CN104878085A - New method for testing authenticity and proportion of parental origin of rape - Google Patents
New method for testing authenticity and proportion of parental origin of rape Download PDFInfo
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Abstract
The invention discloses a new method for testing the authenticity and the proportion of a parental origin of rape and belongs to the technical field of biology. The method comprises the following steps: obtaining mutation sites between different rape varieties; determining a test region according to the mutation sites; extracting the DNA of a sampled sample; preparing a PCR primer of the test region; constructing a high-flux sequencing library; performing high-flux sequencing on the high-flux sequencing library to obtain a sequencing fragment group; analyzing the sequencing fragment group to obtain the genotype and the parental genotype of a rape variety to be tested; determining the authenticity of the parental origin of the rape variety to be tested and calculating the proportion of the parental origin according to the genotype and the parental genotype of the rape variety to be tested. The method is capable of accurately, quickly and simply determining the authenticity and the proportion of the parental origin.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of rape parental source verity and ratio test novel method thereof.
Background technology
China's crop varieties carries out authorization system, for the object of intellectual property protection, requires that the kind of participating in authorization provides parental source and breeding process.Parental source and breeding process are also the auxiliary foundations of qualification Essentially derived variety.But owing to relating to commercial benefits, breed of variety person not necessarily provides real parental source and breeding process, examination department is needed to identify.Wherein, the qualification of breeding process verity can be inferred by the ratio of parent's blood relationship in rape variety to be measured.
But, at present, there is no a kind of reliable method qualification parental source verity and the ratio of parent's blood relationship in rape variety to be measured.
Summary of the invention
Identifying the insecure problem of the method for rape variety parental source verity and parent's blood relationship to solve in prior art, present embodiments providing a kind of rape parental source verity and ratio test novel method thereof.Described technical scheme is as follows:
Present embodiments provide a kind of rape parental source verity and ratio test novel method thereof, described method comprises:
Obtain the variant sites between different rape variety;
According to described variant sites determination test zone;
Respectively the parent of rape variety to be measured and described rape variety to be measured is sampled, extract and obtain the DNA of the sampling sample of the DNA of the sampling sample of described rape variety to be measured and the parent of described rape variety to be measured;
The primer of the described test zone of preparation amplification;
Described primer is utilized to increase to the DNA of the sampling sample of the DNA of the sampling sample of described rape variety to be measured and the parent of described rape variety to be measured respectively, obtain the amplified production of the amplified production of described rape variety to be measured and the parent of described rape variety to be measured respectively, and build the high-throughput sequencing library of the high-throughput sequencing library of described rape variety to be measured and the parent of described rape variety to be measured respectively with the described amplified production obtained;
Respectively high-flux sequence is carried out to the high-throughput sequencing library of the high-throughput sequencing library of described rape variety to be measured and the parent of described rape variety to be measured, obtain the sequenced fragments group of the sequenced fragments group of described rape variety to be measured and the parent of described rape variety to be measured;
Analyze the sequenced fragments group of the sequenced fragments group of described rape variety to be measured and the parent of described rape variety to be measured, obtain rape variety genotype to be measured and parent genotype, described rape variety genotype to be measured is that described rape variety to be measured makes a variation the combination of base in described test zone, and genotypic frequency >=30% of described rape variety to be measured, described parent genotype is that described parent makes a variation the combination of base in described test zone, and frequency >=30% of described parent genotype;
According to described rape variety genotype to be measured and described parent genotype, judge the verity of the parental source of described rape variety to be measured and calculate the ratio of parental source.
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 described genotype of described hybrid strain genotype and described rape variety to be measured or described distinguishing base.
Particularly, to the method that the parent of described rape variety to be measured and described rape variety to be measured samples be respectively: after the sample of the rape variety described to be measured of random selecting more than 100 and the parent of described rape variety to be measured mixes respectively, obtain the sampling sample of the sampling sample of described rape variety to be measured and the parent of described rape variety to be measured.
Particularly, the method for the verity of the parental source of described judgement rape variety to be measured is: if there is non-parent's genotype in described rape variety to be measured, then the parental source of described rape variety to be measured is untrue; If there is not described non-parent's genotype in described rape variety to be measured, then the parental source of described rape variety to be measured is true; Described non-parent's genotype is described rape variety genotype to be measured, and distinguishing base number >=2 of described non-parent's genotype and any described parent genotype.
Particularly, the formula calculating the ratio of parental source is: the ratio of parental source
wherein, n is the number of the peculiar test zone of parent; I is i-th peculiar test zone of described parent; Si is in i-th peculiar test zone of described parent, genotypic number identical between the peculiar genotype of parent with described rape variety genotype to be measured; Ti is the genotypic number of rape variety to be measured described in i-th peculiar test zone of described parent; The peculiar genotype of described parent is the described parent genotype only occurred in described parent, and the peculiar test zone of described parent refers to have the peculiar genotypic described test zone of described parent.
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.
Particularly, the degree of depth >=5000 times of described high-flux sequence.
Particularly, described primer is the multiplex amplification primer that provides of match Mo Feishier company of the U.S..
The beneficial effect that the technical scheme that the present embodiment provides is brought is: the method that the present embodiment provides is increased and high-flux sequence by multidigit point, ensure the large sample sampling of the test zone of rape variety to be measured, successfully achieve the target of accurately test parental source verity and ratio thereof, 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 rape variety " Soviet Union 2051 " parental source verity and ratio
The rape variety to be measured that the present embodiment provides is rape variety " Soviet Union 2051 ", needs to identify the parental source of its " P65 " and " Shanghai 8920 " of being whether, above parent be disclose, known kind.
One, the variant sites between different rape variety is obtained.
Variant sites between different rape 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 rape and the genome sequence with reference to rape variety being compared, obtain the variant sites between a large amount of different rape varieties.
Further, the method obtaining the genome sequence of different rape variety is as follows:
The genome sequence of the different rape varieties of the present embodiment shows two kinds of sources, the first is the genomic high-flux sequence sequence to 10 rape varieties such as Huang, and pertinent literature information is as follows: Huang et al.:Identification of genome-wide single nucleotide polymorphisms in allopolyploid crop Brassica napus.BMC Genomics 201314:717.The genome sequence of these 10 rape varieties is published in NCBI Short Read Archive (http://www.ncbi.nlm.nih.gov/sra), and reception number is SRA057227; The second is for having carried out high-flux sequence by the method provided in the above-mentioned article delivered of Huang etc. to " 430AB ", " P65 " and cross-fertilize seed " peaceful assorted No. 9 ".The present embodiment obtains the genomic high-flux sequence sequence of 13 rape 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 13 rape 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.Utilize Frederick Sanger comparison software (version number is 0.4) by the genomic high-flux sequence sequence alignment of these 13 rape varieties to rape cell core reference genome (version: Release v1.01, 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 for Biotechnology Information, US National Biotechnology Information center) on reception number be respectively NC_016734.1 and AP006444.1.During contrast, Insert Fragment length is set to 500bp, and other parameter settings are default value.The Ssaha Pileup software package (version number is 0.5) adopted identifies the SNP site of each 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 present embodiment is not for comprise the uncertain base pair of above-mentioned difference.By the definition of above SNP site, the present embodiment obtains 911346 SNP site altogether between all 13 rape varieties, and wherein 18543 SNP site are positioned on cytoplasmic skeleton, and remaining SNP site is positioned on nuclear genome.Namely the genotype hereinafter mentioned refers to the combination of multiple SNP site in test zone, and nuclear gene type refers to that genotype is positioned on nuclear genome, and plasmagene type refers to that genotype is positioned on cytoplasmic skeleton.Such as, in table 1, the 1st test zone is positioned on nuclear genome, and be nuclear gene type, this test zone has 3 SNP site, and the genotype of this test zone is the combination of these 3 SNP site.
Two, according to variant sites determination test zone, concrete grammar is as follows:
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, and the both sides to single nucleotide variations site respectively extend 1/2 of survey sequence length as the window detected.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 parental source verity and ratio thereof 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 911346 SNP site obtained, 911346 variation windows can be obtained, calculate the discrimination of these variation window areas
such as, in the 1st variation window area, detect a=13 kind altogether, total k=2 kind genotype CCA, TTA, their kind number is respectively b1=4 and b2=7, therefore,
its implication is: by the 1st variation window area, the breed combination of 65% in 13 kinds can be distinguished, the breed combination of other 35% cannot distinguish, and needs more variation window just can distinguish.After the same method, the discrimination of whole 911346 the variation windows of calculating acquisition is also therefrom chosen and is arranged in 6000 maximum variation windows of nuclear genome discrimination, 100 the make a variation windows maximum with being arranged in cytoplasmic skeleton discrimination.Check 6000 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 200K (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 200K.The criterion distance of 200K is selected to be because rapeseed gene group size is about 930M (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 about 500K, but because seldom there is variant sites in such as kinetochore etc., some specific regions, therefore, mean distance should be less than 500K.By above method, have selected the variation window that 4367 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 4467 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, respectively the parent of rape variety to be measured and rape variety to be measured is sampled, extract and obtain the DNA of the sampling sample of the DNA of the sampling sample of rape variety to be measured and the parent of rape variety to be measured, the preparation method of sampling sample is: after the sample of the rape variety to be measured of random selecting more than 100 and the parent of rape variety to be measured mixes respectively, obtain the sampling sample of the sampling sample of rape variety to be measured and the parent of rape variety to be measured.
In the present embodiment, have chosen 10000 seed germinations of rape variety to be measured " Soviet Union 2051 ", the roughly equal bud mixing of random selecting 8000 sizes is placed in mortar, fully pulverizes after adding liquid nitrogen in mortar.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 rape variety to be measured " Soviet Union 2051 " 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 Assay Kit (article No. is Q32852) and specification sheets thereof carry out quantitatively to the DNA obtained, and the DNA dilution that the rape variety to be measured quantitatively " is revived 2051 " is 10.00ng/ μ l.
After the same method, respectively parent " P65 " and " Shanghai 8920 " sampled respectively and extract DNA, equally the parent " P65 " after quantitatively and " Shanghai 8920 " DNA being diluted respectively for 10.00ng/ μ l.
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 match Mo Feishier company of the U.S. to provide, and it can arrange the heavy PCR primer of as many as 12000.
Primer acquisition process is as follows: log in match Mo Feishier company multiple PCR primer Photographing On-line webpage https: //ampliseq.com/protected/help/pipelineDetails.action, submit 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 rape variety 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 4467 test zones, design and demonstrate 2302 pairs of multiple PCR primers, for corresponding 2302 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 rape, 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 rape variety to be measured or distinguishing base.Because genomic DNA source is in same strain rape 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, except for except the present invention, can also do the calculating of hybrid strain rate, achieves 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.2302 test zones of above-mentioned successful design multiple PCR primer are the test zone finally detected for rape variety to be measured, and wherein, 55 test zones are positioned on cytoplasmic skeleton, remaining 2247 test zones are positioned on nuclear genome.The method of existing Molecular Identification kind does not comprise thin chest plasmagene substantially, but plasmone affects breediness equally, should be included among cultivar identification.
Five, the DNA of the sampling sample of the DNA of the sampling sample of primer pair rape variety to be measured and the parent of rape variety to be measured is utilized to increase, obtain the amplified production of the amplified production of rape variety to be measured and the parent of rape variety to be measured respectively, and the high-throughput sequencing library of the high-throughput sequencing library of rape variety to be measured and the parent of rape variety to be measured is built respectively with the amplified production obtained, method is as follows:
After utilizing library construction Kit 2.0 (match Mo Feishier company by the U.S. to produce, 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
tMlibrary Preparation " (publication number: MAN0006735, version: A.0) carry out.By multiplexed PCR amplification 2302 test zones, the amplification system of multiplex PCR is as follows: 5 × Ion AmpliSeq
tMthe DNA 10ng of the test zone primer mixed solution 4 μ l of HiFi Mix 4 μ l, preparation, rape variety to be measured " Soviet Union 2051 " 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, to the structure that parent " P65 " and " Shanghai 8920 " carry out high-throughput sequencing library respectively, the high-throughput sequencing library that concentration is about the parent of 100pM is obtained equally.
Six, respectively high-flux sequence is carried out to the high-throughput sequencing library of the high-throughput sequencing library of rape variety to be measured and the parent of rape variety to be measured, obtain the sequenced fragments group of the sequenced fragments group of rape variety to be measured and the parent of rape variety to be measured, 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 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 2302 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, the sequenced fragments group of the sequenced fragments group of rape variety to be measured and the parent of rape variety to be measured is analyzed, obtain rape variety genotype to be measured and parent genotype, rape variety genotype to be measured is that rape variety to be measured makes a variation the combination of base in test zone, and genotypic frequency >=30% of rape variety to be measured, parent genotype is that parent makes a variation the combination of base in test zone, and frequency >=30% of parent genotype, method is as follows:
By the comparison of sequenced fragments group to all test zones, and add up the sequenced fragments number in each test zone, remove the test zone of sequenced fragments number≤1000, remaining test zone is for detecting successful test zone.In the present embodiment, obtain 2117 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.Rape variety genotype to be measured is the combination of variation base in test zone, and genotypic frequency >=30% of rape variety to be measured.In general, in the sample extracted, the amount of hybrid is not higher than 15%, order-checking mistake is no more than 1%, the two total is no more than 16%, therefore, for site of isozygotying, rape variety genotype to be measured only has one, and its frequency should be greater than 84%, and for heterozygous sites, rape variety genotype to be measured has 2 kinds, and its ratio should be greater than 42%, therefore, regulation row surveys frequency >=30% of variety and genetype, can get rid of and have hybrid strain and to the genotypic interference of rape variety to be measured because mixing in check order mistake and rape variety to be measured.
Such as, in sequenced fragments group, the sequenced fragments in the 1st order-checking region adds up to 11230 articles, there are TTA, CCA, TTT, TTG ... totally 29 kinds of genotype, represent these genotypic sequenced fragments numbers 10840,281,2,2 respectively ..., these genotypic frequencies are 10840/11230=96.52%, 281/11230=2.50%, 2/11230=0.02%, 2/11230=0.02% ...By the genotypic definition of rape variety to be measured, TTA is rape variety to be measured " Soviet Union 2051 " 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 2117 rape variety to be measured " Soviet Union 2051 " genotype detecting successful test zones.
By with the rape variety to be measured method that " to revive 2051 " identical, in parent " P65 " and " Shanghai 8920 ", obtain 2117 equally detect successful test zones and parent " P65 " and " Shanghai 8920 " genotype at the successful test zone of all detections, partial results is in table 1.The present embodiment does not have completely to list whole rape 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 1 is rape variety genotype to be measured and parent genotype and relevant information
Eight, according to rape variety genotype to be measured and parent genotype, judge the verity of the parental source of rape variety to be measured and calculate the ratio of parental source, concrete grammar is:
Judge that the method for the verity of the parental source of rape variety to be measured is: if there is non-parent's genotype in rape variety to be measured, then parental source is untrue; If there is not non-parent's genotype in rape variety to be measured, then parental source is true.Non-parent's genotype is rape variety genotype to be measured, and distinguishing base number >=2 between the genotype of non-parent's genotype and arbitrarily parent.The reason of regulation distinguishing base number >=2 is as follows: in plant self-sow reproductive process, is the natural mutation that there is DNA, in most cases, natural mutation is nonsense mutation, do not change the proterties of rape variety to be measured, therefore, do not form new variety.Therefore, can not cultivate parent in rape variety testing process to be measured, the DN A sudden change in parent or rape variety to be measured is judged as non-parent's genotype.In the experiment in our early stage, carry out high-throughput to paddy rice 9311 to resurvey sequence, and compare with the reference genome of 9311, 33538 variant sites are found altogether, because the genome of paddy rice is about 500M, therefore, the natural mutation frequency of each base is roughly: 33538/ (500*1000000)=0.0067076%, by average each test zone genotype number 10 calculation, in so each test zone genotype, occur that the probability of 2 mutating alkali yls is: BINOM.DIST (10-2, 10, 1-0.0067076%, TRUE)=2.02391123634714E-07.Test zone is by 10000 calculations, and all test zones all do not occur because of sudden change that the genotypic probability of non-parent is: BINOM.DIST (10000,10000,1-2.02391123634714E-07, FALSE)=99.80%.Visible, this parameter designing of distinguishing base number is >=2 mistakes judged parental source verity almost can avoiding completely being caused by natural mutation.Above-mentioned parameter is all very strict, such as, our high-flux sequence adopt 9311 and with reference between genomic 9311 every a lot of year, namely suddenly change and be accumulated a lot of year, general rape variety to be measured all can not be so long from cultivation to detection time, and the sudden change of accumulation also can not be so many, in addition, further comprises a large amount of order-checking mistakes in the frequency of 0.0067076%, therefore, real mutation rate should far below this value.So the actual accuracy of method provided by the invention to the judgement of parental source verity should be greater than 99.80%.
In order to more convenient explanation present method, table 2 lists the example of a supposition.In this supposition example, need to judge whether rape variety to be measured truly derives from parent 1, parent 2 and parent 3.In the 1st test zone, rape variety genotype to be measured is AA, and it compares with parent 1 frequency of genotypes AA, and have 0 distinguishing base, this quantity is less than 2, and therefore, the frequency of genotypes AA in the 1st test zone of rape variety to be measured is not non-parent genotype.In the 3rd test zone, the genotype TT of rape variety frequency of genotypes AA to be measured and parent 2 and parent 3 has the difference of 2 bases, but with the difference of the genotype TA of parent 1 only 1 base, therefore, the AA genotype in the 3rd test zone of rape variety to be measured is not non-parent's genotype yet.In the 4th test zone, rape variety genotype to be measured is AA, the genotype of all 3 parents is TT, and difference is 2, therefore, in 4 test zones, rape variety frequency of genotypes AA to be measured is non-parent's genotype, also, in the 4th test zone, rape variety frequency of genotypes AA to be measured can not derive from any one in these 3 parents, and namely the parental source of rape variety to be measured is untrue.In test zone 6-12, have as many as 3 genotype in rape variety to be measured or parent, wherein, there are 2 genotype and be mostly that, because this test zone is heterozygous sites, 3 and above genotype may occur in polyploid rape in same test zone.Polyploid rape is uncommon, just enumerates out by various possible situation and judgement example thereof here.
Table 2 is a supposition example
In the present embodiment, in 1st test zone, rape variety genotype to be measured is TTA, distinguishing base number=0 <2 that exists between the genotype TTA in itself and parent " P65 " and " Shanghai 8920 ", therefore, in 1st test zone, in parent to be measured, there is not non-parent's genotype.By identical method, analyze all test zones successively, result shows: in the 5th test zone, rape variety genotype to be measured is AGA, the genotype in its parent " P65 " and " Shanghai 8920 " is TAA, therefore, in 5th test zone, genotype AGA in parent to be measured and the distinguishing base number that exists between the genotype of any one parent all=2 >=2, also be, in 5th test zone, rape variety genotype to be measured can not come from parent " P65 " and " Shanghai 8920 ", and also can not be produced by natural mutation, so, judge that rape variety parental source to be measured is untrue.
According to rape variety genotype to be measured and parent genotype, calculate the ratio of parental source.The formula calculating the ratio of parental source is: the ratio of parental source
wherein, n is the number of the peculiar test zone of parent; I is i-th peculiar test zone of parent; Si is in i-th peculiar test zone of parent, genotypic number identical between the peculiar genotype of parent with rape variety genotype to be measured; Ti is in i-th peculiar test zone of parent, the genotypic number of rape variety to be measured.The parent genotype that the peculiar genotype of parent only occurs in this parent, the peculiar test zone of parent refers to have the peculiar genotypic test zone of parent.
In the supposition example of table 2, the genotype TT of parent 1 in test zone 2 only occurs in parent 1, therefore genotype TT is the peculiar genotype of parent of parent 1, test zone 2 is the peculiar test zone of parent of parent 1, in this peculiar test zone, genotypic number identical between the peculiar genotype TT of parent with rape variety frequency of genotypes AA to be measured is 0, so, Si=0, in this peculiar test zone, the number of rape variety frequency of genotypes AA to be measured is 1, so, Ti=1, so, Si/Ti=0/1=0, its implication is in test zone 2, in rape variety genotype to be measured, there is no the genotype of parent 1, namely the blood relationship of parent 1 is not had.In the 12nd test zone, the genotype TT of parent 1 only occurs in parent 1, therefore genotype TT is the peculiar genotype of parent of the parent 1 of parent 1, test zone 12 is the peculiar test zone of parent of parent 1, in this peculiar test zone, genotypic number identical between the peculiar genotype TT of parent with rape variety frequency of genotypes AA/TT/GG to be measured is 1, so, Si=1, in this peculiar test zone, the number of rape variety frequency of genotypes AA/TT/GG to be measured is 3, so, Ti=3, so, Si/Ti=1/3=0.33, its implication is in test zone 12, the rape variety genotype to be measured of 1/3 derives from parent 1, namely in this test zone, rape variety to be measured has the blood relationship of the parent 1 of 1/3.In table 2, the peculiar test zone of the parent of parent 1 is test zone 2,5 ... 12, number is n=9 altogether, and the Si/Ti value of its correspondence is respectively 0,0.....0.33, so in rape variety to be measured, and the ratio of the parental source of parent 1
namely judge in rape variety to be measured, roughly have the blood relationship of 35.19% to come from parent 1 from all test zones.List the situation of various parent's idiotype in table 2, wherein, when Si/Ti value is not empty, corresponding parent genotype is parent's idiotype.
In the present embodiment, the genotype TTA of parent " Shanghai 8920 " in test zone 1 all occurs in parent " Shanghai 8920 " and " P65 ", therefore genotype TTA is not the peculiar genotype of parent in " Shanghai 8920 ", and test zone 1 is not also the peculiar test zone of parent in " Shanghai 8920 ".The genotype GCA of parent " Shanghai 8920 " in test zone 2 only occurs in parent " Shanghai 8920 ", therefore genotype GCA is the peculiar genotype of parent in " Shanghai 8920 ", test zone 2 is the peculiar test zone of parent in " Shanghai 8920 ", in this peculiar test zone, genotypic number identical between the peculiar genotype GCA of parent with rape variety genotype GCA to be measured is 1, so, Si=1, in this peculiar test zone, the number of rape variety genotype GCA to be measured is 1, so, Ti=1, so, Si/Ti=1/1=1, its implication is in test zone 1, in rape variety genotype to be measured, the blood relationship of 100% is from " Shanghai 8920 " parent, by identical method, judge in all test zones, whether parent " Shanghai 8920 " exists the peculiar genotype of parent, if there is the peculiar genotype of parent, the then corresponding test zone then peculiar test zone of parent, calculate in the peculiar test zone of this parent, the value of Si/Ti.Result shows: detect in successful test zone at 2117, have the peculiar test zone of parent in 69 " Shanghai 8920 ", in the value of their Si/Ti, be 0 have 65, be 1 have 4, so in rape variety to be measured, the ratio of the parental source of parent " Shanghai 8920 "
its implication is: judge from all test zones, has the blood relationship of 5.79% to come from " Shanghai 8920 " in rape variety to be measured.In the same way, calculate in rape variety to be measured, ratio=23.19% of the parental source of parent " P65 ", its implication is: judge from all test zones, has the blood relationship of 23.19% to come from " P65 " in rape variety to be measured.It should be noted that, because parental source is untrue, so the ratio of parental source calculated here can only, as a kind of reference, be not the actual proportions of parent's blood relationship in rape variety to be measured.
Result verification
A kind of method of standard is not had to judge source authenticity and the ratio thereof of parent at present, but the breeding process of the parent that the present embodiment provides " P65 " is: after rape variety " Soviet Union 2051 " is hybridized with " Shanghai 8920 ", rape variety to be measured " P65 " is obtained by systematic breeding, so, rape variety to be measured " Soviet Union 2051 " is the parent of " P65 ", instead of the parent of rape variety to be measured " Soviet Union 2051 " is " P65 ", so, judge that the parent of rape variety to be measured " Soviet Union 2051 " originates as " P65 " and " Shanghai 8920 " false conclusion is correct.It should be noted that, in variety certification process, in order to avoid the problem of intellecture property, variety right people provides a kind of approximate but parental source that the is property right dispute that is ignorant is comparatively general phenomenon, and the present embodiment therewith situation is similar.Because parental source is untrue, so parental source ratio calculates also can not be entirely true, can only as a reference, so, there is no need to verify the result of this ratio.
Embodiment two measures rape variety " 430AB/ Soviet Union 2051 " parental source verity and ratio
Whether the rape variety to be measured that the present embodiment provides is rape variety " 430AB/ Soviet Union 2051 ", need the parental source " 430AB " detecting this rape variety true with " reviving 2051 ".
By the method identical with the rape variety to be measured in embodiment one, extract the DNA of rape variety to be measured " 430AB/ Soviet Union 2051 " and parent " 430AB " thereof and " reviving 2051 ", utilize multiplex amplification primer identical in embodiment one and method to build high-throughput sequencing library, high-flux sequence, analysis sequenced fragments group, revive 2051 at rape variety 430AB/ to be measured " and parent " 430AB " and " reviving 2051 " in all have successfully been obtained 2117 and detect successful test zone and their genotype, partial results is in table 3.
Table 3 is rape variety to be measured and parent genotype thereof and relevant information
By the method identical with embodiment one, analyze all test zones, all do not find non-parent's genotype, therefore, the parental source of rape variety to be measured is true.By the method identical with embodiment one, judge in all test zones, whether parent " 430AB " exists the peculiar genotype of parent, if there is the peculiar genotype of parent, then corresponding test zone is the peculiar test zone of parent, calculates in the peculiar test zone of this parent, the value of Si/Ti.Result shows: detect in successful test zone at 2117, have the peculiar test zone of parent of 279 " 430AB ", wherein, the value of 271 Si/Ti is 1/2=0.5, the value of 8 Si/Ti is 1/1=1, so in rape variety to be measured, and the ratio of the parental source of parent " 430AB "
its implication is: judge from all test zones, has the blood relationship of 51.43% to come from " 430AB " in rape variety to be measured.In the same way, calculate in rape variety to be measured, the ratio of the parental source of parent " Soviet Union 2051 ", result shows, has the peculiar test zone of parent of 279 " Soviet Unions 2051 ", wherein, the value of 271 Si/Ti is 1/2=0.5, the value of 8 Si/Ti is 0/1=0, so in rape variety to be measured, and the ratio of the parental source of parent " Soviet Union 2051 "
its implication is: judge from all test zones, has the blood relationship of 48.57% to come from " Soviet Union 2051 " in rape variety to be measured.
Result verification
The breeding process of the rape variety to be measured that the present embodiment provides is: rape variety " 430AB " is for maternal, and " Soviet Union 2051 " is paternal hybrid, and assembly becomes rape variety to be measured " 430AB/ Soviet Union 2051 ".As can be seen here, judge rape variety parental source to be measured as " Soviet Union 2051 " and the conclusion of " 430AB " be correct.Due in cross-fertilize seed, Parent respectively provides a set of karyomit(e), therefore, in nuclear genome, " Soviet Union 2051 " and " 430AB " ratio in rape variety to be measured respectively should account for 50%, but due to the female parent of rape variety to be measured be " 430AB ", so, the blood relationship of the cytoplasmic DNA in rape variety to be measured is maternal, therefore, maternal occupy larger be rational.In general, the present embodiment has correctly judged the verity of parental source in rape variety to be measured and ratio thereof.
The present embodiment, by high-flux sequence and the amplification of multidigit point, achieves the large sample sampling in rape variety build-in test region to be measured, ensure that the accuracy of detection.Meanwhile, the embodiment of the present invention utilizes multidigit point amplification technique not only accurate, and method is simple, quick.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, is that other detection method is all incomparable.
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 (8)
1. rape parental source verity and a ratio test novel method thereof, it is characterized in that, described method comprises:
Obtain the variant sites between different rape variety;
According to described variant sites determination test zone;
Respectively the parent of rape variety to be measured and described rape variety to be measured is sampled, extract and obtain the DNA of the sampling sample of the DNA of the sampling sample of described rape variety to be measured and the parent of described rape variety to be measured;
The primer of the described test zone of preparation amplification;
Described primer is utilized to increase to the DNA of the sampling sample of the DNA of the sampling sample of described rape variety to be measured and the parent of described rape variety to be measured respectively, obtain the amplified production of the amplified production of described rape variety to be measured and the parent of described rape variety to be measured respectively, and build the high-throughput sequencing library of the high-throughput sequencing library of described rape variety to be measured and the parent of described rape variety to be measured respectively with the described amplified production obtained;
Respectively high-flux sequence is carried out to the high-throughput sequencing library of the high-throughput sequencing library of described rape variety to be measured and the parent of described rape variety to be measured, obtain the sequenced fragments group of the sequenced fragments group of described rape variety to be measured and the parent of described rape variety to be measured;
Analyze the sequenced fragments group of the sequenced fragments group of described rape variety to be measured and the parent of described rape variety to be measured, obtain rape variety genotype to be measured and parent genotype, described rape variety genotype to be measured is that described rape variety to be measured makes a variation the combination of base in described test zone, and genotypic frequency >=30% of described rape variety to be measured, described parent genotype is that described parent makes a variation the combination of base in described test zone, and frequency >=30% of described parent genotype;
According to described rape variety genotype to be measured and described parent genotype, judge the verity of the parental source of described rape variety to be measured and calculate the ratio of parental source.
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;
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 described genotype of described hybrid strain genotype and described rape variety to be measured or described distinguishing base.
3. method according to claim 1, it is characterized in that, to the method that the parent of described rape variety to be measured and described rape variety to be measured samples be respectively: after the sample of the rape variety described to be measured of random selecting more than 100 and the parent of described rape variety to be measured mixes respectively, obtain the sampling sample of the sampling sample of described rape variety to be measured and the parent of described rape variety to be measured.
4. method according to claim 1, is characterized in that, the method for the verity of the parental source of described judgement rape variety to be measured is: if there is non-parent's genotype in described rape variety to be measured, then the parental source of described rape variety to be measured is untrue; If there is not described non-parent's genotype in described rape variety to be measured, then the parental source of described rape variety to be measured is true; Described non-parent's genotype is described rape variety genotype to be measured, and distinguishing base number >=2 of described non-parent's genotype and any described parent genotype.
5. method according to claim 1, is characterized in that, the formula calculating the ratio of parental source is: the ratio of parental source
wherein, n is the number of the peculiar test zone of parent; I is i-th peculiar test zone of described parent; Si is in i-th peculiar test zone of described parent, genotypic number identical between the peculiar genotype of parent with described rape variety genotype to be measured; Ti is the genotypic number of rape variety to be measured described in i-th peculiar test zone of described parent; The peculiar genotype of described parent is the described parent genotype only occurred in described parent, and the peculiar test zone of described parent refers to have the peculiar genotypic described test zone of described parent.
6. method according to claim 1, is characterized in that, determines 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.
7. method according to claim 1, is characterized in that, the degree of depth >=5000 times of described high-flux sequence.
8. method according to claim 1, is characterized in that, the multiplex amplification primer that described primer provides for match Mo Feishier company of the U.S..
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Application publication date: 20150902 |