CN104805185B

CN104805185B - A kind of method of test plants kind substance derived relation

Info

Publication number: CN104805185B
Application number: CN201510148657.7A
Authority: CN
Inventors: 彭海; 张静; 陈红; 张继
Original assignee: Agriculture Ministry Technology Development Center; Jianghan University
Current assignee: Agriculture Ministry Technology Development Center; Jianghan University
Priority date: 2015-03-31
Filing date: 2015-03-31
Publication date: 2018-02-13
Anticipated expiration: 2035-03-31
Also published as: CN104805185A

Abstract

The invention discloses a kind of method of test plants kind 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 kinds to be measured are used to build two kinds 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 kinds to be measured；Two sequencing fragment groups of analysis, obtain two variety and genetypes to be measured respectively；Compare two variety and genetypes to be measured, obtain the ratio of Differences genotype to be measured；According to the ratio of Differences genotype to be measured, the substantive derived relation of two kinds to be measured is judged.This method can accurately, quickly and easily judge interracial substantive derived relation to be measured.

Description

A kind of method of test plants kind substance derived relation

Technical field

The present invention relates to biological technical field, more particularly to a kind of method of test plants kind substance derived relation.

Background technology

UPOV(International Union for the Protection of New Varieties of Plants：UPOV) pact text in 1991 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 plant varieties, conversely, then it is assumed that there is substantive derive to close between two kinds System.

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 kind to be measured is expanded, then pass through electrophoresis or each test section of generation sequencing detection acquisition The genotype in domain, according to genotype, judge interracial substantive derived relation to be measured.

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

, could accurate judgement two after substantive derived relation is needed substantial amounts of gene loci detects kind to be measured Whether there is substantive derived relation between individual kind.In the substantive method for deriving from sexual intercourse of existing detection, detection site Cause the judgement conclusion of substantive derived relation inaccurate less.Meanwhile existing SSR marker and SNP marker are independent due to needing Expand and individually detect each test zone, therefore, test zone number is excessive, inevitably results in workload and is significantly greatly increased, because This, existing method testing number of regions is all within 300, it is impossible to the full gene type of kind to be measured is completely represented, from And causing testing result inaccurate, the judgement conclusion of substantive derived relation is also inaccurate.

The content of the invention

In order to solve the problems of the prior art, the embodiments of the invention provide a kind of substantive derivation of test plants kind The method of relation.The technical scheme is as follows：

The embodiments of the invention provide a kind of method of test plants kind substance derived relation, methods described includes：

Obtain the variant sites in kind between different cultivars belonging to two kinds to be measured；

Test zone is determined by the variant sites, passes through discriminationThe value of discrimination is calculated, 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 glycosides Centered on sour variant sites, 1/2 conduct for respectively extending sequence length to be measured to the both sides in the single nucleotide variations site detects Window, the test zone is that the discrimination is big on the big region of discrimination or nuclear genome on cytoplasmic skeleton And equally distributed region, wherein, the genotype is the combination in multiple single nucleotide variations sites in the test zone；

Kind to be measured described to two is sampled respectively, is extracted and is obtained the sampling samples of two kinds to be measured DNA；

Prepare the primer for expanding the test zone；

The DNA of the sampling samples is expanded respectively using the primer, respectively obtains two kinds to be measured In the amplified production of the test zone, the amplified production is respectively used to build the high-flux sequence of two kinds to be measured Library；

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

The sequencing fragment group of two kinds to be measured is analyzed, obtains two variety and genetypes to be measured respectively, it is described to be measured Variety and genetype is the combination for becoming isobase in the test zone, and frequency >=30% of the variety and genetype to be measured；

Compare two variety and genetypes to be measured, obtain the ratio of Differences genotype to be measured；

According to the ratio of the Differences genotype to be measured, judge that substantive derive from of two kinds to be measured is closed System.

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 all genes of the hybrid strain genotype and the kind to be measured There are insertion or the missing of discontinuous base in quantity >=2 of distinguishing base between type or the distinguishing base.

Specifically, the number of the test zone meets following condition：BINOMDIST(SD*TN,TN,0.80*SD, TRUE) >=95%, wherein, TN is the number of the test zone, and SD is decision threshold；What the number of the test zone met Condition implication is：When the number of the test zone is TN, the decision threshold is SD and the Differences gene to be measured When the ratio of type is 0.80*SD, judge that the ratio of the Differences genotype to be measured is general less than the decision threshold SD Rate guarantee is more than or equal to 95%.

Specifically, the method that kind to be measured described to two is sampled respectively is：Kind difference to be measured described to two Randomly select more than 100 sample mixing after obtain two kinds to be measured sampling samples.

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

As the ratio ＜ SD of the Differences genotype to be measured, two kinds to be measured have substantive derive from Relation；As ratio >=SD of the Differences genotype to be measured, two kinds to be measured derive from without substantive Relation, wherein, SD is decision threshold.

Further, if judge that two kinds to be measured have substantive derived relation, the correct probability of conclusion >= BINOMDIST(SD*TRN,TRN,OD,TRUE)；If judge that two kinds to be measured do not have substantive derived relation, knot By correct probability >=BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE)；Wherein, TRN is two kinds to be measured Shared test zone number, OD be the Differences genotype to be measured ratio, BINOMDIST is excel 2010 Function, BINOMDIST (SD*TRN, TRN, OD, TRUE) implication are：When the number of the shared test zone is TRN, institute The ratio OD for stating Differences genotype to be measured is less than the probability of the decision threshold SD；BINOMDIST((1-SD)*TRN, TRN, 1-OD, TRUE) implication is：When the number of the shared test zone is TRN, the Differences genotype to be measured Ratio OD be more than the decision threshold SD probability.

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 kind to be measured, be successfully realized accurate judgement The target of product to be tested inter-species substance derived relation, 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 one determines the substantive derived relation between rice varieties " R7723 " and " R8377 "

Kind to be measured provided in an embodiment of the present invention is rice varieties " R7723 " and " R8377 ", both at open, public The kind known.The source of rice varieties " R7723 " is rice varieties " R8377 " with being wheel with " R8377 " after " IRBB23 " hybridization Parent is returned, objective trait is wanted based on bacterial blight-resisting character, cultivates and forms by the method for back cross breeding.

First, the variant sites belonging to kind to be measured in kind between different cultivars are obtained.

The kind of kind to be measured is rice, therefore, obtains the variant sites belonging to kind to be measured in kind between different cultivars and then should To obtain the variant sites between different rice varieties, the variant sites between different rice varieties can be from the documents and materials announced Middle acquisition, but the results contrast that this method is obtained is fragmentary, in the present embodiment, by by the genome sequence of different rice with It is compared with reference to the genome sequence of rice varieties, the variant sites between substantial amounts of different rice varieties is obtained, wherein joining It can be " Japanese eyeball " rice to examine rice varieties, and " Japanese eyeball " rice could alternatively be other known and refer to rice varieties.

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

The genome sequence of the different rice varieties of the present embodiment shows three kinds of sources, and the first is Han Bin to 1082 rice The high-flux sequence sequence of the genome of kind, pertinent literature information are as follows：Huang XH et al.A map of rice genome variation reveals the origin of cultivated rice.Nature.2012；7:497–503. The genome sequence of 1082 rice varieties is published in European NucleotideArchive (http:// Www.ebi.ac.uk/ena/), reception number is ERP001143, ERP000729 and ERP000106；Second be Xu Xun to 50 The high-flux sequence sequence of the genome of rice varieties, pertinent literature information are as follows：Xun X et al.Resequencing 50accessions of cultivated and wild rice yields markers for identifying agronomically important genes.Nat Biotechnol.2011,30(1):105-11,50 rice varieties Genome sequence be published in NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/sra), connect Collect the digits for SRA023116；The third be by the method provided in the above-mentioned articles delivered of Han Bin to " R8377 ", " Jin Ke 1A ", " " D excellent 527 " has carried out high-flux sequence to Jin Ke 1A/R7723 " with hybrid strain kind for " IRBB23 ", cenospecies.The present embodiment obtains altogether The high-flux sequence sequence of the genome of 1137 rice varieties.

Further, variant sites are obtained using the genome sequence of different cultivars.

Specifically, because the sequencing depth of this 1137 rice varieties is not high, it is only capable of identifying single nucleotide variations (SNP) Site, other variation types are as repeated number variation, due to a low credibility, without identification.Utilize Frederick Sanger ratios To software (version number 0.4) by the high-flux sequence sequence alignment of the genome of this 1137 rice varieties to " Japanese eyeball " water (version is IRGSP 4.0 to rice nucleus reference gene group, download address：http://www.ncbi.nlm.nih.gov) and it is thin In kytoplasm reference gene group, the cytoplasm reference gene group includes mitochondria reference gene group and chloroplaset reference gene group, its In NCBI (National Center for Biotechnology Information, in US National Biotechnology Information The heart) on reception number be respectively NC_011033 and NC_001320.During contrast, Insert Fragment length is set to 500bp, other specification It is set as default value.The Ssaha Pileup software kits (version number 0.5) used identify the SNP site of each kind.The SNP Site is defined as base-pair, the insertion of single base or the missing of single base of difference determination.The base-pair that the difference determines refers to Do not include the uncertain base-pair of difference, it is base-pair between some degeneracy bases that the uncertain base-pair of difference, which refers to, as R is represented 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 indefinite between A and R, is not mutually SNP.Therefore, the SNP site in the embodiment of the present invention is not include the uncertain base-pair of above-mentioned difference.By above SNP site Definition, the embodiment of the present invention obtains 7236888 SNP sites altogether between all 1137 rice varieties, wherein 59503 SNP site is located on cytoplasmic skeleton, and remaining SNP site is located on nuclear genome.Genotype referred to hereafter is Refer to the combination of multiple SNP sites in test zone, karyogene type refers to genotype and is located on nuclear genome, and matter genotype is Refer to genotype to be located on cytoplasmic skeleton.For example, the 8th test zone is located on nuclear genome in table 1, it is karyogene Type, the test zone share 9 SNP sites, and the genotype of the test zone is the combination of this 9 SNP sites.

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, base referred to hereafter inside it 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 8th test zone is located on nuclear genome in table 1, it is Karyogene type, the test zone share 9 SNP sites, and the genotype of the test zone is the combination of this 9 SNP sites.Mesh The preceding high flux detection state-of-the-art method of SNP site is genetic chip, and each pair chip probe can only detect 1 pair of SNP site, this hair The bright combination (genotype) that have detected multiple SNP sites in test zone.

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

For rice varieties to be measured " R7723 " and " R8377 ", test zone is the area that discrimination is big on cytoplasmic skeleton Discrimination is big on domain or nuclear genome and the equally distributed region of SNP site, wherein, discrimination Wherein, a is the kind sum being detected in variation window area, and bi is the kind of i-th kind of genotype in variation window area Number, and bi>1, k is the number of the genotype comprising more than a kind, and variation window area is with each single nucleotide variations position Centered on point (SNP site), respectively extend 1/2 window as detection of sequence length to be measured to the both sides in single nucleotide variations site Mouthful；Test zone is that discrimination is big and equally distributed on the region that discrimination is big on cytoplasmic skeleton or nuclear genome Region.The Computing Principle of discrimination is as follows：All interracial number of combinations areWherein, the different cultivars in same gene type Between combination be undistinguishable, its number isSo, the ratio for the breed combination that can not be distinguished isCan The ratio for the breed combination being distinguished i.e. discriminationAs can be seen here, discrimination is bigger, more can be by different product Kind distinguishes, and test of the big variation window area of discrimination to substantive derived relation is more effective.If on nuclear genome Variation window area skewness, some regions can be caused adjacent, so as to linkage inheritance, information is easily overlapping, therefore, carefully The principle of compositionality of test zone is selected on karyon genome is：Discrimination is big and SNP site is uniformly distributed.Cytoplasmic skeleton without Linkage inheritance problem, so, the big region of selective discrimination degree is only needed on cytoplasmic skeleton.

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 7236888 of acquisition SNP sites, 7236888 variation windows can be obtained, calculate these variation windows The discrimination in regionFor example, in the 1st variation window area, a=520 kind is detected altogether, altogether There are k=3 kind genotype ACCT, CGTT, ACCC, their kind number is respectively b1=10, b2=30 and b3=431, Therefore, It is meant that：, can be by 520 product by the 1st variation window area 31% breed combination in kind distinguishes, and 79% breed combination cannot be distinguished by out, it is necessary to more make a variation window just in addition It can distinguish.After the same method, calculate and obtain all discriminations of 7236888 variation windows and therefrom choose positioned at thin 6800 maximum variation windows of discrimination and 200 maximum changes of discrimination in cytoplasmic skeleton in karyon genome Different window.Check one by one in 6800 variation windows of nuclear genome, each window and next variation window of making a variation Mouthful between distance, if distance exceed 100K (1K=1000 base), abandon wherein discrimination it is less make a variation window after Reexamine, untill the adjacent distance for looking into variation window is all higher than 100K.Selection 100K criterion distance is because rice base Because a group size is about 500M (ten thousand bases of 1M=100), by final selected 2000 test zones for being located at nuclear genome Meter, distance is 250K between average test zone, but due to few variant sites such as some specific regions such as centromeres, because This, average distance should be less than 250K.By the above process, 4061 variation windows for being located at nuclear genome are have selected, it With acquisition be located at 200 maximum variation windows of discrimination in cytoplasmic skeleton together with totally 4261 variation window conducts Selected test zone.Wherein, 200 maximum variation windows of selective discrimination degree, are empirical value, the quantity can be according to specific Situation is modified.

3rd, two kinds to be measured are sampled respectively, extract and obtain the DNA of the sampling samples of two kinds to be measured, The preparation method of sampling samples is：After two kinds to be measured are randomly selected with the sample mixing of more than 100 respectively, two are obtained The sampling samples of kind to be measured.

In the present embodiment, 50000 germinations of kind to be measured " R7723 " are have chosen, are randomly selected 30000 big It is placed in after small roughly equal bud mixing in mortar, powder is fully ground into after adding liquid nitrogen into mortar.Given birth to using Beijing Tiangeng The plant genome DNA extracts kit that the article No. for changing Science and Technology Ltd.'s production is DP305 is extracted and obtains kind to be measured The DNA of " R7723 " mixing sample, DNA extraction method are carried out by the operation manual of the kit.It is public using U.S. Invitrigen Department's productionDsDNA HS Assay Kit (article No. Q32852) and its specification are determined the DNA of acquisition Amount, the DNA of the kind to be measured " R7723 " after quantifying are diluted to 10.00ng/ μ l.

After the same method, rice varieties to be measured " R8377 " are sampled and extract DNA, after equally quantifying The DNA of rice varieties " R8377 " to be measured is diluted to 10.00ng/ μ l.

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

Test zone is carried out using multiplex PCR (Polymerase Chain Reaction, PCR) technology 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；BINOMDIST (SD*TN, TN, 0.80*SD, TRUE) implication is： When the number of test zone is TN, the ratio of Differences genotype to be measured is 0.80*SD and decision threshold is SD, judge The probability that the ratio of Differences genotype to be measured is less than decision threshold is ensured more than or equal to 95%.The condition is meant that： When the ratio of Differences genotype to be measured is the 80% of judgment threshold, the judgement determined by the number of test zone is to be measured Kind has accuracy >=95% of substantive derived relation.The judgment threshold of substantive derived relation is the breeding according to various countries Present situation, mark mode, require Stringency and artificially determine.In the present embodiment, SD is defined as 3%.Progressively increase test The number TN in region had found, as TN >=1934, above-mentioned formula is set up, therefore, the number of test zone should >=1934.To For some SSR and SNP test, 200 test zones have calculated that it is enough, if the ratio of Differences genotype to be measured is sentences The 80% of disconnected threshold value, its accuracy be only its accuracy only >=BINOMDIST (3%*200,200,0.80*3%, TRUE)= 79%, therefore, the method that this implementation provides, more accurate conclusion can be obtained.

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 kind to be measured and cytoplasm reference gene group are permeated file, and in " Select the After selecting " Custom " in genome you wish to use " options, the file of fusion is uploaded as design multiple PCR primer When reference gene group." Standard DNA ", in Add Hotspot options, addition needs are set the selection of DNA type options The positional information of SNP site in the universal test region of meter, including chromosome information, SNP initiation site and SNP End locus, its certain embodiments are shown in Table 1.Finally click on the " multiplex PCR that Submit targets " buttons are submitted and designed Primer.In the present embodiment, from all 4261 test zones, design and be successfully authenticated 2231 pairs of multiple PCR primers, be used for Expand corresponding 2231 test zones.The method for verifying multiple PCR primer is by method provided by the invention, extracts same strain Leaves genomic DNA on rice, and using design multiple PCR primer the genomic DNA of acquisition is expanded, build storehouse, High-flux sequence simultaneously analyzes sequencing fragment group, removes the corresponding primer of following test zone：The sequencing segments of the test zone Less than 1000 or hybrid strain genotype be present, the primer remained is the multiple PCR primer being proved to be successful.So test section Domain 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 are insertion or the missing of discontinuous base in quantity >=2 of distinguishing base between all genotype of kind or distinguishing base. Because genomic DNA source is in same strain rice leaf, it is impossible to hybrid strain kind be present, therefore, hybrid strain genotype is by testing PCR caused by the special construction in region or sequencing Preference mistake, remove these test zones and avoid such system mistake.Rule Determining test zone not include another purpose for the test zone that amplification produces hybrid strain genotype is：The test section remained Domain can also make the calculating of hybrid strain rate, realize same in addition to the test as interracial substantive derived relation to be measured The multiple use of set test primer.The multiple PCR primer being proved to be successful provides in fluid form after also being mixed by the said firm Used to client.2231 test zones of above-mentioned successful design multiple PCR primer are to be detected eventually for kind to be measured Test zone, wherein, 100 test zones are located on cytoplasmic skeleton, and remaining 2131 test zones are located at cell On Matrix attachment region.Existing Essentially derived variety judges all not using the test site on cytoplasmic skeleton, and cytoplasm Difference, can equally produce different varietal character performances, it should the judgement for Essentially derived variety relation.

5th, the DNA of two sampling samples is expanded respectively using primer, respectively obtains two kinds to be measured and testing The amplified production in region, amplified production are respectively used to build the high-throughput sequencing library of two kinds to be measured, and specific method 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 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 2231 test zones of multiplexed PCR amplification, the amplification system of multiplex PCR：5×Ion AmpliSeq^TMμ l of HiFi Mix 4, μ l of test zone primer mixed liquor 4, the DNA of rice varieties to be measured " R7723 " prepared 10ng and 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 Individual circulation；10 DEG C of insulations.After primer unnecessary in multiplexed PCR amplification product is digested using FuPa reagents, then phosphorylation is carried out, Specific method is：2 μ L FuPa reagents are added into the amplified production of multiplex PCR, it is anti-by following program in PCR instrument after mixing Should：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 to contain process The amplified production solution of phosphorylation.By the upper sequence measuring joints of amplified production connection of phosphorylation, specific method is：Into mixture a μ L of transferring reagent 4, the μ L of sequence measuring joints solution 2 and the μ L of DNA ligase 2 are added, after mixing, is 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.Mixed liquor is purified using the ethanol precipitation methods of standard 10 μ L are dissolved in after b without in enzyme water.Utilize the production of Invitrigen companies of the U.S. dsDNA HS Assay Kit (article No. Q32852) and it is measured according to its specification, and after obtaining mixed liquor b mass concentration, will mixed liquor after purification B is diluted to 15ng/ml, obtains the high-throughput sequencing library of concentration about 100pM test zone.

After the same method, rice varieties to be measured " R8377 " are carried out with the structure of high-throughput sequencing library, is similarly obtained 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 kinds to be measured, respectively obtain two it is to be measured The sequencing fragment group of kind, 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 works For 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 OT2 200 of all test zones of acquisition Kit v2 (invirtrigen companies of the U.S. produce, article No. 4485146) be sequenced before ePCR (Emulsion PCR, breast Change polymerase chain reaction) expand, operating method is carried out by the operation manual of the kit.Utilize ePCR products and kit Ion The Kit v2 of PI Sequencing 200 (invirtrigen companies of the U.S. produce, article No. 4485149) are high in the generations of Proton bis- High-flux sequence is carried out on flux sequenator, operating method is carried out by the operation manual of the kit.In the present embodiment, high pass Amount sequencing throughput is arranged to average 30000 times of coverage test region.

A large amount sequencing result is pre-processed

High-flux sequence fragment is compared and arrives all 2231 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 kinds to be measured is analyzed, obtains two variety and genetypes to be measured, kind base to be measured respectively Because type is to become the combination of isobase in test zone, and frequency >=30% of variety and genetype to be measured, specific method are 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, 2029 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.Variety and genetype to be measured is the combination for becoming isobase in test zone, and Frequency >=30% of variety and genetype 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%, and therefore, for homozygous site, variety and genetype to be measured only has one Kind, its frequency should be more than 89%, and for heterozygous sites, variety and genetype to be measured has 2 kinds, and its ratio should be more than 45.5%, therefore, it is specified that frequency >=30% of variety and genetype to be measured, can exclude to mix because being sequenced in wrong and to be measured kind There is hybrid strain and to the interference of variety and genetype to be measured.

For example, in fragment group is sequenced, the sequencing fragment sum in the 1st sequencing region is 29888 articles, have ACCC, CGTT, CCCC, GCCC ... totally 41 kinds of genotype, represent these genotype sequencing segments distinguish 29603,14,2,1 Bar ..., the frequency of these genotype is 29603/29888=99.05%, 14/29888=0.05%, 2/29888= 0.007%th, 1/29888=0.003% ....By the definition of variety and genetype to be measured, ACCC is the to be measured of the 1st test zone Kind " R7723 " genotype, other genotype are genotype caused by sequencing mistake or hybrid strain.By identical method, judge simultaneously Obtain kind to be measured " R7723 " genotype of all 2029 successful test zones of detection.

By with rice varieties to be measured " R7723 " identical method, equally obtain the 2029 of rice varieties to be measured " R8377 " The successful test zone of individual detection, and rice varieties to be measured " R8377 " are in the genotype of all successful test zones of detection, Partial results are shown in Table 1.The present embodiment does not list whole kinds to be measured completely in all test zone genes as space is limited, Type, only list certain embodiments.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.

Table 1 is variety and genetype to be measured and its relevant information

8th, compare two variety and genetypes to be measured, obtain the ratio of Differences genotype to be measured, specific method is such as Under：

If in the test, for all variety and genetypes to be measured without missing, the test zone is referred to as being total to for kind to be measured There is test zone.In shared test zone, if product to be tested inter-species genotype is incomplete same, the genotype is referred to as product to be tested Interspecific difference genotype, if for example, kind A to be measured genotype is AA, AA, AA, AA, AA/CC, AA/CC, B pairs of kind to be measured The genotype answered is AA, CC, AA/CC, CC/TT, AA/CC, CC/TT, then the Differences to be measured in this 6 test zones Genotype judges as follows：It is not, is, being, being, is not and is.It is miscellaneous as gene that "/" in above genotype represents the test zone Site is closed, therefore detects "/" both front and back genotype.The ratio of Differences genotype to be measured=possess differential gene The number of the number of the test zone of type/shared test zone.

In the present embodiment, rice varieties " R8377 " to be measured and the shared test zone of rice varieties to be measured " R7723 " are 2029, in the 1st shared test zone, their genotype is respectively ACCC and ACCC, and they are identical, therefore, It is not interracial Differential genotype to be measured.According to said method, judge one by one in all 2029 shared test zones, if exist Differential genotype, as a result for：In all shared test zones, the test zone number for having differences genotype is 0, so, The ratio OD=0/2029=0% of Differences genotype to be measured.

According to the ratio and decision threshold of Differences genotype to be measured, the substantive derivation of two kinds to be measured is judged Relation, the method for judging the substantive derived relation of two kinds to be measured are：As the ratio ＜ of Differences genotype to be measured During SD, two kinds to be measured have substantive derived relation；As ratio >=SD of Differences genotype to be measured, two are treated Survey kind and do not have substantive derived relation, wherein, SD is decision threshold.

In the present embodiment, ratio=0% of Differences genotype to be measured<SD=3%, therefore judge kind to be measured There is substantive derived relation between " R8377 " and kind to be measured " R7723 ".

If two when judging that kind to be measured has substantive derived relation, the correct probability of conclusion >=BINOMDIST (SD* TRN,TRN,OD,TRUE)；If judge that two kinds to be measured do not have substantive derived relation, the correct probability of conclusion >= BINOMDIST((1-SD)*TRN,TRN,1-OD,TRUE)；Wherein, TRN is the number of shared test zone, and SD is decision threshold Value, OD are the ratio of Differences genotype to be measured, and BINOMDIST is the functions of excel 2010, BINOMDIST (SD*TRN, TRN, OD, TRUE) implication be：When the number of shared test zone is TRN, the ratio OD of Differences genotype to be measured Less than decision threshold SD probability；BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) implication is：When shared test zone Number when being TRN, the ratio OD of Differences genotype to be measured is more than decision threshold SD probability.

In the present embodiment, judge that there is two kinds to be measured the number of substantive derived relation and shared test zone to be 2029, therefore, correct probability >=BINOMDIST (SD*TRN, TRN, OD, the TRUE)=BINOMDIST (3%* of conclusion 2029,2029,0%, TRUE)=100%, therefore, in this implementation, judge that there is two rice varieties to be measured substantive derive to close The conclusion of system is very accurate.

9th, 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, rice varieties " R7723 " to be measured are to be used as recurrent parent by rice varieties to be measured " R8377 ", backcrossing Seed selection and come, so, by the description of UPOV texts, the two rice varieties to be measured should have substantive derived relation, table Bright, the conclusion of the present embodiment is correct.

Embodiment two determines the substantive derived relation between corn variety " GL95 " and " 1102 "

Kind to be measured provided in an embodiment of the present invention is corn variety " GL95 " and " 1102 ", both at open, known Kind.Because the method in the present embodiment is similar with the method in embodiment one, therefore, identical place is just no longer repeated, Only list different places.

Variant sites between different corn 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 corns with reference to corn variety genome sequence Row are compared, and obtain the variant sites between substantial amounts of different corn varieties, wherein can be that " B73 " is beautiful with reference to corn variety Rice, " B73 " corn could alternatively be other known and refer to corn variety.

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

The genome sequence of the different corn varieties of the present embodiment shows two kinds of sources, and the first is Chia etc. to 103 jade The high-flux sequence sequence of the genome of rice kind, pertinent literature information are as follows：Chia JM et al.Maize HapMap2 Identifies extant variation from a genome in flux.Nat Genet.2012,44 (7):803-7. The genome sequence of 103 corn varieties is published in NCBI Short Read Archive (http:// Www.ncbi.nlm.nih.gov/sra), reception number is SRA051245；Second is by Chia etc. the above-mentioned article delivered To " GL95 ", " 1102 " and cenospecies, " height relies 145 " to carry out high-flux sequence to the method for middle offer.The present embodiment obtains altogether The high-flux sequence sequence of the genome of 106 corn varieties.

Specifically, because the sequencing depth of this 106 corn varieties is not high, it is only capable of identifying single nucleotide variations (SNP) Site, other variation types are as repeated number variation, due to a low credibility, without identification.Utilize Frederick Sanger ratios It is to software (version number 0.4) that the high-flux sequence sequence alignment of the genome of this 106 corn varieties is thin to " B73 " corn (version is IRGSP 4.0 to karyon reference gene group, download address：http://www.ncbi.nlm.nih.gov) and cytoplasm In reference gene group, the cytoplasm reference gene group includes mitochondria reference gene group and chloroplaset reference gene group, its NCBI (National Center for Biotechnology Information, US National Biotechnology Information center) On reception number be respectively NC_011033 and NC_001320.The parameter of contrast is identical with embodiment one with method, and the present invention is real Apply example and obtain 53855606 SNP sites altogether between all 106 corn varieties, wherein 9005 SNP sites are located at cytoplasm On genome, remaining SNP site is located on nuclear genome.

2nd, the test zone of two kinds to be measured is determined respectively by variant sites, specific method is as follows：

For corn variety to be measured " GL95 " and " 1102 ", test zone is the region that discrimination is big on cytoplasmic skeleton Or discrimination is big and the equally distributed region of SNP site on nuclear genome, wherein, discriminationIts In, a is the kind sum being detected in variation window area, and bi is the kind number of i-th kind of genotype in variation window area, And bi>1, k is the number of the genotype comprising more than a kind.The Computing Principle of discrimination is as follows：All interracial combinations Number isWherein, the combination between the different cultivars in same gene type is undistinguishable, and its number isThat , 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 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 53855606 of acquisition SNP sites, 53855606 variation windows can be obtained, calculate these variation windows The discrimination in mouth region domainFor example, in the 1st variation window area, a=102 kind is detected altogether, Shared k=3 kind genotype CCA, TCA, TCG, their kind number are respectively b1=5, b2=11 and b3=76, because This, It is meant that：, can be by 102 kinds by the 1st variation window area 43% breed combination distinguish, in addition 47% breed combination cannot be distinguished by out, it is necessary to more make a variation window could area Separate.After the same method, calculate to obtain all discriminations of 53855606 variation windows and therefrom choose and be located at cell 8000 maximum variation windows of discrimination and 100 maximum variations of discrimination in cytoplasmic skeleton in Matrix attachment region Window.Check one by one in 8000 variation windows of nuclear genome, each window and next variation window of making a variation Between distance, if distance exceed 500K (1K=1000 base), abandon wherein discrimination it is less make a variation window afterwards again Check, untill the adjacent distance for looking into variation window is all higher than 500K.Selection 500K criterion distance is because corn gene Group size is about 2300M (ten thousand bases of 1M=100), by final selected 2400 test zones for being located at nuclear genome Meter, distance is 1M between average test zone, but due to few variant sites such as some specific regions such as centromeres, therefore, Average distance should be less than 1M.By the above process, 5030 variation windows for being located at nuclear genome are have selected, they are with obtaining Be located at 100 maximum variation windows of discrimination in cytoplasmic skeleton together totally 5130 variation windows as being selected in Test zone.Wherein, 100 maximum variation windows of selective discrimination degree, are empirical value, the quantity can be entered as the case may be Row modification.

3rd, two kinds to be measured are sampled respectively, extract and obtain the DNA of the sampling samples of two kinds to be measured, The preparation method of sampling samples is：After two kinds to be measured are randomly selected with the sample mixing of more than 100 respectively, sampled Sample.

In the present embodiment, 5000 germinations of corn variety to be measured " GL95 " are have chosen, randomly select 4000 It is placed in after the bud mixing being substantially equal to the magnitudes in mortar, powder is fully ground into after adding liquid nitrogen into mortar.Using Beijing Tiangeng The plant genome DNA extracts kit that the article No. of biochemical technology Co., Ltd production is DP305 is extracted and obtains corn to be measured The DNA of kind " GL95 " mixing sample, DNA extraction method are carried out by the operation manual of the kit.Utilize the U.S. The production of Invitrigen companiesDsDNA HS Assay Kit (article No. Q32852) and its specification are to obtaining DNA quantified, will it is quantitative after the DNA of corn variety to be measured " GL95 " be diluted to 10.00ng/ μ l.

After the same method, corn variety to be measured " 1102 " is sampled and extracts DNA, treated after equally quantifying The DNA for surveying corn variety " 1102 " is diluted to 10.00ng/ μ l.

By with the identical method of embodiment one, in the present embodiment, from all 5130 test zones, design and demonstrate 2506 pairs of multiple PCR primers, for expanding corresponding 2506 test zones.

By with the identical method of embodiment one, to corn variety to be measured " GL95 " carry out high-throughput sequencing library structure, Obtain the high-throughput sequencing library of concentration about 100pM test zone.

After the same method, the structure of high-throughput sequencing library is carried out to corn variety to be measured " 1102 ", is similarly obtained The high-throughput sequencing library of concentration about 100pM test zone.

High-flux sequence is carried out using high-throughput sequencing library

Utilize the high-throughput sequencing library and kit Ion PI Template OT2 200 of all test zones of acquisition Kit v2 (invirtrigen companies of the U.S. produce, article No. 4485146) be sequenced before ePCR (Emulsion PCR, breast Change polymerase chain reaction) expand, operating method is carried out by the operation manual of the kit.Utilize ePCR products and kit Ion The Kit v2 of PI Sequencing 200 (invirtrigen companies of the U.S. produce, article No. 4485149) are high in the generations of Proton bis- High-flux sequence is carried out on flux sequenator, operating method is carried out by the operation manual of the kit.In the present embodiment, high pass Amount sequencing throughput 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 2506 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 2 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.

By with the identical method of embodiment one, judge and obtain the jade to be measured of all 2465 successful test zones of detection Rice kind " GL95 " genotype.

By DNA, the structure high pass that corn variety " 1102 " to be measured is extracted with corn variety to be measured " GL95 " identical method Sequencing library, high-flux sequence, analysis sequencing fragment group are measured, equally obtains 2465 successful test zones of detection and to be measured Corn variety " 1102 " is shown in Table 2 in all genotype for detecting successful test zones, partial results.As space is limited, originally Embodiment does not list all corn varieties to be measured completely and in all test zone genotype, only lists certain embodiments.Equally Limited based on length, also have some areas also only to list part related example in the present embodiment, remaining unlisted data can root According to the method completion of the present embodiment.

Table 2 is variety and genetype to be measured and its relevant information

8th, compare two variety and genetypes to be measured, obtain the ratio of Differences genotype to be measured, method is as follows：

In the present embodiment, corn variety " 1102 " to be measured and the shared test zone of corn variety to be measured " GL95 " are 2465, in the 1st shared test zone, their genotype is respectively TCG and TCG, and they are identical, therefore, no For interracial Differential genotype to be measured.According to said method, judge one by one in all 2465 shared test zones, if difference be present Allogene type, as a result for：In all shared test zones, the test zone number for having differences genotype is 63, so, treat Survey the ratio OD=63/2465=2.56% of Differences genotype.

In the present embodiment, ratio=2.56% of Differences genotype to be measured<SD=3%, therefore judge jade to be measured There is substantive derived relation between rice kind " 1102 " and corn variety to be measured " GL95 ".

In the present embodiment, judge that there is two product to be tested inter-species the number of substantive derived relation and shared test zone to be 2465, therefore, correct probability >=BINOMDIST (SD*TRN, TRN, OD, the TRUE)=BINOMDIST (3%* of conclusion 2465,2465,2.56%, TRUE)=90.53%, therefore, in this implementation, judge that two kinds to be measured have substantive derive from The conclusion of relation is more accurately.

9th, result verification

At present, without a kind of detection method of substantive derived relation between kind of standard, international New variety protection alliance In the definition that 1991 give substantive derived relation, according to this definition, if two Differences of A and B are smaller, show blood Edge relation is nearer, it is possible to determine that has substantive derived relation.According to this definition, in the present embodiment, by two kinds to be measured Press《New variety of plant specificity, uniformity and stability test guide-corn》In method plant and observe two jade to be measured The interracial multiple situations of rice, in 70 characters investigated, only 3 Traits changes reach the level of signifiance, show the two Differences to be measured are little, it is likely that have substantive derived relation, be consistent with the conclusion that the present embodiment obtains.

The embodiment of the present invention is expanded by high-flux sequence and more sites, realizes the full-page proof of test zone in kind to be measured This sampling, it ensure that the accuracy of substantive derived relation detection.Meanwhile utilize more site amplification techniques, multiple test zones Detection workload be substantially equivalent to traditional SSR or SNP detection a test zone workload, therefore, the present embodiment It is not only accurate, and method is simple, quick.By taking embodiment one as an example, if equally 2029 test zones of detection, using traditional (SSR is by electrophoresis method with the detection of the amplified production of 2029 times, it is necessary to carry out 2029 PCR amplifications for SSR or SNP detection method Detection, SNP are detected by the method for generation test), its workload, speed and testing cost are all can not be received.Except this Outside, the sequence of each base in test zone is obtained in the present embodiment, resolution ratio has reached ultimate attainment, and information content is also It is maximum, be that other detection methods are all incomparable, for example, SSR obtain be fragment length information, it is understood that there may be the problem of It is：Fragment length difference is too small, may can't detect, even the fragment of equal length, its internal base composition may also It is different, but SSR equally can't detect.SNP detection can only detect a pair of SNP sites, and in the present embodiment, it can detect All SNP of test zone, it has also been found that the variation in test zone outside SNP, such as repeat number, missing and insertion, these All single SNP detections can not be found.In addition, in addition to using generation sequencing detection SNP, also have and detected by the way of hybridization SNP, in this case, what is obtained is hybridization signal, and noise is big and can not find unknown variation, and a probe is only capable of detection one To SNP, resolution ratio is 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

A kind of 1. method of test plants substance derived relation, it is characterised in that methods described includes：

Obtain the variant sites in kind between different cultivars belonging to two kinds to be measured；

Test zone is determined by the variant sites, passes through discriminationThe value of discrimination is calculated, wherein, a For the kind sum being detected in variation window area, bi is the kind number of i-th kind of genotype in the variation window area, And bi>1, k is the number of the genotype comprising more than a kind, and the variation window area is with each single nucleotide variations Centered on site, respectively extend 1/2 window as detection of sequence length to be measured to the both sides in the single nucleotide variations site, The test zone is the 6800 variation windows and the area in cytoplasmic skeleton that discrimination is maximum in cytoplasmic skeleton 200 maximum variation windows of indexing, wherein, the genotype is multiple single nucleotide variations sites in the test zone Combination, the number of the test zone meet following condition：BINOMDIST (SD*TN, TN, 0.80*SD, TRUE) >=95%, its In, TN is the number of the test zone, and SD is decision threshold；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 SD and the ratio of the Differences genotype to be measured is During 0.80*SD, judge that probability guarantee of the ratio less than the decision threshold SD of the Differences genotype to be measured is more than Equal to 95%；

Kind to be measured described to two is sampled respectively, is extracted and is obtained the DNA of the sampling samples of two kinds to be measured；

Prepare the primer for expanding the test zone；

The DNA of the sampling samples is expanded respectively using the primer, respectively obtains two kinds to be measured in institute The amplified production of test zone is stated, the amplified production is respectively used to the high-flux sequence text of two kinds to be measured of structure Storehouse；

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

The sequencing fragment group of two kinds to be measured is analyzed, obtains two variety and genetypes to be measured, the kind to be measured respectively Genotype is to become the combination of isobase in the test zone, and frequency >=30% of the variety and genetype to be measured；

Compare two variety and genetypes to be measured, obtain the ratio of Differences genotype to be measured；

According to the ratio of the Differences genotype to be measured, the substantive derived relation of two kinds to be measured is judged； As the ratio ＜ SD of the Differences genotype to be measured, two kinds to be measured have substantive derived relation；When During ratio >=SD of the Differences genotype to be measured, two kinds to be measured do not have substantive derived relation, its In, SD is decision threshold, if judge that two kinds to be measured have substantive derived relation, the correct probability of conclusion >= BINOMDIST(SD*TRN,TRN,OD,T RUE)；If judge that two kinds to be measured do not have substantive derived relation, Correct probability >=the BINOMDIST of conclusion ((1-SD) * TRN, TRN, 1-OD, TRUE)；Wherein, TRN is two products to be tested The number of the shared test zone of kind, OD are the ratio of the Differences genotype to be measured, and BINOMDI ST are excel 2010 functions, BINOMDIST (SD*TRN, TRN, OD, TRUE) implication are：When the number of the shared test zone is TRN When, the ratio OD of the Differences genotype to be measured is less than the probability of the decision threshold SD；BINOMDIST((1-SD)* TRN, TRN, 1-OD, TRUE) implication is：When the number of the shared test zone is TRN, the Differences base to be measured Because the ratio OD of type is more than the probability of the decision threshold SD.
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 between all genotype of the hybrid strain genotype and the kind to be measured Distinguishing base quantity >=2 or the distinguishing base in have insertion or the missing of discontinuous base.
3. according to the method for claim 1, it is characterised in that the method that kind to be measured described to two is sampled respectively For：Kind to be measured described to two obtains two kinds to be measured after randomly selecting the sample mixing of more than 100 respectively Sampling samples.