current two-line hybrid rice parent sterile line, and the reason producing study on temperature sensitive male sterility is this gene 71 bit base there occurs by the sudden change of C to A, cause premature termination (Zhou etc., RNaseZ (S1) processesUbL40mRNAsandcontrolsthermosensitivegenicmalest erilityinrice.NatCommun.2014,5:4884.); For this functional site design KASP mark, detect this gene and just can judge whether it is two-line hybrid rice or two-line sterile line.

(2) gene haplotype mark

In paddy rice, a lot of Main Agronomic Characters related gene is not all single copy, and such as most blast resistant gene all belongs to NBS-LRR genoid family.For this genoid, be difficult to specific amplification or design dna Probe Hybridization.For this type of constant gene segment C, the polymorphism mark of application kind and doubtful kind in gene upstream and downstream certain limit (as: each 200kb of upstream and downstream) can be provided, be combined into constant gene segment C haplotype-tag, with this prove application kind and doubtful kind really variant.

(3) constant gene segment C restructuring mark

The process of crossbreeding inherently genetic recombination and selection, and genetic recombination is random, utilizes genomic segment mark of recombinating to be marked with clear superiority as characteristic molecular.When utilizing molecular mark to import specific objective gene, can restructuring be produced in gene both sides donor parents and receptor parent genome, restructuring mark can be developed near restructuring breakpoint.Because genetic recombination is random, so any one restructuring breakpoint is all different theoretically, namely specificity is very high.

(4) parent's specific genetic mark

Parent's specific genetic mark refers to the specific group echo of this parent of reflection screened from one group of genetic diversity mark that breed standard genetic fingerprints detect, or the one group of specific mark developed in the Different regions that standard gene fingerprint detection goes out, such as embodiment 5.

(5) other marks

Other can reflect the mark of kind singularity, as the mark on mark near the insertion point of transformed variety and the target gene that proceeds to, and the mark etc. on the mutational site utilizing radioinduction kind corresponding with mutant phenotype.

" rice varieties authenticate technology code SSR marker method " (standard No. NY/T1433-2014) utilization variance SSR marker number judges consistance between kind, " rice varieties qualification SNP marker method " (standard No. NY/T2745-2015) utilizes genetic similarty to judge consistance between kind, and what it calculated publicity reflection is the ratio that identical number of alleles accounts for total number of alleles.And the application replaces the existing degree differentiation judging kind with same tag number or ratio with " genome similarity ".

" genome similarity " as used herein, refers to that homologous genes type sector number accounts for the number percent of the total sector number of full-length genome, utilizes formula (1) to calculate genome similarity:

GI=(1-x/n) × 100% formula (1)

Wherein GI is genome similarity (GenomeIdentify), x is differential gene group number of sections, and n is the total number of sections of full-length genome.

" genotype of genetic diversity molecular labeling detects and there are differences " as used herein, refers to that having the effective genetic diversity molecular labeling of Genotyping (i.e. high-quality site) through experimental test verification can detect difference.In the method for the application, in each section, at least one (namely >=1) high-quality Site discrepancy detects, then think and there are differences between this section.

" paddy rice full-length genome breeding chip Rice6K " as used herein, it is the applicant's paddy rice full-length genome breeding chip disclosed in Chinese patent application CN102747138A, it is the paddy rice full-length genome breeding chip be most widely used at present, have the advantages that detection speed is fast, precision is high, flux is large, price is low, extensive for directions such as the fingerprint analysis of Rice Germplasm Resources molecular labeling, seed authenticity detection, the detection of seed homozygosity, filial generation Genotypings at present.

" paddy rice full-length genome breeding chip Rice60K " as used herein is the applicant's paddy rice full-length genome breeding chip disclosed in PCT application WO/2014/121419.

Following examples are only illustrative rather than definitive thereof the object of the application's scope.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (SambrookJ & RussellDW, Molecularcloning:alaboratorymanual, 2001) condition of, or according to manufacturer's instructions advising.

The rice varieties material information used in the application all can see rice in China kind and pedigree database (http://www.ricedata.cn/variety/index.htm) thereof.

The rice genome physical location mentioned in the application all annotates the 6.1st edition (http://rice.plantbiology.msu.edu/) with reference to the fine genome MSU/TIGR of paddy rice Japan.

Embodiment 1 rice varieties standard gene fingerprint detection parameter is determined

1.SNP number of labels

On the one hand, SNP marker number is more, and the resolution distinguishing DNA difference between kind is higher; On the other hand, SNP marker number is more, and determination and analysis cost is higher.

In order to determine the suitableeest number of labels that can meet interracial DNA differential identification, in the present embodiment, the paddy rice full-length genome breeding chip Rice60K coming from the 195 parts of Mini core collection resources in the whole world is utilized to detect data analysis (Chen etc., Ahigh-densitySNPgenotypingarrayforricebiologyandmolecula rbreeding.MolPlant.2014,7:541-553).

Total high-quality SNP site about 4.4 ten thousand on paddy rice full-length genome breeding chip Rice60K chip, therefrom random sampling 1000 ~ 30000 site is detected, and calculates the genetic distance of kind between two, is sample value; The genetic distance of kind is between two calculated for standard value with 4.4 ten thousand SNP marker testing results.Relatively sample value and standard value, represents difference between the two with " (sample value-standard value)/standard value ".

The results are shown in Figure transverse axis in 1, figure is SNP marker number, and the longitudinal axis is the difference average of sample value and standard value.Result display is reduced along with the difference between the increase sampling results of SNP marker number and standard value, when SNP marker number 4000 ~ 5000 time and standard value difference within 10%.Illustrate that the standard value of the sample value of 4000 ~ 5000 SNP marker and 4.4 ten thousand SNP marker is basically identical, and be the minimum SNP marker number that sample value is consistent with standard value.Therefore think that number of loci 4000 ~ 5000 is for most suitable.

2. genome similarity

The application utilize genome similarity replace existing with same tag number or ratio to judge kind degree differentiation.In order to show to utilize genomic segment to do the advantage of cultivar identification, the present embodiment to be resurveyed order sequenced data (Huang etc., Genome-wideassociationstudiesof14agronomictraitsinricela ndraces at about 5000 portions of paddy rice; Huang etc., Amapofricegenomevariationrevealstheoriginofcultivatedric e; Zhao etc., RiceVarMap:acomprehensivedatabaseofricegenomicvariations .NucleicAcidsResearch; The3,000ricegenomesproject1,2,3* the3,000ricegenomesproject) in have selected 137 representative kinds, respectively add up:

(1) with all SNP of the gained that checks order for data set, calculate the genetic distance between two between kind by SNP number difference;

(2) with about 5000SNP for data set, calculate the genetic distance between two between kind by difference SNP number;

(3) with about 5000SNP for data set, by section difference calculate between two before genetic distance.

The results are shown in Figure the dark-grey color dot of upper triangular in 2, figure is with about 5000SNP for data set, the genetic distance before calculating between two by section difference; Middle circular light grey point for about 5000SNP for data set, calculate the genetic distance between two between kind by difference SNP number; Below Dark grey Diamond spot be with all SNP of gained that check order for data set, calculate the genetic distance between two between kind by SNP number difference; Straight line and formula are the result of linear analogue.Straight slope in Fig. 2 is the index weighing group structure, when slope is close to when namely straight line is tending towards parallel, represents that group structure is more close.Because sequencing result is comprehensive, true, reliable, acquired results is closest to actual group structure.Result shows, the group structure that the group structure calculated by section difference calculates closer to foundation sequencing data of whole genome than the group structure calculated by SNP number.

Therefore, utilization variance genomic segment number replaces difference number of labels, and the DNA difference namely utilizing genome similarity to replace genetic similarty to come between identification of species is more reliable.

3. genomic segment divides

When carrying out section partition to genome, desirable mode is that linkage site is divided to same section, and non-chain site is divided to different section.LD (LinkageDisequilibrium, linkage disequilibrium) is usually with R ²value is measured: for A, B two sites, and the frequency that the frequency that A1, B1 two allele linkages occur is PII, A1, B1, A2, B2 occur separately is respectively p1, q1, p2, q2; R ²=(PII-pIqI)/PA*PB*Pa*Pb; R ²be worth larger explanation two site linkage degree stronger.

The present embodiment is with the fine genome of Japan for reference, and the SNP utilizing the sequencing data random selecting of about 5000 parts of rice varieties in common data not wait at a distance of 1bp ~ 10Mb (10,000,000bp), then calculates R between SNP site between two ²value, then result is averaged according to every 10bp, obtain a result and do simulation curve.The results are shown in Figure 3, wherein transverse axis is physical distance on chromosome, and the longitudinal axis is R ²value, grey lines are simulation curve.Result display point of inflexion on a curve is at about 750kb ~ 1.0Mb; On the left of flex point, LD decays rapidly, and on the right side of flex point, LD tends to balance; This illustrates that SNP spacing linkage degree when being less than flex point is being greater than the linkage degree of flex point much larger than SNP spacing.Therefore think that flex point distance is the suitable distance dividing section; For convenience of calculating, preferably arranging demarcation interval size is 1.0Mb.

4. sample preparation methods

Current high flux detects SNP marker and mainly contains order-checking and chip two kinds of methods.In order to Criterion genetic fingerprints database, need number of labels relatively fixing, best bet adopts SNP chip technology for detection.Higher with SNP chip technology for detection cost, preferably adopt biased sample to detect when breed standard genetic fingerprints build sampling Detection, and require that testing result can the genotype of most of seed in representative sample seed.According to crop seeds quality national standard, the purity of all rice paddy seeds all requires more than 95%, namely allows 1 external seed (genotype is inconsistent) (" crop seeds quality standard cereal crops seed Part I: Cereal " GB4404.1-2008) in 20 seeds.

In order to verify this point, the present embodiment utilizes paddy rice full-length genome breeding chip Rice6K to have detected following 3 samples:

I) biased sample KJ1-20, japonica rice variety no loadtransformer mixes according to the ratio of 1:19 with the seed of rice variety gold 23B, i.e. 1 no loadtransformer and 19 golden 23B seed mixing;

Ii) parental animal KY131, the mixing of 20, no loadtransformer seed;

Iii) parental animal J23B, the mixing of 20, golden 23B seed.

Testing result shows, the mark verification and measurement ratio (Callrate) of 3 samples, all higher than 90%, is thought and detected successfully.Analyze and find, on paddy rice full-length genome breeding chip Rice6K, about 4500 high-quality SNP site only have 6 marker genetypes inconsistent in the genotype of KJ1-20 and J23B, show that KJ1-20 almost can replace the genotype of golden 23B.

Due to blade extracting DNA easier than seed, so the biased sample of 20 plant leafs is feasible equally.When sampling number is less than 20, probably take out less than mixing sample; Sampling number more than 20 time, consistent with the result of mixed sample 20, but DNA extracting link can complicated a lot.

As can be seen here, the biased sample of 20 seeds or the biased sample of 20 strain blades is preferably used when building standard gene finger print data.

5. result judgment threshold

In testing result statement, the most important thing is the judgment threshold will determining same breed and different cultivars genome similarity.

Judgment threshold in industry standard NY/T1433-2014 is that 48 SSR marker have 3 marker genetype differences, similarity is about (48-3)/48 × 100%=93.75%; And judgment threshold is genetic similarty 95% in industry standard NY/T2745-2015.

These two industry standards comprehensive, the application intends determining that judgment threshold is 95%.

In order to verify that whether this threshold value is suitable, the present embodiment have purchased more than 100 part of Hybrid Rice Varieties from the market of all parts of the country, therefrom random selecting 47 kinds, each kind gets 2 seeds more at random, paddy rice full-length genome breeding chip Rice6K is utilized to detect, the genome similarity of two seeds of more same kind.Result shows: two seed cdna group similarities in 18 kinds are 100%; Two seeds in 20 kinds have a small amount of genomic fragment there are differences, genome similarity >95%; Two seed cdna group similarity <95% in other 9 kinds, show as more and larger genomic fragment and there are differences.Labor shows: in the kind of genome similarity >95%, the difference of two seeds is kind variations inner itself, and have one to be maternal selfing in kind two seeds of genome similarity <95%, string powder or mix.

Therefore, determine that judgment threshold is 95%, i.e. genome similarity > 95%, result is judged to be no significant difference, and genome similarity≤95%, result has been judged to be notable difference.

Embodiment 2 utilizes paddy rice full-length genome breeding chip Rice6K build rice varieties standard gene fingerprint database and carry out DNA identity authentication

Mark on paddy rice full-length genome breeding chip Rice6K screens from 520 parts of germ plasm resources resurvey order sequenced data, and represent genetic diversity, be uniformly distributed in the chromosomal group echo of 12, paddy rice, high-quality reference numerals is about 4500.

This chip is the InfiniumSNP chip platform of Illumina company, the maximum feature of this platform is that result is reliable and stable, for the high-quality site selected after tested, accuracy rate is 99.9% (Yu etc., Awhole-genomeSNParray (Rice6K) forgenomicbreedinginrice.PlantBiotechnolJ.2014,12:28-37).According to the detected parameters that embodiment 1 is determined, this breeding chip meets the condition of examination criteria genetic fingerprints completely.

In the present embodiment, utilize paddy rice full-length genome breeding chip Rice6K to build rice varieties standard gene fingerprint database specifically to comprise the following steps:

1. test specimen

Test specimen is rice paddy seed or its brood body, and its quality meets the requirement to rice seed purity in standard GB/T 4404.1.Carry out sample according to standard GB/T/T3543.2 (" crop seeds inspection procedure sample "), and from sample sample, get 20 seeds or blade be at random mixed into one and detect sample, carry out according to following steps.

2.DNA extracts

Conveniently DNA extraction process (as: CTAB method, SDS method, ammonium acetate method etc.) or DNA extraction kit (as: health is that complicated Plant Genome extracts kit, Promega Plant Genome extraction agent box etc.) extract test specimen genome DNA, and remove RNA.Detect extracted DNA quality respectively with agarose electrophoresis and ultraviolet spectrophotometer, the DNA quality of extraction should meet relevant quality requirements: agarose electrophoresis display DNA band is single, does not have obvious degradation; Ultraviolet light absorbance A 260/280 is between 1.8-2.0, and A260/230 is between 1.8-2.0; DNA concentration >30ng/ μ l, total amount >500ng.Chip detection DNA working concentration is 30 ~ 50ng/ μ l, ampoule 4 μ l.

3. chip detection

According to IlluminaInfinium genechip detection normal process operation (InfiniumHDAssayUltraProtocolGuide, http://www.illumina.com/).

4. data analysis and expression

Original gene type is analyzed with GenomeStudio software (http://www.illumina.com/).Original gene type is converted to standard gene fingerprint, and specific practice is as follows:

(1) marker genetype is extracted.With the fine genome of Japan for reference, screen high-quality site on paddy rice full-length genome breeding chip Rice6K chip, remove and detect the site that Japanese fine sample genotype is disappearance or heterozygosis, residue reference numerals is 4453.Extract the genotype of test specimen 4453 mark.Fine genotype is consistent is designated as " A " with reference gene group Japan, inconsistent and isozygoty and be designated as " B ", and heterozygosis is designated as " H ", and disappearance is designated as "-" (result is not shown).

(2) analyzing gene group section genotype.For rice genome, according to average every 1Mb, 12 chromosomes are divided into 379 sections (end of chromosome can be regarded as a section less than 1Mb), calculate the reference numerals in each section respectively, in 379 sections, have 360 segment mark number >=5.The section that reference numerals is less than the section of 5 reference numerals adjacent with front and back less merges, and merges further again, only merge adjacent section at every turn if number of labels is still less than 5, until meet the requirement that each segment mark number is no less than 5.Rice genome divide into 363 sections the most at last, respectively called after Bin001 ~ Bin363, maximum section 3Mb, and minimum 5 of segment mark number, maximum 20, average each section 12.3 mark, is marked at full-length genome and is uniformly distributed.4453 of test specimen marker genetypes are grouped in corresponding section according to genomic locations, in table 1, illustrated therein is the standard gene finger print information of representative rice varieties.

5. database sharing

The standard gene finger print data of rice varieties is stored and builds database.By data importing to MySQL/Oracle or other types relevant database.This database has inquiry, than reciprocity basic function.

6. expression of results

Utilize standard gene finger print data identification of species DNA identity.Test specimen and standard model Identification authenticity, or with known control sample multilevel iudge consistance.

In the present embodiment, the step of the rice varieties standard gene fingerprint database qualification rice varieties DNA identity constructed by utilization comprises:

(1) Different regions judges.Rice genome 363 sections (Bin), have 1 or more SNP site there are differences, then think that this section there are differences (ignoring deletion Genotype) in section.

(2) statistical discrepancy number of sections.

(3) genome similarity is calculated according to formula (1).

The qualification result of different testing goal is reported as:

(1) test specimen and control sample Identification authenticity (whether test specimen and control sample exist notable difference), expression of results is:

A) genome similarity > 95% between test specimen and control sample, conclusion is not for notable difference to be detected;

B) genome similarity≤95% between test specimen and check variety, conclusion is for notable difference being detected.

If genome similarity > 95% between test specimen and control sample detected, additive method can be increased if desired and identify further.

(2) test specimen and control sample multilevel iudge consistance (test specimen whether be same breed with control sample), expression of results is:

A) genome similarity > 95% between testing sample and control sample, is judged to be " same breed ";

B) genome similarity≤95% between testing sample and check variety, is judged to be " different cultivars ".

If genome similarity > 95% between testing sample and control sample detected, additive method can be increased if desired and identify further.

Embodiment 3 utilizes standard gene fingerprint identification rice varieties " imperial round-grained rice 31 "

1. material explanation

Conventional japonica rice " no loadtransformer " originates in Japan.Nineteen ninety introduces also seed selection by rice research institute of Heilongjiang Academy of Land Reclamation Sciences from Jilin academy of agricultural sciences and forms, and for cultivating mirror 90 ~ 31, is authorized in 2000 years by Heilungkiang for primary number.

Another conventional japonica rice " imperial round-grained rice 31 " is that for maternal, " cultivating rice No. 8 " is male parent, inoculates its F1 vitro anther culture, forms by the seed selection of pedigree method, is authorized in 2011 years by Heilungkiang with " dragon flower 96-1513 ".

Performance during variety certification: with contrast " no loadtransformer " compared with, output and the rice blast resistance of " imperial round-grained rice 31 " obviously strengthen, other phenotypic characters such as strain Leaf pattern with contrasted significant difference.According to Plant new variety protection and variety certification relevant regulations, " imperial round-grained rice 31 " and " no loadtransformer " should belong to different cultivars.

2. use conventional genetic similarity to identify

Paddy rice full-length genome breeding chip Rice6K is utilized to obtain the genotype of kind to be measured " imperial round-grained rice 31 " and check variety " no loadtransformer " according to embodiment 2.Both 4453 be marked with 84 SNP site genotype and there are differences, conveniently difference number of labels calculates, and genetic similarty is 98.1% ([1-(4453-84)/4453] × 100%).

Conclusion is: kind to be measured " imperial round-grained rice 31 " and check variety " no loadtransformer " genetic similarty >95%, be judged to be same breed.

3. use the genome similarity of the application to identify

Paddy rice full-length genome breeding chip Rice6K is utilized to obtain kind to be measured " imperial round-grained rice 31 " and check variety " no loadtransformer " standard gene fingerprint according to embodiment 2.Both have 41 section genotype there are differences by 363 genomic segment, and genome similarity is 88.7% ([1-(363-41)/363] × 100%).

Conclusion is: kind to be measured " imperial round-grained rice 31 " and check variety " no loadtransformer " genome similarity <95%, be judged to be different cultivars.

Fig. 4 is " imperial round-grained rice 31 " and " no loadtransformer " paddy rice full-length genome breeding chip Rice6K testing result, and wherein A identifies for utilizing conventional genetic similarity, and black lines represents two breed difference SNP marker positions; B identifies for utilizing the application's genome similarity, and black region represents two breed difference sections.In Fig. 4, square frame represents paddy rice 12 chromosomes successively, and ordinate numeral is the physical location on rice genome.

Visible, the method for use the application carries out qualification and actual conditions match, more more scientific than general survey method, accurate.

Embodiment 4 utilizes standard gene fingerprint and characterizing gene fingerprint identification rice varieties " 9311 " and " peace selects No. 6 "

1. material source

Rice varieties " 93-11 " (crying again " raising rice No. 6 ") is not only the excellent indica conventional rice of China, or the restorer parent of many superior hybrid crosses rice.2002, China scientist utilizes shotgun sequencing to obtain the whole genome sequence sketch (Yu etc. of this kind, Adraftsequenceofthericegenome (OryzasativaL.ssp.Indica) .Science.2002,296:79-92).

Another rice varieties " peace selects No. 6 " (crying again " Anhui rice 115 ") is the natural mutant selected from 9311, through systematic breeding Xian kind in the routine of incubation in 1998.

" 93-11 " is almost consistent with the phenotype of " peace selects No. 6 ", and the most significantly difference is that " 93-11 " has awns, and " peace selects No. 6 " is without awns, and as shown in Figure 5, A is for there being awns kind " 93-11 ", and B is awnless variety " peace selects No. 6 ".

2. utilize standard gene fingerprint identification

Paddy rice full-length genome breeding chip Rice6K is utilized to build the standard gene fingerprint of " 93-11 " and " peace selects No. 6 " according to embodiment 2.

By analysis, " 93-11 " and " peace selects No. 6 " always has 13 sections (Bin241 ~ Bin253) at Chr8 and there are differences, and the results are shown in Figure 6.In Fig. 6, black region represent 9311 and peace select No. 6 Different regions; Square frame represents paddy rice 12 chromosomes successively, and ordinate numeral is the physical location on rice genome.

Calculating genome similarity by formula (1) is 96.4%>95%, and conclusion is for detecting no significant difference.Because standard gene fingerprint cannot be distinguished " 93-11 " and " peace selects No. 6 ", and they are different cultivars, and phenotype has significant difference, need to utilize characterizing gene fingerprint to distinguish further.

3. set up characterizing gene fingerprint

Above-mentioned standard gene fingerprint detection " 93-11 " and " peace selects No. 6 " always have 13 sections at Chr8 and there are differences, difference reference numerals in these 13 sections is 53, these 53 SNP marker can be used as the characteristic molecular mark of differentiation " 93-11 " and " peace selects No. 6 ", and the genotype of molecular labeling is respectively the characterizing gene fingerprint of " 93-11 " and " peace selects No. 6 ".

Conveniently detect, 10 marks be distributed in different section can be selected in 53 SNP marker, utilize KASP unit point to react detection of platform, the results are shown in Table 2.

Table 2 " 93-11 " and " peace selects No. 6 " characterizing gene fingerprint

Visible, standard gene fingerprint detection without significant difference or prior known standard gene fingerprint without significant difference time, extracting data parent specific mark can be detected from breed standard genetic fingerprints to mark as varietal characteristic, utilize characterizing gene fingerprint to identify rice varieties further.

Embodiment 5 utilizes standard gene fingerprint and characterizing gene fingerprint identification " no loadtransformer " to improve strain K1 ~ K4

1. material explanation

" no loadtransformer " is the maximum kind of Heilungkiang popularizing area, but recent years is due to rice blast resistance problem, and its cultivated area declines year by year.Therefore, in order to improve the rice blast resistance of " no loadtransformer ", the not iso-allele being arranged in paddy rice the 6th chromosome Pi2 interval is imported " no loadtransformer " kind, as (Dai etc., 2010 such as Pi2, Pi9; Qu etc., 2006; Wang etc., 2012; Zhou etc., 2006; Xiao etc., 2012; Liu etc., 2002; Liu etc., 2011; Jiang etc., 2012; Zhu etc., 2012; Deng etc., 2006).

Obtain at present and imported target gene pack section <500kb, improve strain for 4 that genetic background is consistent with no loadtransformer height, be respectively K1 ~ No. K4, target gene Pi9 (the .Thebroad-spectrumblastresistancegenePi9encodesanucleoti de-bindingsite-leucine-richrepeatproteinandisamemberofam ultigenefamilyinrice.Genetics.2006 such as Qu wherein in K1 and K2 Introduced into Rice " 75-1-127 ", 172:1901-1914.), target gene Pi2 (the .Theeightamino-aciddifferenceswithinthreeleucine-richrep eatsbetweenPi2andPiz-tresistanceproteinsdeterminetheresi stancespecificitytoMagnaporthegrisea.MolPlantMicrobeInte ract.2006 such as Zhou in K3 and K4 Introduced into Rice " C101A51 ", 19:1216-1228).

2. utilize standard gene fingerprint identification

Paddy rice full-length genome breeding chip Rice6K is utilized to build the standard gene fingerprint of no loadtransformer and Improved lines K1 ~ K4 thereof according to embodiment 2.

By analysis, the genome difference sector number of K1 ~ K4 and no loadtransformer is 1, calculates genome similarity be 99.7% by formula (1).Conclusion is: testing sample K1, K2, K3, K4 and control sample no loadtransformer genome similarity >95%, be judged to be same breed.

But improvement strain has obvious rice blast resistance advantage, according to new variety of plant application requirement, as long as there is an obvious phenotypic character difference just can become new varieties compared with no loadtransformer.For the new varieties (being) utilizing this special breeding method to cultivate, varietal characteristic genetic fingerprints can be utilized to identify its identity.

3. development features molecular labeling

According to the feature of 4 Improved lines, functional gene haplotype-tag and genomic segment restructuring marker combination is selected to mark as characteristic molecular.

For functional gene haplotype-tag, compare donor parents " 75-1-127 ", " C101A51 " and receptor parent " no loadtransformer " sequence difference at target gene Pi2/Pi9, develop 3-5 mark, different haplotypes is distinguished in the combination utilizing these to mark.

For genomic segment restructuring mark, be import the distribution of target gene restructuring breakpoint location according to K1 ~ K4 tetra-, design can distinguish the restructuring mark of different breakpoint.

According to this principle, the present embodiment designs 4 SNP marker altogether as functional gene haplotype-tag, designs 28 SNP marker as restructuring mark.ThermoFisher company these 32 SNP marker is submitted to make the OpenArray chip (or 32 unit points detect) in 32 sites

4. construction feature genetic fingerprints

To the donor material " 75-1-127 " of Pi2/Pi9 gene, " C101A51 " and acceptor material " no loadtransformer " and 4 improvement strains extracting DNA respectively, the normal process detected according to OpenArray chip (or 32 unit points detect) carries out detecting (http://tools.lifetechnologies.com/content/sfs/manuals/4478673.p df), QuantStudio12KFlexReal-TimePCRSystem instrument reads data and carries out Genotyping.

Take no loadtransformer as reference, the genotype results of each kind (being) is in table 3, as can be seen from the table, 4 haplotype-tag (KY131X15, KY131X16, KY131X17, KY131X18) are with target gene close linkage or just on gene.If the testing result of 4 marks is AAAA, be exactly that no loadtransformer is not containing disease-resistant gene haplotype; If testing result is BAAB, derive from " 75-1-127 " haplotype containing Pi9 gene exactly; If testing result is BBBB, derive from " C101A51 " haplotype containing Pi2 gene exactly.Clearly K1 and K2 contains Pi9, K3 and K4 contains Pi2.Other 28 marks (each 14 of upstream and downstream) can distinguish different restructuring breakpoints, if it is that B represents and comes from donor parents pack section that genotype is detected in these sites, testing result is that A represents original " no loadtransformer " fragment.Downstream totally 8 restructuring breakpoints fastened by 4 improved goodss, and each restructuring breakpoint has at least 2 marks to distinguish.32 genotypic combinations of marker site constitute the characterizing gene fingerprint of each kind (being).

The characterizing gene fingerprint of table 3 no loadtransformer and blast resisting improved materials

Visible, standard gene fingerprint detection without significant difference or prior known standard gene fingerprint without significant difference time, functional gene haplotype-tag and genomic segment restructuring marker combination can be selected to mark as characteristic molecular, utilize characterizing gene fingerprint to identifying rice varieties further.

The application of embodiment 6 rice varieties DNA identity identifying method in new varieties are assert

When Plant new variety protection and variety certification, need first to determine whether new varieties.At present, country adopts DUS test judgement, but needs to utilize DNA identity identifying method tentatively to judge before DUS test.

In the present embodiment, the method utilizing the application to provide assert that new rice variety comprises the following steps:

1. build known kind genetic fingerprints database

The genetic fingerprints database of known kind (comprise and apply for Plant new variety protection and passed through variety certification) is built according to the method for embodiment 2.Known kind has a characterizing gene fingerprint or characteristics of needs genetic fingerprints material is distinguished, and characterizing gene fingerprint can be added, supplementing as breed standard genetic fingerprints.

2. utilize standard gene fingerprint to judge

The standard gene fingerprint of testing sample is built according to the method for embodiment 2.All kinds in the standard gene fingerprint of testing sample and standard gene fingerprint database are compared, finds out the immediate kind of genome similarity kind in contrast.If genome similarity≤95% between testing sample and control sample, then directly can regard as new varieties; If the genome similarity >95% between testing sample and control sample, then judge by the following method further.

3. increase characterizing gene fingerprint to judge

Distinguish the signature of testing sample and control sample according to the method exploitation of embodiment 3, construction feature genetic fingerprints, as embodiment 5 and 6.If the varietal characteristic genetic fingerprints of testing sample are obviously different from control sample, then new varieties can be regarded as; If cannot obtain testing sample signature, and applicant can produce evidence to prove that testing sample and control sample have significant difference in phenotype, then judge by the following method further.

4. increase additive method to judge

Whether the proof validation testing sample provided according to applicant and control sample have significant difference in phenotype, and the method that usually can specify according to DUS manual testing performs, and the condition can specified according to applicant is if desired identified.If qualification result shows that testing sample and control sample have significant difference in phenotype, meet new varieties and assert condition, can new varieties be regarded as, otherwise can not new varieties be regarded as.For to regard as new varieties according to step 2 and step 3, DUS can be utilized to test checking further.

The application of embodiment 7 rice varieties DNA identity identifying method in seed supervision and management

In seed supervision and management, need to judge that whether the variety name marked in seed produces business process is consistent with the standard model of this kind.

Standard gene fingerprint is utilized to detect according to the method for embodiment 2.By test specimen and standard model Identification authenticity: if genome similarity > 95% between testing sample and control sample, conclusion is not for notable difference to be detected, and sell goods are consistent with standard model; If genome similarity≤95% between testing sample and check variety, conclusion for notable difference being detected, sell goods and standard model inconsistent, can pseudosperm be judged to be.

The application of embodiment 8 rice varieties DNA identity identifying method in right-safeguarding

If suspect illegal distribution and the known mandate kind of breeding, in order to safeguard the legitimate rights and interests of variety right people, method described in the application can be utilized to carry out DNA identity authentication.First utilize standard gene fingerprint detection, testing sample and known variety protection are judged consistance: if genome similarity > 95% between testing sample and control sample, be judged to be " same breed "; If genome similarity≤95% between testing sample and check variety, is judged to be " different cultivars ".

If genome similarity > 95% to be detected between testing sample and known kind or known in advance testing sample and known kind closely similar, characterizing gene fingerprint can be utilized to identify: if the characterizing gene fingerprint no significant difference of testing sample and control sample, be judged to be " same breed "; If testing sample and check variety characterizing gene fingerprint have notable difference, be judged to be " different cultivars ".For utilizing standard gene fingerprint and characterizing gene fingerprint all to can't detect notable difference, additive method can be utilized to prove, as phenotypic evaluation.Example below illustrates and utilizes characterizing gene fingerprint to carry out kind right-safeguarding.

Characterizing gene fingerprint protection 4 the blast resisting improvement strain K1 ~ K4 in embodiment 5 can be utilized.The present embodiment profit carries out characterizing gene fingerprint detection to certain the doubtful infringement kind (numbering ZD001) being planted in northeast build space in this way.

First utilize 20 seed mixtures to detect 32 SNP site, find that the signal value in a lot of site is abnormal, A genotype or 1 B gene type cannot be judged, neither heterozygous genotypes.Again get 20 seed mixtures and detect the same result of appearance, suspection may be mixing genotype, so adopt single seed to detect.Get 48 independent extracting DNA of seed obtain every seed characterizing gene fingerprint according to the method for embodiment 5 at random, found that the genotype of 30 seeds is identical with K1, remain 18 seeds identical with " no loadtransformer ", obvious ZD001 is the mixed system that " no loadtransformer " original seed and blast resistant gene Pi9 improve version K1.

Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (10)

1. identify the method for paddy DNA identity, it comprises by detecting the genotype being distributed in one group of genetic diversity molecular labeling of paddy rice full-length genome, obtains the standard gene finger print data of paddy rice, identifies the DNA identity of described paddy rice thus.

2. the method for claim 1, wherein utilizes genome similarity to identify the DNA identity of described paddy rice.

3. method as claimed in claim 2, it comprises the following steps:

(1) detect the genotype being distributed in one group of genetic diversity molecular labeling of paddy rice full-length genome respectively, obtain the standard gene finger print data of at least two kinds of paddy rice;

(2) full-length genome of described paddy rice is divided into multiple section;

(3) the standard gene finger print data of obtained at least two kinds of paddy rice is compared by divided section, determine whether the genotype of the genetic diversity molecular labeling in each section there are differences respectively; And

(4) the differential gene group number of sections that the genotype of adding up genetic diversity molecular labeling there are differences, is calculated as follows genome similarity, identifies the DNA identity of described paddy rice thus:

GI＝(1-x/n)×100％

Wherein GI is genome similarity, and x is differential gene group number of sections, and n is the total number of sections of full-length genome.

4. the method as described in any one of claims 1 to 3, the number of wherein detected genetic diversity molecular labeling is 3000 ~ 6000, preferably 4000 ~ 5000;

Optionally, with 750kb ~ 1.0Mb, preferably the full-length genome of described paddy rice is divided into 300 ~ 400 sections by the physical distance of about 1.0Mb, preferably 350 ~ 400 sections, more preferably 360 ~ 380 sections, the number of genetic diversity molecular labeling that wherein average each section comprises is at least 5, preferably 5 ~ 20, more preferably 10 ~ 15.

5. the method as described in claim 3 or 4, wherein in step (1), detects the genotype of described genetic diversity molecular labeling by SNP chip;

Optionally, described SNP chip is paddy rice full-length genome breeding chip Rice6K.

6. the method according to any one of claim 3 to 5, wherein in step (4), if genome similarity > 95%, then described paddy rice is accredited as no significant difference; Or if genome similarity≤95%, then described paddy rice has been accredited as notable difference.

7. method as claimed in claim 6, if when described paddy rice is accredited as no significant difference, described method also comprises the genotype of the characteristic molecular mark by detecting one or more groups reflection rice varieties feature, obtain the characterizing gene finger print data of paddy rice, identify the DNA identity of paddy rice thus further;

Optionally, described characteristic molecular mark comprises one or more in gene function molecular labeling, gene haplotype molecular labeling, constant gene segment C recombinant molecule mark, parent's specific genetic molecular labeling or other marks;

Optionally, described characteristic molecular mark is selected from the genetic diversity molecular labeling in the differential gene group section that detects from described standard gene finger print data;

Optionally, the genotype of described characteristic molecular mark is wherein detected by unit point or multidigit point detection platform;

Optionally, described unit point or multidigit point detection platform are TaqMan/KASPSNP mark or OpenArray chip.

8. identify the method for paddy DNA identity, it comprises the following steps:

(1) paddy rice full-length genome breeding chip Rice6K is used, detect 3000 ~ 6000 that are distributed in paddy rice full-length genome respectively, the preferably genotype of 4000 ~ 5000 genetic diversity molecular labelings, obtains the standard gene finger print data of at least two kinds of paddy rice;

(2) with 750kb ~ 1.0Mb, the preferably physical distance of about 1.0Mb, the full-length genome of described paddy rice is divided into 300 ~ 400 sections, preferably 350 ~ 400 sections, more preferably 360 ~ 380 sections, the number of genetic diversity molecular labeling that wherein average each section comprises is at least 5, preferably 5 ~ 20, more preferably 10 ~ 15;

(3) the standard gene finger print data of obtained at least two kinds of paddy rice is compared by divided section, if have the genotype of at least 1 genetic diversity molecular labeling to detect in the section wherein divided there are differences, then determine that this section is differential gene group section; And

(4) statistical discrepancy genomic segment number, is calculated as follows genome similarity, thus identifies the DNA identity of described paddy rice:

GI＝(1-x/n)×100％

Wherein GI is genome similarity, and x is differential gene group number of sections, and n is the total number of sections of full-length genome;

Wherein, if genome similarity > 95%, then described paddy rice is accredited as no significant difference; Or if genome similarity≤95%, then described paddy rice has been accredited as notable difference;

Optionally, if when described paddy rice is accredited as no significant difference, described method also comprises:

(5) by detecting the genotype of the characteristic molecular mark of one or more groups reflection rice varieties feature, obtaining the characterizing gene finger print data of paddy rice, identifying the DNA identity of paddy rice thus further;

Optionally, described characteristic molecular mark comprises one or more in gene function molecular labeling, gene haplotype molecular labeling, constant gene segment C recombinant molecule mark, parent's specific genetic molecular labeling or other marks;

Optionally, described characteristic molecular mark is selected from the genetic diversity molecular labeling in the differential gene group section that detects from described standard gene finger print data;

Optionally, marked by TaqMan/KASPSNP or described in OpenArray chip detection characteristic molecular mark genotype.

9. method according to any one of claim 1 to 8, wherein detects the brood body compound sample of paddy rice; Optionally, at least 20 of paddy rice, the preferably compound sample of 20 seeds, or at least 20 strains, preferably the compound sample of 20 strain blades detects; Optionally, the single seed of paddy rice or single-strain blade are detected.

10. the method according to any one of claim 1 to 9, may be used in new rice variety identification, rice paddy seed supervision and management or rice varieties right-safeguarding.