CN104805187B - A kind of method of the specificity for testing pure lines new soybean varieties, uniformity and stability - Google Patents

A kind of method of the specificity for testing pure lines new soybean varieties, uniformity and stability Download PDF

Info

Publication number
CN104805187B
CN104805187B CN201510150173.6A CN201510150173A CN104805187B CN 104805187 B CN104805187 B CN 104805187B CN 201510150173 A CN201510150173 A CN 201510150173A CN 104805187 B CN104805187 B CN 104805187B
Authority
CN
China
Prior art keywords
hybrid strain
genotype
measured
soybean varieties
rate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201510150173.6A
Other languages
Chinese (zh)
Other versions
CN104805187A (en
Inventor
崔野韩
唐浩
陈红
杨坤
徐岩
卢新
杨旭红
堵苑苑
杨扬
侯耀华
温雯
邓超
彭海
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ministry Of Agriculture's Development In Science And Technology Center
Jianghan University
Original Assignee
Ministry Of Agriculture's Development In Science And Technology Center
Jianghan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ministry Of Agriculture's Development In Science And Technology Center, Jianghan University filed Critical Ministry Of Agriculture's Development In Science And Technology Center
Priority to CN201510150173.6A priority Critical patent/CN104805187B/en
Publication of CN104805187A publication Critical patent/CN104805187A/en
Application granted granted Critical
Publication of CN104805187B publication Critical patent/CN104805187B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a kind of method of specificity for testing pure lines new soybean varieties, uniformity and stability.Methods described includes：Obtain variant sites；Determine the test zone of soybean varieties to be measured；Build database；After determining amount of sampling, random sampling mixes and extracts the DNA of mixing sample；Prepare primer；Expanded using the DNA of primer pair mixing sample, amplified production is used to build high-throughput sequencing library；High-flux sequence is carried out to high-throughput sequencing library, obtains that fragment group is sequenced；Analysis sequencing fragment group, obtains soybean varieties genotype and hybrid strain genotype to be measured；Compare and obtain approximate kind, variant sites and variant sites rate；By hybrid strain genotype compared with the genotype in database, after obtaining hybrid strain kind, hybrid strain rate is calculated；Using variant sites, variant sites rate and hybrid strain rate, soybean varieties specificity, uniformity and stability to be measured are judged.This method can accurately, intactly judge the specificity, stability and uniformity of soybean varieties to be measured.

Description

A kind of method of the specificity for testing pure lines new soybean varieties, uniformity and stability
Technical field
The present invention relates to biological technical field, more particularly to a kind of specificity, uniformity for testing pure lines new soybean varieties With the method for stability.
Background technology
As a kind of intellectual property of specialization, new variety of plant has become a company and competing to a national core Strive power.The solution that new variety of plant authorizes account and relative legal problems is tested dependent on DUS, i.e. the specificity to soybean varieties to be measured (Distinctness), the field trapping test or molecules inside of uniformity (Uniformity) and stability (Stability) Marker Identification.Field trapping test flow is：Soybean varieties to be measured and approximate kind are planted in field simultaneously, in 2 years and more than The season of growth in, observe their multiple characters, difference of the soybean varieties to be measured with approximate kind judged according to trait expression Conspicuousness, i.e., it is specific, while judge hybrid strain ratio in colony, i.e. uniformity and stability；The stream of molecules inside Marker Identification Cheng Wei：Individual plant is divided to extract DNA of the soybean varieties to be measured with each sample in approximate kind, and each survey to each sample respectively Performing PCR (Polymerase Chain Reaction, polymerase chain reaction) is entered in examination region, and carries out electrophoresis to each PCR primer Or generation sequencing detection, according to testing result, the difference site ratio of soybean varieties to be measured and approximate kind is obtained, according to difference Site ratio, judge the specificity of soybean varieties to be measured.
The shortcomings that field trapping test is：Cycle is long, workload is big, environmental impact shape, causes to judge inaccuracy.It is indoor The shortcomings that molecular markers for identification is：Need to handle each test zone of each sample respectively, workload is big, it is impossible to sample with Test zone bulk sampling, hybrid strain rate can not be calculated, thus the test of stability and uniformity can not be carried out.Field trapping test Common drawback with molecules inside Marker Identification is：Due to workload, can not from existing kind objective selection it is near Like kind, applicant can only be weighed by kind and provided, and the approximate kind provided based on the motivations such as commercial interest, kind power applicant May be untrue, so as to cause the legal consequence of wrong kind mandate.
The content of the invention
In order to solve the problems of the prior art, the embodiments of the invention provide a kind of spy for testing pure lines new soybean varieties The method of the opposite sex, uniformity and stability.The technical scheme is as follows：
The embodiments of the invention provide the side of a kind of specificity for testing pure lines new soybean varieties, uniformity and stability Method, methods described include：
Obtain the variant sites that soybean varieties to be measured belong between interior different cultivars of the same race；
The test zone of soybean varieties to be measured is determined by the variant sites, the test zone includes universal test area Domain, at least partly described variant sites are included in the universal test region, determined by the variant sites described general The method of test zone is：Pass through discriminationThe value of discrimination is calculated, wherein, a is in variation window area The kind sum being detected, bi are the kind number of i-th kind of genotype in the variation window area, and bi>1, k is comprising big In the number of the genotype of a kind, the variation window area is centered on each single nucleotide variations site, to described The both sides in single nucleotide variations site respectively extend 1/2 window as detection of sequence length to be measured, the universal test region For the discrimination on the big region of discrimination on cytoplasmic skeleton or nuclear genome is big and equally distributed region, its In, the genotype is the combination in multiple single nucleotide variations sites in the test zone；
Structure includes database of the different soybean varieties in the genotype of all test zones；
After the amount of sampling SN for determining the soybean varieties to be measured, random sampling mixes and extracts the DNA of mixing sample；
The primer for expanding the test zone is prepared, institute's primer includes universal test region primer；
Expanded using the DNA of mixing sample described in the primer pair, obtain the amplified production of the test zone, institute Amplified production is stated as high-throughput sequencing library；
High-flux sequence is carried out to the high-throughput sequencing library, obtains that fragment group is sequenced；
The sequencing fragment group is analyzed, obtains soybean varieties genotype and hybrid strain genotype to be measured；
By the soybean varieties genotype to be measured compared with the genotype of the different cultivars in the database, obtain Approximate kind, variant sites and the variant sites rate of the soybean varieties to be measured；
By the hybrid strain genotype compared with the genotype of the different cultivars in the database, hybrid strain kind is obtained Afterwards, hybrid strain rate is calculated；
Using the variant sites, the variant sites rate and the hybrid strain rate, the spy of the soybean varieties to be measured is judged The opposite sex, uniformity and stability.
Specifically, the 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, and M is described to take out to judge threshold value selected when the uniformity and stability Sample amount SN meet condition implication be：Even if 15% of judgment threshold M when the hybrid strain rate only exceeds uniformity and stability, The amount of sampling can correctly judge the stability and uniformity of the soybean varieties to be measured in the case where 95% probability ensures.
Specifically, the depth CF of the 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 depth of the high-flux sequence, to judge threshold value selected when the uniformity and stability, BINOM.DIST is M Function in excel 2010, the condition implication that the depth CF of the high-flux sequence meets are：In the hybrid strain rate as little as 0.1%th, the hybrid strain kind averagely only has 20 difference sites between 10 and the hybrid strain kind and the soybean varieties Under the conditions of, probability >=99.9% of the whole hybrid strain kinds of detection determined by the depth CF of the high-flux sequence；Institute The kind for stating database averagely only has the bar in 20 difference sites between 10000 and the hybrid strain kind and the soybean varieties Under part, probability≤0.1% of the hybrid strain kind is judged in the presence determined by the depth CF of the high-flux sequence by accident；Described miscellaneous When strain kind is 10 and true hybrid strain rate exceeds only the 10% of threshold value selected when judging specific, measured by the high pass Correct probability >=95.0% of the judgement conclusion to stability and uniformity that the depth CF of sequence is determined.
Specifically, the test zone also includes non-universal test zone, and the primer also includes non-universal test zone Primer.
Further, the non-universal test zone primer includes the first primer and the second primer, the first primer bag The first forward primer and the first reverse primer are included, second primer includes the second forward primer and the second reverse primer, described First primer and second primer carry out individually amplification and obtain the amplified production of two non-universal test zones respectively, will The amplified production mixed in equal amounts of two non-universal test zones is used to build the high-throughput sequencing library individually expanded；
5 ' end connections of first forward primer are just like SEQ ID NO in sequence table:Sequence 1 shown in 1, described first 5 ' end connections in reverse primer are just like SEQ ID NO in sequence table:Sequence 2 shown in 2；
5 ' end connections of second forward primer are just like SEQ ID NO in sequence table:Sequence 2 shown in 2, described second 5 ' end connections of reverse primer are just like SEQ ID NO in sequence table:Sequence 1 shown in 1.
Specifically, using the variant sites, the variant sites rate and the hybrid strain rate, the soybean product to be measured are judged The method of species specificity, uniformity and stability includes：
It is described to be measured big when the variant sites rate >=non-universal test zones of SD or described have the variant sites Beans kind has specificity, when the variant sites rate ＜ SD and the variant sites are not present in the non-universal test zone When middle, the soybean varieties to be measured do not have specificity, wherein, SD is threshold value selected when judging specific；
As the hybrid strain rate≤M of the soybean varieties to be measured, the soybean varieties to be measured have uniformity and stably Property, when the hybrid strain rate of the soybean varieties to be measured is more than ＞ M, the soybean varieties to be measured are without uniformity and stably Property, M is to judge threshold value selected when the uniformity and stability；
The hybrid strain rate R=R1+R2-R3-R4, wherein：
Wherein, n1 is the number of nucleus hybrid strain kind, and t1 is all special hybrid strains of the i-th 1 nucleus hybrid strain kinds The number of karyogene type, i1j1 are that all special hybrid strain karyogene types of the i-th 1 nucleus hybrid strain kinds press frequency After sorting from low to high, the special hybrid strain karyogene type of jth 1, R1i1j1 is the i-th 1j1 special hybrid strain karyogenes The frequency of type；R1 is the summation of the hybrid strain rate of the nucleus hybrid strain kind calculated by hybrid strain karyogene type, described thin The hybrid strain rate of karyon hybrid strain kind is to remove the institute of 80% and highest 10% minimum in the nucleus hybrid strain kind After the frequency for stating special hybrid strain karyogene type, 2 times of the average value of the frequency of the remaining special hybrid strain karyogene type；
Wherein, t2 is described in addition to the hybrid strain karyogene type that the nucleus hybrid strain kind possesses and frequency >=0.17% The number of hybrid strain karyogene type, i2 are all institutes in addition to the hybrid strain karyogene type that the nucleus hybrid strain kind possesses State after hybrid strain karyogene type sorts from low to high by frequency, the i-th 2 hybrid strain karyogene types, R2i2 is described miscellaneous for the i-th 2 The frequency of strain karyogene type；R2 is to utilize to remove the described of the hybrid strain karyogene type calculating that the nucleus hybrid strain kind possesses Hybrid strain rate, R2 are 80% He minimum in the frequency for remove the hybrid strain karyogene type possessed except the nucleus hybrid strain kind After the value of highest 10%, 2 times of the average value of surplus value；
Wherein, N2 is the number of cytoplasm hybrid strain kind, and R3i3 is the hybrid strain rate of the i-th 3 cytoplasm hybrid strain kinds, R3ic i3 R3i3 value during=ic, ic be when the soybean varieties to be measured are nucleo_cytoplasmic interaction sterile line or maintainer, it is corresponding described The cytoplasm hybrid strain kind of maintainer or the sterile line, t3 are all special of the i-th 3 cytoplasm hybrid strain kinds The number of hybrid strain matter genotype, i3j3 are that all special hybrid strain matter genotype of the i-th 3 cytoplasm hybrid strain kinds are pressed After frequency sorts from low to high, the special hybrid strain matter genotype of jth 3, R3i3j3 is the i-th 3j3 special hybrid strain matter The frequency of genotype, R3ic refer to the hybrid strain rate for the maintainer being mixed into the sterile line or the institute being mixed into the maintainer State the hybrid strain rate of sterile line；R3 is the total of the hybrid strain rate of the cytoplasm hybrid strain kind calculated by hybrid strain matter genotype With the hybrid strain rate of the cytoplasm hybrid strain kind is to remove 80% and highest 10% minimum in the cytoplasm hybrid strain kind The special hybrid strain matter genotype frequency after, the average value of the frequency of the remaining special hybrid strain matter genotype；
Wherein, t4 is in addition to the hybrid strain matter genotype that the cytoplasm hybrid strain kind possesses and frequency >=0.17% The number of the hybrid strain matter genotype, i4 are the institute in addition to the hybrid strain matter genotype that the cytoplasm hybrid strain kind possesses After having the hybrid strain matter genotype to be sorted from low to high by frequency, the i-th 4 hybrid strain matter genotype, R4i4 is the i-th 4 institutes State the frequency of hybrid strain matter genotype；R4 is to utilize the hybrid strain matter genotype possessed except the cytoplasm hybrid strain kind to calculate The hybrid strain rate, R4 are minimum in the frequency for remove the hybrid strain matter genotype possessed except the cytoplasm hybrid strain kind 80% and highest 10% value after, the average value of surplus value；
Int () is bracket function；
The nucleus hybrid strain kind refers to calculate the hybrid strain kind obtained, the cytoplasm merely with karyogene type Hybrid strain kind refers to calculate the hybrid strain kind obtained merely with matter genotype；The special hybrid strain karyogene type refers to All hybrid strain karyogene types of one nucleus hybrid strain kind；The special hybrid strain matter genotype refers to only one All hybrid strain matter genotype of the cytoplasm hybrid strain kind；The hybrid strain karyogene type refers to that the hybrid strain genotype is The karyogene type, the karyogene type refer to the genotype and are located on nuclear genome；The hybrid strain matter genotype refers to The hybrid strain genotype is the matter genotype, and the matter genotype refers to that the genotype is located on cytoplasmic skeleton；Base Because the frequency of type refers in the sequencing fragment group that the sequencing segments for representing the genotype is accounted for described in where the genotype The ratio of the sequencing fragment sum of test zone.
Further, methods described also includes the uniformity and stably for judging the soybean varieties to be measured in the following ways The correct probability of conclusion of property is：When the soybean varieties to be measured have uniformity and stability, the correct probability of conclusion >= BINOM.DIST(M*SN,SN,R,TRUE)*BINOM.DIST(∑SeN*M,∑SeN,R,TRUE)；When the soybean product to be measured When kind not having the uniformity and stability, the correct probability >=BINOM.DIST of conclusion ((1-M) * SN, SN, (1-R), TRUE)*BINOM.DIST(∑SeN*(1-M),∑SeN,1-R,TRUE)；Wherein, ∑ SeN is used to calculate the hybrid strain to be all The summation of the sequencing fragment of the test zone where the frequency of the rate R genotype, M are to judge the uniformity and stably Property when selected threshold value, BINOM.DIST (M*SN, SN, R, TRUE) is that the soybean varieties to be measured have carried out SN time and sampled, The hybrid strain rate R being actually pumped is less than the probability of the threshold value M, BINOM.DIST (∑ SeN*M, ∑ SeN, R, TRUE) meaning Justice is：The soybean varieties to be measured are carried out with SeN sampling of ∑, the hybrid strain rate R being actually pumped is general less than threshold value M's Rate.
Further, when the variant sites are not present in the non-universal test zone, if judging the soybean to be measured Kind has specificity, the correct probability >=BINOMDIST of conclusion ((1-SD) * TRN, TRN, 1-OD, TRUE)；If described in judging Soybean varieties to be measured do not have specific, the correct probability >=BINOMDIST (SD*TRN, TRN, OD, TRUE) of conclusion, wherein, TRN is the number for detecting successful test zone, and OD is the variant sites rate, and SD is threshold selected when judging specific Value, BINOMDIST are the function in excel 2010, and the correct probability of conclusion, which is expressed as working as, judges the soybean product to be measured When kind having specific, the variant sites rate is more than SD probability, when judging the soybean varieties to be measured without specific When, the variant sites rate is less than SD probability, after the successful test zone of detection is by analyzing the sequencing fragment group Obtain.
Specifically, obtaining the method for the hybrid strain kind includes：The hybrid strain kind is to be present in the database Kind, and the potential hybrid strain genotype of the hybrid strain kind is with there is the test section of phase homogenic type between the hybrid strain genotype The number in domain accounts for total ratio >=60% that the hybrid strain kind has the test zone of the potential hybrid strain genotype； The hybrid strain genotype refers to the potential hybrid strain genotype of frequency >=0.02%；
Quantity >=2 of distinguishing base between the potential hybrid strain genotype and all genotype of the soybean varieties to be measured There are insertion or the missing of discontinuous base in individual or described distinguishing base.
The beneficial effect that technical scheme provided in an embodiment of the present invention is brought is：Method provided in an embodiment of the present invention passes through High-flux sequence and the amplification of more sites, realize large sample sampling and the full-page proof of inter-species individual test zone of soybean varieties to be measured This sampling, recycle and define hybrid strain genotype, define cytoplasm hybrid strain kind and define the comprehensive means such as hybrid strain rate calculation formula, The target of specificity that is accurate, intactly judging soybean varieties to be measured, stability and uniformity is successfully realized, and tests speed Degree faster, can be completed within 10 days.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, embodiment of the present invention will be made into one below It is described in detail on step ground.
Embodiment determines soybean varieties ' northern 93-406 ' specificity, uniformity and stability
Soybean varieties to be measured provided in an embodiment of the present invention are soybean varieties " northern 93-406 ", soybean varieties " northern 93-406 " For pure lines soybean and it is that disclosure uses kind, itself and " rich No. 8 of north " hybridize, and it is big to have cultivated " cultivating mirror beans 26 " by systematic breeding Beans kind.The method for then determining the specificity of the soybean varieties, uniformity and stability comprises the following steps.
First, the variant sites between different soybean varieties are obtained.
The variant sites of different soybean varieties can obtain from the documents and materials announced, but the knot that this method is obtained Fruit is more fragmentary, in the present embodiment, substantial amounts of different soybean is obtained by the genome sequence of more different soybean varieties Interracial variant sites.
Further, the method for 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, and the first is Lam etc. to 31 soybean The high-flux sequence sequence of the genome of kind, pertinent literature information are as follows：Lam HM et al. Resequencing of 31wild and cultivated soybean genomes identifies patterns of genetic diversity and selection.Nat Genet 2010,42:1053–1059.The genome sequence of 31 soybean varieties Row are published in NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/sra), reception number is SRA020131；It is for second to " north rich No. 8 " and " to cultivate the beans 26 that reflect by the method provided in Lam etc. the above-mentioned article delivered Number " carry out high-flux sequence.The present embodiment obtains the high-flux sequence sequence of the genome of 33 soybean varieties altogether.
Further, variant sites are obtained using the genome sequence of different cultivars.
Specifically, because the sequencing depth of this 33 soybean varieties is not high, it is only capable of identifying single nucleotide variations (SNP) position Point, if the sequencing depth of soybean varieties is sufficiently high, other variation types can be identified as repeated number variation, due to confidence level It is low, without identification.Software (version number 0.4) is compared by the gene of this 33 soybean varieties using Frederick Sanger The high-flux sequence sequence alignment of group is to " Williams_82 " soya cells core reference gene group (version：Release v1.01, Download address：http://genome.jgi-psf.org/) and cytoplasm reference gene group on, the cytoplasm reference gene group bag Mitochondria reference gene group and chloroplaset reference gene group are included, it is in 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 specification is set as default value.The Ssaha of use Pileup software kits (version number 0.5) identify the SNP site of each soybean varieties.The SNP site is defined as difference determination The missing of base-pair, the insertion of single base or single base.The base-pair that the difference determines refers to not include the uncertain alkali of difference Base pair, the uncertain base-pair of difference refer to the base-pair between some degeneracy bases, therefore, can between A and R as R represents A or G It can have differences, it is also possible to which in the absence of difference, therefore, difference is indefinite between A and R, is not mutually SNP.Therefore, the embodiment of the present invention In SNP site not include the uncertain base-pair of above-mentioned difference.By the definition of above SNP site, the embodiment of the present invention exists 6350046 SNP sites are obtained altogether between all 33 soybean varieties, wherein 31937 SNP sites are located at cytoplasmic skeleton On, remaining SNP site is located on nuclear genome.Genotype referred to hereafter refers to multiple SNP positions in test zone The combination of point, karyogene type refer to genotype and are located on nuclear genome, and matter genotype refers to that genotype is located at cytogene In group.For example, the 1st test zone is located on nuclear genome in table 1, it is karyogene type, the test zone shares 3 SNP site, the genotype of the test zone are the combination of this 3 SNP sites.
2nd, the test section of soybean varieties to be measured is determined by variant sites, test zone includes universal test region, at least Meristic variation site is included in universal test region, and its method includes：
Determine universal test region
Universal test region be on cytoplasmic skeleton on the big region of discrimination or nuclear genome discrimination it is big and Equally distributed region, wherein, discriminationWherein, a is that the kind being detected in variation window area is total Number, bi are the kind number of i-th kind of genotype in variation window area, and bi>1, k is the genotype comprising more than a kind Number, variation window area are centered on each single nucleotide variations site (SNP site), to single nucleotide variations site Both sides respectively extend 1/2 window as detection of sequence length to be measured；Test zone is that discrimination is big on cytoplasmic skeleton Discrimination is big on region or nuclear genome and equally distributed region.The Computing Principle of discrimination is as follows：Between all kinds Number of combinations beWherein, the combination between the different cultivars in same gene type is undistinguishable, and its number is So, the ratio for the breed combination that can not be distinguished isThe ratio for the breed combination that can be distinguished i.e. discriminationAs can be seen here, discrimination is bigger, can more distinguish different cultivars, the big variation window area of discrimination It is more effective to DUS tests.If the variation window area skewness on nuclear genome, can cause some regions adjacent, So as to linkage inheritance, information is easily overlapping, and therefore, the principle of compositionality in universal test region is selected on nuclear genome is：Area Indexing is big and SNP site is uniformly distributed.Cytoplasmic skeleton without linkage inheritance problem, so, on cytoplasmic skeleton only need The big region of selective discrimination degree.
High-flux sequence is carried out using Proton high-flux sequences instrument in the embodiment of the present invention, the test section of detection is sequenced in it Length of field can reach 200bp, and in order to obtain maximum fault information, the most long test zone in the present embodiment is also 200bp.Therefore, The variant sites that the present embodiment is mentioned refer to whole test zone, may include multiple SNP sites inside it.
First, centered on each SNP site of acquisition, respectively extend 99bp and 100bp to the left and right, form 200bp change Different window.According to the 6350046 of acquisition SNP sites, 6350046 variation windows can be obtained, calculate these variation windows The discrimination in regionFor example, in the 1st variation window area, a=29 kind is detected altogether, shares k =2 kinds of genotype GTT, ACC, their kind number are respectively b1=22, b2=5, therefore, It is meant that：By the 1st variation window area, 41% breed combination in 29 kinds can be distinguished, in addition 59% breed combination cannot be distinguished by out, it is necessary to which more variation window can just distinguish.After the same method, calculate and obtain The all discrimination of 6350046 variation windows and therefrom 8000 changes of the selection positioned at discrimination maximum in nuclear genome Different window and 100 maximum variation windows of discrimination in cytoplasmic skeleton.Check one by one and be located at nuclear genome 8000 variation windows in, each distance to make a variation between window and next variation window, if distance is more than 300K (1K= 1000 bases), then abandon reexamining after the less variation window of wherein discrimination, until the adjacent distance for looking into variation window Untill being all higher than 300K.Selection 300K criterion distance is because soybean gene group size is about 975M (ten thousand alkali of 1M=100 Base), by final selected 2000 based on the universal test region of nuclear genome, the interregional distance of average universal test About 500K, but due to few variant sites such as some specific regions such as centromeres, therefore, average distance should be less than 500K.By the above process, 4987 variation windows for being located at nuclear genome are have selected, they are located at cytoplasm with what is obtained Totally 5087 variation windows pass through test zone to 100 maximum variation windows of discrimination as selected together in genome. Wherein, 200 maximum variation windows of selective discrimination degree, are empirical value, the quantity can modify as the case may be.
The test zone can also include non-universal test zone, and specific method is as follows：
Determine non-universal test zone：
Non-universal test zone refers to the non-universal site that special kinds needs detect.DUS tests need detection fixed point to change The special site made, fixed point transformation are the technological means commonly used in modern breeding, such as back cross breeding, transgenic breeding, are pinpointed Transformation kind can also have specificity because of it and turn into new varieties.It is non-through based on the specific decision principle of New variety protection It should not be included in universal test region with test zone and the site of qualitative character is controlled for known to.In the present embodiment, due to Soybean varieties to be measured are come by pinpointing transformation, need to detect without non-universal site, therefore, universal test area nothing but Domain.
3rd, the primer in amplification assay region is prepared, test zone primer includes universal test region primer, specific as follows：
Universal test region primer is prepared, the universal test region primer is directed to all kinds, specifically：
Universal test region is detected using multiple PCR technique, and multiple PCR technique refers in same PCR reacts Add multiple PCR primers, while multiple sites in amplification gene group.The key of the technology is to design and synthesize multiplex PCR to draw Thing, the present embodiment use the multiple PCR technique that LifeTechnology companies of the U.S. provide, and it can set up to 12000 weights PCR primer.
Primer acquisition process is as follows：Log in LifeTechnology companies multiple PCR primer Photographing On-line webpage https://ampliseq.com/protected/help/pipelineDetails.action, related letter is submitted by its requirement Breath.In the present embodiment, " Application type " options select " DNA Hotspot designs (single- pool)”.If selecting multi-pool, multiplex PCR will divide multitube to carry out, and cost can increased, and single-pool Primer only needs a multiplex PCR, saves cost, and shortcoming is that some universal test regions design of primers may fail, but Alternative universal test region on genome is more, therefore, abandons some alternative universal test regions and has no effect on result. The nucleus reference gene group of soybean varieties to be measured and cytoplasm reference gene group are permeated file, and in " Select After selecting " Custom " in the genome you wish to use " options, the file of fusion is uploaded as design multiplex PCR Reference gene group during primer." Standard DNA ", in Add Hotspot options, addition needs the selection of DNA type options The positional information of SNP site in the universal test region to be designed, including chromosome information, SNP initiation site and SNP end locus, its certain embodiments are shown in Table 1." Submit targets " buttons are submitted and designed more for finally click Weight PCR primer.In the present embodiment, from all 5087 universal test regions, design and be successfully authenticated 2488 pairs of multiplex PCRs Primer, for expanding corresponding 2488 universal test regions.The method for demonstrating multiple PCR primer is the said firm by the present invention The method of offer, extract the leaves genomic DNA on same strain soybean, and the base using the multiple PCR primer designed to acquisition Because a group DNA expanded, build storehouse, high-flux sequence and analyzes sequencing fragment group, remove the corresponding primer of following test zone：Should The sequencing segments of test zone is less than 1000 or hybrid strain genotype be present, and the primer remained is as proved to be successful multiple PCR primer.Because genomic DNA source is in same strain soybean leaves, it is impossible to hybrid strain kind be present, therefore, hybrid strain genotype It is PCR or sequencing Preference mistake as caused by the special construction of test zone, removes these test zones and avoid such system System mistake.The multiple PCR primer being proved to be successful is supplied to client to use in fluid form after also being mixed by the said firm.It is above-mentioned 2488 universal test regions of successful design multiple PCR primer are the general survey eventually for soybean varieties to be measured detection Region is tried, meanwhile, each kind in the database of structure also contains above-mentioned 2488 universal test regions, wherein, 47 Universal test region is located on cytoplasmic skeleton, and remaining 2441 universal test regions are located on nuclear genome.
It should be noted that：The number requirement >=900 in universal test region, reason is as follows：If less than 900, exist The probability of the hybrid strain kind of erroneous judgement will be more than 1%, and the projectional technique of the threshold value is shown in Table 2.Due to there may be the survey of detection failure Region is tried, therefore, test zone number is general >=and 1000.
Test zone primer can also include non-universal test zone primer, and the non-universal test zone primer is for be measured Soybean varieties, it is specific as follows：
Prepare non-universal test zone primer：
The primer of non-universal test zone includes the first primer and the second primer, the first primer include the first forward primer and First reverse primer, the second primer include the second forward primer and the second reverse primer, and the first primer and the second primer enter respectively Individually amplification obtains the amplified production of two non-universal test zones to row, by the amplified production equivalent of two non-universal test zones It is mixed for building the high-throughput sequencing library individually expanded.5 ' end connections of the first forward primer are just like SEQ ID in sequence table NO:Sequence 1 shown in 1,5 ' end connections in the first reverse primer are just like SEQ ID NO in sequence table:Sequence 2 shown in 2；The 5 ' end connections of two forward primers are just like SEQ ID NO in sequence table:Sequence 2 shown in 2,5 ' end connections of the second reverse primer Just like SEQ ID NO in sequence table:Sequence 1 shown in 1.
The design process of non-universal test zone primer is as follows：The first step, it is no more than 200bp and comprising non-by amplification length The requirement of all SNP sites in universal test region, by common PCR primers design method, design expands non-universal test zone PCR forward primer and reverse primer；Second step, 5 ' ends of designed forward primer and reverse primer are connected into sequence respectively SEQ ID NO in list:1 and sequence table in SEQ ID NO:2, the forward primer and first primer of the first primer are obtained respectively Reverse primer；3rd step, by SEQ ID NO in 5 ' ends of designed forward primer and reverse primer respectively catenation sequence table:2 With SEQ ID NO in sequence table:1, the forward primer of the second primer and the reverse primer of the second primer are obtained respectively.In sequence table SEQ ID NO:1 and sequence table in SEQ ID NO:2 be the joint sequence used in high-flux sequence, thereby using PCR primer band There is the joint sequence of high-flux sequence, after establishing sequencing library after directly being mixed with the product in the general sequencing region of amplification Together be sequenced, without by fragmentation, jointing etc. it is cumbersome build storehouse step, improve operating efficiency and reduce into This.It is to be sequenced from the both ends of non-universal test zone simultaneously to make two pairs of only different primers of joint.
Soybean varieties to be measured in the present embodiment are due to no non-universal test zone, it is therefore not necessary to non-universal test section Domain primer.
4th, structure is as follows in the method for the database of the genotype of all test zones comprising different soybean varieties：
This example obtains 2488 universal test region primers and 0 non-universal test zone primer, corresponding to them Amplification region is the test zone of soybean varieties to be measured.The genotype of 2488 test zones of the structure comprising 33 kinds and its The database of SNP positional information, partial results are shown in Table 1.
Table 1 is database variety and genetype and its position, soybean varieties genotype to be measured, hybrid strain genotype and its frequency Certain embodiments
In table 1, the position that '-' represents the SNP site lacks in reference gene group；In addition to ATGC, other letters represent Degeneracy base.If genotype is made up of degeneracy base N entirely, claim corresponding test zone genotype and SNP shortage of data, the base of missing During because of type or SNP compared with any genotype or SNP, make indifference processing.Can be by detection soybean to be measured provided by the invention The genotype of the method Test database kind of variety and genetype and completion missing.
The present embodiment does not list all database content completely as space is limited, only lists wherein 5 kinds The information of 10 test zones.Equally limited based on length, also have some areas also only to list part in the present embodiment related real Example, remaining unlisted data can be according to the method completion of the present embodiment.
5th, after the amount of sampling SN for determining soybean varieties to be measured, random sampling mixes and extracts the DNA of mixing sample, method It 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 to judge one Selected threshold value when cause property and stability, BINOM.INV are the function in excel 2010, its application method and excel Definition in 2010 is identical, and its implication is so that the functional value of accumulation bi-distribution is more than or equal to the smallest positive integral of critical value. The condition that amount of sampling SN meets is meant that：15% of judgment threshold M when even if hybrid strain rate only exceeds uniformity and stability, should Amount of sampling can correctly judge the stability and uniformity of soybean varieties to be measured in the case where 95% probability ensures.M values are according to crop What the conditions such as species, type, specific requirement artificially determined.In New variety protection office of the Ministry of Agriculture issues《Plant is new VarietY specificity, uniformity and stability test guide-soybean》Middle regulation：For conventional kind, during consistency checking, use 0.5% population norms and at least 95% acceptance probability.Therefore, in the present embodiment, M values are used as from median 0.5%.By After step increases SN values, above-mentioned formula discovery is calculated, as SN >=25218, BINOM.INV (SN, 0.5%, 0.95)/SN≤ 1.15*0.5% is set up.Therefore, the soybean varieties amount of sampling to be measured in the present embodiment answers >=25218.
Random sampling mixes and extracts the DNA of mixing sample
In the present embodiment, 30000 germinations are have chosen, 26000 buds being substantially equal to the magnitudes is randomly selected and mixes It is placed in after conjunction in mortar, powder is fully ground into after adding liquid nitrogen into mortar.Given birth to using Beijing Tiangeng biochemical technology Co., Ltd The article No. of production is that DP305 plant genome DNA extracts kit is extracted and obtains the DNA of soybean varieties mixing sample to be measured, DNA extraction method is carried out by the operation manual of the kit.Utilize the production of Invitrigen companies of the U.S.dsDNA HS Assay Kit (article No. Q32852) and its specification quantify to the DNA of acquisition, the soybean product to be measured after quantifying Kind DNA is diluted to 10.00ng/ μ l.
6th, expanded using the DNA of primer pair mixing sample, obtain the amplified production of test zone, amplified production is made It is as follows for the method for high-throughput sequencing library：
High-throughput sequencing library includes：The high pass of the high-throughput sequencing library in universal test region and non-universal test zone Sequencing library is measured, in the present embodiment, builds 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 for building the high-throughput sequencing library in universal test region is as follows：
It is multiple using library construction Kit 2.0 (being produced by LifeTechnology companies of the U.S., article No. 4475345) Behind PCR amplification universal tests region, high-throughput sequencing library is built using amplified production.The kit includes following reagent：5× Ion AmpliSeqTMHiFi Mix, FuPa reagents, transferring reagent, sequence measuring joints solution and DNA ligase.The side of library construction Method presses the operation manual of the kit《Ion AmpliSeqTMLibrary Preparation》(publication number：MAN0006735, Version：A.0) carry out.It is as follows by 2488 universal test regions of multiplexed PCR amplification, the amplification system of multiplex PCR：5×Ion AmpliSeqTMμ l of HiFi Mix 4, μ l of universal test region primer mixed liquor 4, the DNA 10ng of soybean varieties to be measured prepared With without the μ l of enzyme water 11.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 follow Ring；10 DEG C of insulations.After primer unnecessary in multiplexed PCR amplification product is digested using FuPa reagents, then phosphorylation is carried out, specifically Method is：2 μ L FuPa reagents are added into the amplified production of multiplex PCR, after mixing, are reacted in PCR instrument by following program： 50 DEG C, 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 by phosphorus The amplified production solution of acidifying.By the upper sequence measuring joints of amplified production connection of phosphorylation, specific method is：Add into mixture a Enter μ L of transferring reagent 4, the μ L of sequence measuring joints solution 2 and the μ L of DNA ligase 2, after mixing, reacted in PCR instrument by following program：22 DEG C, 30 minutes；72 DEG C, 10 minutes；10 DEG C of preservations, obtain mixed liquor b.Utilize the ethanol precipitation methods purifying mixed liquor b of standard After be dissolved in 10 μ L without in enzyme water.Utilize the production of Invitrigen companies of the U.S.DsDNA HS Assay Kit (goods Number be Q32852) and be measured according to its specification, and after obtaining mixed liquor b mass concentration, will after purification mixed liquor b it is dilute Release to 15ng/ml, obtain the high-throughput sequencing library in concentration about 100pM universal test region.
The method for building the high-throughput sequencing library of non-universal test zone is as follows：
Using the DNA of soybean varieties to be measured as template, the first primer of the non-universal test zone of above-mentioned preparation and are utilized Two primers carry out independent PCR amplifications respectively, and the high-flux sequence text of non-universal test zone is obtained after mixed in equal amounts amplified production Storehouse.Concrete operations are pressed《Ion Amplicon Library Preparation(Fusion Method)》(publication number： 4468326) carry out, substantially process is as follows：The forward primer of first primer and reverse primer are dissolved as with water to 10 μM of concentration Afterwards, isometric mixing, obtains the first primer solution.It is formulated as follows PCR reaction systems：μ L of first primer solution 1,30ng are to be measured big Beans kind DNA and PCR high-fidelity mixture (invirtrigen companies of the U.S. produce, article No. 12532016) 45 μ L, are mixed Afterwards, reacted in PCR instrument by following program：94 DEG C, 3 minutes；(94 DEG C, 30 seconds；58 DEG C, 30 seconds；68 DEG C, 1 minute) × 40 Circulation；4 DEG C of insulations.Pcr amplification product is dissolved in 10 μ L water after purification by the method for the ethanol precipitation of standard, utilizes DNA On the biological analyser (model 2100) that 1000 kits (article No. 5067-1504) produce in Agilent company of the U.S., press After the kit specification determines and obtains the molar concentration of amplified production, 200pM, the amplification production of as the first primer are diluted to Thing.Using identical method, amplified production of the concentration for 200pM the second primer is obtained.By the amplified production of the first primer with The amplified production of second primer mixes in equal volume, obtains the non-universal test zone high-throughput sequencing library that concentration is 100pM.This In embodiment, due to universal test region nothing but, therefore, without the high-throughput sequencing library for building non-universal test zone.
Obtain the high-throughput sequencing library of all test zones
In universal test region number and non-universal test zone number ratio mixing equimolar concentration it is general The high-throughput sequencing library of the high-throughput sequencing library of test zone and non-universal test zone, obtained mixture are all The high-throughput sequencing library of test zone.In the present embodiment, because of the high-throughput sequencing library in universal test region nothing but, because This, the high-throughput sequencing library of structure is the high-throughput sequencing library in the universal test region that concentration is 100pM.
7th, high-flux sequence is carried out to high-throughput sequencing library, obtains that fragment group is sequenced, method is as follows：
Determine the principle of high-flux sequence depth：The 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 depth of high-flux sequence, namely averagely the capped multiple of each test zone, M are Judge threshold value selected when uniformity and stability, BINOM.DIST is the function in excel 2010, its application method with Definition in excel 2010 is identical, and what it was returned is the probability of binomial distribution.The meaning of three functions is：In hybrid strain rate As little as 0.1%, the hybrid strain condition wide in variety up to average only 20 difference sites between 10 and hybrid strain kind and soybean varieties to be measured Under, probability >=99.9% of the whole hybrid strain kinds of detection determined by high-flux sequence depth；In database kind up to 10000 It is individual and between hybrid strain kind and soybean varieties to be measured under conditions of average only 20 difference sites, determined by high-flux sequence depth In the presence of probability≤0.1% of erroneous judgement hybrid strain kind；It is wide in variety up to 10 and true hybrid strain rate exceeds only judgement specificity in hybrid strain When selected threshold value 10% when, the judgement conclusion to stability and uniformity determined by high-flux sequence depth is correct Probability >=95.0%.Conditions above is very strict, and therefore, true effect is better than above-mentioned threshold value.The projectional technique of above probability is shown in Table 2.
Table 2 is the computational methods of the present embodiment dependent probability
Table 2 is that the tables of data of Excel 2010, its function, cell etc. is identical with Excel 2010 definition.Wherein, " judging threshold value selected when uniformity and stability (M) " for cell B31, other cell numberings are pressed using B31 as reference Excel 2010 rule defines, such as the cell where " hybrid strain rate (R) " adds 4 rows 1 on the basis of B2 and arranged, therefore Numbering is C35, and other cell coding rules are identical with this.
The determination method of the present embodiment high-flux sequence depth is：After M=0.5% is substituted into above three formula, progressively It when increasing sequencing depth CF to 6001, can set up above three equation, therefore, the present embodiment sequencing depth is defined as >=6001 Times.
High-flux sequence is carried out using high-throughput sequencing library
Utilize the high-throughput sequencing library and kit Ion PI Template of all test zones of acquisition OT2200Kit v2 (invirtrigen companies of the U.S. produce, article No. 4485146) be sequenced before ePCR (Emulsion PCR, emulsion polymerization enzyme chain reaction) expand, operating method is carried out by the operation manual of the kit.Utilize ePCR products and reagent Box Ion PI Sequencing 200Kit v2 (invirtrigen companies of the U.S. produce, article No. 4485149) are in Proton High-flux sequence is carried out on two generation high-flux sequence instrument, operating method is carried out by the operation manual of the kit.In the present embodiment In, high-flux sequence flux is arranged to average 10000 times of coverage test region.
High-flux sequence result is pre-processed
First determine whether high-flux sequence the quality of data whether >=Q20, if<Q20 (this situation is few), then as stated above High-flux sequence is re-started, until quality requirement reaches Q20 standards, Q20 standards, which are met in table 2, " to be sequenced wrong to be specific The requirement of the probability of base "≤0.33%.The high-flux sequence fragment for being up to quality requirement is compared to all 2488 tests Region, remove after comparing the unsuccessful and infull sequencing fragment of genotype detection, remaining all sequencing fragments are referred to as piece is sequenced Section group.The incomplete sequencing fragment of genotype detection refers to could not be by table 1 shown in " positions of the SNP in reference gene group " The reason for all SNP sites in sequencing region where the sequencing fragment detect, genotype detection is not complete is sequencing fragment Too short, it is that sequencing fragment is mostly non-specific amplification product to compare unsuccessful reason.
8th, analysis sequencing fragment group, it is as follows to obtain soybean varieties genotype and hybrid strain genotype, method to be measured：
Sequencing fragment group is compared and arrives all test zones, and counts the sequencing segments in each test zone, is removed The test zone of segments≤1000 is sequenced, remaining test zone is the successful test zone of detection.In the present embodiment, 2406 successful test zones of detection are obtained altogether.The fragment for comparing test zone is referred to as the sequencing fragment of the test zone, The base composition that the position in table 1 shown in " positions of the SNP in reference gene group " is extracted from sequencing fragment is referred to as the sequencing The genotype of fragment.The frequency of genotype refers to be sequenced in fragment group, and the sequencing segments for representing the genotype accounts for the genotype The ratio of the sequencing fragment sum of place test zone.The maximum genotype of frequency is referred to as soybean varieties genotype to be measured.Hybrid strain Genotype refers to the potential hybrid strain genotype of frequency >=0.02%, wherein, potential hybrid strain genotype is all with soybean varieties to be measured There are insertion or the missing of discontinuous base in quantity >=2 of distinguishing base between genotype or distinguishing base.Hybrid strain genotype The principle of definition is：In high-flux sequence, insertion or missing errors are extremely rare, and the 2 fixed difference caused by mistake is sequenced The probability of base as little as (1%/3) 2=0.0011%, and require hybrid strain genotype frequency >=0.02%, limited in these conditions Under, even 30000 sequencing depth, because the probability that sequencing mistake produces certain hybrid strain genotype is only 0.0001% (calculating 2) method is shown in Table.0.02% frequency meets most strict DUS testing standards at present, i.e., as little as 2 detected from 10,000 seeds Hybrid.If distinguishing base quantity=1, whole test zones can all produce wrong hybrid strain genotype, and (computational methods are shown in Table 2), if during distinguishing base quantity >=3, hybrid strain genotype quantity is drastically reduced, it is difficult to accurate calculating hybrid strain rate R is therefore, poor The threshold value of isobase quantity >=2 is optimal.
For example, in fragment group is sequenced, the sequencing fragment sum in the 1st sequencing region is 9987 articles, have TCA, TCG, TCC, TCT ... totally 25 kinds of genotype, represent these genotype sequencing segments distinguish 9612,218,1,2 Bar ..., the frequencies of these genotype is 9612/9987=96.25%, 218/9987=2.18%, 1/9987=0.01%, 2/9987=0.02% ....By the definition of soybean varieties genotype to be measured and hybrid strain genotype, TCA should be soybean product to be measured Kind the 1st test zone soybean varieties genotype to be measured, and TCG frequency is more than 0.02%, but with soybean varieties to be measured Genotype TCA relatively has 1<The difference of 2 bases, therefore TCG is not hybrid strain genotype, other genotype are sequencing mistake production Raw genotype.Hybrid strain karyogene type refers to that hybrid strain genotype is karyogene type, and hybrid strain matter genotype refers to that hybrid strain genotype is Matter genotype.By this definition, first test zone also without hybrid strain karyogene type.By identical method, judge and obtain complete Soybean varieties genotype to be measured, hybrid strain genotype and its frequency of the 2406 successful test zones of detection in portion, and judge to obtain Hybrid strain genotype be hybrid strain karyogene type or hybrid strain matter genotype.As a result show：291 hybrid strain genotype are obtained altogether, its In, 286 are hybrid strain karyogene type, and 5 are hybrid strain matter genotype.
The standard sample detection method in the present embodiment is following is a brief introduction of, a kind is taken from soybean varieties to be measured Son, after sowing and growing up to seedling, pressed using the blade of seedling and extract genomic DNA with soybean varieties identical method to be measured, should DNA is referred to as the standard sample of soybean varieties to be measured.With soybean varieties to be measured simultaneously and by the parallel structure standard sample of same procedure High-throughput sequencing library and high-flux sequence.Wherein, the maximum genotype of frequency is referred to as standard sample genotype, standard sample In quantity >=2 or distinguishing base of frequency >=0.02% of hybrid strain genotype and the distinguishing base between standard sample genotype There are insertion or the missing of discontinuous base.Successful test section is each detected by with soybean varieties identical method to be measured, acquisition Standard sample genotype and standard sample hybrid strain genotype in domain.If standard sample genotype and soybean varieties genotype to be measured Identical test zone accounts for ratio that standard sample and soybean varieties to be measured detect successful test zone more than 90%, then marks Quasi- sample is correct, otherwise, takes 1 seed from soybean varieties to be measured again, repeats above procedure, until obtaining correct standard Sample.By the hybrid strain genotype of correct standard sample compared with the hybrid strain genotype of the corresponding test zone of soybean varieties to be measured, Identical hybrid strain genotype is obtained, removes identical hybrid strain genotype in soybean varieties to be measured, correct soybean varieties to be measured are miscellaneous Pnca gene type is retained and is used for subsequent analysis.Above measure eliminates the hybrid strain gene caused by Systematic selection mistake Type, Systematic selection mistake are mainly the PCR selectivity mistake amplifications caused by the special construction of gene order.Need what is illustrated It is：When database is wide in variety, can represent different cultivars genotype extensively, hybrid strain genotype and database kind can be required Some genotype is identical, can equally play with standard sample identical function, in this case, it is possible to not examination criteria sample Product, reach the purpose for mitigating workload.Result is in the present embodiment：From the 291 hybrid strain genotype obtained, 2 are eliminated altogether Individual hybrid strain genotype, wherein 2 are hybrid strain karyogene type, 0 is hybrid strain matter genotype, the 289 hybrid strain bases remained Because type is used for subsequent analysis, partial results are shown in Table 1.
9th, by soybean varieties genotype to be measured compared with the genotype of the different cultivars in database, obtain approximate kind, Variant sites and variant sites rate, method are as follows：
If in the test, soybean varieties to be measured claim the test zone with the genotype of database kind without missing For soybean varieties to be measured and the shared test zone of the database kind.In shared test zone, if soybean varieties to be measured with The genotype of database kind is incomplete same, then the test zone where the incomplete same genotype is referred to as soybean to be measured The difference site of kind and the database kind, corresponding genotype Differential genotype each other, difference site rate=difference site Number/shared test zone number.The minimum kind of difference bit rate is obtained from database and is referred to as soybean varieties to be measured Approximate kind, corresponding difference site are referred to as variant sites, number/shared test zone of variant sites rate=variant sites Number.
In the present embodiment, the shared test zone number of the 1st kind " rich No. 8 of north " of soybean varieties and database to be measured For 2335.In the 1st shared test zone, soybean varieties to be measured and " north rich No. 8 " genotype are respectively GTT and GTT, and two Person is identical, and therefore, the 1st shared test zone is not also or not soybean varieties to be measured and the difference site in " rich No. 8 of north ", GTT Soybean varieties to be measured and the Differential genotype in " rich No. 8 of north ".It is to be measured big by all shared test zones by identical method For beans kind compared with " rich No. 8 of north " genotype, discovery shares 190 difference sites, difference site rate=190/2335= 8.14%.By identical method, soybean varieties to be measured and all 33 interracial difference sites rate in database are obtained, and obtain The minimum kind of difference site rate is obtained as " cultivating mirror beans 26 ", difference site rate is 3.24%.Therefore, " mirror beans 26 are cultivated " to treat The approximate kind of soybean varieties is surveyed, the variant sites rate of soybean varieties to be measured is 3.24%.
Tenth, by hybrid strain genotype compared with the genotype of the different cultivars in database, after obtaining hybrid strain kind, calculate miscellaneous Strain rate, method are as follows：
Obtain hybrid strain kind：The kind that hybrid strain kind is present in database, and the potential hybrid strain genotype of hybrid strain kind Having the number of the test zone of phase homogenic type to account for hybrid strain kind between hybrid strain genotype has the test of potential hybrid strain genotype Total ratio >=60% in region, wherein, the difference between all genotype of potential hybrid strain genotype and soybean varieties to be measured There are insertion or the missing of discontinuous base in quantity >=2 of base or distinguishing base.Hybrid strain kind is divided into nucleus hybrid strain product Kind and cytoplasm hybrid strain kind, wherein, nucleus hybrid strain kind refers to calculate the hybrid strain kind obtained merely with karyogene type, carefully Kytoplasm hybrid strain kind refers to calculate the hybrid strain kind obtained merely with matter genotype.For example, it is assumed that the base of the kind in database Because type is respectively that ("/" represents that the test zone is heterozygous genotypes, the different gene of "/" both front and back be present by AA, AA, AA/TT Type), AA/TT, AA/TT, AA/TT and AA when, the corresponding genotype of soybean varieties to be measured is respectively AA, AA/TT, TT, AA, TT/ CC, GG/CC and during-A, corresponding potential hybrid strain genotype is：Nothing, nothing, AA, TT, AA, AA/TT and AA.In general pure line cultivar In the absence of heterozygous genotypes, but only a few site there may be, in addition, hybrid strain is mostly cenospecies, heterozygous sites are more typical, therefore List various possible situations.Parameter 60% can ensure that whole hybrid strain kind detection probabilities are 100% and the miscellaneous of erroneous judgement be present The probability of strain kind is 0%, and the determination method of the parameter value is shown in Table 2.
In the present embodiment, in the 1st test zone, first kind " rich No. 8 of north " and soybean product to be measured in database The genotype of kind is respectively GTT and GTT, and the difference without base between the two, therefore, GTT are not potential hybrid strain genotype, thus the In 1 test zone, " north rich No. 8 " be also not present with the potential hybrid strain genotype of hybrid strain genotype identical, by identical method, In the test zone for judging all karyogene types one by one, whether the genotype of first kind " rich No. 8 of north " is latent in database In hybrid strain genotype, if potential hybrid strain genotype, then judge whether to have between potential hybrid strain genotype and hybrid strain genotype identical Genotype, the results showed that, " north rich No. 8 " shares 134 test zones with potential hybrid strain genotype, they and same test The test zone number for having phase homogenic type between the hybrid strain genotype in region is 133, and its ratio is 133/134=99.25%> 60%, therefore, judge " rich No. 8 of north " as nucleus hybrid strain kind.In a similar manner, the test section of all matter genotype is utilized Domain, judge " rich No. 8 of north " not as cytoplasm hybrid strain kind.By identical method, judge in database whether is all other kind For nucleus hybrid strain kind or cytoplasm hybrid strain kind, the results showed that：Only " rich No. 8 of north " is nucleus hybrid strain kind, is not sent out Existing cytoplasm hybrid strain kind.It these results suggest that：" rich No. 8 of north " may be by flyings pollination rather than mechanical admixture, by base Because type has been mixed into soybean varieties to be measured.
Obtain special hybrid strain genotype：Special hybrid strain genotype refers to the hybrid strain gene that only a hybrid strain kind is all Type, it includes special hybrid strain karyogene type and special hybrid strain matter genotype；Special hybrid strain karyogene type refers to an only cell All hybrid strain karyogene types of core hybrid strain kind, special hybrid strain matter genotype refer to that only a cytoplasm hybrid strain kind is all Hybrid strain matter genotype.In the present embodiment, 291 hybrid strain genotype are obtained altogether, wherein, 286 are hybrid strain karyogene type, and 5 are Hybrid strain matter genotype.First hybrid strain karyogene type ATGA is only that nucleus hybrid strain kind " rich No. 8 of north " is all, so, ATGA For the special hybrid strain karyogene type in " rich No. 8 of north ".By identical method, 286 hybrid strain genotype of all acquisitions are judged one by one In, 133 special hybrid strain karyogene types possessed for " rich No. 8 of north ".In a similar manner, 5 hybrid strain matter genotype are judged It is not special hybrid strain matter genotype.
Hybrid strain rate R principles are calculated, it is specific as follows：
Hybrid strain rate R=R1+R2-R3-R4, wherein：Wherein, n1 For the number of nucleus hybrid strain kind, t1 is the number of all special hybrid strain karyogene types of the i-th 1 nucleus hybrid strain kinds, I1j1 is jth 1 after all special hybrid strain karyogene types of the i-th 1 nucleus hybrid strain kinds sort from low to high by its frequency Special hybrid strain karyogene type, R1i1j1 are the frequency of the i-th 1j1 special hybrid strain karyogene types；R1 is by hybrid strain karyogene type meter The summation of the hybrid strain rate of the nucleus hybrid strain kind of calculation, the hybrid strain rate of nucleus hybrid strain kind are to remove in nucleus hybrid strain kind After the frequency of the special hybrid strain karyogene type of minimum 80% and highest 10%, the frequency of remaining special hybrid strain karyogene type 2 times of average value；Wherein, t2 possesses except nucleus hybrid strain kind The number of outside hybrid strain karyogene type and frequency >=0.17% hybrid strain karyogene type, i2 are except nucleus hybrid strain kind is gathered around After all hybrid strain karyogene types outside some hybrid strain karyogene types sort from low to high by its frequency, the i-th 2 hybrid strain core bases Because of type, R2i2 is the frequency of the i-th 2 hybrid strain karyogene types；R2 is to utilize the hybrid strain core base possessed except nucleus hybrid strain kind Because of the hybrid strain rate that type calculates, it is minimum in the frequency for remove the hybrid strain karyogene type possessed except nucleus hybrid strain kind 80% and highest 10% value after, 2 times of the average value of surplus value；Its In,N2 is the number of cytoplasm hybrid strain kind, and R3i3 is thin for the i-th 3 The hybrid strain rate of kytoplasm hybrid strain kind, R3i3 value when R3ic is i3=ic, ic be when soybean varieties to be measured be nucleo_cytoplasmic interaction not Educate and be or during maintainer, the cytoplasm hybrid strain kind of corresponding maintainer or sterile line, t3 is the i-th 3 cytoplasm hybrid strain kinds All special hybrid strain matter genotype number, i3j3 is all special hybrid strain matter genotype of the i-th 3 cytoplasm hybrid strain kinds After being sorted from low to high by its frequency, the special hybrid strain matter genotype of jth 3, R3i3j3 is the i-th 3j3 special hybrid strain matter genes The frequency of type, the hybrid strain rate of sterile line that R3ic refers to the hybrid strain rate for the maintainer being mixed into sterile line or is mixed into maintainer；R3 For the summation of the hybrid strain rate of cytoplasm hybrid strain kind calculated by hybrid strain matter genotype, the hybrid strain rate of cytoplasm hybrid strain kind is goes It is remaining special after falling the frequency of special hybrid strain matter genotype of 80% and highest 10% minimum in cytoplasm hybrid strain kind The average value of the frequency of hybrid strain matter genotype；Wherein, t4 is except cytoplasm is miscellaneous Outside the hybrid strain matter genotype that strain kind possesses and the hybrid strain matter genotype of frequency >=0.17% number, i4 are except cytoplasm After all hybrid strain matter genotype outside the hybrid strain matter genotype that hybrid strain kind possesses sort from low to high by its frequency, the i-th 4 Hybrid strain matter genotype, R4i4 are the frequency of the i-th 4 hybrid strain matter genotype；R4 be utilize except cytoplasm hybrid strain kind possess it is miscellaneous The hybrid strain rate that strain matter genotype calculates, it is minimum in the frequency for remove the hybrid strain matter genotype possessed except cytoplasm hybrid strain kind 80% and highest 10% value after, the average value of surplus value；Int () is bracket function, returns to the integer of the number in bracket Part.
The flyings pollination that hybrid strain in soybean varieties to be measured comes from reproductive process mixes and mechanical admixture, wherein, fly Flower pollination mix be hybrid strain variet complexity main source.Flyings pollination, which mixes, refers to that the pollen of hybrid strain kind is passed by wind-force etc. To soybean varieties to be measured and the hybrid seed for formation of pollinating, flyings pollination can not possibly introduce cytoplasm, therefore can only cause hybrid strain Karyogene type, its hybrid strain rate are 2 times of hybrid strain karyogene type frequency.It is to be measured that mechanical admixture refers to that hybrid strain variety seeds are directly mixed in In soybean varieties, while nucleus and cytoplasm are introduced, while form hybrid strain karyogene type and hybrid strain matter genotype, its hybrid strain Rate should be the frequency of hybrid strain matter genotype.In hybrid strain rate R calculation formula, R1+R2 over-evaluates the hybrid strain rate of mechanical admixture 1 times, need to correct, the R=R1+R2-R3-R4 after correction.It is a technical barrier to distinguish mechanical admixture with flyings pollination to mix, The present invention solves this problem.
In hybrid strain rate R calculation formula, the hybrid strain rate of nucleus hybrid strain kind is all 2 × hybrid strain karyogene type frequency, Its reason is as follows：Diploid or allopolyploid soybean are 2 copies, therefore, hybrid strain in the test zone of nuclear genome Rate is 2 times of corresponding hybrid strain karyogene type frequency.If the test zone of the nuclear genome of N parts copy must be selected, Then coefficient should be adjusted to N, if copy number is indefinite, make N=2 processing, if wrong, it will when calculating R, by removing 80% The mode of low extremum excludes them.
In hybrid strain rate R calculation formula, merely with 10% of hybrid strain genotype frequency value in centre count Calculate, its principle is：The different hybrid strain genotype of same hybrid strain kind are determined by the hybrid strain rate of the hybrid strain kind, so the phase of frequency Prestige value is equal, and the difference between frequency is expanded by PCR, the error during high-flux sequence causes.Pass through hybrid strain gene The definition of type and soybean varieties standard sample to be measured, substantially eliminate these improper values, remove 10% extremum and are enough Remove the test zone that very small amount deviates true hybrid strain rate.Why remove the 80% of minimum, and it is maximum then only remove 10%, Principle is as follows：(1) worst error source is sequencing mistake, and it is very low that hybrid strain genotype frequency caused by mistake is sequenced；(2) except In the frequency of hybrid strain genotype outside hybrid strain kind, high level is more likely the common hybrid strain genotype of different hybrid strains, is represent Real hybrid strain rate.
When soybean varieties to be measured are nucleo_cytoplasmic interaction sterile line, if being wherein mixed with maintainer hybrid strain corresponding to the sterile line Kind, then, it is miscellaneous by cytoplasm is detected as because the cytoplasm of the maintainer hybrid strain kind and soybean varieties to be measured are different Strain kind, but because the nucleus of sterile line and maintainer is just the same, nucleus hybrid strain kind will not be detected as, because This, R3ic value is not calculated in R1+R2, but is calculated in R3i3, therefore, it is necessary to subtract 2 in R3 × R3ic is imitated just.Same reason, when soybean varieties to be measured are nucleo_cytoplasmic interaction maintainer, it is also desirable to subtracted in R3 pair 2 × R3ic of the sterile line hybrid strain kind answered is imitated just.Obviously, when soybean varieties to be measured are neither nucleo_cytoplasmic interaction sterile line When not being nucleo_cytoplasmic interaction maintainer yet, R3ic=0.
In R2 and R4 calculation formula, it is desirable to which frequency >=0.17% of hybrid strain genotype, its principle are as follows：Work as database In kind number and detection site when reaching 10000,149 hybrid strain genotype erroneous judgements will be averagely produced, when setting hybrid strain During genotype frequency >=0.17%, probability >=99.98% (projectional technique is shown in Table 2) of the hybrid strain genotype of no erroneous judgement just can be accurate Really calculate the value to R2 and R4.It has been the limit in reality that kind number in database and detection site, which reach 10000, because This, the threshold value of frequency >=0.17% of hybrid strain genotype goes for various situations.R2 and R4 introducing so that energy of the present invention It is 0 enough in database kind, i.e., in the case that no database is supported, calculates hybrid strain rate R.
Especially, if hybrid strain kind A all hybrid strain genotype are possessed by hybrid strain kind B and other hybrid strain kinds, because And hybrid strain kind A is without special hybrid strain genotype.Now, when calculating hybrid strain rate R, hybrid strain kind A and hybrid strain kind B are not calculated Hybrid strain rate, and calculate hybrid strain kind AB hybrid strain rate.Hybrid strain kind AB hybrid strain VDA genotypes are：Hybrid strain kind A with it is miscellaneous Hybrid strain genotype common to strain kind B.
Hybrid strain rate R calculation formula is general formula, and soybean varieties to be measured typically only mix a kind of hybrid strain product in reality Kind.
Calculate hybrid strain rate R hypothesis example
Table 3 assumes a hybrid strain rate calculated examples, to become apparent from illustrating hybrid strain rate R calculating process.
Table 3 assumes example to calculate one of hybrid strain rate R
In table 3, nucleus hybrid strain kind common A and B two, so n1=2, cytoplasm hybrid strain kind number only C mono-, so N2=1.By the definition of special hybrid strain karyogene type, the special hybrid strain karyogene type for obtaining hybrid strain kind A is that numbering is No. 1-10 Hybrid strain karyogene type AA, TT, TCC, GG, AC, TTC, TCCC, GGC, ACC and AG, so, t1=10, they frequency difference For 0.10%, 1.20%, 0.10%, 0.10%, 0.02%, 0.10%, 0.10%, 0.10%, 0.10% and 0.10%, to this It is R11111=0.02%, R11121=0.02%, R11131 after 10 special hybrid strain karyogene type frequencies sort from low to high =0.10%, R11141=0.10%, R11151=0.10%, R11161=0.10%, R11171=0.10%, R11181= 0.10%th, R11191=0.10% and R111101=1.20%.From j1=Int (0.8 × t1)+1=Int (0.8 × 10)+1= 9 to j1=t1-Int (0.1 × t1)=10-Int (0.1 × 10)+1=9 R111j1 value is R11191=0.10%, so Nucleus hybrid strain kind A hybrid strain rate isIn the same way, nucleus hybrid strain is obtained Kind B hybrid strain rate isThus, nucleus hybrid strain kind is obtained In a similar manner, R2=0.02%, cytoplasm hybrid strain product are obtained The hybrid strain rate of kindR4=0.04%.Therefore, hybrid strain rate R=R1+ in the hypothesis example R2-R3-R4=0.60%+0.02%-0.10%-0.04%=0.48%.
With reference to above-mentioned hypothesis example, the hybrid strain rate R in the present embodiment is calculated：In the present embodiment, hybrid strain kind is only " north Rich No. 8 " and be nucleus hybrid strain kind, R2, R3 and R4 are 0, thus, R=R1=R111." rich No. 8 of north " shares 133 spies Different hybrid strain karyogene type, frequency are：1.02%th, 1.03%...... (certain embodiments are shown in Table 1), by R computation rule, remove most After the frequency values of small 80% (106) and 10% (13) of minimum, the average value of remaining 14 frequencies is hybrid strain rate R =1.03%.
11, using variant sites, variant sites rate and hybrid strain rate, specificity, the uniformity of soybean varieties to be measured are judged And stability, method are as follows：
Wherein, SD is threshold value selected when judging specific, and M is to judge threshold selected when uniformity and stability Value.The method for judging soybean varieties to be measured specificity, uniformity and stability is：When variant sites rate >=SD or non-universal tests When region has variant sites, soybean varieties to be measured have specificity, and as variant sites rate ＜ SD and variant sites are not present in When in non-universal test zone, soybean varieties to be measured are without specificity；It is to be measured as hybrid strain rate≤M of soybean varieties to be measured Soybean varieties have uniformity and stability, and when the hybrid strain rate of soybean varieties to be measured is more than ＞ M, soybean varieties to be measured do not have Uniformity and stability.With M values, SD values be according to breeding level, desired Stringency, mark characteristic etc. it is many because Element, artificially determine.In the present embodiment, SD selects 1% standard.
In the present embodiment, variant sites rate is 3.24%>SD=1%, therefore, it is special to judge that soybean varieties to be measured have Property；Hybrid strain 1.03%≤M=0.5% of rate of soybean varieties to be measured, therefore, judge soybean varieties to be measured without uniformity and steady It is qualitative.
Further, after specific soybean varieties to be measured, uniformity and stability is judged, the accuracy of judgement is carried out Estimation, method are as follows：
Pure lines new soybean varieties in the present invention refer to be sheerly genotype as target and the conventional kind of seed selection, self-mating system, The types such as restorer, maintainer, sterile line.
Specific accuracy calculates：When variant sites are not present in non-universal test zone, if judging soybean varieties to be measured With specificity, the correct probability >=BINOM.DIST of conclusion ((1-SD) * TRN, TRN, 1-OD, TRUE)；If judge to be measured big Beans kind does not have specific, the correct probability >=BINOM.DIST (SD*TRN, TRN, OD, TRUE) of conclusion, wherein, TRN is The number for the test zone that success detects, OD are variant sites rate, and BINOM.DIST is the function in excel 2010, and it is used Method is identical with the definition in excel 2010, and what it was returned is the probability of binomial distribution.What above-mentioned probability actually calculated It is：When judging to have specific, variant sites rate is more than SD probability；When judging not having specific, variant sites rate Probability less than SD, successful test zone is detected by being obtained after analyzing sequencing fragment group.
In the present embodiment, soybean varieties to be measured are judged using variant sites rate has specificity, therefore, specificity knot By correct probability >=BINOM.DIST ((1-1%) * 2406,2406,1-3.24%, TRUE)=100.00%, it is seen that this reality It is very high to the accuracy of special sex determination conclusion to apply example.
Uniformity calculates with stability accuracy
The correct probability of conclusion for judging the uniformity and stability of soybean varieties to be measured is：When soybean varieties to be measured have When uniformity and stability, correct probability >=BINOM.DIST (M*SN, SN, R, TRUE) * BINOM.DIST (the Σ SeN* of conclusion M,ΣSeN,R,TRUE)；When soybean varieties to be measured do not have uniformity and stability, the correct probability of conclusion >= BINOM.DIST ((1-M) * SN, SN, (1-R), TRUE) * BINOM.DIST (Σ SeN* (1-M), Σ SeN, 1-R, TRUE), its In, Σ SeN are the summation of all sequencing fragments for being used for test zone where calculating hybrid strain rate R genotype frequency, namely go After falling 80% minimum value and 10% maximum, remain test zone for calculating hybrid strain rate test fragment it is total With, M to judge threshold value selected when uniformity and stability.Judge uniformity and stability accuracy depend entirely on it is miscellaneous The accuracy of strain rate, and the positive rate of hybrid strain rate really depends on the accuracy of following three steps：First, soybean varieties sampling to be measured Accuracy, second, the accuracy of detection hybrid strain kind, the 3rd from extraction sample, utilize the hybrid strain kind of detection to calculate hybrid strain The accuracy of rate.Therefore, the accuracy for judging soybean varieties uniformity and stability to be measured is the product of the step accuracy of the above three.By Even in the present invention under the conditions of most stringent of, the accuracy of detection hybrid strain kind also controls more than 99.9%, actually absolutely Major part is close to 100%.For example, in the present embodiment, whole hybrid strain kind detection probabilities are deposited more than 100.0000% Erroneous judgement hybrid strain kind probability below 0.0000% (circular is shown in Table 2).Therefore, soybean varieties to be measured are judged The accuracy of uniformity and stability can be estimated as the product of the accuracy of the first step and the 3rd step, its be respectively in above-mentioned formula before The value that latter two function is calculated.For example, BINOM.DIST (M*SN, SN, R, TRUE) meaning is：Soybean varieties to be measured are carried out SN sampling, the hybrid strain rate R that is actually pumped are less than threshold value M probability；For calculating each of soybean varieties hybrid strain rate to be measured Soybean varieties to be measured have substantially also quite been carried out single sample by individual sequencing fragment, therefore, BINOM.DIST (Σ SeN*M, Σ SeN, R, TRUE) meaning be：Soybean varieties to be measured are carried out with SeN sampling of Σ, the hybrid strain rate R being actually pumped is less than threshold Value M probability.
In the present embodiment, after removing the 80% of minimum and the hybrid strain genotype frequency of maximum 10%, 6 hybrid strain genes are shared Type frequency be used to calculate hybrid strain rate R, and the sequencing fragment sum of test zone corresponding to them is 138586, so Σ SeN =138586, also that is, 26000 samples being pumped have been carried out with 138586 sampling, the mistake of so big amount of sampling again Difference is fairly small.In the present embodiment, judge that soybean varieties to be measured do not have uniformity and stability, therefore, the judgement conclusion Correct probability >=BINOM.DIST ((1-0.5%) * 26000,26000, (1-1.03%), TRUE) * BINOM.DIST (138586* (1-0.5%), 138586,1-1.03%, TRUE)=100.0000%.It can be seen that this implementation is to soybean varieties to be measured Uniformity and the judgement of stability be also very accurate.
Result verification
Press《New variety of plant specificity, uniformity and stability test guide-soybean》In method plant and observe and treat Survey soybean varieties and its approximate kind " cultivating mirror beans 26 ", find soybean varieties to be measured in multiple characters such as leaf color with approximate product Notable difference be present in kind.《New variety of plant specificity, uniformity and stability test guide-soybean》Middle regulation：At least one When there is obvious and reproducible difference with approximate kind in individual character, you can judge that the soybean varieties to be measured of application possess specifically Property.Therefore, judge that soybean varieties to be measured have specificity.In experimentation, planted altogether 300 plants of soybean varieties to be measured with it is near Like kind (150 plants of cells, totally 2 repetitions), 7 plants of special-shaped strains are found,《New variety of plant specificity, uniformity and stably Property Testing Guideline-soybean》Middle regulation：When sample size is 300 plants, 4 plants of special-shaped strains are at most allowed for, thus judge to treat Survey soybean varieties and do not have uniformity.It is believed that the kind does not possess stability due to not possessing the kind of uniformity.Thus sentence Fixed, soybean varieties to be measured do not have stability yet.Experiment shows more than：To the special of soybean varieties to be measured in the present embodiment The judgement of property, stability and uniformity is correct.
The embodiment of the present invention is expanded by high-flux sequence and more sites, realizes the large sample sampling of soybean varieties to be measured Sampled with the large sample of inter-species individual test zone, recycle and define hybrid strain genotype, define cytoplasm hybrid strain kind and definition The comprehensive means such as hybrid strain rate calculation formula, successfully realize it is accurate, quick, intactly judge the special of soybean varieties to be measured The target of property, stability and uniformity, it has the technical effect that what existing DUS method of testings did not all reach.Existing molecule DUS detections Technology such as chip only detects fixed test zone, it is impossible to according to case, flexibly selects non-universal test zone.And present invention detection Be PCR primer, non-universal test zone can be detected easily according to case flexible design primer.In addition, the present invention is real Example is applied for 26000 individual amount of samplings for traditional DUS measuring technologies, work is big, can not complete, for example, field Between in DUS tests, 26000 plants of soybean of sampling need more than 2 mu of plantation, and need to plant 2 years, and annual every plant of soybean need to investigate it is more Individual character., it is necessary to be 26000 DNA extractions, 26000*2488 PCR respectively in widely used SSR molecules DUS tests (assuming that as the present embodiment, have detected 2488 universal test regions) is detected with 26000*2488 PCR primer.Therefore, Because workload is excessive, existing molecule DUS tests there all are not measuring stability and uniformity, although DUS test detections in field are consistent Property and stability, but sampling samples amount, all below 1000 plants, 26000 plants of soybean and the present embodiment has been sampled, its accuracy shows It is so higher.Why the present embodiment can increase amount of sampling, be because all 26000 samples are used as a sample after all mixing Processing, and field DUS test and comparisons, workload is equivalent to being reduced to 1/26000；Further, all 2488 universal tests Mixed once amplification is all only done in region and high-flux sequence detects, and SSR molecule DUS test and comparisons, workload equivalent to It is reduced to 1/ (26000*2488).Therefore, the present invention realizes large sample and more sites in the case where workload significantly mitigates Detection, make DUS tests not only accurate but also simple.Database variety and genetype is base composition in the embodiment of the present invention simultaneously, Very standard, same breed is detected in the present inventive method under different experimental conditions, can obtain identical genotype, because And, it is not necessary under different conditions repeat DUS test, therefore, the embodiment of the present invention can directly with database variety and genetype Compare, objectively select the approximate kind of soybean varieties to be measured.And existing DUS measuring technologies are not up to standard, it is necessary to put down simultaneously Row ground carries out DUS tests to soybean varieties to be measured with approximate kind, just reliable conclusion can be obtained, in order to mitigate workload, no Obtain not by providing approximate kind by kind power applicant, if approximate kind mistake, there may be the legal consequence of erroneous grants.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent substitution and improvements made etc., it should be included in the scope of the protection.

Claims (7)

1. a kind of method of specificity for testing pure lines new soybean varieties, uniformity and stability, it is characterised in that methods described Including：
Obtain the variant sites between different soybean varieties；
The test zone of soybean varieties to be measured is determined by the variant sites, the test zone includes universal test region, At least partly described variant sites are included in the universal test region, and the universal test is determined by the variant sites The method in region is：Pass through discriminationThe value of discrimination is calculated, wherein, a is tested in variation window area The kind sum measured, bi are the kind number of i-th kind of genotype in the variation window area, and bi>1, k is comprising more than 1 The number of the genotype of individual kind, the variation window area is centered on each single nucleotide variations site, to the list The both sides in nucleotide diversity site respectively extend 1/2 window as detection of sequence length to be measured, and the universal test region is In nuclear genome 8000 maximum variation windows of discrimination and discrimination is maximum in cytoplasmic skeleton 100 Make a variation window, wherein, the genotype is the combination in multiple single nucleotide variations sites in the test zone；
Structure includes database of the different soybean varieties in the genotype of all test zones；
After the amount of sampling SN for determining the soybean varieties to be measured, random sampling mixes and extracts the DNA of mixing sample, the sampling Amount SN meets following condition：BINOM.INV (SN, M, 0.95)/SN≤1.15*M, wherein BINOM.INV are in excel 2010 Function, to judge threshold value selected when the uniformity and stability, the condition implication of the amount of sampling SN satisfactions is M： Even if 15% of judgment threshold M when the hybrid strain rate only exceeds uniformity and stability, probability of the amount of sampling 95% Under guarantee, the stability and uniformity of the soybean varieties to be measured can be correctly judged；
The primer for expanding the test zone is prepared, the primer includes universal test region primer；
Expanded using the DNA of mixing sample described in the primer pair, obtain the amplified production of the test zone, the expansion Increase production thing as high-throughput sequencing library；
High-flux sequence is carried out to the high-throughput sequencing library, obtains that fragment group, the depth CF of the high-flux sequence is sequenced Meet 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 depth of the high-flux sequence, and M is to judge the uniformity With threshold value selected during stability, BINOM.DIST be excel 2010 in function, the depth CF of the high-flux sequence The condition implication of satisfaction is：The hybrid strain rate as little as 0.1%, the hybrid strain kind be 10 and the hybrid strain kind with it is described Under conditions of averagely only having 20 difference sites between soybean varieties, the detection determined by the depth CF of the high-flux sequence is whole Probability >=99.9% of the hybrid strain kind；The database kind for 10000 and the hybrid strain kind with it is described big Under conditions of averagely only having 20 difference sites between beans kind, institute is judged in the presence determined by the depth CF of the high-flux sequence by accident State probability≤0.1% of hybrid strain kind；The hybrid strain kind is 10 and true hybrid strain rate exceeds only when that judges specific institute The threshold value of selection 10% when, by the depth CF of the high-flux sequence determine to the judgement conclusion of stability and uniformity just True probability >=95.0%；
The sequencing fragment group is analyzed, obtains soybean varieties genotype and hybrid strain genotype to be measured；
By the soybean varieties genotype to be measured compared with the genotype of the different cultivars in the database, described in acquisition Approximate kind, variant sites and the variant sites rate of soybean varieties to be measured；
By the hybrid strain genotype compared with the genotype of the different cultivars in the database, after obtaining hybrid strain kind, Calculate hybrid strain rate；
Using the variant sites, the variant sites rate and the hybrid strain rate, judge the soybean varieties to be measured specificity, Uniformity and stability.
2. according to the method for claim 1, it is characterised in that the test zone also includes non-universal test zone, institute Stating primer also includes non-universal test zone primer.
3. according to the method for claim 2, it is characterised in that the non-universal test zone primer include the first primer and Second primer, first primer include the first forward primer and the first reverse primer, and it is positive that second primer includes second Primer and the second reverse primer, first primer and second primer carry out respectively individually amplification obtain two it is described non-through With the amplified production of test zone, the amplified production mixed in equal amounts of two non-universal test zones is used to build independent expansion The high-throughput sequencing library of increasing；
5 ' end connections of first forward primer are just like SEQ ID NO in sequence table:Sequence 1 shown in 1, described first is reverse 5 ' end connections in primer are just like SEQ ID NO in sequence table:Sequence 2 shown in 2；
5 ' end connections of second forward primer are just like SEQ ID NO in sequence table:Sequence 2 shown in 2, described second is reverse 5 ' end connections of primer are just like SEQ ID NO in sequence table:Sequence 1 shown in 1.
4. according to the method for claim 2, it is characterised in that utilize the variant sites, the variant sites rate and institute Hybrid strain rate is stated, judging the method for the soybean varieties specificity to be measured, uniformity and stability includes：
When the variant sites be present in the variant sites rate >=non-universal test zones of SD or described, the soybean product to be measured Kind there is specificity, when the variant sites rate ＜ SD and the variant sites are not present in the non-universal test zone, The soybean varieties to be measured do not have specificity, wherein, SD is threshold value selected when judging specific；
As the hybrid strain rate≤M of the soybean varieties to be measured, the soybean varieties to be measured have uniformity and stability, when When the hybrid strain rate of the soybean varieties to be measured is more than ＞ M, the product to be tested Soybean Species do not have uniformity and stability, M Selected threshold value during to judge the uniformity and stability；
The hybrid strain rate R=R1+R2-R3-R4, wherein：
Wherein, n1 is the number of nucleus hybrid strain kind, and t1 is all special hybrid strains of the i-th 1 nucleus hybrid strain kinds The number of karyogene type, i1j1 are that all special hybrid strain karyogene types of the i-th 1 nucleus hybrid strain kinds press frequency After sorting from low to high, the special hybrid strain karyogene type of jth 1, R1i1j1 is the i-th 1j1 special hybrid strain karyogenes The frequency of type；R1 is the summation of the hybrid strain rate of the nucleus hybrid strain kind calculated by hybrid strain karyogene type, described thin The hybrid strain rate of karyon hybrid strain kind is to remove the institute of 80% and highest 10% minimum in the nucleus hybrid strain kind After the frequency for stating special hybrid strain karyogene type, 2 times of the average value of the frequency of the remaining special hybrid strain karyogene type；
Wherein, t2 is described in addition to the hybrid strain karyogene type that the nucleus hybrid strain kind possesses and frequency >=0.17% The number of hybrid strain karyogene type, i2 are all institutes in addition to the hybrid strain karyogene type that the nucleus hybrid strain kind possesses State after hybrid strain karyogene type sorts from low to high by frequency, the i-th 2 hybrid strain karyogene types, R2i2 is described miscellaneous for the i-th 2 The frequency of strain karyogene type；R2 is to utilize to remove the described of the hybrid strain karyogene type calculating that the nucleus hybrid strain kind possesses Hybrid strain rate, R2 are 80% He minimum in the frequency for remove the hybrid strain karyogene type possessed except the nucleus hybrid strain kind After the value of highest 10%, 2 times of the average value of surplus value；
Wherein, N2 is the number of cytoplasm hybrid strain kind, and R3i3 is the hybrid strain rate of the i-th 3 cytoplasm hybrid strain kinds, R3ic i3 R3i3 value during=ic, ic be when the soybean varieties to be measured are nucleo_cytoplasmic interaction sterile line or maintainer, it is corresponding described The cytoplasm hybrid strain kind of maintainer or the sterile line, t3 are all special of the i-th 3 cytoplasm hybrid strain kinds The number of hybrid strain matter genotype, i3j3 are that all special hybrid strain matter genotype of the i-th 3 cytoplasm hybrid strain kinds are pressed After frequency sorts from low to high, the special hybrid strain matter genotype of jth 3, R3i3j3 is the i-th 3j3 special hybrid strain matter The frequency of genotype, R3ic refer to the hybrid strain rate for the maintainer being mixed into the sterile line or the institute being mixed into the maintainer State the hybrid strain rate of sterile line；R3 is the total of the hybrid strain rate of the cytoplasm hybrid strain kind calculated by hybrid strain matter genotype With the hybrid strain rate of the cytoplasm hybrid strain kind is to remove 80% and highest 10% minimum in the cytoplasm hybrid strain kind The special hybrid strain matter genotype frequency after, the average value of the frequency of the remaining special hybrid strain matter genotype；
Wherein, t4 is in addition to the hybrid strain matter genotype that the cytoplasm hybrid strain kind possesses and frequency >=0.17% The number of the hybrid strain matter genotype, i4 are the institute in addition to the hybrid strain matter genotype that the cytoplasm hybrid strain kind possesses After having the hybrid strain matter genotype to be sorted from low to high by frequency, the i-th 4 hybrid strain matter genotype, R4i4 is the i-th 4 institutes State the frequency of hybrid strain matter genotype；R4 is to utilize the hybrid strain matter genotype possessed except the cytoplasm hybrid strain kind to calculate The hybrid strain rate, R4 are minimum in the frequency for remove the hybrid strain matter genotype possessed except the cytoplasm hybrid strain kind 80% and highest 10% value after, the average value of surplus value；
Int () is bracket function；
The nucleus hybrid strain kind refers to calculate the hybrid strain kind obtained, the cytoplasm hybrid strain merely with karyogene type Kind refers to calculate the hybrid strain kind obtained merely with matter genotype；The special hybrid strain karyogene type refers to only one All hybrid strain karyogene types of the nucleus hybrid strain kind；The special hybrid strain matter genotype refers to only described in one All hybrid strain matter genotype of cytoplasm hybrid strain kind；The hybrid strain karyogene type refers to that the hybrid strain genotype is described Karyogene type, the karyogene type refer to the genotype and are located on nuclear genome；The hybrid strain matter genotype refers to described Hybrid strain genotype is the matter genotype, and the matter genotype refers to that the genotype is located on cytoplasmic skeleton.
5. according to the method for claim 4, it is characterised in that methods described also includes treating described in judgement in the following ways The correct probability of conclusion of uniformity and stability for surveying soybean varieties is：When the soybean varieties to be measured have uniformity and steady When qualitative, correct probability >=BINOM.DIST (M*SN, SN, R, TRUE) * BINOM.DIST of conclusion (∑ SeN*M, ∑ SeN, R, TRUE)；When the soybean varieties to be measured do not have the uniformity and stability, the correct probability >=BINOM.DIST of conclusion ((1-M)*SN,SN,(1-R),TRUE)*BINOM.DIST(∑SeN*(1-M),∑SeN,1-R,TRUE)；Wherein, ∑ SeN is The summation of all sequencing fragments for being used for the test zone where calculating the frequency of the genotype of the hybrid strain rate R, M are Judge threshold value selected when the uniformity and stability, BINOM.DIST (M*SN, SN, R, TRUE) is the soybean to be measured Kind has carried out SN sampling, and the hybrid strain rate R being actually pumped is less than the probability of the threshold value M, BINOM.DIST (∑ SeN* M, ∑ SeN, R, TRUE) meaning be：The soybean varieties to be measured are carried out with SeN sampling of ∑, what is be actually pumped is described miscellaneous Strain rate R is less than threshold value M probability.
6. according to the method for claim 5, it is characterised in that when the change dystopy is not present in the non-universal test zone During point, if it is specific to judge that the soybean varieties to be measured have, the correct probability >=BINOMDIST of conclusion ((1-SD) * TRN, TRN,1-OD,TRUE)；If judge the soybean varieties to be measured without specificity, the correct probability >=BINOMDIST of conclusion (SD*TRN, TRN, OD, TRUE), wherein, TRN is the number for detecting successful test zone, and OD is the variant sites rate, SD Selected threshold value during to judge specific, BINOMDIST be excel 2010 in function, the correct probability tables of conclusion It is shown as when judging that the soybean varieties to be measured have specific, the variant sites rate is more than SD probability, when described in judgement When soybean varieties to be measured do not have specific, the variant sites rate is less than SD probability, the successful test zone of detection By being obtained after analyzing the sequencing fragment group.
7. according to the method for claim 1, it is characterised in that obtaining the method for the hybrid strain kind includes：The hybrid strain Kind is the kind being present in the database, and the potential hybrid strain genotype of the hybrid strain kind and the hybrid strain genotype Between have phase homogenic type the number of the test zone account for the hybrid strain kind there is the described of the potential hybrid strain genotype Total ratio >=60% of test zone；The hybrid strain genotype refers to the potential hybrid strain genotype of frequency >=0.02%；
Quantity >=2 of distinguishing base between the potential hybrid strain genotype and all genotype of the soybean varieties to be measured or There are insertion or the missing of discontinuous base in the distinguishing base.
CN201510150173.6A 2015-03-31 2015-03-31 A kind of method of the specificity for testing pure lines new soybean varieties, uniformity and stability Active CN104805187B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510150173.6A CN104805187B (en) 2015-03-31 2015-03-31 A kind of method of the specificity for testing pure lines new soybean varieties, uniformity and stability

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510150173.6A CN104805187B (en) 2015-03-31 2015-03-31 A kind of method of the specificity for testing pure lines new soybean varieties, uniformity and stability

Publications (2)

Publication Number Publication Date
CN104805187A CN104805187A (en) 2015-07-29
CN104805187B true CN104805187B (en) 2018-02-13

Family

ID=53690383

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510150173.6A Active CN104805187B (en) 2015-03-31 2015-03-31 A kind of method of the specificity for testing pure lines new soybean varieties, uniformity and stability

Country Status (1)

Country Link
CN (1) CN104805187B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111524022B (en) * 2020-03-12 2023-04-25 中国农业科学院蔬菜花卉研究所 Plant variety DUS testing method

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2148885A4 (en) * 2007-05-31 2010-12-15 Monsanto Technology Llc Soybean polymorphisms and methods of genotyping
BRPI0704999A2 (en) * 2007-12-21 2009-08-11 Tmg Tropical Melhoramento E Ge genotypes, alleles and molecular markers associated with Asian soybean rust, as well as methods, processes and uses thereof
CN102329869B (en) * 2011-10-05 2013-04-03 吉林省农业科学院 Method for identifying hybrid soybeans seeds by molecular markers
CN102329876B (en) * 2011-10-14 2014-04-02 深圳华大基因科技有限公司 Method for measuring nucleotide sequence of disease associated nucleic acid molecules in sample to be detected
WO2014048062A1 (en) * 2012-09-28 2014-04-03 未名兴旺系统作物设计前沿实验室（北京）有限公司 Snp loci set and usage method and application thereof
CN102943111B (en) * 2012-11-16 2014-07-16 北京爱普益生物科技有限公司 Application of high-pass DNA (Deoxyribonucleic Acid) sequencing method on determination of short tandem repeat gene locus in human genome and method
CN104328507B (en) * 2014-10-11 2016-03-30 中国水稻研究所 A kind of SNP chip, Preparation method and use for rice varieties qualification

Also Published As

Publication number Publication date
CN104805187A (en) 2015-07-29

Similar Documents

Publication Publication Date Title
CN104846076B (en) A method of specificity, consistency and the stability of measurement cross-bred rape new varieties
Williams et al. [51] Genetic analysis using random amplified polymorphic DNA markers
CN104830975A (en) Novel method for testing corn parent source authenticity and proportion
CN104805187B (en) A kind of method of the specificity for testing pure lines new soybean varieties, uniformity and stability
CN105603074A (en) Qualitative and quantitative microorganism detecting method
CN104805191B (en) A kind of method of the specificity for testing pure lines corn variety, uniformity and stability
CN104805190B (en) A kind of method of the specificity for determining hybrid maize variety, uniformity and stability
CN104805184B (en) A kind of method of the specificity for testing pure lines new rice variety, uniformity and stability
CN104805182B (en) A kind of method for the specificity, uniformity and stability for determining new hybrid rice varieties
CN104805189B (en) A kind of method of the specificity for determining hybrid plant new varieties, uniformity and stability
CN105624298A (en) Method for detecting genetically modified components of rape
CN105603075A (en) Qualitative and quantitative wheat microorganism detecting method
CN104846077B (en) A method of specificity, consistency and the stability of test pure lines new rape variety
CN104805186B (en) A kind of method for testing corn variety substance derived relation
CN104573409B (en) The multiple check method of the assignment of genes gene mapping
CN104805188B (en) A kind of method for testing soybean varieties substance derived relation
CN104805185B (en) A kind of method of test plants kind substance derived relation
CN105603076A (en) Qualitative and quantitative soil microorganism detecting method
CN104805193A (en) Method for testing substantive derivation relation of rice varieties
CN104805195A (en) Novel method for testing rice parental source authenticity and proportion of rice parental source
CN104805183A (en) Method for testing distinctness, uniformity and stability of pure-line plant new variety
CN113862383B (en) MNP (MNP) marking site of bacillus subtilis, primer composition and application of MNP marking site
CN117757979B (en) Primer group, kit and identification method for identifying soybean varieties
CN105586410B (en) Detection method of drug resistance genes of soil microorganisms
CN105586413A (en) Detection method of transgenic components in potatoes

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
EXSB Decision made by sipo to initiate substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant