CN104805185A

CN104805185A - Method for testing substantive derivation relation of plant varieties

Info

Publication number: CN104805185A
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: 2015-07-29
Anticipated expiration: 2035-03-31
Also published as: CN104805185B

Abstract

The invention discloses a method for testing the substantive derivation relation of plant varieties. The method comprises the following steps: obtaining variation points; determining a testing area; sampling, extracting and obtaining DNA in the samples; preparing primers; respectively amplifying the DNA in the two samples by using the primers, and respectively obtaining amplification products of the two varieties to be tested in the testing area for establishing high-throughput sequencing libraries of the two varieties to be tested; performing high-throughput sequencing on the two high-throughput sequencing libraries, so as to respectively obtain sequencing fragment groups of the two varieties to be tested; analyzing the two sequencing fragment groups, so as to respectively obtain the genotypes of the two varieties to be tested; comparing the genotypes of the two varieties to be tested, so as to obtain the proportion of differential genotypes between the varieties to be tested; judging the substantive derivation relation of the two varieties to be tested according to the proportion of differential genotypes between the varieties to be tested. The method can be used for accurately, quickly and simply judging the substantive derivation relation between the varieties to be tested.

Description

The method of the substantive derived relation of a kind of test plants kind

Technical field

The present invention relates to biological technical field, particularly the method for the substantive derived relation of a kind of test plants kind.

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; namely Essentially derived variety refers to that the B kind obtained by A breed breeding does not have substantial change; B kind is called the Essentially derived variety of A kind, has substantive derived relation between A kind and B kind.The method judging whether to have between two kinds substantive derived relation detects genotypic difference ratio between these two kinds, when this difference ratio exceedes certain value, can think, between two plant varieties, not there is substantive derived relation, on the contrary, then think, between two kinds, there is substantive derived relation.

The method that current detection substance derives from sexual intercourse is also little, only methodical roughly flow process is: by SSR marker or SNP marker, each test zone of the kind to be measured that increases, the genotype of each test zone obtained is detected again by electrophoresis or generation order-checking, according to genotype, judge interracial substantive derived relation to be measured.

Realizing in process of the present invention, contriver finds that prior art at least exists following problem:

After substantive derived relation needs to detect gene locuss a large amount of between kind to be measured, accurately could judge, between two kinds, whether there is substantive derived relation.Detect substantive derivation in the method for sexual intercourse existing, detection site causes the judgement conclusion of substantive derived relation inaccurate less.Simultaneously, existing SSR marker and SNP marker are due to needs amplification and each test zone of independent detection separately, therefore, test zone number is too much, workload will inevitably be caused greatly to increase, therefore, existing method testing number of regions is all within 300, completely can not represent the full gene type of kind to be measured, thus cause detected result inaccurate, the judgement conclusion of substantive derived relation is also inaccurate.

Summary of the invention

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

Embodiments provide the method for the substantive derived relation of a kind of test plants kind, described method comprises:

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

By described variant sites determination test zone;

Respectively two described kinds to be measured are sampled, extract and obtain the DNA of the sampling sample of two described kinds to be measured;

The primer of the described test zone of preparation amplification;

Utilize described primer to increase to the DNA of described sampling sample respectively, obtain the amplified production of two described kinds to be measured at described test zone respectively, described amplified production is respectively used to the high-throughput sequencing library of the described kind to be measured of structure two;

Respectively high-flux sequence is carried out to the described high-throughput sequencing library of two described kinds to be measured, obtains the sequenced fragments group of two described kinds to be measured respectively;

Analyze the sequenced fragments group of two described kinds to be measured, obtain two variety and genetypes to be measured respectively, described variety and genetype to be measured is the combination of variation base in described test zone, and genotypic frequency >=30% of described kind to be measured;

Relatively two described variety and genetypes to be measured, obtain the genotypic ratio of product to be tested difference between species;

According to the genotypic ratio of described product to be tested difference between species, judge the substantive derived relation of two described kinds to be measured.

Particularly, described test zone does not comprise the amplification generation genotypic region of hybrid strain;

Described hybrid strain genotype refers to frequency >=0.02%, and has insertion or the disappearance of discontinuous base in quantity >=2 of distinguishing base between all genotype of described hybrid strain genotype and described kind to be measured or described distinguishing base.

Particularly, the number of described test zone meets the following conditions: BINOMDIST (SD*TN, TN, 0.80*SD, TRUE) >=95%, and wherein, TN is the number of described test zone, and SD is decision threshold; The condition implication that the number of described test zone meets is: when the number of described test zone be TN, described decision threshold be SD and the genotypic ratio of described product to be tested difference between species is 0.80*SD time, judge that the probability guarantee that the genotypic ratio of described product to be tested difference between species is less than described decision threshold SD is more than or equal to 95%.

Particularly, to the method that two described kinds to be measured are sampled be respectively: the sampling sample obtaining two described kinds to be measured after the sample mixing to two described kinds difference random selecting more than 100 to be measured.

Particularly, judge that the method for the substantive derived relation of two described kinds to be measured is:

As the genotypic ratio < SD of described product to be tested difference between species, two described kinds to be measured have substantive derived relation; As the genotypic ratio >=SD of described product to be tested difference between species, two described kinds to be measured do not have substantive derived relation, and wherein, SD is decision threshold.

Further, if when judging that two described kinds to be measured have substantive derived relation, probability >=BINOMDIST (SD*TRN, TRN, OD, TRUE) that conclusion is correct; If when judging that two described kinds to be measured do not have substantive derived relation, probability >=BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) that conclusion is correct; Wherein, TRN is the number of the total test zone of two described kinds to be measured, OD is the genotypic ratio of described product to be tested difference between species, BINOMDIST is excel 2010 function, BINOMDIST (SD*TRN, TRN, OD, TRUE) implication is: when the number of described total test zone is TRN, and the genotypic ratio OD of described product to be tested difference between species is less than the probability of described decision threshold SD; BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) implication is: when the number of described total test zone is TRN, the genotypic ratio OD of described product to be tested difference between species is greater than the probability of described decision threshold SD.

Particularly, determine that the method for described test zone is by described variant sites:

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

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

The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: the method that the embodiment of the present invention provides is increased and high-flux sequence by multidigit point, ensure the large sample sampling of the test zone of kind to be measured, successfully achieve the target accurately judging substantive derived relation between kind to be measured, and test is simple, quick.

Embodiment

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

Embodiment one measures the substantive derived relation between rice varieties " R7723 " and " R8377 "

The kind to be measured that the embodiment of the present invention provides is rice varieties " R7723 " and " R8377 ", and the two is open, known kind.The source of rice varieties " R7723 " is after rice varieties " R8377 " is hybridized with " IRBB23 ", with " R8377 " for recurrent parent, with resisting bacterial leaf-blight proterties for major objective proterties, cultivates form by the method for back cross breeding.

One, the variant sites between different varieties in kind belonging to kind to be measured is obtained.

The kind of kind to be measured is paddy rice, therefore, obtain the variant sites of planting between interior different varieties belonging to kind to be measured and then should be the variant sites obtained between different rice varieties, variant sites between different rice varieties can obtain from the documents and materials announced, but the results contrast that the method obtains is fragmentary, in the present embodiment, by the genome sequence of different paddy rice and the genome sequence with reference to rice varieties are compared, obtain the variant sites between a large amount of different rice varieties, wherein reference rice varieties can be " Japanese eyeball " paddy rice, should can replace with other known reference rice varieties by " Japanese eyeball " paddy rice.

Further, the method 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, the first is the genomic high-flux sequence sequence of Han Bin to 1082 rice varieties, and pertinent literature information is as follows: Huang XH et al.Amap of rice genome variation reveals the origin of cultivated rice.Nature.2012; 7:497 – 503.The genome sequence of these 1082 rice varieties is published in EuropeanNucleotideArchive (http://www.ebi.ac.uk/ena/), and reception number is ERP001143, ERP000729 and ERP000106; The second is the genomic high-flux sequence sequence of Xu Xun to 50 rice varieties, pertinent literature information is as follows: Xun X et al.Resequencing 50accessions of cultivated and wildrice yields markers for identifying agronomically important genes.Nat Biotechnol.2011,30 (1): 105-11, the genome sequence of these 50 rice varieties is published in NCBI Short ReadArchive (http://www.ncbi.nlm.nih.gov/sra), and reception number is SRA023116; The third is for having carried out high-flux sequence by the method provided in the above-mentioned article delivered of Han Bin to " R8377 ", " Jin Ke 1A ", " IRBB23 ", cross-fertilize seed " Jin Ke 1A/R7723 " and hybrid strain kind " D excellent 527 ".The present embodiment obtains the genomic high-flux sequence sequence of 1137 rice varieties altogether.

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

Particularly, because the order-checking degree of depth of these 1137 rice varieties is not high, only can identify single nucleotide variations (SNP) site, such as the repeat number of other variation type makes a variation, and due to a low credibility, does not identify.To " Japanese eyeball " rice cell core, with reference to genome, (version is IRGSP 4.0 by the genomic high-flux sequence sequence alignment of these 1137 rice varieties to utilize Frederick Sanger comparison software (version number is 0.4), download address: http://www.ncbi.nlm.nih.gov) and tenuigenin with reference on genome, this tenuigenin comprises plastosome with reference to genome and chloroplast(id) reference genome with reference to genome, it is at NCBI (National Center forBiotechnology Information, US National Biotechnology Information center) on reception number be respectively NC_011033 and NC_001320.During contrast, Insert Fragment length is set to 500bp, and other parameter settings are default value.The Ssaha Pileup software package (version number is 0.5) adopted identifies the SNP site of each kind.This SNP site is defined as base pair that difference determines, the insertion of single base or the disappearance of single base.The base pair that this difference is determined refers to and does not comprise the uncertain base pair of difference, the uncertain base pair of difference refers to it is the base pair between some degeneracy base, as R represents A or G, therefore, may there are differences between A and R, also may not there are differences, therefore, between A and R, difference is indefinite, is not SNP mutually.Therefore, the SNP site in the embodiment of the present invention is not for comprise the uncertain base pair of above-mentioned difference.By the definition of above SNP site, the embodiment of the present invention obtains 7236888 SNP site altogether between all 1137 rice varieties, and wherein 59503 SNP site are positioned on cytoplasmic skeleton, and remaining SNP site is positioned on nuclear genome.Namely the genotype hereinafter mentioned refers to the combination of multiple SNP site in test zone, and nuclear gene type refers to that genotype is positioned on nuclear genome, and plasmagene type refers to that genotype is positioned on cytoplasmic skeleton.Such as, in table 1, the 8th test zone is positioned on nuclear genome, and be nuclear gene type, this test zone has 9 SNP site, and the genotype of this test zone is the combination of these 9 SNP site.

Adopt Proton high-flux sequence instrument to carry out high-flux sequence in the embodiment of the present invention, the test zone length that its order-checking detects can reach 200bp, and in order to obtain maximum fault information, the longest test zone in the present embodiment is also 200bp.Therefore, the variant sites that the present embodiment is mentioned refers to whole test zone, its inside may comprise multiple SNP site, namely the genotype hereinafter mentioned refers to the combination of multiple SNP site in test zone, nuclear gene type refers to that genotype is positioned on nuclear genome, and plasmagene type refers to that genotype is positioned on cytoplasmic skeleton.Such as, in table 1, the 8th test zone is positioned on nuclear genome, and be nuclear gene type, this test zone has 9 SNP site, and the genotype of this test zone is the combination of these 9 SNP site.The state-of-the-art method of current high throughput testing SNP site is gene chip, and often pair of chip probe can only detect 1 pair of SNP site, and the present invention have detected the combination (genotype) of multiple SNP site in test zone.

Two, by variant sites determination test zone, concrete grammar is as follows:

For rice varieties to be measured " R7723 " and " R8377 ", test zone is the large and equally distributed region of SNP site of discrimination on region or nuclear genome that on cytoplasmic skeleton, discrimination is large, wherein, and discrimination wherein, a is the kind sum be detected in variation window area, bi is i-th kind of genotypic kind number in variation window area, and bi>1, k comprises the genotypic number being greater than a kind, variation window area is centered by each single nucleotide variations site (SNP site), and the both sides to single nucleotide variations site respectively extend 1/2 of survey sequence length as the window detected; Test zone is the large and equally distributed region of discrimination on region or nuclear genome that on cytoplasmic skeleton, discrimination is large.The Computing Principle of discrimination is as follows: all interracial number of combinations are wherein, the combination between the different varieties in same gene type is undistinguishable, and its number is so, can not be by the ratio of the breed combination distinguished can by the ratio of breed combination distinguished and discrimination as can be seen here, discrimination is larger, more different varieties can be distinguished, and the test of variation window area to substantive derived relation that discrimination is large is more effective.If the variation window area skewness on nuclear genome, can cause some region adjacent, thus linkage inheritance, information is easily overlapping, therefore, nuclear genome is selected the principle of compositionality of test zone be: the large and SNP site of discrimination is uniformly distributed.Cytoplasmic skeleton without linkage inheritance problem, so, cytoplasmic skeleton only needs the region that selection area calibration is large.

First, centered by each SNP site obtained, respectively extend 99bp and 100bp to the left and right, form the variation window of 200bp.According to 7236888 SNP site obtained, 7236888 variation windows can be obtained, calculate the discrimination of these variation window areas such as, in the 1st variation window area, detect a=520 kind altogether, total k=3 kind genotype ACCT, CGTT, ACCC, their kind number is respectively b1=10, b2=30 and b3=431 is individual, therefore, its implication is: by the 1st variation window area, the breed combination of 31% in 520 kinds can be distinguished, the breed combination of other 79% cannot distinguish, and needs more variation window just can distinguish.After the same method, the discrimination of whole 7236888 the variation windows of calculating acquisition is also therefrom chosen and is arranged in 6800 maximum variation windows of nuclear genome discrimination, 200 the make a variation windows maximum with being arranged in cytoplasmic skeleton discrimination.Check 6800 the variation windows being arranged in nuclear genome one by one, each variation window and the next distance made a variation between window, if distance is more than 100K (1K=1000 base), reexamine after then abandoning the less variation window of wherein discrimination, till the adjacent distance looking into variation window is all greater than 100K.The criterion distance of 100K is selected to be because rice genome size is about 500M (1M=,100 ten thousand bases), the test zone that 2000 are positioned at nuclear genome is selected in by final, average test zone spacing is 250K, but because seldom there is variant sites in such as kinetochore etc., some specific regions, therefore, mean distance should be less than 250K.By above method, have selected the variation window that 4061 are positioned at nuclear genome, they with obtain to be arranged in together with 200 maximum windows that make a variations of cytoplasmic skeleton discrimination totally 4261 windows that make a variation as the test zone be selected in.Wherein, 200 variation windows that selection area calibration is maximum, be empirical value, this quantity can be modified as the case may be.

Three, respectively two kinds to be measured are sampled, extract and obtain the DNA of the sampling sample of two kinds to be measured, the preparation method of sampling sample is: after the sample mixing to two kinds difference random selecting more than 100 to be measured, obtain the sampling sample of two kinds to be measured.

In the present embodiment, have chosen 50000 seed germinations of kind to be measured " R7723 ", random selecting 30000 sizes roughly equal bud mixing be placed in mortar, fully pulverize add liquid nitrogen in mortar after.The article No. adopting Beijing Tian Gen biochemical technology company limited to produce is that the plant genome DNA extraction test kit of DP305 extracts and obtains the DNA of kind to be measured " R7723 " mixing sample, and DNA extraction method is undertaken by the operational manual of this test kit.American I nvitrigen company is utilized to produce dsDNA HS AssayKit (article No. is Q32852) and specification sheets thereof carry out quantitatively to the DNA obtained, and are 10.00ng/ μ l by the DNA dilution of the kind to be measured " R7723 " after quantitatively.

After the same method, sampling to rice varieties to be measured " R8377 " and extract DNA, is 10.00ng/ μ l by the DNA dilution of the rice varieties to be measured " R8377 " after quantitatively equally.

Four, the primer in amplification assay region is prepared, specific as follows:

Test zone adopts multiplex PCR (Polymerase Chain Reaction, polymerase chain reaction) technology to detect, and multiple PCR technique refers to and add multiple PCR primer, the multiple sites simultaneously in amplification gene group in same PCR reaction.The key of this technology designs and synthesizes multiple PCR primer, the multiple PCR technique that the present embodiment adopts LifeTechnology company of the U.S. to provide, and it can arrange the heavy PCR primer of as many as 12000.

The number of test zone meets the following conditions: BINOMDIST (SD*TN, TN, 0.80*SD, TRUE) >=95%, and wherein, 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 be TN, the genotypic ratio of product to be tested difference between species be 0.80*SD and decision threshold is SD time, judge that the probability guarantee that the genotypic ratio of product to be tested difference between species is less than decision threshold is more than or equal to 95%.The implication of this condition is: when the genotypic ratio of product to be tested difference between species is 80% of judgment threshold, and the judgement kind to be measured determined by the number of test zone has accuracy >=95% of substantive derived relation.The judgment threshold of substantive derived relation is Breeding Situation, mark mode according to various countries, requires Stringency and artificially determine.In the present embodiment, SD is defined as 3%.The number TN progressively strengthening test zone finds, when TN >=1934, above-mentioned formula is set up, therefore, the number of test zone should >=1934.Concerning existing SSR and SNP test, 200 test zones have calculated that it is enough, if the genotypic ratio of product to be tested difference between species is 80% of judgment threshold, its accuracy be only its accuracy only >=BINOMDIST (3%*200,200,0.80*3%, TRUE)=79%, therefore, the method that this enforcement provides, can obtain conclusion more accurately.

Primer acquisition process is as follows: log in LifeTechnology company multiple PCR primer Photographing On-line webpage https: //ampliseq.com/protected/help/pipelineDetails.action, submits relevant information to by its requirement.Wherein, in the present embodiment, " Application type " option selects " DNA Hotspot designs (single-pool) ".If select multi-pool, then multiplex PCR will divide multitube to carry out, cost can increase to some extent, and the primer of single-pool only needs a multiplex PCR, save cost, shortcoming is that some universal test regions design of primers may failure, but alternative universal test region on genome is more, therefore, abandon some alternative universal test regions and do not affect result.Permeate the nucleus of kind to be measured reference genome and tenuigenin reference genome a file, and select " Custom " in " Select the genome you wish to use " option after, upload the file of fusion as reference genome during design multiple PCR primer.DNA type option selects " Standard DNA ", in Add Hotspot option, add the positional information of the SNP site in the universal test region needing design, comprise chromosome information, the initiation site of SNP and the end locus of SNP, its certain embodiments is in table 1.Finally click " Submit targets " button to submit to and the multiple PCR primer obtaining design.In the present embodiment, from all 4261 test zones, design and be successfully authenticated 2231 pairs of multiple PCR primers, for corresponding 2231 test zones that increase.The method of checking multiple PCR primer is for pressing method provided by the invention, extract the leaves genomic DNA on same strain paddy rice, and utilize the multiple PCR primer of design to increase to the genomic dna obtained, build storehouse, high-flux sequence analyze sequenced fragments group, remove the corresponding primer of following test zone: the sequenced fragments number of this test zone is less than 1000 or there is hybrid strain genotype, and the primer remained is the multiple PCR primer be proved to be successful.So, test zone does not comprise amplification and produces the genotypic region of hybrid strain, hybrid strain genotype refers to frequency >=0.02%, and has insertion or the disappearance of discontinuous base in quantity >=2 of distinguishing base between all genotype of hybrid strain genotype and kind to be measured or distinguishing base.Because genomic DNA source is in same strain rice leaf, can not there is hybrid strain kind, therefore, hybrid strain genotype is the PCR or order-checking Preference mistake that are caused by the special construction of test zone, removes these test zones and avoids this type of system mistake.Regulation test zone is another object not comprising the genotypic test zone of amplification generation hybrid strain: the test zone remained is except being used as the test of interracial substantive derived relation to be measured, the calculating of hybrid strain rate can also be done, achieve the multiple use of same set of test primer.The multiple PCR primer be proved to be successful is supplied to client in fluid form and uses after also being mixed by the said firm.2231 test zones of above-mentioned successful design multiple PCR primer are the test zone finally detected for kind to be measured, and wherein, 100 test zones are positioned on cytoplasmic skeleton, remaining 2131 test zones are positioned on nuclear genome.Existing Essentially derived variety judges the test site all do not adopted on cytoplasmic skeleton, and cytoplasmic difference, different varietal character performances can be produced equally, the judgement of Essentially derived variety relation should be used for.

Five, utilize primer to increase to the DNA of two sampling samples respectively, obtain the amplified production of two kinds to be measured at test zone respectively, amplified production is respectively used to the high-throughput sequencing library of structure two kinds to be measured, and concrete grammar is as follows:

After utilizing library construction Kit 2.0 (produced by LifeTechnology company of the U.S., article No. is 4475345) multiplexed PCR amplification test zone, amplified production is utilized to build high-throughput sequencing library.This test kit comprises following reagent: 5 × Ion AmpliSeq ^tMhiFi Mix, FuPa reagent, transferring reagent, sequence measuring joints solution and DNA ligase.The method of library construction presses operational manual " the Ion AmpliSeq of this test kit ^tMlibraryPreparation " (publication number: MAN0006735, version: A.0) carry out.By multiplexed PCR amplification 2231 test zones, the amplification system of multiplex PCR is as follows: 5 × Ion AmpliSeq ^tMthe test zone primer mixed solution 4 μ l of HiFi Mix 4 μ l, preparation, the DNA 10ng of rice varieties to be measured " R7723 " and without enzyme water 11 μ l.The amplification program of multiplex PCR is as follows: 99 DEG C, 2 minutes; (99 DEG C, 15 seconds; 60 DEG C, 4 minutes) × 25 circulations; 10 DEG C of insulations.After utilizing FuPa reagent to digest primer unnecessary in multiplexed PCR amplification product, then carry out phosphorylation, concrete grammar is: in the amplified production of multiplex PCR, add 2 μ L FuPa reagent, after mixing, by following program reaction in PCR instrument: 50 DEG C, and 10 minutes; 55 DEG C, 10 minutes; 60 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixture a, and mixture a is containing the amplified production solution through phosphorylation.The amplified production of phosphorylation is connected upper sequence measuring joints, and concrete grammar is: in mixture a, add transferring reagent 4 μ L, sequence measuring joints solution 2 μ L and DNA ligase 2 μ L, after mixing, by following program reaction in PCR instrument: 22 DEG C, and 30 minutes; 72 DEG C, 10 minutes; 10 DEG C of preservations, obtain mixed solution b.10 μ L are dissolved in without in enzyme water after utilizing the ethanol precipitation methods purifying mixed solution b of standard.American I nvitrigen company is utilized to produce dsDNA HS Assay Kit (article No. is Q32852) also measures according to its specification sheets, and after obtaining the mass concentration of mixed solution b, mixed solution b after purifying is diluted to 15ng/ml, obtains the high-throughput sequencing library that concentration is about the test zone of 100pM.

After the same method, rice varieties to be measured " R8377 " is carried out to the structure of high-throughput sequencing library, obtain the high-throughput sequencing library that concentration is about the test zone of 100pM equally.

Six, carry out high-flux sequence respectively to the high-throughput sequencing library of two kinds to be measured, obtain the sequenced fragments group of two kinds to be measured respectively, method is as follows:

Determine the high-flux sequence degree of depth: the degree of depth >=5000 times of high-flux sequence, i.e. segments >=5000 fragment in average coverage test district, 5000 times is an empirical value, can adjust according to practical situation.Why specify this value, be because the order-checking amount cost of 5000 times not high but be enough to accurately calculate 30% testing gene type frequency, therefore, specify 5000 times of degree of depth as high-flux sequence.

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

Utilize high-throughput sequencing library and test kit Ion PI Template OT2200Kit v2 (invirtrigen company of the U.S. production of all test zones obtained, article No. is 4485146) check order before ePCR (Emulsion PCR, emulsion polymerization enzyme chain reaction) amplification, working method is undertaken by the operational manual of this test kit.(invirtrigen company of the U.S. produces to utilize ePCR product and test kit Ion PI Sequencing 200Kit v2, article No. is 4485149) on Proton bis-generation high-flux sequence instrument, carry out high-flux sequence, working method is undertaken by the operational manual of this test kit.In the present embodiment, high-flux sequence flux is set to 30000 times, average coverage test region.

Pre-treatment is carried out to a large amount sequencing result

By the comparison of high-flux sequence fragment to all 2231 test zones, after removing the sequenced fragments that comparison is unsuccessful and genotype detection is incomplete, remaining all sequenced fragments are called sequenced fragments group.The incomplete sequenced fragments of genotype detection refers to the sequenced fragments that all SNP site shown in " position of SNP on reference genome " in table 1 could not be detected, the reason that genotype detection is incomplete is that sequenced fragments is too short, and the unsuccessful reason of comparison is that sequenced fragments mostly is non-specific amplification product.

Seven, analyze the sequenced fragments group of two kinds to be measured, obtain two variety and genetypes to be measured respectively, variety and genetype to be measured is the combination of variation base in test zone, and genotypic frequency >=30% of kind to be measured, concrete grammar is as follows:

By the comparison of sequenced fragments group to all test zones, and add up the sequenced fragments number in each test zone, remove the test zone of sequenced fragments number≤1000, remaining test zone is for detecting successful test zone.In the present embodiment, obtain 2029 altogether and detect successful test zone.Comparison is called the sequenced fragments of this test zone to the fragment of test zone, and the base composition extracting in table 1 position shown in " SNP with reference to the position on genome " from sequenced fragments is called the genotype of this sequenced fragments.Genotypic frequency refers in sequenced fragments group, represents the ratio that this genotypic sequenced fragments number accounts for the sequenced fragments sum of this genotype place test zone.Variety and genetype to be measured is the combination of variation base in test zone, and genotypic frequency >=30% of kind to be measured.In general, in the sample extracted, the amount of hybrid is not higher than 10%, order-checking mistake is no more than 1%, the two total is no more than 11%, therefore, for site of isozygotying, variety and genetype to be measured only has one, and its frequency should be greater than 89%, and for heterozygous sites, variety and genetype to be measured has 2 kinds, and its ratio should be greater than 45.5%, therefore, specify genotypic frequency >=30% of kind to be measured, can get rid of and have hybrid strain and interference to variety and genetype to be measured because mixing in check order mistake and kind to be measured.

Such as, in sequenced fragments group, the sequenced fragments in the 1st order-checking region adds up to 29888 articles, there are ACCC, CGTT, CCCC, GCCC ... totally 41 kinds of genotype, represent these genotypic sequenced fragments numbers 29603,14,2,1 respectively ..., these genotypic frequencies are 29603/29888=99.05%, 14/29888=0.05%, 2/29888=0.007%, 1/29888=0.003% ...By the genotypic definition of kind to be measured, ACCC is kind to be measured " R7723 " genotype of the 1st test zone, and other genotype is the genotype that order-checking mistake or hybrid strain cause.By identical method, judge and obtain whole 2029 to detect kind to be measured " R7723 " genotype of successful test zones.

By the method identical with rice varieties to be measured " R7723 ", 2029 that obtain rice varieties to be measured " R8377 " are equally detected successful test zone, and rice varieties to be measured " R8377 " is in the genotype of the successful test zone of all detections, partial results is in table 1.The present embodiment does not have completely to list whole kind to be measured in all test zone genotype as space is limited, only lists certain embodiments.Equally based on length restriction, also have some areas also only to list part related example in the present embodiment, all the other unlisted data can according to the method completion of the present embodiment.

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

Eight, compare two variety and genetypes to be measured, obtain the genotypic ratio of product to be tested difference between species, concrete grammar is as follows:

If in the test, all variety and genetypes to be measured, all without disappearance, claim this test zone to be the total test zone of kind to be measured.In total test zone, if genotype is incomplete same between kind to be measured, this genotype is then claimed to be product to be tested difference between species genotype, such as, if the genotype of kind A to be measured is AA, AA, AA, AA, AA/CC, AA/CC, the genotype that kind B to be measured is corresponding is AA, CC, AA/CC, CC/TT, AA/CC, CC/TT, and so in these 6 test zones, product to be tested difference between species genotype judges as follows: be not, be, be, be, be not and be.It is genetic heterozygosis site that "/" in above genotype represents this test zone, therefore detects latter two genotype before "/".The number of the number/total test zone of the genotypic ratio of product to be tested difference between species=the have test zone of Differential genotype.

In the present embodiment, rice varieties to be measured " R8377 " is 2029 with the total test zone of rice varieties to be measured " R7723 ", in the 1st total test zone, their genotype is respectively ACCC and ACCC, they are identical, therefore, are not interracial Differential genotype to be measured.According to said method, judge, in all 2029 total test zones, whether to there are differences genotype one by one, result is: in all total test zones, there are differences genotypic test zone number is 0, so, the genotypic ratio OD=0/2029=0% of product to be tested difference between species.

According to the genotypic ratio of product to be tested difference between species and decision threshold, judge the substantive derived relation of two kinds to be measured, judge that the method for the substantive derived relation of two kinds to be measured is: as the genotypic ratio < SD of product to be tested difference between species, two kinds to be measured have substantive derived relation; As the genotypic ratio >=SD of product to be tested difference between species, two kinds to be measured do not have substantive derived relation, and wherein, SD is decision threshold.

In the present embodiment, the genotypic ratio=0%<SD=3% of product to be tested difference between species, therefore judges to have substantive derived relation between kind to be measured " R8377 " and kind to be measured " R7723 ".

If two when judging that kind to be measured has substantive derived relation, probability >=BINOMDIST (SD*TRN, TRN, OD, TRUE) that conclusion is correct; If when judging that two kinds to be measured do not have substantive derived relation, probability >=BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) that conclusion is correct; Wherein, TRN is the number of total test zone, SD is decision threshold, OD is the genotypic ratio of product to be tested difference between species, and BINOMDIST is excel 2010 function, BINOMDIST (SD*TRN, TRN, OD, TRUE) implication be: when the number of total test zone is TRN, the genotypic ratio OD of product to be tested difference between species is less than the probability of decision threshold SD; BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) implication is: when the number of total test zone is TRN, the genotypic ratio OD of product to be tested difference between species is greater than the probability of decision threshold SD.

In the present embodiment, judge that two kinds to be measured have substantive derived relation and the number of total test zone is 2029, therefore, the probability that conclusion is correct >=BINOMDIST (SD*TRN, TRN, OD, TRUE)=BINOMDIST (3%*2029,2029,0%, TRUE)=100%, therefore, in this enforcement, judge that the conclusion that two rice varieties to be measured have substantive derived relation is very accurately.

Nine, result verification

At present, the standard method that between the whole world kind that also neither one is generally acknowledged, substantive derived relation is assert, but 1991 of UPOV have such description in text: " Essentially derived variety can by such as natural or bring out saltant type, somaclonal variation type selection; from the selection of the variation strain original variety plant, and backcross or genetic engineering transform obtain ".In the present embodiment, rice varieties to be measured " R7723 " is by rice varieties to be measured " R8377 " as recurrent parent, backcross breeding and come, so by the description of UPOV text, these two rice varieties to be measured should have substantive derived relation, show, the conclusion of the present embodiment is correct.

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

The kind to be measured that the embodiment of the present invention provides is corn variety " GL95 " and " 1102 ", and the two is open, known kind.Because the method in the method in the present embodiment and embodiment one is similar, therefore, identical place is just no longer repeated, and only lists different places.

Variant sites between different corn variety can obtain from the documents and materials announced, but the results contrast that the method obtains is fragmentary, in the present embodiment, by the genome sequence of different corn and the genome sequence with reference to corn variety are compared, obtain the variant sites between a large amount of different corn varieties, wherein reference corn variety can be " B73 " corn, should can replace with other known reference corn varieties by " B73 " corn.

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

The genome sequence of the different corn varieties of the present embodiment shows two kinds of sources, the first is the genomic high-flux sequence sequence to 103 corn varieties such as Chia, pertinent literature information is as follows: Chia JM et al.MaizeHapMap2 identifies extant variation from a genome in flux.Nat Genet.2012,44 (7): 803-7.The genome sequence of these 103 corn varieties is published in NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/sra), and reception number is SRA051245; The second is for having carried out high-flux sequence by the method provided in the above-mentioned article delivered of Chia etc. to " GL95 ", " 1102 " and cross-fertilize seed " high bad 145 ".The present embodiment obtains the genomic high-flux sequence sequence of 106 corn varieties altogether.

Particularly, because the order-checking degree of depth of these 106 corn varieties is not high, only can identify single nucleotide variations (SNP) site, such as the repeat number of other variation type makes a variation, and due to a low credibility, does not identify.To " B73 " maize cell core, with reference to genome, (version is IRGSP 4.0 by the genomic high-flux sequence sequence alignment of these 106 corn varieties to utilize Frederick Sanger comparison software (version number is 0.4), download address: http://www.ncbi.nlm.nih.gov) and tenuigenin with reference on genome, this tenuigenin comprises plastosome with reference to genome and chloroplast(id) reference genome with reference to genome, it is at NCBI (National Center forBiotechnology 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 embodiment of the present invention obtains 53855606 SNP site altogether between all 106 corn varieties, and wherein 9005 SNP site are positioned on cytoplasmic skeleton, and remaining SNP site is positioned on nuclear genome.

Two, determined the test zone of two kinds to be measured respectively by variant sites, concrete grammar is as follows:

For corn variety to be measured " GL95 " and " 1102 ", test zone is the large and equally distributed region of SNP site of discrimination on region or nuclear genome that on cytoplasmic skeleton, discrimination is large, wherein, and discrimination wherein, a is the kind sum be detected in variation window area, and bi is i-th kind of genotypic kind number in variation window area, and bi>1, k comprise the genotypic number being greater than a kind.The Computing Principle of discrimination is as follows: all interracial number of combinations are wherein, the combination between the different varieties in same gene type is undistinguishable, and its number is so, can not be by the ratio of the breed combination distinguished can by the ratio of breed combination distinguished and discrimination as can be seen here, discrimination is larger, more different varieties can be distinguished, and the test of variation window area to substantive derived relation that discrimination is large is more effective.If the variation window area skewness on nuclear genome, can cause some region adjacent, thus linkage inheritance, information is easily overlapping, therefore, nuclear genome is selected the principle of compositionality of test zone be: the large and SNP site of discrimination is uniformly distributed.Cytoplasmic skeleton without linkage inheritance problem, so, cytoplasmic skeleton only needs the region that selection area calibration is large.

First, centered by each SNP site obtained, respectively extend 99bp and 100bp to the left and right, form the variation window of 200bp.According to 53855606 SNP site obtained, 53855606 variation windows can be obtained, calculate the discrimination of these variation window areas such as, in the 1st variation window area, detect a=102 kind altogether, total k=3 kind genotype CCA, TCA, TCG, their kind number is respectively b1=5, b2=11 and b3=76 is individual, therefore, its implication is: by the 1st variation window area, the breed combination of 43% in 102 kinds can be distinguished, the breed combination of other 47% cannot distinguish, and needs more variation window just can distinguish.After the same method, the discrimination of whole 53855606 the variation windows of calculating acquisition is also therefrom chosen and is arranged in 8000 maximum variation windows of nuclear genome discrimination, 100 the make a variation windows maximum with being arranged in cytoplasmic skeleton discrimination.Check 8000 the variation windows being arranged in nuclear genome one by one, each variation window and the next distance made a variation between window, if distance is more than 500K (1K=1000 base), reexamine after then abandoning the less variation window of wherein discrimination, till the adjacent distance looking into variation window is all greater than 500K.The criterion distance of 500K is selected to be because Maize genome size is about 2300M (1M=,100 ten thousand bases), the test zone that 2400 are positioned at nuclear genome is selected in by final, average test zone spacing is 1M, but because seldom there is variant sites in such as kinetochore etc., some specific regions, therefore, mean distance should be less than 1M.By above method, have selected the variation window that 5030 are positioned at nuclear genome, they with obtain to be arranged in together with 100 maximum windows that make a variations of cytoplasmic skeleton discrimination totally 5130 windows that make a variation as the test zone be selected in.Wherein, 100 variation windows that selection area calibration is maximum, be empirical value, this quantity can be modified as the case may be.

Three, sample to two kinds to be measured respectively, extract and obtain the DNA of the sampling sample of two kinds to be measured, the preparation method of sampling sample is: after the sample mixing to two kinds to be measured random selecting more than 100 respectively, obtain sample of sampling.

In the present embodiment, have chosen 5000 seed germinations of corn variety to be measured " GL95 ", random selecting 4000 sizes roughly equal bud mixing be placed in mortar, fully pulverize add liquid nitrogen in mortar after.The article No. adopting Beijing Tian Gen biochemical technology company limited to produce is that the plant genome DNA extraction test kit of DP305 extracts and obtains the DNA of corn variety to be measured " GL95 " mixing sample, and DNA extraction method is undertaken by the operational manual of this test kit.American I nvitrigen company is utilized to produce dsDNA HSAssay Kit (article No. is Q32852) and specification sheets thereof carry out quantitatively to the DNA obtained, and are 10.00ng/ μ l by the DNA dilution of the corn variety to be measured " GL95 " after quantitatively.

After the same method, sampling to corn variety to be measured " 1102 " and extract DNA, is 10.00ng/ μ l by the DNA dilution of the corn variety to be measured " 1102 " after quantitatively equally.

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

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

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

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

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

Pre-treatment is carried out to a large amount sequencing result

By the comparison of high-flux sequence fragment to all 2506 test zones, after removing the sequenced fragments that comparison is unsuccessful and genotype detection is incomplete, remaining all sequenced fragments are called sequenced fragments group.The incomplete sequenced fragments of genotype detection refers to the sequenced fragments that all SNP site shown in " position of SNP on reference genome " in table 2 could not be detected, the reason that genotype detection is incomplete is that sequenced fragments is too short, and the unsuccessful reason of comparison is that sequenced fragments mostly is non-specific amplification product.

By the method identical with embodiment one, judge and obtain corn variety to be measured " GL95 " genotype of whole 2465 successful test zones of detection.

By the method identical with corn variety to be measured " GL95 " extract corn variety to be measured " 1102 " DNA, build high-throughput sequencing library, high-flux sequence, analysis sequenced fragments group, obtain 2465 equally and detect successful test zones and corn variety to be measured " 1102 " genotype at the successful test zone of all detections, partial results is in table 2.The present embodiment does not have completely to list whole corn variety to be measured in all test zone genotype as space is limited, only lists certain embodiments.Equally based on length restriction, also have some areas also only to list part related example in the present embodiment, all the other unlisted data can according to the method completion of the present embodiment.

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

Eight, compare two variety and genetypes to be measured, obtain the genotypic ratio of product to be tested difference between species, method is as follows:

In the present embodiment, corn variety to be measured " 1102 " is 2465 with the total test zone of corn variety to be measured " GL95 ", in the 1st total test zone, their genotype is respectively TCG and TCG, they are identical, therefore, are not interracial Differential genotype to be measured.According to said method, judge, in all 2465 total test zones, whether to there are differences genotype one by one, result is: in all total test zones, there are differences genotypic test zone number is 63, so, the genotypic ratio OD=63/2465=2.56% of product to be tested difference between species.

In the present embodiment, the genotypic ratio=2.56%<SD=3% of product to be tested difference between species, therefore judges to have substantive derived relation between corn variety to be measured " 1102 " and corn variety to be measured " GL95 ".

In the present embodiment, judge, between two kinds to be measured, there is substantive derived relation and the number of total test zone is 2465, therefore, the probability that conclusion is correct >=BINOMDIST (SD*TRN, TRN, OD, TRUE)=BINOMDIST (3%*2465,2465,2.56%, TRUE)=90.53%, therefore, in this enforcement, judge that the conclusion that two kinds to be measured have substantive derived relation is more accurately.

Nine, result verification

At present; there is no the detection method of substantive derived relation between a kind of kind of standard; international New variety protection alliance gave the definition of substantive derived relation in 1991; according to this definition; if A and B two Differences are less; show that genetic connection is comparatively near, can judge that there is substantive derived relation.According to this definition, in the present embodiment, two kinds to be measured are planted by the method in " new variety of plant specificity, consistence and stability test guide-corn " and observed the multiple situations between two corn varieties to be measured, in investigated 70 proterties, only 3 Traits change reach conspicuous level, show that these two product to be tested difference between speciess are little, probably there is substantive derived relation, conform to the conclusion that the present embodiment obtains.

The embodiment of the present invention, by high-flux sequence and the amplification of multidigit point, achieves the large sample sampling in kind build-in test region to be measured, ensure that the accuracy that substantive derived relation detects.Meanwhile, utilize multidigit point amplification technique, the testing quality entity of multiple test zone is equivalent to the workload of the test zone that traditional SSR or SNP detects, therefore, the present embodiment is not only accurate, and method is simple, quick.For embodiment one, 2029 test zones are detected if same, utilize traditional SSR or SNP detection method, (SSR detects by electrophoresis method to need to carry out the detection of the amplified production of 2029 pcr amplifications and 2029 times, SNP detects by the method for generation test), its workload, speed and testing cost are all can not be received.In addition, obtain the sequence of each base in test zone in the present embodiment, resolving power has reached ultimate attainment, and quantity of information is also maximum, that other detection method is all incomparable, such as, the length information of fragment that what SSR obtained is, possible Problems existing is: fragment length difference is too small, may can't detect, even the fragment of equal length, the based composition of its inside also may be different, but SSR can't detect equally.A SNP detects and can only detect a pair SNP site, and in the present embodiment, can detect all SNP of test zone, can also find the variation in test zone outside SNP, and as repeat number, disappearance and insertion etc., these single SNP detect and cannot find.In addition, except the order-checking of an employing generation detects except SNP, also have and adopt the mode of hybridization to detect SNP, in this case, what obtain is hybridization signal, and noise is large and cannot find unknown variation, and a probe only can detect a pair SNP, resolving power and quantity of information are also much smaller than the present invention.

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

Claims

1. a method for the substantive derived relation of test plants, it is characterized in that, described method comprises:

By described variant sites determination test zone;

The primer of the described test zone of preparation amplification;

2. method according to claim 1, is characterized in that, described test zone does not comprise amplification and produces the genotypic region of hybrid strain;

3. method according to claim 1, is characterized in that, the number of described test zone meets the following conditions: BINOMDIST (SD*TN, TN, 0.80*SD, TRUE) >=95%, and wherein, TN is the number of described test zone, and SD is decision threshold; The condition implication that the number of described test zone meets is: when the number of described test zone be TN, described decision threshold be SD and the genotypic ratio of described product to be tested difference between species is 0.80*SD time, judge that the probability guarantee that the genotypic ratio of described product to be tested difference between species is less than described decision threshold SD is more than or equal to 95%.

4. method according to claim 1, it is characterized in that, to the method that two described kinds to be measured are sampled be respectively: the sampling sample obtaining two described kinds to be measured after the sample mixing to two described kinds difference random selecting more than 100 to be measured.

5. method according to claim 1, is characterized in that, judges that the method for the substantive derived relation of two described kinds to be measured is:

6. method according to claim 5, is characterized in that, if when judging that two described kinds to be measured have substantive derived relation, and probability >=BINOMDIST (SD*TRN, TRN, OD, TRUE) that conclusion is correct; If when judging that two described kinds to be measured do not have substantive derived relation, probability >=BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) that conclusion is correct; Wherein, TRN is the number of the total test zone of two described kinds to be measured, OD is the genotypic ratio of described product to be tested difference between species, BINOMDIST is excel 2010 function, BINOMDIST (SD*TRN, TRN, OD, TRUE) implication is: when the number of described total test zone is TRN, and the genotypic ratio OD of described product to be tested difference between species is less than the probability of described decision threshold SD; BINOMDIST ((1-SD) * TRN, TRN, 1-OD, TRUE) implication is: when the number of described total test zone is TRN, the genotypic ratio OD of described product to be tested difference between species is greater than the probability of described decision threshold SD.

7. method according to claim 1, is characterized in that, determines that the method for described test zone is by described variant sites: