CN104805187A - Method for testing distinctness, uniformity and stability of pure-line soybean new variety - Google Patents

Abstract

The invention discloses a method for testing the distinctness, uniformity and stability of a pure-line soybean new variety. The method comprises the following steps: obtaining variation points; determining a testing area of the soybean variety to be tested; establishing a database; after determining the sampling amount, randomly sampling, mixing samples, and extracting DNA in the mixed sample; preparing primers; amplifying the DNA in the mixed sample by using the primers, and establishing a high-throughput sequencing library by using the product generated after amplification; performing high-throughput sequencing on the high-throughput sequencing library, so as to obtain a sequencing fragment group; analyzing the sequencing fragment group, so as to obtain the genotype of the soybean variety to be tested and the genotype of an abnormal plant; obtaining similar varieties, variation points and variation point rate through comparison; after obtaining an abnormal plant variety through comparing the genotype of the abnormal plant and genotypes in the database, computing the abnormal plant rate; judging the distinctness, uniformity and stability of the soybean variety to be tested according to the variation points, variation point rate and abnormal plant rate. The method can be used for accurately and completely judging the distinctness, uniformity and stability of the plant variety to be tested.

Description

A kind of method of testing specificity, consistence and the stability being sheerly new soybean varieties

Technical field

The present invention relates to biological technical field, particularly a kind of method of testing specificity, consistence and the stability being sheerly new soybean varieties.

Background technology

As a kind of intellecture property of specialization, new variety of plant has become a company and to a national core competitiveness.The solution that new variety of plant authorizes account and relative legal problems depends on DUS test, namely to field trapping test or the molecules inside Marker Identification of the specificity (Distinctness) of soybean varieties to be measured, consistence (Uniformity) and stability (Stability).Field trapping test flow process is: plant soybean varieties to be measured and approximate kind in field simultaneously, in 2 years and the above season of growth, observe their multiple proterties, the significance of difference of soybean varieties to be measured and approximate kind is judged according to the Characters, i.e. specificity, judge hybrid strain ratio in colony, i.e. consistence and stability simultaneously; The flow process of molecules inside Marker Identification is: point individual plant extracts the DNA of each sample in soybean varieties to be measured and approximate kind, and respectively PCR (Polymerase Chain Reaction is carried out to each test zone of each sample, polymerase chain reaction), and electrophoresis or generation order-checking detection are carried out to each PCR primer, according to detected result, obtain the difference site ratio of soybean varieties to be measured and approximate kind, according to difference site ratio, judge the specificity of soybean varieties to be measured.

The shortcoming of field trapping test is: the cycle is long, workload large, environmental influence proterties, causes judging inaccurate.The shortcoming of molecules inside Marker Identification is: need each test zone processing each sample respectively, and workload is large, to sample and test zone bulk sampling, can not cannot calculate hybrid strain rate, thus cannot carry out stability and conforming test.The common drawback of field trapping test and molecules inside Marker Identification is: all due to the reason of workload, kind cannot be similar to by objective selection from existing kind, applicant can only be weighed by kind to provide, and based on motivations such as commercial benefitss, the approximate kind that kind power applicant provides may be untrue, thus the legal consequence of the kind mandate that makes the mistake.

Summary of the invention

In order to solve the problems of the prior art, embodiments provide a kind of method of testing specificity, consistence and the stability being sheerly new soybean varieties.Described technical scheme is as follows:

Embodiments provide a kind of method of testing specificity, consistence and the stability being sheerly new soybean varieties, described method comprises:

Obtain soybean varieties to be measured and belong to variant sites between interior different varieties of the same race;

Determined the test zone of soybean varieties to be measured by described variant sites, described test zone comprises universal test region, and at least part of described variant sites is included in described universal test region;

Build and comprise the genotypic database of described different soybean varieties at all described test zones;

After determining the amount of sampling SN of described soybean varieties to be measured, stochastic sampling mixes the DNA of united extraction mixing sample;

The primer of the described test zone of preparation amplification, institute's primer comprises described universal test region primer;

Utilize the DNA of mixing sample described in described primer pair to increase, obtain the amplified production of described test zone, described amplified production is as high-throughput sequencing library;

High-flux sequence is carried out to described high-throughput sequencing library, obtains sequenced fragments group;

Analyze described sequenced fragments group, obtain soybean varieties genotype to be measured and hybrid strain genotype;

Described soybean varieties genotype to be measured is compared with the genotype of the described different varieties in described database, obtains the approximate kind of described soybean varieties to be measured, variant sites and variant sites rate;

The genotype of described hybrid strain genotype with the described different varieties in described database is compared, after obtaining hybrid strain kind, calculates hybrid strain rate;

Utilize described variant sites, described variant sites rate and described hybrid strain rate, judge the specificity of described soybean varieties to be measured, consistence and stability.

Particularly, described amount of sampling SN meets following condition: BINOM.INV (SN, M, 0.95)/SN≤1.15*M, wherein BINOM.INV is the function in excel 2010, M is threshold value selected when judging described consistence and stability, the condition implication that described amount of sampling SN meets is: even if 15% of judgment threshold M when described hybrid strain rate only exceeds consistence and stability, described amount of sampling, under the probability guarantee of 95%, correctly can judge stability and the consistence of described soybean varieties to be measured.

Particularly, the degree of depth CF of described high-flux sequence meets following condition: BINOM.DIST (10, 10, BINOM.DIST (8, 20, BINOM.DIST (0, CF, 0.1%, TRUE), TRUE), FALSE) >=99.9%, 1-BINOM.DIST (10000, 10000, 1-BINOM.DIST (8, 20, 1-BINOM.DIST (99.99%*CF, CF, 99.9989%, TRUE), TRUE), FALSE)≤0.1% and BINOM.DIST (10* (1-M) * CF, 10*CF, 1-110%*M, TRUE) >=95.0%, wherein, CF is the degree of depth of described high-flux sequence, M is threshold value selected when judging described consistence and stability, BINOM.DIST is the function in excel 2010, the condition implication that the degree of depth CF of described high-flux sequence meets is: be low to moderate 0.1% in described hybrid strain rate, described hybrid strain kind is 10 and under on average only having the condition in 20 difference sites between described hybrid strain kind and described soybean varieties, probability >=99.9% detecting whole described hybrid strain kind determined by the degree of depth CF of described high-flux sequence, under the kind of described database is 10000 and on average only has the condition in 20 difference sites between described hybrid strain kind and described soybean varieties, probability≤0.1% of described hybrid strain kind is judged in the existence determined by the degree of depth CF of described high-flux sequence by accident, described hybrid strain kind be 10 and true hybrid strain rate exceed only judge specificity time selected threshold value 10% time, by the degree of depth CF of described high-flux sequence determine to stability and correct probability >=95.0% of conforming judgement conclusion.

Particularly, described test zone also comprises non-universal test zone, and described primer also comprises non-universal test zone primer.

Further, described non-universal test zone primer comprises the first primer and the second primer, described first primer comprises the first forward primer and the first reverse primer, described second primer comprises the second forward primer and the second reverse primer, described first primer and described second primer carry out amplification separately respectively and obtain the amplified production of two described non-universal test zones, the amplified production balanced mix of two described non-universal test zones are used for the high-throughput sequencing library building amplification separately;

5 ' of described first forward primer holds the sequence 1 be connected with as shown in SEQ ID NO:1 in sequence table, and 5 ' in described first reverse primer holds the sequence 2 be connected with as shown in SEQ ID NO:2 in sequence table;

5 ' of described second forward primer holds the sequence 2 be connected with as shown in SEQ ID NO:2 in sequence table, and 5 ' of described second reverse primer holds the sequence 1 be connected with as shown in SEQ ID NO:1 in sequence table.

Particularly, utilize described variant sites, described variant sites rate and described hybrid strain rate, judge that the method for described soybean varieties specificity to be measured, consistence and stability comprises:

When there is described variant sites in described variant sites rate >=SD or described non-universal test zone, described soybean varieties to be measured has specificity, as described variant sites rate < SD and described variant sites is not present in described non-universal test zone time, described soybean varieties to be measured does not have specificity, wherein, SD is threshold value selected when judging specificity;

As the described hybrid strain rate≤M of described soybean varieties to be measured, described soybean varieties to be measured has consistence and stability, when the described hybrid strain rate of described soybean varieties to be measured is greater than > M, described soybean varieties to be measured does not have consistence and stability, and M is threshold value selected when judging described consistence and stability;

Described hybrid strain rate R=R1+R2-R3-R4, wherein:

$R1={\mathrm{\&Sigma;}}_{i1=1}^{n1}\frac{{\mathrm{\&Sigma;}}_{j1=\mathrm{Int}(0.8\&times;t1)+1}^{t1-\mathrm{Int}(0.1\&times;t1)}2\&times;R1i1j1}{t1-\mathrm{Int}(0.8\&times;t1)-\mathrm{Int}(0.1\&times;t1)},$ Wherein, n1 is the number of nucleus hybrid strain kind, t1 is the number of all special hybrid strain nuclear gene type of the i-th 1 described nucleus hybrid strain kinds, i1j1 is after all described special hybrid strain nuclear gene type of the i-th 1 described nucleus hybrid strain kinds sorts from low to high by frequency, the described special hybrid strain nuclear gene type of jth 1, R1i1j1 is the frequency of the i-th 1j1 described special hybrid strain nuclear gene type; R1 is the summation of the described hybrid strain rate of the described nucleus hybrid strain kind calculated by hybrid strain nuclear gene type, the described hybrid strain rate of described nucleus hybrid strain kind is after the frequency of the described special hybrid strain nuclear gene type removing in described nucleus hybrid strain kind minimum 80% and the highest 10%, 2 times of the mean value of the frequency of remaining described special hybrid strain nuclear gene type;

wherein, t2 is number that is except the hybrid strain nuclear gene type that described nucleus hybrid strain kind has and the described hybrid strain nuclear gene type of frequency>=0.17%, i2 is after all described hybrid strain nuclear gene type except the described hybrid strain nuclear gene type that described nucleus hybrid strain kind has sorts from low to high by frequency, the i-th 2 described hybrid strain nuclear gene types, R2i2 is the frequency of the i-th 2 described hybrid strain nuclear gene types; R2 is the described hybrid strain rate utilizing the described hybrid strain nuclear gene type had except described nucleus hybrid strain kind to calculate, R2 is after the value of in the frequency removing the described hybrid strain nuclear gene type had except described nucleus hybrid strain kind minimum 80% and the highest 10%, 2 times of the mean value of residual value shi;

$R3={\mathrm{\&Sigma;}}_{i3=1}^{n2}R3i3-2\&times;R3\mathrm{ic},$ Wherein, $R3i3=\frac{{\mathrm{\&Sigma;}}_{j3=\mathrm{Int}(0.8\&times;t3)+1}^{t3-\mathrm{Int}(0.3\&times;t3)}R3i3j3}{t3-\mathrm{Int}(0.8\&times;t3)-\mathrm{Int}(0.1\&times;t3)},$ N2 is the number of tenuigenin hybrid strain kind, R3i3 is the described hybrid strain rate of the i-th 3 described tenuigenin hybrid strain kinds, the value of R3i3 when R3ic is i3=ic, ic for when described soybean varieties to be measured be nucleo_cytoplasmic interaction sterile line or maintenance line time, corresponding described maintenance line or the described tenuigenin hybrid strain kind of described sterile line, t3 is the number of all special hybrid strain plasmagene type of the i-th 3 described tenuigenin hybrid strain kinds, i3j3 is after all described special hybrid strain plasmagene type of the i-th 3 described tenuigenin hybrid strain kinds sorts from low to high by frequency, the described special hybrid strain plasmagene type of jth 3, R3i3j3 is the frequency of the i-th 3j3 described special hybrid strain plasmagene type, R3ic refers to the hybrid strain rate of the described maintenance line be mixed in described sterile line or is mixed into the hybrid strain rate of the described sterile line in described maintenance line, R3 is the summation of the described hybrid strain rate of the described tenuigenin hybrid strain kind calculated by hybrid strain plasmagene type, the hybrid strain rate of described tenuigenin hybrid strain kind is after the frequency of the described special hybrid strain plasmagene type removing in described tenuigenin hybrid strain kind minimum 80% and the highest 10%, the mean value of the frequency of remaining described special hybrid strain plasmagene type,

wherein, t4 is number that is except the described hybrid strain plasmagene type that described tenuigenin hybrid strain kind has and the described hybrid strain plasmagene type of frequency>=0.17%, i4 is after all described hybrid strain plasmagene type except the described hybrid strain plasmagene type that described tenuigenin hybrid strain kind has sorts from low to high by frequency, the i-th 4 described hybrid strain plasmagene types, R4i4 is the frequency of the i-th 4 described hybrid strain plasmagene types; R4 is the described hybrid strain rate utilizing the described hybrid strain plasmagene type had except described tenuigenin hybrid strain kind to calculate, R4 is after the value of in the frequency removing the described hybrid strain plasmagene type had except described tenuigenin hybrid strain kind minimum 80% and the highest 10%, the mean value of residual value shi;

Int () is bracket function;

Described nucleus hybrid strain kind refers to and only utilizes nuclear gene type to calculate the described hybrid strain kind obtained, and described tenuigenin hybrid strain kind refers to and only utilizes plasmagene type to calculate the described hybrid strain kind obtained; Described special hybrid strain nuclear gene type refers to and is only all described hybrid strain nuclear gene types of a described nucleus hybrid strain kind; Described special hybrid strain plasmagene type refers to and is only all described hybrid strain plasmagene types of a described tenuigenin hybrid strain kind; Described hybrid strain nuclear gene type refers to that described hybrid strain genotype is described nuclear gene type, and described nuclear gene type refers to that described genotype is positioned on nuclear genome; Described hybrid strain plasmagene type refers to that described hybrid strain genotype is described plasmagene type, and described plasmagene type refers to that described genotype is positioned on cytoplasmic skeleton; Genotypic frequency refers in described sequenced fragments group, represents the ratio that described genotypic sequenced fragments number accounts for the sequenced fragments sum of test zone described in described genotype place.

Further, described method also comprise the correct probability of the conclusion of consistence and the stability judging described soybean varieties to be measured in the following ways as: when described soybean varieties to be measured has consistence and stability, the probability that conclusion is correct >=BINOM.DIST (M*SN, SN, R, TRUE) * BINOM.DIST (Σ SeN*M, Σ SeN, R, TRUE), when described soybean varieties to be measured does not have described consistence and stability, probability >=BINOM.DIST ((1-M) * SN that conclusion is correct, SN, (1-R), TRUE) * BINOM.DIST (Σ SeN* (1-M), Σ SeN, 1-R, TRUE), wherein, Σ SeN is the summation of the sequenced fragments of test zone described in all described genotypic frequency places for calculating described hybrid strain rate R, M is threshold value selected when judging described consistence and stability, BINOM.DIST (M*SN, SN, R, TRUE) for described soybean varieties to be measured has carried out SN sampling, the actual described hybrid strain rate R taken out is less than the probability of described threshold value M, BINOM.DIST (Σ SeN*M, Σ SeN, R, TRUE) meaning is: carried out Σ SeN sampling to described soybean varieties to be measured, the actual described hybrid strain rate R taken out is less than the probability of threshold value M.

Further, when described non-universal test zone does not exist described variant sites, if judge, described soybean varieties to be measured has specificity, probability >=BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) that conclusion is correct, if judge, described soybean varieties to be measured does not have specificity, the probability that conclusion is correct >=BINOMDIST (SD*TRN, TRN, OD, TRUE), wherein, TRN is the number detecting successful test zone, OD is described variant sites rate, SD is threshold value selected when judging specificity, BINOMDIST is the function in excel 2010, the probability that described conclusion is correct is expressed as when judging that described soybean varieties to be measured has specificity, described variant sites rate is greater than the probability of SD, when judging that described soybean varieties to be measured does not have specificity, described variant sites rate is less than the probability of SD, the successful test zone of described detection obtains after analyzing described sequenced fragments group.

Particularly, the method obtaining described hybrid strain kind comprises: described hybrid strain kind is be present in the kind in described database, and has the number of the described test zone of homologous genes type to account for ratio >=60% that described hybrid strain kind has the sum of the genotypic described test zone of described potential hybrid strain between the potential hybrid strain genotype of described hybrid strain kind and described hybrid strain genotype; Described hybrid strain genotype refers to the described potential hybrid strain genotype of frequency >=0.02%;

Insertion or the disappearance of discontinuous base is had in quantity >=2 of the distinguishing base between all genotype of described potential hybrid strain genotype and described soybean varieties to be measured or described distinguishing base.

Particularly, determine that the method in described universal test region is by described variant sites:

Pass through discrimination calculate the value of discrimination, wherein, a is the kind sum be detected in variation window area, bi is i-th kind of genotypic kind number in described variation window area, and bi>1, k comprises the genotypic number being greater than a kind, and described variation window area is centered by each single nucleotide variations site, and the both sides to described single nucleotide variations site respectively extend 1/2 of survey sequence length as the window detected;

Described universal test region is the large and equally distributed region of described discrimination on region or nuclear genome that on cytoplasmic skeleton, discrimination is large.

The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: the method that the embodiment of the present invention provides is by high-flux sequence and the amplification of multidigit point, the large sample achieving individual test zone between the large sample sampling of soybean varieties to be measured and kind is sampled, the comprehensive means such as recycling definition hybrid strain genotype, definition tenuigenin hybrid strain kind and definition hybrid strain rate calculation formula, successfully achieve the specificity, stability and the conforming target that accurately, intactly judge soybean varieties to be measured, and test speed is faster, can complete within 10 days.

Embodiment

For making the object, technical solutions and advantages of the present invention clearly, below embodiment of the present invention is described further in detail.

Embodiment. measure soybean varieties ' specificity of northern 93-406 ', consistence and stability

The soybean varieties to be measured that the embodiment of the present invention provides is soybean varieties " northern 93-406 ", soybean varieties " northern 93-406 " is for pure lines soybean and for openly to use kind, it is hybridized with " rich No. 8 of north ", has cultivated " cultivating mirror No. 26, beans " soybean varieties by systematic breeding.The method then measuring the specificity of this soybean varieties, consistence and stability comprises the following steps.

One, the variant sites between different soybean varieties is obtained.

The variant sites of different soybean varieties can obtain from the documents and materials announced, but the results contrast that the method obtains is fragmentary, in the present embodiment, the variant sites between a large amount of different soybean varieties is obtained by the genome sequence of more different soybean varieties.

Further, the method obtaining the genome sequence of different soybean varieties is as follows:

The genome sequence of the different soybean varieties of the present embodiment shows two kinds of sources, the first is the genomic high-flux sequence sequence to 31 soybean varieties such as Lam, pertinent literature information is as follows: Lam HM et al.Resequencing of 31wild and cultivated soybean genomes identifies patterns ofgenetic diversity and selection.Nat Genet 2010,42:1053 – 1059.The genome sequence of these 31 soybean varieties is published in NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/sra), and reception number is SRA020131; The second is for having carried out high-flux sequence by the method provided in the above-mentioned article delivered of Lam etc. to " rich No. 8 of north " and " cultivating mirror No. 26, beans ".The present embodiment obtains the genomic high-flux sequence sequence of 33 soybean varieties altogether.

Further, the genome sequence of different varieties is utilized to obtain variant sites.

Particularly, because the order-checking degree of depth of these 33 soybean varieties is not high, single nucleotide variations (SNP) site can only be identified, if the order-checking degree of depth of soybean varieties is enough high, then can identify such as the repeat number of other variation type to make a variation, due to a low credibility, not identify.Utilize Frederick Sanger comparison software (version number is 0.4) by the genomic high-flux sequence sequence alignment of these 33 soybean varieties to " Williams_82 " soya cells core reference genome (version: Release v1.01, download address: http://genome.jgi-psf.org/) and tenuigenin with reference on genome, this tenuigenin comprises plastosome with reference to genome and chloroplast(id) reference genome with reference to genome, it is at NCBI (National Center for Biotechnology Information, US National Biotechnology Information center) on reception number be respectively JX463295.1 and NC_007942.1.During contrast, Insert Fragment length is set to 500bp, and other parameter settings are default value.The Ssaha Pileup software package (version number is 0.5) adopted identifies the SNP site of each soybean varieties.This SNP site is defined as base pair that difference determines, the insertion of single base or the disappearance of single base.The base pair that this difference is determined refers to and does not comprise the uncertain base pair of difference, the uncertain base pair of difference refers to the base pair between some degeneracy base, as R represents A or G, therefore, may there are differences between A and R, also may not there are differences, therefore, between A and R, difference is indefinite, is not SNP mutually.Therefore, the SNP site in the embodiment of the present invention is not for comprise the uncertain base pair of above-mentioned difference.By the definition of above SNP site, the embodiment of the present invention obtains 6350046 SNP site altogether between all 33 soybean varieties, and wherein 31937 SNP site are positioned on cytoplasmic skeleton, and remaining SNP site is positioned on nuclear genome.Namely the genotype hereinafter mentioned refers to the combination of multiple SNP site in test zone, and nuclear gene type refers to that genotype is positioned on nuclear genome, and plasmagene type refers to that genotype is positioned on cytoplasmic skeleton.Such as, in table 1, the 1st test zone is positioned on nuclear genome, and be nuclear gene type, this test zone has 3 SNP site, and the genotype of this test zone is the combination of these 3 SNP site.

Two, determined the test zone of soybean varieties to be measured by variant sites, test zone comprises universal test region, and at least part of variant sites is included in universal test region, and its method comprises:

Determine universal test region

Universal test region is the large and equally distributed region of discrimination on region or nuclear genome that on cytoplasmic skeleton, discrimination is large, wherein, and discrimination wherein, a is the kind sum be detected in variation window area, bi is i-th kind of genotypic kind number in variation window area, and bi>1, k comprises the genotypic number being greater than a kind, variation window area is centered by each single nucleotide variations site (SNP site), and the both sides to single nucleotide variations site respectively extend 1/2 of survey sequence length as the window detected; Test zone is the large and equally distributed region of discrimination on region or nuclear genome that on cytoplasmic skeleton, discrimination is large.The Computing Principle of discrimination is as follows: all interracial number of combinations are wherein, the combination between the different varieties in same gene type is undistinguishable, and its number is so, can not be by the ratio of the breed combination distinguished can by the ratio of breed combination distinguished and discrimination as can be seen here, discrimination is larger, more different varieties can be distinguished, and the variation window area that discrimination is large is more effective to DUS test.If the variation window area skewness on nuclear genome, some region can be caused adjacent, thus linkage inheritance, information is easily overlapping, therefore, nuclear genome is selected the principle of compositionality in universal test region to be: the large and SNP site of discrimination is uniformly distributed.Cytoplasmic skeleton without linkage inheritance problem, so, cytoplasmic skeleton only needs the region that selection area calibration is large.

Adopt Proton high-flux sequence instrument to carry out high-flux sequence in the embodiment of the present invention, the test zone length that its order-checking detects can reach 200bp, and in order to obtain maximum fault information, the longest test zone in the present embodiment is also 200bp.Therefore, the variant sites that the present embodiment is mentioned refers to whole test zone, and its inside may comprise multiple SNP site.

First, centered by each SNP site obtained, respectively extend 99bp and 100bp to the left and right, form the variation window of 200bp.According to 6350046 SNP site obtained, 6350046 variation windows can be obtained, calculate the discrimination of these variation window areas such as, in the 1st variation window area, detect a=29 kind altogether, total k=2 kind genotype GTT, ACC, their kind number is respectively b1=22, b2=5, therefore, its implication is: by the 1st variation window area, the breed combination of 41% in 29 kinds can be distinguished, the breed combination of other 59% cannot distinguish, and needs more variation window just can distinguish.After the same method, the discrimination of whole 6350046 the variation windows of calculating acquisition is also therefrom chosen and is arranged in 8000 maximum variation windows of nuclear genome discrimination, 100 the make a variation windows maximum with being arranged in cytoplasmic skeleton discrimination.Check 8000 the variation windows being arranged in nuclear genome one by one, each variation window and the next distance made a variation between window, if distance is more than 300K (1K=1000 base), reexamine after then abandoning the less variation window of wherein discrimination, till the adjacent distance looking into variation window is all greater than 300K.The criterion distance of 300K is selected to be because soybean gene group size is about 975M (1M=,100 ten thousand bases), the universal test region that 2000 are positioned at nuclear genome is selected in by final, the interregional distance of average universal test is about 500K, but because seldom there is variant sites in such as kinetochore etc., some specific regions, therefore, mean distance should be less than 500K.By above method, have selected the variation window that 4987 are positioned at nuclear genome, they pass through test zone with totally 5087 windows that make a variation that to be arranged in together with 100 maximum windows that make a variations of cytoplasmic skeleton discrimination obtained as what be selected in.Wherein, 200 variation windows that selection area calibration is maximum, be empirical value, this quantity can be modified as the case may be.

This test zone can also comprise non-universal test zone, and concrete grammar is as follows:

Determine non-universal test zone:

Non-universal test zone refers to that special kinds needs the non-universal site detected.DUS test needs the special site detecting fixed point transformation, and fixed point transformation is technique means conventional in modern breeding, and as back cross breeding, transgenic breeding etc., fixed point transformation kind also can become new variety because it has specificity.Based on the specific decision principle of New variety protection, non-universal test zone should not included in universal test region and be the site of known control qualitative character.In the present embodiment, because soybean varieties to be measured is not by fixed point transformation, need to detect without non-universal site, therefore, universal test region nothing but.

Three, prepare the primer in amplification assay region, test zone primer comprises universal test region primer, specific as follows:

Prepare universal test region primer, this universal test region primer for all kinds, particularly:

Universal test region adopts multiple PCR technique to detect, and multiple PCR technique refers to and add multiple PCR primer, the multiple sites simultaneously in amplification gene group in same PCR reaction.The key of this technology designs and synthesizes multiple PCR primer, the multiple PCR technique that the present embodiment adopts LifeTechnology company of the U.S. to provide, and it can arrange the heavy PCR primer of as many as 12000.

Primer acquisition process is as follows: log in LifeTechnology company multiple PCR primer Photographing On-line webpage https: //ampliseq.com/protected/help/pipelineDetails.action, submits relevant information to by its requirement.In the present embodiment, " Application type " option selects " DNA Hotspot designs (single-pool) ".If select multi-pool, then multiplex PCR will divide multitube to carry out, cost can increase to some extent, and the primer of single-pool only needs a multiplex PCR, save cost, shortcoming is that some universal test regions design of primers may failure, but alternative universal test region on genome is more, therefore, abandon some alternative universal test regions and do not affect result.Permeate the nucleus of soybean varieties to be measured reference genome and tenuigenin reference genome a file, and select " Custom " in " Select the genome you wish to use " option after, upload the file of fusion as reference genome during design multiple PCR primer.DNA type option selects " Standard DNA ", in Add Hotspot option, add the positional information of the SNP site in the universal test region needing design, comprise chromosome information, the initiation site of SNP and the end locus of SNP, its certain embodiments is in table 1.Finally click " Submit targets " button to submit to and the multiple PCR primer obtaining design.In the present embodiment, from all 5087 universal test regions, design and be successfully authenticated 2488 pairs of multiple PCR primers, for corresponding 2488 universal test regions of increasing.The method demonstrating multiple PCR primer is that the said firm is by method provided by the invention, extract the leaves genomic DNA on same strain soybean, and utilize the multiple PCR primer of design to increase to the genomic dna obtained, build storehouse, high-flux sequence analyze sequenced fragments group, remove the corresponding primer of following test zone: the sequenced fragments number of this test zone is less than 1000 or there is hybrid strain genotype, and the primer remained is the multiple PCR primer be proved to be successful.Because genomic DNA source is in same strain soybean leaves, can not there is hybrid strain kind, therefore, hybrid strain genotype is the PCR or order-checking Preference mistake that are caused by the special construction of test zone, removes these test zones and avoids this type of system mistake.The multiple PCR primer be proved to be successful is supplied to client in fluid form and uses after also being mixed by the said firm.2488 universal test regions of above-mentioned successful design multiple PCR primer are the universal test region finally detected for soybean varieties to be measured, simultaneously, each kind in the database built also contains above-mentioned 2488 universal test regions, wherein, 47 universal test regions are positioned on cytoplasmic skeleton, and remaining 2441 universal test regions are positioned on nuclear genome.

It should be noted that: the number in universal test region requires >=900, and reason is as follows: if lower than 900, and the probability that there is the hybrid strain kind of erroneous judgement will more than 1%, and the projectional technique of this threshold value is in table 2.Detect failed test zone owing to existing, therefore, test zone number generally >=1000.

Test zone primer can also comprise non-universal test zone primer, and this non-universal test zone primer is for soybean varieties to be measured, specific as follows:

Preparation non-universal test zone primer:

The primer of non-universal test zone comprises the first primer and the second primer, first primer comprises the first forward primer and the first reverse primer, second primer comprises the second forward primer and the second reverse primer, first primer and the second primer carry out amplification separately respectively and obtain the amplified production of two non-universal test zones, the amplified production balanced mix of two non-universal test zones are used for the high-throughput sequencing library building amplification separately.5 ' of first forward primer holds in sequence 1, first reverse primer be connected with as shown in SEQ ID NO:1 in sequence table 5 ' to hold the sequence 2 be connected with as shown in SEQ ID NO:2 in sequence table; 5 ' end of the second forward primer is connected with 5 ' of sequence 2, second reverse primer as shown in SEQ ID NO:2 in sequence table and holds the sequence 1 be connected with as shown in SEQ ID NO:1 in sequence table.

The design process of non-universal test zone primer is as follows: the first step, be no more than 200bp by amplification length and comprise the requirement of all SNP site in non-universal test zone, by common PCR primers method of design, the forward primer of the PCR of design amplification non-universal test zone and reverse primer; Second step, holds SEQ IDNO:2 in SEQ ID NO:1 and sequence table in catenation sequence table respectively, obtains the forward primer of the first primer and the reverse primer of the first primer respectively by 5 ' of the forward primer designed and reverse primer; 3rd step, holds SEQ ID NO:1 in SEQ ID NO:2 and sequence table in catenation sequence table respectively, obtains the forward primer of the second primer and the reverse primer of the second primer respectively by 5 ' of the forward primer designed and reverse primer.In sequence table, in SEQ ID NO:1 and sequence table, SEQ ID NO:2 is high-flux sequence joint sequence used, thus use PCR primer with the joint sequence of high-flux sequence, together check order after setting up sequencing library after can directly mixing with the product in the general order-checking region of amplification, and need not through fragmentation, jointing etc. loaded down with trivial details build storehouse step, improve working efficiency and reduce cost.Making two to the only different primer of joint is to check order from the two ends of non-universal test zone simultaneously.

Soybean varieties to be measured in the present embodiment owing to there is no non-universal test zone, therefore, without the need to non-universal test zone primer.

Four, build that to comprise different soybean varieties as follows in the method for the genotypic database of all test zones:

This example obtains 2488 universal test region primers and 0 non-universal test zone primer, and the amplification region of their correspondences is the test zone of soybean varieties to be measured.Build and comprise the genotype of 2488 test zones of 33 kinds and the database of the positional information of SNP thereof, partial results is in table 1.

Table 1 is the certain embodiments of database variety and genetype and position thereof, soybean varieties genotype to be measured, hybrid strain genotype and frequency thereof

In table 1, the position that '-' represents this SNP site is lacking with reference on genome; Except ATGC, other letter represents degeneracy base.If genotype is made up of degeneracy base N entirely, claim corresponding test zone genotype and SNP shortage of data, when the genotype of disappearance or SNP compare with any genotype or SNP, all do indifference process.Can by detection provided by the invention soybean varieties to be measured genotypic method Test database kind and completion disappearance genotype.

The present embodiment does not have completely to list all database content as space is limited, only lists the information of 10 test zones of wherein 5 kinds.Equally based on length restriction, also have some areas also only to list part related example in the present embodiment, all the other unlisted data can according to the method completion of the present embodiment.

Five, after determining the amount of sampling SN of soybean varieties to be measured, stochastic sampling mixes the DNA of united extraction mixing sample, and method is as follows:

Calculate soybean varieties amount of sampling to be measured

Amount of sampling SN should meet following condition: BINOM.INV (SN, M, 0.95)/SN≤1.15*M, wherein, M is threshold value selected when judging consistence and stability, BINOM.INV is the function in excel 2010, and its using method is identical with the definition in excel 2010, and its implication is the smallest positive integral making the functional value of accumulation binominal distribution be more than or equal to threshold value.The condition implication that amount of sampling SN meets is: even if 15% of judgment threshold M when hybrid strain rate only exceeds consistence and stability, this amount of sampling 95% probability ensure under, correctly can judge stability and the consistence of soybean varieties to be measured.M value is artificially determined according to conditions such as crop species, labeling pattern, specific requirements.Regulation in " new variety of plant specificity, consistence and stability test guide-soybean " in New variety protection office of the Ministry of Agriculture issues: for routine kind, during consistency checking, adopt the population norms of 0.5% and the acceptance probability of at least 95%.Therefore, in the present embodiment, select intermediate value 0.5% as M value.After progressively strengthening SN value, calculate above-mentioned formula and find, when SN >=25218, BINOM.INV (SN, 0.5%, 0.95)/SN≤1.15*0.5% sets up.Therefore, the soybean varieties amount of sampling to be measured in the present embodiment answers >=25218.

Stochastic sampling mixes the DNA of united extraction mixing sample

In the present embodiment, have chosen 30000 seed germinations, random selecting 26000 sizes roughly equal bud mixing be placed in mortar, fully pulverize add liquid nitrogen in mortar after.The article No. adopting Beijing Tian Gen biochemical technology company limited to produce is that the plant genome DNA extraction test kit of DP305 extracts and obtains the DNA of soybean varieties mixing sample to be measured, and DNA extraction method is undertaken by the operational manual of this test kit.American I nvitrigen company is utilized to produce dsDNA HS Assay Kit (article No. is Q32852) and specification sheets thereof carry out quantitatively to the DNA obtained, and dilute the soybean varieties DNA to be measured quantitatively for 10.00ng/ μ l.

Six, utilize the DNA of primer pair mixing sample to increase, obtain the amplified production of test zone, amplified production is as follows as the method for high-throughput sequencing library:

High-throughput sequencing library comprises: the high-throughput sequencing library in universal test region and the high-throughput sequencing library of non-universal test zone, in the present embodiment, build the high-throughput sequencing library of universal test region and non-universal test zone respectively, the two is mixed, obtains the high-throughput sequencing library of all test zones.

The method building the high-throughput sequencing library in universal test region is as follows:

After utilizing library construction Kit 2.0 (produced by LifeTechnology company of the U.S., article No. is 4475345) multiplexed PCR amplification universal test region, amplified production is utilized to build high-throughput sequencing library.This test kit comprises following reagent: 5 × Ion AmpliSeq ^tMhiFi Mix, FuPa reagent, transferring reagent, sequence measuring joints solution and DNA ligase.The method of library construction presses the operational manual " IonAmpliSeq of this test kit ^tMlibrary Preparation " (publication number: MAN0006735, version: A.0) carry out.By multiplexed PCR amplification 2488 universal test regions, the amplification system of multiplex PCR is as follows: 5 × IonAmpliSeq ^tMthe DNA 10ng of the universal test region primer mixed solution 4 μ l of HiFi Mix 4 μ l, preparation, soybean varieties to be measured and without enzyme water 11 μ l.The amplification program of multiplex PCR is as follows: 99 DEG C, 2 minutes; (99 DEG C, 15 seconds; 60 DEG C, 4 minutes) × 25 circulations; 10 DEG C of insulations.After utilizing FuPa reagent to digest primer unnecessary in multiplexed PCR amplification product, then carry out phosphorylation, concrete grammar is: in the amplified production of multiplex PCR, add 2 μ L FuPa reagent, after mixing, by following program reaction in PCR instrument: 50 DEG C, and 10 minutes; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixture a, and mixture a is containing the amplified production solution through phosphorylation.The amplified production of phosphorylation is connected upper sequence measuring joints, and concrete grammar is: in mixture a, add transferring reagent 4 μ L, sequence measuring joints solution 2 μ L and DNA ligase 2 μ L, after mixing, by following program reaction in PCR instrument: 22 DEG C, and 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixed solution b.10 μ L are dissolved in without in enzyme water after utilizing the ethanol precipitation methods purifying mixed solution b of standard.American I nvitrigen company is utilized to produce dsDNA HS Assay Kit (article No. is Q32852) also measures according to its specification sheets, and after obtaining the mass concentration of mixed solution b, mixed solution b after purifying is diluted to 15ng/ml, obtains the high-throughput sequencing library that concentration is about the universal test region of 100pM.

The method building the high-throughput sequencing library of non-universal test zone is as follows:

With the DNA of soybean varieties to be measured for template, utilize the first primer of the non-universal test zone of above-mentioned preparation and the second primer to carry out independent pcr amplification respectively, after balanced mix amplified production, obtain the high-throughput sequencing library of non-universal test zone.Concrete operations are by " Ion Amplicon Library Preparation (FusionMethod) " (publication number: 4468326) carry out, roughly process is as follows: be after the concentration of 10 μMs by the forward primer of the first primer and reverse primer water dissolution, equal-volume mixes, and obtains the first primer solution.Be formulated as follows PCR reaction system: (invirtrigen company of the U.S. produces for first primer solution 1 μ L, 30ng soybean varieties DNA to be measured and PCR high-fidelity mixture, article No. is 12532016) 45 μ L, after mixing, by following program reaction in PCR instrument: 94 DEG C, 3 minutes; (94 DEG C, 30 seconds; 58 DEG C, 30 seconds; 68 DEG C, 1 minute) × 40 circulations; 4 DEG C of insulations.Pcr amplification product is by being dissolved in 10 μ L water after the method purifying of the alcohol settling of standard, on the biological analyser (model is 2100) utilizing DNA 1000 test kit (article No. is 5067-1504) to produce in Agilent company of the U.S., to measure by this test kit specification sheets and after obtaining the volumetric molar concentration of amplified production, dilution is 200pM, is the amplified production of the first primer.Adopt identical method, obtain the amplified production that concentration is second primer of 200pM.The amplified production of the first primer is mixed with the amplified production equal-volume of the second primer, obtains the non-universal test zone high-throughput sequencing library that concentration is 100pM.In the present embodiment, due to universal test region nothing but, therefore, also without the need to building the high-throughput sequencing library of non-universal test zone.

Obtain the high-throughput sequencing library of all test zones

In the number in universal test region and the high-throughput sequencing library in universal test region of the volumetric molar concentration such as the ratio of the number of non-universal test zone mixes and the high-throughput sequencing library of non-universal test zone, the mixture obtained is the high-throughput sequencing library of all test zones.In the present embodiment, because of the high-throughput sequencing library in universal test region nothing but, therefore, the high-throughput sequencing library of structure is the high-throughput sequencing library that concentration is the universal test region of 100pM.

Seven, carry out high-flux sequence to high-throughput sequencing library, obtain sequenced fragments group, method is as follows:

Determine the principle of the high-flux sequence degree of depth: the degree of depth of high-flux sequence meets following condition: BINOM.DIST (10, 10, BINOM.DIST (8, 20, BINOM.DIST (0, CF, 0.1%, TRUE), TRUE), FALSE) >=99.9%, 1-BINOM.DIST (10000, 10000, 1-BINOM.DIST (8, 20, 1-BINOM.DIST (99.99%*CF, CF, 99.9989%, TRUE), TRUE), FALSE)≤0.1% and BINOM.DIST (10* (1-M) * CF, 10*CF, 1-110%*M, TRUE) >=95.0%, wherein, CF is the degree of depth of high-flux sequence, also the multiple that namely average each test zone is capped, M is threshold value selected when judging consistence and stability, BINOM.DIST is the function in excel 2010, its using method is identical with the definition in excel 2010, the probability of binomial distribution that what it returned is.The meaning of these three functions is: be low to moderate 0.1% in hybrid strain rate, hybrid strain kind reach 10 and between hybrid strain kind and soybean varieties to be measured average only 20 difference sites condition under, probability >=99.9% detecting whole hybrid strain kind determined by the high-flux sequence degree of depth; Under the condition in database average only 20 difference sites to 10000 and between hybrid strain kind and soybean varieties to be measured wide in variety, probability≤0.1% of the existence erroneous judgement hybrid strain kind determined by the high-flux sequence degree of depth; Hybrid strain kind reach 10 and true hybrid strain rate exceed only judge specificity time selected threshold value 10% time, by the high-flux sequence degree of depth determine to stability and correct probability >=95.0% of conforming judgement conclusion.Above condition is very strict, and therefore, true effect is better than above-mentioned threshold value.The projectional technique of above probability is in table 2.

Table 2 is the method for calculation of the present embodiment dependent probability

Table 2 is Excel 2010 data sheet, and its function, cell etc. are all identical with the definition of Excel 2010.Wherein, " threshold value (M) selected when judging consistence and stability " is as cell B31, other cell numbering is reference with B31, by the rule definition of Excel 2010, the such as cell at " hybrid strain rate (R) " place adds 4 row 1 and arranges on the basis of B2, therefore be numbered C35, other cell coding rule is identical therewith.

The defining method of the present embodiment high-flux sequence degree of depth is: after M=0.5% being substituted into above-mentioned three formula, when progressively strengthening order-checking degree of depth CF to 6001, above-mentioned three equations can be made to set up, therefore, the present embodiment order-checking degree of depth is defined as >=and 6001 times.

High-throughput sequencing library is utilized to carry out high-flux sequence

Utilize high-throughput sequencing library and test kit Ion PI Template OT2200Kit v2 (invirtrigen company of the U.S. production of all test zones obtained, article No. is 4485146) check order before ePCR (Emulsion PCR, emulsion polymerization enzyme chain reaction) amplification, working method is undertaken by the operational manual of this test kit.(invirtrigen company of the U.S. produces to utilize ePCR product and test kit Ion PI Sequencing 200Kit v2, article No. is 4485149) on Proton bis-generation high-flux sequence instrument, carry out high-flux sequence, working method is undertaken by the operational manual of this test kit.In the present embodiment, high-flux sequence flux is set to 10000 times, average coverage test region.

Pre-treatment is carried out to high-flux sequence result

First judge the quality of data of high-flux sequence whether >=Q20, if <Q20 (this situation is few), then re-start high-flux sequence as stated above, until specification of quality reaches Q20 standard, Q20 standard meets the requirement of " order-checking mistake is the probability of particular bases "≤0.33% in table 2.To reach the high-flux sequence fragment comparison of specification of quality to all 2488 test zones, after removing the sequenced fragments that comparison is unsuccessful and genotype detection is incomplete, remaining all sequenced fragments are called sequenced fragments group.The incomplete sequenced fragments of genotype detection refers to and all SNP site in the order-checking region at this sequenced fragments place shown in " position of SNP on reference genome " in table 1 could not be detected, the reason that genotype detection is incomplete is that sequenced fragments is too short, and the unsuccessful reason of comparison is that sequenced fragments mostly is non-specific amplification product.

Eight, analyze sequenced fragments group, obtain soybean varieties genotype to be measured and hybrid strain genotype, method is as follows:

By the comparison of sequenced fragments group to all test zones, and add up the sequenced fragments number in each test zone, remove the test zone of sequenced fragments number≤1000, remaining test zone is for detecting successful test zone.In the present embodiment, obtain 2406 altogether and detect successful test zone.Comparison is called the sequenced fragments of this test zone to the fragment of test zone, and the base composition extracting in table 1 position shown in " SNP with reference to the position on genome " from sequenced fragments is called the genotype of this sequenced fragments.Genotypic frequency refers in sequenced fragments group, represents the ratio that this genotypic sequenced fragments number accounts for the sequenced fragments sum of this genotype place test zone.The maximum genotype of frequency is called soybean varieties genotype to be measured.Hybrid strain genotype refers to the potential hybrid strain genotype of frequency >=0.02%, wherein, has insertion or the disappearance of discontinuous base in quantity >=2 of the distinguishing base between all genotype of potential hybrid strain genotype and soybean varieties to be measured or distinguishing base.The principle of hybrid strain VDA genotypes is: in high-flux sequence, insert or missing errors very rare, and cause the probability of 2 fixing distinguishing base to be low to moderate (1%/3) 2=0.0011% because of the mistake that checks order, and require hybrid strain genotype frequency >=0.02%, under the restriction of these conditions, even the order-checking degree of depth of 30000, be only 0.0001% (method of calculation are in table 2) because the mistake that checks order produces the genotypic probability of certain hybrid strain.The frequency of 0.02% meets the strictest DUS testing standard at present, and what namely detect from 10,000 seeds is low to moderate 2 hybrid.If distinguishing base quantity=1, then all test zone all can produce the hybrid strain genotype (method of calculation are in table 2) of mistake, if during distinguishing base quantity >=3, hybrid strain genotype quantity sharply reduces, be difficult to accurately calculate hybrid strain rate R, therefore, the threshold value of distinguishing base quantity >=2 is optimum.

Such as, in sequenced fragments group, the sequenced fragments in the 1st order-checking region adds up to 9987 articles, there are TCA, TCG, TCC, TCT ... totally 25 kinds of genotype, represent these genotypic sequenced fragments numbers 9612,218,1,2 respectively ..., these genotypic frequencies are 9612/9987=96.25%, 218/9987=2.18%, 1/9987=0.01%, 2/9987=0.02% ...By soybean varieties genotype to be measured and the genotypic definition of hybrid strain, TCA should be the to be measured soybean varieties genotype of soybean varieties to be measured at the 1st test zone, and the frequency of TCG is more than 0.02%, but compare with soybean varieties genotype TCA to be measured the difference having 1 <2 base, therefore TCG is not hybrid strain genotype, and other genotype is the genotype that order-checking mistake produces.Hybrid strain nuclear gene type refers to that hybrid strain genotype is nuclear gene type, and hybrid strain plasmagene type refers to that hybrid strain genotype is plasmagene type.By this definition, first test zone also without hybrid strain nuclear gene type.By identical method, judge and obtain whole 2406 to detect the soybean varieties genotype to be measured of successful test zones, hybrid strain genotype and frequency thereof, and judge that the hybrid strain genotype obtained is hybrid strain nuclear gene type or hybrid strain plasmagene type.Result shows: obtain 291 hybrid strain genotype altogether, wherein, 286 is hybrid strain nuclear gene type, and 5 is hybrid strain plasmagene type.

Simply introduce the standard model detection method in the present embodiment below, 1 seed is got from soybean varieties to be measured, to sow and after growing up to seedling, utilize the blade of seedling to extract genomic dna by the method identical with soybean varieties to be measured, this DNA is called the standard model of soybean varieties to be measured.With soybean varieties to be measured simultaneously and by the high-throughput sequencing library of the parallel structure standard model of same procedure and high-flux sequence.Wherein, the maximum genotype of frequency is called standard model genotype, genotypic frequency >=0.02% of standard model hybrid strain and and have insertion or the disappearance of discontinuous base in quantity >=2 of distinguishing base between standard model genotype or distinguishing base.By the method identical with soybean varieties to be measured, obtain the standard model genotype in the successful test zone of each detection and standard model hybrid strain genotype.If the test zone that standard model genotype is identical with soybean varieties genotype to be measured accounts for standard model and soybean varieties to be measured all detects the ratio of successful test zone more than 90%, then standard model is correct, otherwise, again from soybean varieties to be measured, 1 seed is got, repeat above process, until obtain correct standard model.The hybrid strain genotype of corresponding with soybean varieties to be measured for the hybrid strain genotype of correct standard model test zone is compared, obtain identical hybrid strain genotype, remove hybrid strain genotype identical in soybean varieties to be measured, correct soybean varieties hybrid strain genotype to be measured is retained and for subsequent analysis.Above measure eliminates the hybrid strain genotype because Systematic selection mistake causes, the PCR selectivity mistake amplification that the special construction of Systematic selection mistake mainly gene order causes.It should be noted that: when database is wide in variety, when extensively can represent different varieties genotype, can require that hybrid strain genotype is identical with certain genotype of database kind, the function identical with standard model can be played equally, in the case, can not examination criteria sample, reach the object alleviating workload.In the present embodiment, result is: from 291 the hybrid strain genotype obtained, eliminate 2 hybrid strain genotype altogether, wherein 2 is hybrid strain nuclear gene type, and 0 is hybrid strain plasmagene type, 289 the hybrid strain genotype remained are for subsequent analysis, and partial results is in table 1.

Nine, soybean varieties genotype to be measured compared with the genotype of the different varieties in database, obtain approximate kind, variant sites and variant sites rate, method is as follows:

If in the test, the genotype of soybean varieties to be measured and database kind, all without lacking, claims this test zone to be the total test zone of soybean varieties to be measured and this database kind.In total test zone, if the genotype of soybean varieties to be measured and database kind is incomplete same, the test zone at the genotype place then claiming this incomplete same is the difference site of soybean varieties to be measured and this database kind, corresponding genotype Differential genotype each other, the number of the number/total test zone in difference site rate=difference site.From database, obtain the approximate kind that the minimum kind of difference bit rate is called soybean varieties to be measured, corresponding difference site is called variant sites, the number of the number/total test zone of variant sites rate=variant sites.

In the present embodiment, the total test zone number of the 1st kind " rich No. 8 of north " of soybean varieties to be measured and database is 2335.In the 1st total test zone, soybean varieties to be measured is respectively GTT and GTT with " rich No. 8 of north " genotype, the two is identical, therefore, 1st total test zone is not the difference site in soybean varieties to be measured and " rich No. 8 of north ", and GTT is not the Differential genotype in soybean varieties to be measured and " rich No. 8 of north " yet.By identical method, by all total test zones, soybean varieties to be measured compares with " rich No. 8 of north " genotype, finds to have 190 difference sites, difference site rate=190/2335=8.14%.By identical method, obtain all 33 interracial difference site rates in soybean varieties to be measured and database, and obtain the minimum kind of difference site rate for " cultivating mirror No. 26, beans ", difference site rate is 3.24%.Therefore, the approximate kind that " cultivating mirror No. 26, beans " is soybean varieties to be measured, the variant sites rate of soybean varieties to be measured is 3.24%.

Ten, the genotype of hybrid strain genotype with the different varieties in database compared, after obtaining hybrid strain kind, calculate hybrid strain rate, method is as follows:

Obtain hybrid strain kind: hybrid strain kind is present in the kind in database, and have the number of the test zone of homologous genes type to account between the potential hybrid strain genotype of hybrid strain kind and hybrid strain genotype ratio >=60% that hybrid strain kind has the sum of the genotypic test zone of potential hybrid strain, wherein, insertion or the disappearance of discontinuous base is had in quantity >=2 of the distinguishing base between all genotype of potential hybrid strain genotype and soybean varieties to be measured or distinguishing base.Hybrid strain kind is divided into nucleus hybrid strain kind and tenuigenin hybrid strain kind, and wherein, nucleus hybrid strain kind refers to and only utilizes nuclear gene type to calculate the hybrid strain kind obtained, and tenuigenin hybrid strain kind refers to and only utilizes plasmagene type to calculate the hybrid strain kind obtained.Such as, the genotype of the kind in tentation data storehouse is respectively AA, AA, AA/TT, and ("/" represents that this test zone is heterozygous genotypes, there is the genotype that latter two before "/" is different), AA/TT, AA/TT, AA/TT and AA time, when the corresponding genotype of soybean varieties to be measured is respectively AA, AA/TT, TT, AA, TT/CC, GG/CC and-A, corresponding potential hybrid strain genotype is: nothing, nothing, AA, TT, AA, AA/TT and AA.There is not heterozygous genotypes in general pure line cultivar, but only a few site may exist, in addition, hybrid strain mostly is cross-fertilize seed, and heterozygous sites is more common, therefore lists various possibility situation.Parameter 60% can ensure that whole hybrid strain kind detection probability is 100% and the probability of the hybrid strain kind of existence erroneous judgement is 0%, and the defining method of this parameter value is in table 2.

In the present embodiment, in 1st test zone, in database, first kind " rich No. 8 of north " is respectively GTT and GTT with the genotype of soybean varieties to be measured, without the difference of base between the two, therefore, GTT is not potential hybrid strain genotype, thus in the 1st test zone, there is not the potential hybrid strain genotype identical with hybrid strain genotype in " rich No. 8 of north " yet, by identical method, judge in the test zone of all nuclear gene types one by one, in database, whether the genotype of first kind " rich No. 8 of north " is potential hybrid strain genotype, if potential hybrid strain genotype, judge whether there is homologous genes type between potential hybrid strain genotype and hybrid strain genotype again, result shows, " rich No. 8 of north " has 134 and has the genotypic test zone of potential hybrid strain, the test zone number of homologous genes type is had to be 133 between the hybrid strain genotype in they and same test region, its ratio is 133/134=99.25%>60%, therefore, judge that " rich No. 8 of north " is as nucleus hybrid strain kind.By similar method, utilize the test zone of all plasmagene types, judge that " rich No. 8 of north " is not as tenuigenin hybrid strain kind.By identical method, judge in database, whether other kinds all are nucleus hybrid strain kind or tenuigenin hybrid strain kind, and result shows: only " rich No. 8 of north " is nucleus hybrid strain kind, does not find tenuigenin hybrid strain kind.These results suggest that: " rich No. 8 of north " may be pollinated instead of mechanical admixture by flyings, genotype has been mixed into soybean varieties to be measured.

Obtain special hybrid strain genotype: special hybrid strain genotype refers to and is only all hybrid strain genotype of a hybrid strain kind, it comprises special hybrid strain nuclear gene type and special hybrid strain plasmagene type; Special hybrid strain nuclear gene type refers to and is only all hybrid strain nuclear gene types of a nucleus hybrid strain kind, and special hybrid strain plasmagene type refers to and is only all hybrid strain plasmagene types of a tenuigenin hybrid strain kind.In the present embodiment, obtain 291 hybrid strain genotype altogether, wherein, 286 is hybrid strain nuclear gene type, and 5 is hybrid strain plasmagene type.First hybrid strain nuclear gene type ATGA is only nucleus hybrid strain kind " rich No. 8 of north " to be owned, so ATGA is the special hybrid strain nuclear gene type in " rich No. 8 of north ".By identical method, judge in 286 hybrid strain genotype of all acquisitions one by one, 133 special hybrid strain nuclear gene types had for " rich No. 8 of north ".By similar method, judge 5 hybrid strain plasmagene types all not as special hybrid strain plasmagene type.

Calculate hybrid strain rate R principle, specific as follows:

Hybrid strain rate R=R1+R2-R3-R4, wherein: wherein, n1 is the number of nucleus hybrid strain kind, t1 is the number of all special hybrid strain nuclear gene type of the i-th 1 nucleus hybrid strain kinds, i1j1 is after all special hybrid strain nuclear gene type of the i-th 1 nucleus hybrid strain kinds sorts from low to high by its frequency, the special hybrid strain nuclear gene type of jth 1, R1i1j1 is the frequency of the i-th 1j1 special hybrid strain nuclear gene type; R1 is the summation of the hybrid strain rate of the nucleus hybrid strain kind calculated by hybrid strain nuclear gene type, the hybrid strain rate of nucleus hybrid strain kind is after the frequency of the special hybrid strain nuclear gene type removing in nucleus hybrid strain kind minimum 80% and the highest 10%, 2 times of the mean value of the frequency of remaining special hybrid strain nuclear gene type; wherein, t2 is number that is except the hybrid strain nuclear gene type that nucleus hybrid strain kind has and the hybrid strain nuclear gene type of frequency>=0.17%, i2 is after all hybrid strain nuclear gene types except the hybrid strain nuclear gene type that nucleus hybrid strain kind has sort from low to high by its frequency, the i-th 2 hybrid strain nuclear gene types, R2i2 is the frequency of the i-th 2 hybrid strain nuclear gene types; R2 is the hybrid strain rate utilizing the hybrid strain nuclear gene type had except nucleus hybrid strain kind to calculate, and it is after the value of in the frequency removing the hybrid strain nuclear gene type had except nucleus hybrid strain kind minimum 80% and the highest 10%, 2 times of the mean value of residual value shi; $R3={\mathrm{\&Sigma;}}_{i3=1}^{n2}R3i3-2\&times;R3\mathrm{ic},$ Wherein, $R3i3=\frac{{\mathrm{\&Sigma;}}_{j3=\mathrm{Int}(0.8\&times;t3)+1}^{t3-\mathrm{Int}(0.1\&times;t3)}R3i3j3}{t3-\mathrm{Int}(0.8\&times;t3)-\mathrm{Int}(0.1\&times;t3)},$ N2 is the number of tenuigenin hybrid strain kind, R3i3 is the hybrid strain rate of the i-th 3 tenuigenin hybrid strain kinds, the value of R3i3 when R3ic is i3=ic, ic for when soybean varieties to be measured be nucleo_cytoplasmic interaction sterile line or maintenance line time, corresponding maintenance line or the tenuigenin hybrid strain kind of sterile line, t3 is the number of all special hybrid strain plasmagene type of the i-th 3 tenuigenin hybrid strain kinds, i3j3 is after all special hybrid strain plasmagene type of the i-th 3 tenuigenin hybrid strain kinds sorts from low to high by its frequency, the special hybrid strain plasmagene type of jth 3, R3i3j3 is the frequency of the i-th 3j3 special hybrid strain plasmagene type, R3ic refers to be mixed into the hybrid strain rate of the maintenance line in sterile line or be mixed into the hybrid strain rate of the sterile line in maintenance line, R3 is the summation of the hybrid strain rate of the tenuigenin hybrid strain kind calculated by hybrid strain plasmagene type, the hybrid strain rate of tenuigenin hybrid strain kind is after the frequency of the special hybrid strain plasmagene type removing in tenuigenin hybrid strain kind minimum 80% and the highest 10%, the mean value of the frequency of remaining special hybrid strain plasmagene type, wherein, t4 is number that is except the hybrid strain plasmagene type that tenuigenin hybrid strain kind has and the hybrid strain plasmagene type of frequency>=0.17%, i4 is after all hybrid strain plasmagene types except the hybrid strain plasmagene type that tenuigenin hybrid strain kind has sort from low to high by its frequency, the i-th 4 hybrid strain plasmagene types, R4i4 is the frequency of the i-th 4 hybrid strain plasmagene types, R4 is the hybrid strain rate utilizing the hybrid strain plasmagene type had except tenuigenin hybrid strain kind to calculate, and it is after the value of in the frequency removing the hybrid strain plasmagene type had except tenuigenin hybrid strain kind minimum 80% and the highest 10%, the mean value of residual value shi, Int () is bracket function, returns the integral part of the number in bracket.

The flyings pollination that hybrid strain in soybean varieties to be measured comes from reproductive process mixes and mechanical admixture, and wherein, it is the main source of hybrid strain variet complexity that flyings pollination mixes.Flyings are pollinated to mix and are referred to that the pollen of hybrid strain kind passes to soybean varieties to be measured by wind-force etc. and the cenospecies of pollination formation, flyings pollination can not introduce tenuigenin, therefore only can cause hybrid strain nuclear gene type, its hybrid strain rate is 2 times of hybrid strain nuclear gene type frequency.Mechanical admixture refers to that hybrid strain variety seeds is directly mixed in soybean varieties to be measured, introduces nucleus and tenuigenin simultaneously, and form hybrid strain nuclear gene type and hybrid strain plasmagene type, its hybrid strain rate should be the frequency of hybrid strain plasmagene type simultaneously.In the calculation formula of hybrid strain rate R, the hybrid strain rate of mechanical admixture has been over-evaluated 1 times by R1+R2, needs to correct, the R=R1+R2-R3-R4 after correction.Distinguish mechanical admixture and flyings and pollinate that to mix be a technical barrier, the invention solves this difficult problem.

In the calculation formula of hybrid strain rate R, the hybrid strain rate of nucleus hybrid strain kind is all 2 × hybrid strain nuclear gene type frequency, its reason is as follows: diploid or allopolyploid soybean are 2 copies at the test zone of nuclear genome, and therefore, hybrid strain rate is 2 times of corresponding hybrid strain nuclear gene type frequency.If the test zone of the nuclear genome that N part must be selected to copy, then coefficient should be adjusted to N, if copy number is indefinite, does N=2 process, if wrong, when calculating R, they will be got rid of by the mode of the low extremum removing 80%.

In the calculation formula of hybrid strain rate R, the carrying out that only make use of hybrid strain genotype frequency value mediates 10% calculates, its principle is: the different hybrid strain genotype of same hybrid strain kind are determined by the hybrid strain rate of this hybrid strain kind, so the expected value of frequency is equal, for the difference between frequency is caused by the error in pcr amplification, high-flux sequence process.By the genotypic definition of hybrid strain and soybean varieties standard model to be measured, substantially to be eliminated by these improper values, the extremum removing 10% is enough to remove the test zone that minute quantity departs from true hybrid strain rate.Why remove minimum 80%, maximum then only removes 10%, and principle is as follows: (1) worst error source is order-checking mistake, and the hybrid strain genotype frequency that order-checking mistake produces is very low; (2) in the genotypic frequency of the hybrid strain except hybrid strain kind, high level may be the more common hybrid strain genotype of different hybrid strain, represents real hybrid strain rate.

When soybean varieties to be measured is nucleo_cytoplasmic interaction sterile line, if be wherein mixed with the maintenance line hybrid strain kind that this sterile line is corresponding, so, due to the tenuigenin of this maintenance line hybrid strain kind and soybean varieties to be measured different, tenuigenin hybrid strain kind will be detected as, but because the nucleus of sterile line and maintenance line is just the same, nucleus hybrid strain kind can not be detected as, therefore, the value of R3ic is not calculated in R1+R2, but calculated in R3i3, therefore, needed in R3, deduct 2 × R3ic and carry out just imitating.Same reason, when soybean varieties to be measured is nucleo_cytoplasmic interaction maintenance line, also needs the 2 × R3ic deducting corresponding sterile line hybrid strain kind in R3 to carry out just imitating.Obviously, when soybean varieties to be measured be neither nucleo_cytoplasmic interaction sterile line also for nucleo_cytoplasmic interaction maintenance line time, R3ic=0.

In the calculation formula of R2 and R4, require genotypic frequency >=0.17% of hybrid strain, its principle is as follows: when the kind number in database and detection site all reach 10000, average by generation 149 hybrid strain genotype erroneous judgements, when arranging hybrid strain genotype frequency >=0.17%, without genotypic probability >=99.98% of hybrid strain (projectional technique is in table 2) of erroneous judgement, the value of R2 and R4 just accurately can be calculated.It has been the limit in reality that kind number in database and detection site all reach 10000, and therefore, the threshold value of genotypic frequency >=0.17% of hybrid strain goes for various situation.The introducing of R2 and R4, makes the present invention can be 0 in database kind, when namely not having database to support, calculates hybrid strain rate R.

Especially, if all hybrid strain genotype of hybrid strain kind A are had by hybrid strain kind B and other hybrid strain kind, thus, hybrid strain kind A is without special hybrid strain genotype.Now, when calculating hybrid strain rate R, not calculating the hybrid strain rate of hybrid strain kind A and hybrid strain kind B, and calculating the hybrid strain rate of hybrid strain kind AB.The hybrid strain VDA genotypes of hybrid strain kind AB is: the common hybrid strain genotype of hybrid strain kind A and hybrid strain kind B.

The calculation formula of hybrid strain rate R is general formula, and in reality, soybean varieties to be measured generally only mixes a kind of hybrid strain kind.

Calculate the supposition example of hybrid strain rate R

Table 3 assumes a hybrid strain rate calculated examples, more to clearly demonstrate the computation process of hybrid strain rate R.

Table 3 is for calculating a supposition example of hybrid strain rate R

In table 3, nucleus hybrid strain kind is A and B two altogether, so n1=2, tenuigenin hybrid strain kind number only C mono-, so n2=1.By the definition of special hybrid strain nuclear gene type, the special hybrid strain nuclear gene type obtaining hybrid strain kind A is be numbered the hybrid strain nuclear gene type AA of No. 1-10, TT, TCC, GG, AC, TTC, TCCC, GGC, ACC and AG, so, t1=10, their frequency is respectively 0.10%, 1.20%, 0.10%, 0.10%, 0.02%, 0.10%, 0.10%, 0.10%, 0.10% and 0.10%, after these 10 special hybrid strain nuclear gene type frequencies are sorted from low to high, for R11111=0.02%, R11121=0.02%, R11131=0.10%, R11141=0.10%, R11151=0.10%, R11161=0.10%, R11171=0.10%, R11181=0.10%, R11191=0.10% and R111101=1.20%.Be R11191=0.10% from the value of the R111j1 of j 1=Int (0.8 × t1)+1=Int (0.8 × 10)+1=9 to j 1=t1-Int (0.1 × t1)=10-Int (0.1 × 10)+1=9, so the hybrid strain rate of nucleus hybrid strain kind A is in the same way, the hybrid strain rate obtaining nucleus hybrid strain kind B is $R121=\frac{2\&times;0.20\%+2\&times;0.20\%}{2-0-0}=0.40\%.$ Thus, nucleus hybrid strain kind is obtained $R1={\mathrm{\&Sigma;}}_{i1=1}^{2}R1i1=$ $R111+R121=0.60\%.$ By similar method, obtain R2=0.02%, the hybrid strain rate of tenuigenin hybrid strain kind r4=0.04%.Therefore, hybrid strain rate R=R1+R2-R3-R4=0.60%+0.02%-0.10%-0.04%=0.48% in this supposition example.

With reference to above-mentioned supposition example, calculate the hybrid strain rate R in the present embodiment: in the present embodiment, hybrid strain kind is only " rich No. 8 of north " and be nucleus hybrid strain kind, R2, R3 and R4 are 0, thus, and R=R1=R111." rich No. 8 of north " has 133 special hybrid strain nuclear gene types, frequency is: 1.02%, 1.03%...... (certain embodiments is in table 1), by the computation rule of R, after removing the frequency values of 80% minimum (106) and minimum 10% (13), the mean value of remaining 14 frequencies is hybrid strain rate R=1.03%.

11, utilize variant sites, variant sites rate and hybrid strain rate, judge the specificity of soybean varieties to be measured, consistence and stability, method is as follows:

Wherein, SD is threshold value selected when judging specificity, and M is threshold value selected when judging consistence and stability.The method of soybean varieties specificity to be measured, consistence and stability that judges is: when variant sites rate >=SD or non-universal test zone exist variant sites, soybean varieties to be measured has specificity, as variant sites rate < SD and variant sites is not present in non-universal test zone time, soybean varieties to be measured does not have specificity; As the hybrid strain rate≤M of soybean varieties to be measured, soybean varieties to be measured has consistence and stability, and when the hybrid strain rate of soybean varieties to be measured is greater than > M, soybean varieties to be measured does not have consistence and stability.The factors such as the same with M value, SD value is the Stringency according to breeding level, requirement, mark characteristic, artificially determine.In the present embodiment, SD selects the standard of 1%.

In the present embodiment, variant sites rate is 3.24%>SD=1%, therefore, judges that soybean varieties to be measured has specificity; The hybrid strain rate 1.03%≤M=0.5% of soybean varieties to be measured, therefore, judges that soybean varieties to be measured does not have consistence and stability.

Further, after judging soybean varieties specificity to be measured, consistence and stability, estimate the accuracy judged, method is as follows:

Pure lines new soybean varieties in the present invention refers to be sheerly genotype for target and the type such as routine kind, self-mating system, restorer, maintenance line, sterile line of seed selection.

Specificity accuracy calculates: when non-universal test zone does not exist variant sites, if judge, soybean varieties to be measured has specificity, probability >=BINOM.DIST ((1-SD) * TRN, TRN, 1-OD, TRUE) that conclusion is correct; If judge, soybean varieties to be measured does not have specificity, the probability that conclusion is correct >=BINOM.DIST (SD*TRN, TRN, OD, TRUE), wherein, TRN is the number of the test zone successfully detected, and OD is variant sites rate, and BINOM.DIST is the function in excel 2010, its using method is identical with the definition in excel 2010, the probability of binomial distribution that what it returned is.In fact above-mentioned probability calculates: when judging to have specificity, variant sites rate is greater than the probability of SD; When judging do not have specificity, variant sites rate is less than the probability of SD, detects successful test zone and obtains after analyzing sequenced fragments group.

In the present embodiment, what adopt variant sites rate to judge soybean varieties to be measured has specificity, therefore, probability >=BINOM.DIST ((1-1%) * 2406 that specificity conclusion is correct, 2406,1-3.24%, TRUE)=100.00%, to specificity, visible the present embodiment judges that the accuracy of conclusion is very high.

Consistence and stability accuracy calculate

Judge probability that the conclusion of the consistence of soybean varieties to be measured and stability is correct as: when soybean varieties to be measured has consistence and stability, the probability that conclusion is correct >=BINOM.DIST (M*SN, SN, R, TRUE) * BINOM.DIST (Σ SeN*M, Σ SeN, R, TRUE); When soybean varieties to be measured does not have consistence and stability, probability >=BINOM.DIST ((1-M) * SN that conclusion is correct, SN, (1-R), TRUE) * BINOM.DIST (Σ SeN* (1-M), Σ SeN, 1-R, TRUE), wherein, Σ SeN is the summation of the sequenced fragments of all genotype frequency place test zones for calculating hybrid strain rate R, also namely remove 80% minimum value and 10% maximum value after, remain the summation of the test fragment of the test zone for calculating hybrid strain rate, M is threshold value selected when judging consistence and stability.Judge that the accuracy of consistence and stability depends on the accuracy of hybrid strain rate completely, and the just rate of hybrid strain rate really depends on the accuracy of following three steps: first, soybean varieties sampling accuracy to be measured, second, from extracting the accuracy detecting hybrid strain kind sample out, 3rd, utilize the hybrid strain kind detected to calculate the accuracy of hybrid strain rate.Therefore, judge that the accuracy of soybean varieties consistence to be measured and stability is the long-pending of above three step accuracy.Even because the present invention is under the strictest condition, the accuracy detecting hybrid strain kind also controls more than 99.9%, and in fact the overwhelming majority is close to 100%.Such as, in the present embodiment, more than 100.0000%, there is the probability (circular is in table 2) below 0.0000% of the hybrid strain kind of erroneous judgement in whole hybrid strain kind detection probability.Therefore, judge that the accuracy of soybean varieties consistence to be measured and stability can be estimated as the long-pending of the accuracy of the first step and the 3rd step, it is respectively the value that in above-mentioned formula, former and later two functions calculate.Such as, the meaning of BINOM.DIST (M*SN, SN, R, TRUE) is: soybean varieties to be measured has carried out SN sampling, and the actual hybrid strain rate R taken out is less than the probability of threshold value M; For calculating each sequenced fragments of soybean varieties hybrid strain rate to be measured, in fact also quite single sample is carried out to soybean varieties to be measured, therefore, BINOM.DIST (Σ SeN*M, Σ SeN, R, TRUE) meaning be: carried out Σ SeN time sampling to soybean varieties to be measured, the actual hybrid strain rate R taken out is less than the probability of threshold value M.

In the present embodiment, after removing the hybrid strain genotype frequency of minimum 80% and maximum 10%, have 6 hybrid strain genotype frequencies to be used to calculate hybrid strain rate R, the sequenced fragments of the test zone of their correspondences adds up to 138586, so Σ SeN=138586, also be namely equivalent to carry out 138586 sampling again to 26000 samples taken out, the error of so large amount of sampling is quite little.In the present embodiment, judge that soybean varieties to be measured does not have consistence and stability, therefore, probability >=BINOM.DIST ((1-0.5%) * 26000,26000, (1-1.03%) that this judgement conclusion is correct, TRUE) * BINOM.DIST (138586* (1-0.5%), 138586,1-1.03%, TRUE)=100.0000%.Visible, the judgement of this enforcement to the consistence of soybean varieties to be measured and stability is also very accurately.

Result verification

Plant by the method in " new variety of plant specificity, consistence and stability test guide-soybean " and observe soybean varieties to be measured and approximate kind " cultivates No. 26, the beans that reflect ", finding that soybean varieties to be measured exists notable difference with approximate kind in multiple proterties such as leaf look.Regulation in " new variety of plant specificity, consistence and stability test guide-soybean ": when at least there is with approximate kind obvious and reproducible difference in a proterties, can judge that the soybean varieties to be measured applied for possesses specificity.Therefore, judge that soybean varieties to be measured has specificity.In experimentation, 300 strain soybean varieties to be measured and approximate kind (150 Zhu Yige communities are planted altogether, totally 2 repetitions), find 7 strain abnormity strains, regulation in " new variety of plant specificity, consistence and stability test guide-soybean ": when sample size is 300 strain, the special-shaped strain of 4 strains can be allowed at most, judged that soybean varieties to be measured does not have consistence thus.Can think that this kind does not possess stability owing to not possessing conforming kind.Judge thus, soybean varieties to be measured does not also have stability.Shown by experiment above: be correct to the specificity of soybean varieties to be measured, stability and conforming judgement in the present embodiment.

The embodiment of the present invention is by high-flux sequence and the amplification of multidigit point, the large sample achieving individual test zone between the large sample sampling of soybean varieties to be measured and kind is sampled, the comprehensive means such as recycling definition hybrid strain genotype, definition tenuigenin hybrid strain kind and definition hybrid strain rate calculation formula, successfully achieve the specificity, stability and the conforming target that accurately, fast, intactly judge soybean varieties to be measured, its technique effect is that existing DUS testing method does not all reach.Existing molecule DUS detection technique such as chip only detects fixing test region, according to case, can not select non-universal test zone flexibly.And the present invention's detection is PCR primer, easily according to case flexible design primer, non-universal test zone can be detected.In addition, the embodiment of the present invention is for 26000 individual amount of samplings for traditional DUS measuring technology, and work is large, cannot complete, such as, during field DUS tests, 26000 strain soybean of sampling need plantation more than 2 mu, and need plant 2 years, and annual every strain soybean need investigate multiple proterties.In the SSR molecule DUS extensively adopted tests, need to do 26000 DNA extraction respectively, 26000*2488 time PCR detects (suppose the same with the present embodiment, have detected 2488 universal test regions) with 26000*2488 PCR primer.Therefore, because workload is excessive, existing molecule DUS test there all is not measuring stability and consistence, although field DUS tests detect consistence and stability, but sampling sample size is all below 1000 strains, and the present embodiment has been sampled 26000 strain soybean, and its accuracy is obviously higher.Why the present embodiment can strengthen amount of sampling, is that with field DUS test and comparison, workload is equivalent to be reduced to 1/26000 as a sample process after all mixing because of all 26000 samples; Further, mixed once amplification is all only done in all 2488 universal test regions and a high-flux sequence detects, and with SSR molecule DUS test and comparison, workload is equivalent to be reduced to 1/ (26000*2488).Therefore, the present invention, when workload significantly alleviates, achieves large sample and the detection of multidigit point, DUS is tested not only accurate but also simple.In the embodiment of the present invention, database variety and genetype is based composition simultaneously, very standard, same breed is detected by method of the present invention under different experimental conditions, identical genotype can be obtained, thus, do not need to repeat DUS test under different conditions, therefore, the embodiment of the present invention can directly compare with database variety and genetype, selects the approximate kind of soybean varieties to be measured objectively.And existing DUS measuring technology is not up to standard, need to carry out DUS test to soybean varieties to be measured and approximate kind abreast simultaneously, just can obtain reliable conclusion, in order to alleviate workload, have to provide approximate kind by weighing applicant by kind, if approximate kind mistake, then may produce the legal consequence of erroneous grants.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.

Claims (10)

1. test a method for the specificity of pure lines new soybean varieties, consistence and stability, it is characterized in that, described method comprises:

Obtain the variant sites between different soybean varieties;

Determined the test zone of soybean varieties to be measured by described variant sites, described test zone comprises universal test region, and at least part of described variant sites is included in described universal test region;

Build and comprise the genotypic database of described different soybean varieties at all described test zones;

After determining the amount of sampling SN of described soybean varieties to be measured, stochastic sampling mixes the DNA of united extraction mixing sample;

The primer of the described test zone of preparation amplification, described primer comprises described universal test region primer;

Utilize the DNA of mixing sample described in described primer pair to increase, obtain the amplified production of described test zone, described amplified production is as high-throughput sequencing library;

High-flux sequence is carried out to described high-throughput sequencing library, obtains sequenced fragments group;

Analyze described sequenced fragments group, obtain soybean varieties genotype to be measured and hybrid strain genotype;

Described soybean varieties genotype to be measured is compared with the genotype of the described different varieties in described database, obtains the approximate kind of described soybean varieties to be measured, variant sites and variant sites rate;

The genotype of described hybrid strain genotype with the described different varieties in described database is compared, after obtaining hybrid strain kind, calculates hybrid strain rate;

Utilize described variant sites, described variant sites rate and described hybrid strain rate, judge the specificity of described soybean varieties to be measured, consistence and stability.

2. method according to claim 1, it is characterized in that, described amount of sampling SN meets following condition: BINOM.INV (SN, M, 0.95)/SN≤1.15*M, wherein BINOM.INV is the function in excel 2010, M is threshold value selected when judging described consistence and stability, the condition implication that described amount of sampling SN meets is: even if 15% of judgment threshold M when described hybrid strain rate only exceeds consistence and stability, described amount of sampling, under the probability guarantee of 95%, correctly can judge stability and the consistence of described soybean varieties to be measured.

3. method according to claim 1, it is characterized in that, the degree of depth CF of described high-flux sequence meets following condition: BINOM.DIST (10, 10, BINOM.DIST (8, 20, BINOM.DIST (0, CF, 0.1%, TRUE), TRUE), FALSE) >=99.9%, 1-BINOM.DIST (10000, 10000, 1-BINOM.DIST (8, 20, 1-BINOM.DIST (99.99%*CF, CF, 99.9989%, TRUE), TRUE), FALSE)≤0.1% and BINOM.DIST (10* (1-M) * CF, 10*CF, 1-110%*M, TRUE) >=95.0%, wherein, CF is the degree of depth of described high-flux sequence, M is threshold value selected when judging described consistence and stability, BINOM.DIST is the function in excel 2010, the condition implication that the degree of depth CF of described high-flux sequence meets is: be low to moderate 0.1% in described hybrid strain rate, described hybrid strain kind is 10 and under on average only having the condition in 20 difference sites between described hybrid strain kind and described soybean varieties, probability >=99.9% detecting whole described hybrid strain kind determined by the degree of depth CF of described high-flux sequence, under the kind of described database is 10000 and on average only has the condition in 20 difference sites between described hybrid strain kind and described soybean varieties, probability≤0.1% of described hybrid strain kind is judged in the existence determined by the degree of depth CF of described high-flux sequence by accident, described hybrid strain kind be 10 and true hybrid strain rate exceed only judge specificity time selected threshold value 10% time, by the degree of depth CF of described high-flux sequence determine to stability and correct probability >=95.0% of conforming judgement conclusion.

4. method according to claim 1, is characterized in that, described test zone also comprises non-universal test zone, and described primer also comprises non-universal test zone primer.

5. method according to claim 4, it is characterized in that, described non-universal test zone primer comprises the first primer and the second primer, described first primer comprises the first forward primer and the first reverse primer, described second primer comprises the second forward primer and the second reverse primer, described first primer and described second primer carry out amplification separately respectively and obtain the amplified production of two described non-universal test zones, the amplified production balanced mix of two described non-universal test zones are used for the high-throughput sequencing library building amplification separately;

5 ' of described first forward primer holds the sequence 1 be connected with as shown in SEQ ID NO:1 in sequence table, and 5 ' in described first reverse primer holds the sequence 2 be connected with as shown in SEQ ID NO:2 in sequence table;

5 ' of described second forward primer holds the sequence 2 be connected with as shown in SEQ ID NO:2 in sequence table, and 5 ' of described second reverse primer holds the sequence 1 be connected with as shown in SEQ ID NO:1 in sequence table.

6. method according to claim 4, is characterized in that, utilizes described variant sites, described variant sites rate and described hybrid strain rate, judges that the method for described soybean varieties specificity to be measured, consistence and stability comprises:

When there is described variant sites in described variant sites rate >=SD or described non-universal test zone, described soybean varieties to be measured has specificity, as described variant sites rate < SD and described variant sites is not present in described non-universal test zone, described soybean varieties to be measured does not have specificity, wherein, SD is threshold value selected when judging specificity;

As the described hybrid strain rate≤M of described soybean varieties to be measured, described soybean varieties to be measured has consistence and stability, when the described hybrid strain rate of described soybean varieties to be measured is greater than > M, described product to be tested Soybean Species does not have consistence and stability, and M is threshold value selected when judging described consistence and stability;

Described hybrid strain rate R=R1+R2-R3-R4, wherein:

wherein, n1 is the number of nucleus hybrid strain kind, t1 is the number of all special hybrid strain nuclear gene type of the i-th 1 described nucleus hybrid strain kinds, i1j1 is after all described special hybrid strain nuclear gene type of the i-th 1 described nucleus hybrid strain kinds sorts from low to high by frequency, the described special hybrid strain nuclear gene type of jth 1, R1i1j1 is the frequency of the i-th 1j1 described special hybrid strain nuclear gene type; R1 is the summation of the described hybrid strain rate of the described nucleus hybrid strain kind calculated by hybrid strain nuclear gene type, the described hybrid strain rate of described nucleus hybrid strain kind is after the frequency of the described special hybrid strain nuclear gene type removing in described nucleus hybrid strain kind minimum 80% and the highest 10%, 2 times of the mean value of the frequency of remaining described special hybrid strain nuclear gene type;

wherein, t2 is number that is except the hybrid strain nuclear gene type that described nucleus hybrid strain kind has and the described hybrid strain nuclear gene type of frequency>=0.17%, i2 is after all described hybrid strain nuclear gene type except the described hybrid strain nuclear gene type that described nucleus hybrid strain kind has sorts from low to high by frequency, the i-th 2 described hybrid strain nuclear gene types, R2i2 is the frequency of the i-th 2 described hybrid strain nuclear gene types; R2 is the described hybrid strain rate utilizing the described hybrid strain nuclear gene type had except described nucleus hybrid strain kind to calculate, R2 is after the value of in the frequency removing the described hybrid strain nuclear gene type had except described nucleus hybrid strain kind minimum 80% and the highest 10%, 2 times of the mean value of residual value shi;

$R3={\mathrm{\&Sigma;}}_{i3=1}^{n2}R3i3-2\&times;R3\mathrm{ic},$ Wherein, $R3i3=\frac{{\mathrm{\&Sigma;}}_{j3=\mathrm{Int}(0.8\&times;t3)+1}^{t3-\mathrm{Int}(0.1\&times;t3)}R3i3j3}{t3-\mathrm{Int}(0.8\&times;t3)-\mathrm{Int}(0.1\&times;t3)},$ N2 is the number of tenuigenin hybrid strain kind, R3i3 is the described hybrid strain rate of the i-th 3 described tenuigenin hybrid strain kinds, the value of R3i3 when R3ic is i3=ic, ic for when described soybean varieties to be measured be nucleo_cytoplasmic interaction sterile line or maintenance line time, corresponding described maintenance line or the described tenuigenin hybrid strain kind of described sterile line, t3 is the number of all special hybrid strain plasmagene type of the i-th 3 described tenuigenin hybrid strain kinds, i3j3 is after all described special hybrid strain plasmagene type of the i-th 3 described tenuigenin hybrid strain kinds sorts from low to high by frequency, the described special hybrid strain plasmagene type of jth 3, R3i3j3 is the frequency of the i-th 3j3 described special hybrid strain plasmagene type, R3ic refers to the hybrid strain rate of the described maintenance line be mixed in described sterile line or is mixed into the hybrid strain rate of the described sterile line in described maintenance line, R3 is the summation of the described hybrid strain rate of the described tenuigenin hybrid strain kind calculated by hybrid strain plasmagene type, the hybrid strain rate of described tenuigenin hybrid strain kind is after the frequency of the described special hybrid strain plasmagene type removing in described tenuigenin hybrid strain kind minimum 80% and the highest 10%, the mean value of the frequency of remaining described special hybrid strain plasmagene type,

wherein, t4 is number that is except the described hybrid strain plasmagene type that described tenuigenin hybrid strain kind has and the described hybrid strain plasmagene type of frequency>=0.17%, i4 is after all described hybrid strain plasmagene type except the described hybrid strain plasmagene type that described tenuigenin hybrid strain kind has sorts from low to high by frequency, the i-th 4 described hybrid strain plasmagene types, R4i4 is the frequency of the i-th 4 described hybrid strain plasmagene types; R4 is the described hybrid strain rate utilizing the described hybrid strain plasmagene type had except described tenuigenin hybrid strain kind to calculate, R4 is after the value of in the frequency removing the described hybrid strain plasmagene type had except described tenuigenin hybrid strain kind minimum 80% and the highest 10%, the mean value of residual value shi;

Int () is bracket function;

Described nucleus hybrid strain kind refers to and only utilizes nuclear gene type to calculate the described hybrid strain kind obtained, and described tenuigenin hybrid strain kind refers to and only utilizes plasmagene type to calculate the described hybrid strain kind obtained; Described special hybrid strain nuclear gene type refers to and is only all described hybrid strain nuclear gene types of a described nucleus hybrid strain kind; Described special hybrid strain plasmagene type refers to and is only all described hybrid strain plasmagene types of a described tenuigenin hybrid strain kind; Described hybrid strain nuclear gene type refers to that described hybrid strain genotype is described nuclear gene type, and described nuclear gene type refers to that described genotype is positioned on nuclear genome; Described hybrid strain plasmagene type refers to that described hybrid strain genotype is described plasmagene type, and described plasmagene type refers to that described genotype is positioned on cytoplasmic skeleton.

7. method according to claim 6, it is characterized in that, described method also comprise the correct probability of the conclusion of consistence and the stability judging described soybean varieties to be measured in the following ways as: when described soybean varieties to be measured has consistence and stability, the probability that conclusion is correct >=BINOM.DIST (M*SN, SN, R, TRUE) * BINOM.DIST (Σ SeN*M, Σ SeN, R, TRUE), when described soybean varieties to be measured does not have described consistence and stability, probability >=BINOM.DIST ((1-M) * SN that conclusion is correct, SN, (1-R), TRUE) * BINOM.DIST (Σ SeN* (1-M), Σ SeN, 1-R, TRUE), wherein, Σ SeN is the summation of the sequenced fragments of test zone described in all described genotypic frequency places for calculating described hybrid strain rate R, M is threshold value selected when judging described consistence and stability, BINOM.DIST (M*SN, SN, R, TRUE) for described soybean varieties to be measured has carried out SN sampling, the actual described hybrid strain rate R taken out is less than the probability of described threshold value M, BINOM.DIST (Σ SeN*M, Σ SeN, R, TRUE) meaning is: carried out Σ SeN sampling to described soybean varieties to be measured, the actual described hybrid strain rate R taken out is less than the probability of threshold value M.

8. method according to claim 6, it is characterized in that, when there is not described variant sites in described non-universal test zone, if judge, described soybean varieties to be measured has specificity, probability >=BINOMDIST ((1-SD) * TRN that conclusion is correct, TRN, 1-OD, TRUE), if judge, described soybean varieties to be measured does not have specificity, the probability that conclusion is correct >=BINOMDIST (SD*TRN, TRN, OD, TRUE), wherein, TRN is the number detecting successful test zone, OD is described variant sites rate, SD is threshold value selected when judging specificity, BINOMDIST is the function in excel 2010, the probability that described conclusion is correct is expressed as when judging that described soybean varieties to be measured has specificity, described variant sites rate is greater than the probability of SD, when judging that described soybean varieties to be measured does not have specificity, described variant sites rate is less than the probability of SD, the successful test zone of described detection obtains after analyzing described sequenced fragments group.

9. method according to claim 1, it is characterized in that, the method obtaining described hybrid strain kind comprises: described hybrid strain kind is be present in the kind in described database, and has the number of the described test zone of homologous genes type to account for ratio >=60% that described hybrid strain kind has the sum of the genotypic described test zone of described potential hybrid strain between the potential hybrid strain genotype of described hybrid strain kind and described hybrid strain genotype; Described hybrid strain genotype refers to the described potential hybrid strain genotype of frequency >=0.02%;

Insertion or the disappearance of discontinuous base is had in quantity >=2 of the distinguishing base between all genotype of described potential hybrid strain genotype and described soybean varieties to be measured or described distinguishing base.

10. method according to claim 1, is characterized in that, determines that the method in described universal test region is by described variant sites:

Pass through discrimination calculate the value of discrimination, wherein, a is the kind sum be detected in variation window area, bi is i-th kind of genotypic kind number in described variation window area, and bi>1, k comprises the genotypic number being greater than a kind, and described variation window area is centered by each single nucleotide variations site, and the both sides to described single nucleotide variations site respectively extend 1/2 of survey sequence length as the window detected;

Described universal test region is the large and equally distributed region of described discrimination on region or nuclear genome that on cytoplasmic skeleton, discrimination is large.