CN104805188B - A kind of method for testing soybean varieties substance derived relation - Google Patents

Abstract

The invention discloses a kind of method for testing soybean varieties substance derived relation.This method includes：Obtain variant sites；Determine test zone；Sampling is extracted and obtains the DNA of sampling samples；Prepare primer；The DNA of two sampling samples is expanded respectively using the primer, respectively obtains the high-throughput sequencing library that two soybean varieties to be measured are used to build two soybean varieties to be measured in the amplified production of test zone；High-flux sequence is carried out respectively to two high-throughput sequencing libraries, respectively obtains the sequencing fragment group of two soybean varieties to be measured；Two sequencing fragment groups of analysis, obtain two soybean varieties genotype to be measured respectively；Compare two soybean varieties genotype to be measured, obtain the ratio of Differential genotype between soybean varieties to be measured；According to the ratio of Differential genotype between soybean varieties to be measured, the substantive derived relation of two soybean varieties to be measured is judged.This method can accurately, quickly and easily judge the substantive derived relation between soybean varieties to be measured.

Description

A kind of method for testing soybean varieties substance derived relation

Technical field

The present invention relates to biological technical field, more particularly to a kind of test new method of soybean Essentially derived variety.

Background technology

UPOV（International Union for the Protection of New Varieties of Plants：UPOV）Pact text in 1991 has done the regulation of principle to Essentially derived variety, The B kinds that i.e. Essentially derived variety refers to be obtained by A breed breedings do not have substantial change, and B kinds are referred to as the reality of A kinds Matter derives from kind, has substantive derived relation between A kinds and B kinds.Judge whether there is substantive group between two kinds The method of raw relation is to detect the difference ratio of genotype between the two kinds, when the difference ratio exceedes certain value, you can Think do not have substantive derived relation between two kinds, conversely, then it is assumed that there is substantive derived relation between two kinds.

The method of the substantive derivation sexual intercourse of detection at present is also seldom, and only methodical substantially flow is：Pass through SSR marker Or SNP marker, each test zone of soybean varieties to be measured is expanded, then pass through electrophoresis or each survey of generation sequencing detection acquisition The genotype in region is tried, according to genotype, judges the substantive derived relation between soybean varieties to be measured.

During the present invention is realized, inventor has found that prior art at least has problems with：

After substantive derived relation is needed substantial amounts of gene loci detects soybean varieties to be measured, could accurately it sentence Whether there is substantive derived relation between two kinds of breaking.In the substantive method for deriving from sexual intercourse of existing detection, detection Site causes the judgement conclusion of substantive derived relation inaccurate less.Meanwhile existing SSR marker and SNP marker are due to needing Individually expand and individually detect each test zone, therefore, test zone number is excessive, inevitably results in workload and greatly increases Add, therefore, existing method testing number of regions is all within 300, it is impossible to completely represents the whole of soybean varieties to be measured Genotype, so as to cause testing result inaccurate, the judgement conclusion of substantive derived relation is also inaccurate.

The content of the invention

In order to solve the problems, such as to detect substantive derived relation inaccuracy in the prior art, the embodiments of the invention provide one The method of kind test soybean varieties substance derived relation.The technical scheme is as follows：

The embodiments of the invention provide a kind of method for testing soybean varieties substance derived relation, methods described includes：

Obtain the variant sites between different soybean varieties；

Test zone is determined by the variant sites, the number of the test zone meets that following condition is： BINOMDIST (SD*TN, TN, 0.80*SD, TRUE) >=95%, wherein, TN is the number of the test zone, and SD is decision threshold Value；The condition implication that the number of the test zone meets is：When the number of the test zone is TN, the decision threshold is When the ratio of Differential genotype is 0.80*SD between SD and the soybean varieties to be measured, difference between the soybean varieties to be measured is judged Probability of the ratio of genotype less than the decision threshold SD, which ensures, is more than or equal to 95%；

Two soybean varieties to be measured are sampled respectively, extracts and obtains the sampling sample of two soybean varieties to be measured This DNA；

Prepare the primer for expanding the test zone；

The DNA of two sampling samples is expanded respectively using the primer, respectively obtain two it is described to be measured Soybean varieties the test zone amplified production, the amplified production be respectively used to build two soybean varieties to be measured High-throughput sequencing library；

The high-throughput sequencing library of soybean varieties to be measured described to two carries out high-flux sequence respectively, respectively obtains The sequencing fragment group of two soybean varieties to be measured；

The sequencing fragment group of two soybean varieties to be measured is analyzed, obtains two soybean varieties genotype to be measured respectively, The soybean varieties genotype to be measured is to become the combination of isobase in the test zone, and the soybean varieties genotype to be measured Frequency >=30%；

Compare two soybean varieties genotype to be measured, obtain the ratio of Differential genotype between soybean varieties to be measured；

According to the ratio of the Differential genotype, the substantive derived relation of two soybean varieties to be measured is judged.

Specifically, the test zone does not include the region that amplification produces hybrid strain genotype；

The hybrid strain genotype refers to frequency >=0.02%, and the hybrid strain genotype and the soybean varieties to be measured is all There are insertion or the missing of discontinuous base in quantity >=2 of distinguishing base between the genotype or the distinguishing base.

Specifically, the method that soybean varieties to be measured described to two are sampled respectively is：Soybean to be measured described to two Kind randomly select respectively more than 100 sample mixing after obtain two soybean varieties to be measured sampling samples.

Specifically, the method for judging the substantive derived relation of two soybean varieties to be measured is：

As the ratio ＜ SD of Differential genotype between the soybean varieties to be measured, two soybean varieties to be measured have real Matter derived relation；As ratio >=SD of Differential genotype between the soybean varieties to be measured, two soybean varieties to be measured Without substantive derived relation, wherein, SD is decision threshold.

Further, if judge that two soybean varieties to be measured have substantive derived relation, conclusion is correctly general Rate >=BINOMDIST (SD*TRN, TRN, OD, TRUE)；If judging, two soybean varieties to be measured derive from without substantive During relation, the correct probability >=BINOMDIST of conclusion ((1-SD) * TRN, TRN, 1-OD, TRUE)；Wherein, TRN is described in two The number of the shared test zone of soybean varieties to be measured, the ratio of OD Differential genotypes between the soybean varieties to be measured, BINOMDIST is the functions of excel 2010, and BINOMDIST (SD*TRN, TRN, OD, TRUE) implication is：When the shared survey When the number for trying region is TRN, the ratio OD of Differential genotype is less than the decision threshold SD's between the soybean varieties to be measured Probability；BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) implication is：When the number of the shared test zone is TRN When, the ratio OD of Differential genotype is more than the probability of the decision threshold SD between the soybean varieties to be measured.

Specifically, the method for determining the test zone by the variant sites is：

Pass through discriminationThe value of discrimination is calculated, wherein, a is to be detected in variation window area Kind sum, bi is the kind number of i-th kind of genotype in the variation window area, and bi>1, k is to include more than 1 product The number of the genotype of kind, the variation window area is centered on each single nucleotide variations site, to the monokaryon glycosides The both sides of sour variant sites respectively extend 1/2 window as detection of sequence length to be measured；

The test zone is the 8000 variation windows or nuclear genome that discrimination is maximum on cytoplasmic skeleton The upper discrimination is maximum and equally distributed 100 variations window.

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 More site amplifications and high-flux sequence, ensure the large sample sampling of the test zone of soybean varieties to be measured, it is accurate to be successfully realized Judge the target of substantive derived relation between soybean varieties to be measured, and test simple, quick.

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 measure soybean varieties " the substantive derived relation between northern 93-406 " and " cultivating mirror beans 26 "

Soybean varieties to be measured provided in an embodiment of the present invention be soybean varieties " northern 93-406 " and " cultivate mirror beans 26 ", the two It is disclosure, known kind.

First, the variant sites between different soybean varieties are obtained.

Variant sites between different soybean varieties can be obtained from the documents and materials announced, but this method is obtained Results contrast is fragmentary, in the present embodiment, by by the genome sequence of different soybean with reference to soybean varieties genome sequence Row are compared, and the variant sites between substantial amounts of different soybean varieties are obtained, wherein can be with reference to soybean varieties " WILLIAMS_82 " soybean, " WILLIAMS_82 " soybean could alternatively be other known and refer to soybean varieties.

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 31 wild and cultivated soybean genomesidentifies patterns of genetic diversity and selection.Nat Genet 2010,42:1053–1059.The genome sequence of 31 soybean varieties is published in NCBI Short Read Archive(http://www.ncbi.nlm.nih.gov/sra), reception number is SRA020131；The Two kinds are to have carried out high pass to " rich No. 8 of north " and " cultivating mirror beans 26 " by the method provided in Lam etc. the above-mentioned article delivered Measure 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, other variation types are as repeated number variation, due to a low credibility, without identification.Compared using Frederick Sanger Software（Version number is 0.4）By the high-flux sequence sequence alignment of the genome of this 33 soybean varieties to " Williams_82 " Soya cells core reference gene group（Version：Release v1.01, download address：http://genome.jgi-psf.org/） In cytoplasm reference gene group, the cytoplasm reference gene group includes mitochondria reference gene group and chloroplaset reference gene Group, it is in NCBI（National Center for Biotechnology Information, US National biotechnology letter Breath center）On reception number be respectively JX463295.1 and NC_007942.1.During contrast, Insert Fragment length is set to 500bp, Other specification is set as default value.The Ssaha Pileup software kits of use（Version number is 0.5）Identify the SNP positions of each kind Point.The SNP site is defined as base-pair, the insertion of single base or the missing of single base of difference determination.The alkali that the difference determines For base to referring to not include the uncertain base-pair of difference, it is base between some degeneracy bases that the uncertain base-pair of difference, which refers to, It is right, as R represents A or G, therefore, difference is there may be between A and R, it is also possible to which, in the absence of difference, therefore, difference is failed to understand between A and R Really, SNP it is not mutually.Therefore, the SNP site in the embodiment of the present invention is not include the uncertain base-pair of above-mentioned difference.By with The definition of upper SNP site, the embodiment of the present invention obtain 6350046 SNP sites altogether between all 33 soybean varieties, wherein 31937 SNP sites are located on cytoplasmic skeleton, and remaining SNP site is located on nuclear genome.Base referred to hereafter Because type is to refer to the combination of multiple SNP sites in test zone, karyogene type refers to genotype and is located on nuclear genome, matter base Because type refers to that genotype is located on cytoplasmic skeleton.For example, the 1st test zone is located on nuclear genome in table 1, it is Karyogene type, the test zone share 3 SNP sites, and the genotype of the test zone is the combination of this 3 SNP sites.

2nd, test zone is determined by variant sites, specific method is as follows：

Test zone is that discrimination is big on the big region of discrimination or nuclear genome and SNP positions on cytoplasmic skeleton The equally distributed region of point, wherein, discriminationWherein, a is the product being detected in variation window area Kind of sum, bi are the kind number of i-th kind of genotype in variation window area, and bi>1, k is the gene comprising more than a kind The number of type, variation window area are with each single nucleotide variations site（SNP site）Centered on, to single nucleotide variations position The both sides of point respectively extend 1/2 window as detection for surveying sequence length；Test zone is that discrimination is big on cytoplasmic skeleton Region or nuclear genome on discrimination is big and equally distributed region.The Computing Principle of discrimination is as follows：All kinds Between number of combinations beWherein, the combination between the different cultivars in same gene type is undistinguishable, and its number isSo, the ratio for the breed combination that can not be distinguished isThe ratio for the breed combination that can be distinguished is distinguished DegreeAs can be seen here, discrimination is bigger, can more distinguish different cultivars, the big variation window region of discrimination Test of the domain to substantive derived relation is more effective.If the variation window area skewness on nuclear genome, can lead Cause some regions adjacent, so as to linkage inheritance, information is easily overlapping, therefore, the comprehensive of test zone is selected on nuclear genome Closing principle is：Discrimination is big and SNP site is uniformly distributed.Cytoplasmic skeleton without linkage inheritance problem, so, cytogene The big region of selective discrimination degree is only needed in group.

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 more variation window can just distinguish.After the same method, calculate and obtain all The discrimination of 6350046 variation windows simultaneously therefrom chooses 8000 windows that make a variation for being located at that discrimination is maximum in nuclear genome Mouth and 100 maximum variation windows of discrimination in cytoplasmic skeleton.Check one by one positioned at nuclear genome In 8000 variation windows, each distance to make a variation between window and next variation window, if distance is more than 300K（1K=1000 Individual base）, then abandon reexamining after the less variation window of wherein discrimination, up to the adjacent distance for looking into variation window is big Untill 300K.Selection 300K criterion distance is because soybean gene group size is about 975M（Ten thousand bases of 1M=100）, press Final to be selected in 2000 test zone meters for being located at nuclear genome, distance is about 500K between average test zone, but by In 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 with obtaining positioned at discrimination in cytoplasmic skeleton most 100 big variation windows together totally 5087 variation windows as the test zone being selected in.Wherein, selective discrimination degree is maximum 100 variation windows, are empirical value, the quantity can modify as the case may be.

3rd, two soybean varieties to be measured are sampled respectively, extract and obtain the sampling sample of two soybean varieties to be measured This DNA, the preparation method of sampling samples are：Two soybean varieties to be measured are randomly selected with the sample of more than 100 respectively to mix After conjunction, the sampling samples of two soybean varieties to be measured are obtained.

In the present embodiment, have chosen soybean varieties to be measured, " northern 93-406 " 30000 germinations, randomly select It is placed in after 26000 bud mixing being substantially equal to the magnitudes in mortar, powder is fully ground into after adding liquid nitrogen into mortar.Using north The plant genome DNA extracts kit that the article No. of capital Tiangeng biochemical technology Co., Ltd production is DP305 is extracted and treated Surveying soybean varieties, " DNA of northern 93-406 " mixing samples, DNA extraction method are carried out by the operation manual of the kit.Utilize U.S. The production of Invitrigen companies of statedsDNA HS Assay Kit（Article No. is Q32852）And its specification is to obtaining The DNA obtained is quantified, and " northern 93-406 " DNA is diluted to 10.00ng/ μ l to the soybean varieties to be measured after quantifying.

After the same method, soybean varieties to be measured " cultivating mirror beans 26 " are sampled and extract DNA, equally will be quantitative The DNA of soybean varieties to be measured " cultivating mirror beans 26 " afterwards is diluted to 10.00ng/ μ l.

4th, the primer in amplification assay region is prepared, it is specific as follows：

Test zone uses multiplex PCR（Polymerase Chain Reaction, PCR）Technology is carried out Detection, multiple PCR technique refer to add multiple PCR primers, while multiple positions in amplification gene group in same PCR reacts Point.The key of the technology is to design and synthesize multiple PCR primer, and the present embodiment is provided using LifeTechnology companies of the U.S. Multiple PCR technique, it can set up to 12000 heavy PCR primers.

The number of test zone meets following condition：BINOMDIST (SD*TN, TN, 0.80*SD, TRUE) >=95%, its In, TN is the number of test zone, and SD is decision threshold；(SD*TN, TN, 0.80*SD, TRUE implication is BINOMDIST：When When the number of test zone is that the ratio of Differential genotype between TN, soybean varieties to be measured is 0.80*SD and decision threshold is SD, treat Probability of the ratio less than decision threshold SD of Differential genotype, which ensures, between survey soybean varieties is more than or equal to 95%.The implication of the condition It is：When the ratio of Differential genotype between soybean varieties to be measured is the 80% of judgment threshold, sentenced by what the number of test zone determined The soybean varieties to be measured that break have accuracy >=95% of substantive derived relation.The judgment threshold of substantive derived relation is basis The Breeding Situations of various countries, mark mode, require Stringency and artificially determine.In the present embodiment, SD is defined as 2.5%.By The number TN that step increases test zone is had found, as TN >=2200, above-mentioned formula is set up, and therefore, the number of test zone should ≥2200.For existing SSR and SNP tests, 200 test zones have calculated that it is enough, if difference between soybean varieties to be measured The ratio of genotype be judgment threshold 80%, its accuracy only >=BINOMDIST (2.5%*200,200,0.80*2.5%, TRUE)=79%, therefore, the method that this implementation provides, can obtain more accurate conclusion.

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.Wherein, 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 test zones, design and demonstrate 2488 pairs of multiple PCR primers, use In corresponding 2488 test zones of amplification.The method for verifying multiple PCR primer is same by method provided by the invention, extraction Leaves genomic DNA on strain soybean, and the genomic DNA of acquisition is expanded, built using the multiple PCR primer of design Storehouse, high-flux sequence simultaneously analyze sequencing fragment group, remove the corresponding primer of following test zone：The sequencing fragment of the test zone Number is less than 1000 or hybrid strain genotype be present, and the primer remained is the multiple PCR primer being proved to be successful.So test Region does not include the region that amplification produces hybrid strain genotype, and hybrid strain genotype refers to frequency >=0.02%, and hybrid strain genotype with it is to be measured There is the insertion of discontinuous base in quantity >=2 of distinguishing base between all genotype of soybean varieties or distinguishing base or lack Lose.Because genomic DNA source is in same strain soybean leaves, it is impossible to hybrid strain kind be present, therefore, hybrid strain genotype is by surveying PCR caused by trying the special construction in region or sequencing Preference mistake, remove these test zones and avoid such system mistake. Regulation test zone is that another purpose for not including the test zone that amplification produces hybrid strain genotype is：The test remained Region can also make the calculating of hybrid strain rate in addition to the test as the substantive derived relation between soybean varieties to be measured, realize The multiple use of same set of test primer.The multiple PCR primer being proved to be successful also mixed by the said firm after with the shape of liquid Formula is supplied to client to use.2488 test zones of above-mentioned successful design multiple PCR primer are eventually for be measured big The test zone of beans kind detection, wherein, 34 test zones are located on cytoplasmic skeleton, remaining 2472 test zones On nuclear genome.Existing Essentially derived variety judgement does not all use the test site on cytoplasmic skeleton, And cytoplasmic difference, it can equally produce different varietal character performances, it should for sentencing for Essentially derived variety relation It is fixed.

5th, the DNA of two sampling samples is expanded respectively using primer, respectively obtains two soybean varieties to be measured and exist The amplified production of test zone, amplified production are respectively used to build the high-throughput sequencing library of two soybean varieties to be measured, specifically Method is as follows：

Utilize library construction Kit 2.0（Produced by LifeTechnology companies of the U.S., article No. 4475345）It is multiple Behind PCR amplification assays region, high-throughput sequencing library is built using amplified production.The kit includes following reagent：5×Ion AmpliSeq^TMHiFi Mix, FuPa reagents, transferring reagent, sequence measuring joints solution and DNA ligase.The method of library construction is pressed The operation manual of the kit《Ion AmpliSeq^TMLibrary Preparation》（Publication number：MAN0006735, version： A.0）Carry out.It is as follows by 2488 test zones of multiplexed PCR amplification, the amplification system of multiplex PCR：5×Ion AmpliSeq^TM μ l of HiFi Mix 4, μ l of test zone primer mixed liquor 4, soybean varieties to be measured " northern 93-406 " the DNA 10ng and nothing prepared 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 circulations；10 DEG C insulation.After primer unnecessary in multiplexed PCR amplification product is digested using FuPa reagents, then carry out phosphorylation, specific method For：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 phosphorylation Amplified production solution.By the upper sequence measuring joints of amplified production connection of phosphorylation, specific method is：Conversion is added into mixture a μ L of 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 Minute；72 DEG C, 10 minutes；10 DEG C of preservations, obtain mixed liquor b.Dissolved after purifying mixed liquor b using the ethanol precipitation methods of standard In 10 μ L without in enzyme water.Utilize the production of Invitrigen companies of the U.S.dsDNA HS Assay Kit（Article No. is Q32852）And be measured according to its specification, and after obtaining mixed liquor b mass concentration, mixed liquor b will be diluted to after purification 15ng/ml, obtain the high-throughput sequencing library of concentration about 100pM test zone.

After the same method, soybean varieties to be measured " cultivating mirror beans 26 " are carried out with the structure of high-throughput sequencing library, together Sample obtains the high-throughput sequencing library of concentration about 100pM test zone.

6th, high-flux sequence is carried out respectively to the high-throughput sequencing library of two soybean varieties to be measured, respectively obtains two The sequencing fragment group of soybean varieties to be measured, specific method are as follows：

Determine high-flux sequence depth：Depth >=5000 times of high-flux sequence, the i.e. average segments in coverage test area >=5000 fragments, 5000 times are an empirical value, can be adjusted according to actual conditions.Why provide this value, be because 5000 times of sequencing amount cost is not high but is enough the testing gene type frequency of accurate calculating 30%, therefore, it is specified that 5000 times of conducts The depth of high-flux sequence.

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）EPCR before being sequenced（Emulsion PCR, emulsion polymerization enzyme chain reaction）Amplification, operating method are 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）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.

A large amount sequencing result is pre-processed

High-flux sequence fragment is compared and arrives all 2488 test zones, removes and compares unsuccessful and genotype detection not After full sequencing fragment, remaining all sequencing fragments are referred to as fragment group is sequenced.Genotype detection it is incomplete sequencing fragment be Refer to the sequencing fragment that all SNP sites in table 1 shown in " positions of the SNP in reference gene group " could not be detected, gene The reason for type detection is not complete is that sequencing fragment is too short, and it is that sequencing fragment is mostly non-specific amplification product to compare unsuccessful reason.

7th, the sequencing fragment group of two soybean varieties to be measured is analyzed, two soybean varieties genotype to be measured is obtained respectively, treats It is to become the combination of isobase in test zone to survey soybean varieties genotype, and frequency >=30% of soybean varieties genotype to be measured, tool Body method is as follows：

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.Soybean varieties genotype to be measured is to become the group of isobase in test zone Close, and frequency >=30% of soybean varieties genotype to be measured.In general, in the sample extracted, the amount of hybrid is not higher than 10%, sequencing mistake is no more than 1%, and the two is total no more than 11%, therefore, for homozygous site, soybean varieties gene to be measured Type only has one kind, and its frequency should be more than 89%, and for heterozygous sites, soybean varieties genotype to be measured has 2 kinds, and it compares Example should be more than 45.5%, therefore, it is specified that frequency >=30% of row survey variety and genetype, can be excluded wrong and to be measured big because being sequenced Hybrid strain is contaminated with beans kind and to the interference of soybean varieties genotype to be measured.

For example, in fragment group is sequenced, the sequencing fragment sum in the 2nd sequencing region is 9987 articles, have CACT, ATGA, CACC, CGCT ... totally 25 kinds of genotype, represent these genotype sequencing segments distinguish 9778,102,2,1 Bar ..., the frequencies of these genotype for 9778/9987=97.91%, 102/9987=1.02%, 2/9987=0.02%, 1/9987= 0.01%…….By the definition of soybean varieties genotype to be measured, CACT is soybean varieties to be measured " the northern 93- of the 2nd test zone 406 " genotype, other genotype are genotype caused by sequencing mistake or hybrid strain.By identical method, judge and obtain whole Soybean varieties to be measured " northern 93-406 " the genotype of 2406 successful test zones of detection.

" northern 93-406 " identicals method, equally obtain extraction soybean varieties to be measured by with soybean varieties to be measured and " cultivate mirror 2406 successful test zones of detection of beans 26 ", and soybean varieties to be measured " cultivating mirror beans 26 " detect successfully all Test zone genotype, partial results are shown in Table 1.The present embodiment is not listed completely all to be measured big as space is limited, Beans kind only lists certain embodiments in all test zone genotype.Equally limited based on length, also have portion in the present embodiment Place is divided also only to list part related example, remaining unlisted data can be according to the method completion of the present embodiment.

Table 1 is soybean varieties genotype to be measured and its relevant information

8th, soybean varieties genotype more to be measured, the ratio of Differential genotype between soybean varieties to be measured is obtained, method is such as Under：

If in the test, the genotype of all soybean varieties to be measured is without missing, the test zone is referred to as to be measured big The shared test zone of beans kind.In shared test zone, if the genotype between soybean varieties to be measured is incomplete same, claim Genotype Differential genotype between soybean varieties to be measured, if for example, soybean varieties A to be measured genotype be AA, AA, AA, AA, AA/CC, AA/CC, genotype corresponding to soybean varieties B to be measured are AA, CC, AA/CC, CC/TT, AA/CC, CC/TT, then at this Differential genotype in 6 test zones between soybean varieties to be measured judges as follows：It is not, is, being, being, is not and is.Above base Because the "/" in type represents the test zone as genetic heterozygosis site, therefore detect "/" both front and back genotype.It is to be measured big The ratio of beans Differences genotype=number for possessing the test zone of Differential genotype/shares the number of test zone.

In the present embodiment, soybean varieties " cultivating mirror beans 26 " to be measured and soybean varieties to be measured " northern 93-406 " shared survey It is 2406 to try region, and in the 1st shared test zone, their genotype is respectively GTT and GTT, and they are identical, Therefore, the not Differential genotype between soybean varieties to be measured.According to said method, all 2406 shared test zones are judged one by one In, if having differences genotype, as a result for：In all shared test zones, the test zone number of genotype is had differences For 78, so, ratio OD=78/2406=3.24% of Differential genotype between soybean varieties to be measured.

According to the ratio and decision threshold of Differential genotype between soybean varieties to be measured, the essence between soybean varieties to be measured is judged Property derived relation, the method for judging the substantive derived relation between two soybean varieties to be measured are：When poor between soybean varieties to be measured During the ratio ＜ SD of allogene type, there is substantive derived relation between two soybean varieties to be measured；When poor between soybean varieties to be measured During ratio >=SD of allogene type, do not have substantive derived relation between two soybean varieties to be measured, wherein, SD is decision threshold Value.

In the present embodiment, ratio=3.24% of Differential genotype between soybean varieties to be measured>SD=2.5%, therefore judge to be measured Soybean varieties " cultivating mirror beans 26 " and soybean varieties to be measured " do not have substantive derived relation between northern 93-406 ".

If there is substantive derived relation between judging soybean varieties to be measured, the correct probability >=BINOMDIST of conclusion (SD*TRN,TRN,OD,TRUE)；If do not have substantive derived relation between judging two soybean varieties to be measured, conclusion is correct Probability >=BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE)；Wherein, TRN is the shared of two soybean varieties to be measured The number of test zone, SD are decision threshold, the ratio of OD Differential genotypes between soybean varieties to be measured, and BINOMDIST is The functions of excel 2010, BINOMDIST (SD*TRN, TRN, OD, TRUE) implication are：When the number of shared test zone is During TRN, the ratio OD of Differential genotype is less than decision threshold SD probability between soybean varieties to be measured；BINOMDIST((1-SD)* TRN, TRN, 1-OD, TRUE) implication is：When the number of shared test zone is TRN, Differential genotype between soybean varieties to be measured Ratio OD be more than decision threshold SD probability.

In the present embodiment, judge there is substantive derived relation between two soybean varieties to be measured and share the number of test zone Mesh is 2406, therefore, the correct probability >=BINOMDIST of conclusion ((1-SD) * TRN, TRN, 1-OD, TRUE)=BINOMDIST ((1-2.5%)*2406,2406,1-3.24%,TRUE)=98.08%.Therefore, in this implementation, judge between two soybean varieties to be measured Conclusion without substantive derived relation is accurate.

Result verification

At present, the standard method of substantive derived relation identification between the kind that the whole world also neither one is generally acknowledged, but UPOV Texts in 1991 in have such description：" Essentially derived variety can be thin by saltant type that is such as natural or inducing, body The selection of born of the same parents' clonal vaviation type, obtained from the selection of the variation strain in original variety plant, and backcrossing or genetic engineering conversion ".In the present embodiment, soybean varieties " cultivating mirror beans 26 " to be measured and soybean varieties to be measured " have no above-mentioned seed selection between northern 93-406 " Relation, so, by the description of UPOV texts, should not have substantive derived relation between the two soybean varieties to be measured, show, The conclusion of the present embodiment is correct.

The embodiment of the present invention is expanded by high-flux sequence and more sites, realizes test zone in soybean varieties to be measured Large sample is sampled, and ensure that the accuracy of substantive derived relation detection.Meanwhile utilize more site amplification techniques, multiple tests The detection workload in region is substantially equivalent to the workload of a test zone of traditional SSR or SNP detection, therefore, this reality It is not only accurate to apply example, and method is simple, quick.If for example, equally detecting 2406 test zones, traditional SSR or SNP are utilized Detection method is, it is necessary to carry out 2406 PCR amplifications and the detection of the amplified production of 2406 times（SSR is detected by electrophoresis method, SNP Detected by the method for generation test）, its workload, speed and testing cost are all can not be received.In addition, this hair Bright to have obtained the sequence of each base in test zone, resolution ratio has reached ultimate attainment, and information content is also maximum, is other Detection method is all incomparable, for example, SSR obtain be fragment length information, it is understood that there may be the problem of be：Fragment length phase Difference is too small, may can't detect, even the fragment of equal length, its internal base composition may also be different, but SSR is same Sample can't detect.SNP detection can only detect a pair of SNP sites, and in the present embodiment, test zone can be detected and owned SNP, it has also been found that the variation in test zone outside SNP, such as repeat number, missing and insertion, these are all single SNP inspections Survey what can not be found.In addition, in addition to using generation sequencing detection SNP, also have and detect SNP, such case by the way of hybridization Under, what is obtained is hybridization signal, and noise is big and can not find unknown variation, and a probe is only capable of detecting a pair of SNP, resolution ratio With information content also much smaller than the present invention.

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 (5)

1. A kind of 1. method for testing soybean varieties substance derived relation, it is characterised in that methods described includes：

   Obtain the variant sites between different soybean varieties；

   Test zone is determined by the variant sites, the number of the test zone meets that following condition is：BINOMDIST (SD*TN, TN, 0.80*SD, TRUE) >=95%, wherein, TN is the number of the test zone, and SD is decision threshold；The survey Examination region number meet condition implication be：When the number of the test zone is TN, the decision threshold is SD and described When the ratio of Differential genotype is 0.80*SD between soybean varieties to be measured, Differential genotype between the soybean varieties to be measured is judged Probability of the ratio less than the decision threshold SD, which ensures, is more than or equal to 95%, passes throughCalculate discrimination Value, wherein, a is the kind sum being detected in variation window area, and bi is i-th kind of genotype in the variation window area Kind number, and bi>1, k is the number of the genotype comprising more than a kind, and the variation window area is with each monokaryon Centered on thuja acid variant sites, 1/2 conduct for respectively extending sequence length to be measured to the both sides in the single nucleotide variations site is examined The window of survey；The test zone is the 8000 variation windows or nuclear genome that discrimination is maximum on cytoplasmic skeleton The upper discrimination is maximum and equally distributed 100 variations window, wherein, the genotype is multiple in the test zone The combination in single nucleotide variations site；

   Two soybean varieties to be measured are sampled respectively, extracts and obtains the sampling samples of two soybean varieties to be measured DNA；

   Prepare the primer for expanding the test zone；

   The DNA of two sampling samples is expanded respectively using the primer, respectively obtains two soybean to be measured Kind the test zone amplified production, the amplified production be respectively used to build two soybean varieties to be measured height Flux sequencing library；

   The high-throughput sequencing library of soybean varieties to be measured described to two carries out high-flux sequence respectively, respectively obtains two The sequencing fragment group of the soybean varieties to be measured；

   The sequencing fragment group of two soybean varieties to be measured is analyzed, obtains two soybean varieties genotype to be measured respectively, it is described Soybean varieties genotype to be measured is to become the combination of isobase in the test zone, and the frequency of the soybean varieties genotype to be measured Rate >=30%；

   Compare two soybean varieties genotype to be measured, obtain the ratio of Differential genotype between soybean varieties to be measured；

   According to the ratio of the Differential genotype, the substantive derived relation of two soybean varieties to be measured is judged.
2. 2. according to the method for claim 1, it is characterised in that the test zone does not include amplification and produces hybrid strain genotype Region；

   The hybrid strain genotype refers to frequency >=0.02%, and the hybrid strain genotype and the soybean varieties to be measured is all described There are insertion or the missing of discontinuous base in quantity >=2 of distinguishing base between genotype or the distinguishing base.
3. 3. according to the method for claim 1, it is characterised in that soybean varieties to be measured described to two are sampled respectively Method is：Soybean varieties to be measured described to two randomly select respectively more than 100 sample mixing after obtain two described in treat Survey the sampling samples of soybean varieties.
4. 4. according to the method for claim 1, it is characterised in that judge the substantive derivation of two soybean varieties to be measured The method of relation is：

   As the ratio ＜ SD of Differential genotype between the soybean varieties to be measured, two soybean varieties to be measured have substance Derived relation；As ratio >=SD of Differential genotype between the soybean varieties to be measured, two soybean varieties to be measured do not have There is substantive derived relation, wherein, SD is decision threshold.
5. 5. according to the method for claim 4, it is characterised in that if judging, two soybean varieties to be measured have substance During derived relation, the correct probability >=BINOMDIST of conclusion (SD*TRN, TRN, OD, T RUE)；If judge two it is described to be measured When soybean varieties do not have substantive derived relation, the correct probability >=BINOMDIST of conclusion ((1-SD) * TRN, TRN, 1-OD, TRUE)；Wherein, TRN is the number of the shared test zone of two soybean varieties to be measured, and OD is the soybean varieties to be measured Between Differential genotype ratio, BINOMDIST is the functions of excel 2010, BINOMDIST's (SD*TRN, TRN, OD, TRUE) Implication is：When the number of the shared test zone is TRN, the ratio OD of Differential genotype is small between the soybean varieties to be measured In the probability of the decision threshold SD；BINOMDIST ((1-SD) * TRN, TRN, 1-OD, T RUE) implication is：When described shared When the number of test zone is TRN, the ratio OD of Differential genotype is more than the decision threshold SD between the soybean varieties to be measured Probability.