CN108220402A - A kind of identification method of Chinese cabbage germplasm and kind genealogical relationship - Google Patents
A kind of identification method of Chinese cabbage germplasm and kind genealogical relationship Download PDFInfo
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Abstract
The invention discloses a kind of Chinese cabbage germplasm and the identification method of kind genealogical relationship.The testing result of the simple codominant marker of banding pattern is converted to tetra- letters of A, G, T, C by the present invention, and effectively the sequence analysis of conventional nucleic acid diversity analysis software such as DNAMAN, clusterW, MEGA etc. are combined with diploid Chinese cabbage codominant marker's Assignment Analysis, the 0 of binary form, 1 data matrix can only be recognized by solving NTSYS, and is unable to interpretation genotype data either NTSYS, Popgene etc. are required for the cumbersome sex chromosome mosaicism that testing result will be marked to be converted by the requirement of software 0/1 matrix or AA/BB letter matrixes.The method of the present invention and NTSYS cluster results are more basically identical, but eliminate the tab file preparation process of NTSYS Software of Fuzzy Clustering Analysis, easy to operate, and this method is suitble to analyze the relationship between cenospecies and pure lines.
Description
Technical field
The invention belongs to field of plant variety breeding technology, especially Chinese cabbage breeding technique, and in particular to a kind of Chinese cabbage kind
The identification method of matter and kind genealogical relationship.
Background technology
Genetic diversity Journal of Sex Research is the important foundation that plant germplasm resource is efficiently used and protected, detection and research plant species
The genetic diversity of matter resource can reveal that the mechanism that genetic diversity is formed and maintained, efficiently be utilized for plant germplasm resource
Formulation with Reasonable Protection strategy provides scientific basis and embodiment.
The tool of plant germplasm resource genetic diversity Journal of Sex Research is in addition to phenotypic markers, and mainly biochemical biomarker is such as work(
Enzyme mark and based on DNA molecular labeling (Zhang Chihong etc., using SSR marker to 61 national barley genetic diversity
Research, plant genetic resources journal, 2008,9 (1):15-19；GarrisAJ, TaiTH, CoburnJ, etal, Genetic
Structure and diversity in Oryza sativa, Genetics, 2005,69:1631-1638).Wherein especially
The development with molecular biology, the sequence difference based on inhereditary material, that is, DNA and the various molecular labelings developed are extensive
Applied to variety of crops or the Relationship iden- tification of resource.Mainly there is molecular labeling tool used by genetic diversity is studied
Dominant and two kinds of codominance, in used molecular labeling, is advisable, such as with being distributed wide, reproducible codominant marker especially
SSR, InDels, SNP etc..Simple sequence repeats (simple sequence repeats, SSRs), also known as microsatellite DNA
(microsatellite DNA) is a kind of DNA sequence dna being made of 1~6 nucleotide tandem sequence repeats, such as (CA) n, (ATG)
N, (TAGG) n etc. is repeated, and length is generally shorter, is distributed widely in the different location of genome, due to repeating primitive number and base
The difference of first base number causes the polymorphism in each site, and SSR marker can be divided into genome SSR and EST-SSR two types,
SSR marker has many advantages, such as codominance, reproducible, rich polymorphism, is easy to detection.Insertion/deletion
(insertion/deletions, InDels) label is since core has occurred in the DNA sequence dna at same site between Different Individual
The insertion/deletion of acid fragments carries out PCR amplification according to the sequence design special primer of target site both sides, amplified fragments
Length polymorphism is InDels labels, and InDel labels are what is developed according to the insertion and deletion difference between homologous sequence, this
Exploitation has certain difficulty for the unknown crop of genome sequence, and InDels is labeled as a codominant marker, has
The advantages that preferable stability and rich polymorphism.
In genetic diversity Journal of Sex Research, dominant molecular labeling two distinct types of with codominance generates different data class
Type, dominant molecular labeling generates binary form data (can use 1/0 label), and codominant marker generates genotype data, number
Various difficulties are brought according to analysis and research of the form difference to genetic diversity.For codominant marker, as SSR,
InDels etc. is for a long time, general using specific when researcher uses the affiliation of these Marker Identification crops germplasm
Software such as NTSYS, Popgene etc. are required for label testing result being converted into 0/1 matrix or AA/BB by the requirement of software
Then alphabetical matrix can just obtain result using complicated calculating.However, during for example, by using NTSYS Software of Fuzzy Clustering Analysis, mark
Remember that file preparation process is cumbersome, and NTSYS can only analyze the relationship between pure lines, not between fit analysis cenospecies and pure lines
Relationship.
Invention content
In view of the above-mentioned problems of the prior art, the present invention develops the mirror of a kind of Chinese cabbage germplasm and kind genealogical relationship
Method is determined, by a kind of codominant marker's assignment method, especially with respect to the assignment method of InDels labels, by same sample
The testing result of codominant markers a series of is converted into " flag sequence " being made of tetra- kinds of basic group letters of A, C, G, T, and combines
Conventional nucleic acid diversity analysis software such as DNAMAN, clusterW, MEGA etc., by the label testing result nucleic acid of different samples
Diversity analysis software is analysed and compared, and draws homology tree, you can intuitively find out the affiliation between each material.
Specifically, the present invention relates to following technical schemes：
First, the invention discloses a kind of Chinese cabbage germplasm and the identification method of kind genealogical relationship, include the following steps：
(1) sample genomic dna extraction to be detected；
(2) selection of codominant marker and screening criteria：Primer is designed according to codominant marker and carries out amplification and PAGE inspections
It surveys, the good primer combination of specificity of the selection with polymorphism carries out PCR amplification and capillary electrophoresis detection；
(3) assignment is marked：The label testing result of all samples to be detected is arranged in order, is marked in a collection of sample
Sequence consensus；If the single labelled target fragment size in all detection samples is more than four kinds, this label is eliminated；It is single
Testing result of a polymorphism mark in all samples is assigned a value of A, G, T, C successively by target fragment is descending；Single mark
Remember that the testing result assignment in specific sample is made of two letters；For the missing shape completely of selected label in detection sample
Condition, missing sample proportion are more than more than 60 the percent of detection sample, eliminate the label, and missing band is represented with NN；
(4) qualification result of germplasm and kind genealogical relationship exports：By generation after above-mentioned label assignment by A/T/G/C letters
One section of " DNA " sequence of composition is divided the label testing result of different samples to be detected with nucleic acid diversity analysis software
Analysis compares, and draws homology tree, obtains the affiliation between each sample to be detected.
The present invention is identified that Chinese cabbage is diploid crop for Chinese cabbage germplasm and kind genealogical relationship, aobvious altogether
Property molecular labeling (such as SSR, InDels) generate genotype data have diversity, most of codominant markers are such as
SSR/InDels effective number of allele be mostly within 4 (instrument pool can etc., Chinese cabbage simple sequence repeats (SSR) and be inserted into/scarce
Lose the exploitation and General Use Analysis of (InDel) label, Journal of Agricultural Biotechnology, 2012,20 (12):1398~1406, this article
Chapter technology contents are included in the application together), since DNA sequence dna comparison software such as DNAMAN can be according to the phase of multiple DNA sequence dnas
Intuitively provide the homology tree file of all sequences like property, the present invention effectively by conventional nucleic acid diversity analysis software such as
The sequence analysis of DNAMAN, clusterW, MEGA etc. are combined with diploid Chinese cabbage codominant marker's Assignment Analysis, to solve
NTSYS can only recognize the 0 of binary form, 1 data matrix, and being unable to interpretation genotype data or NTSYS, Popgene etc. all needs
Label testing result is converted into the cumbersome sex chromosome mosaicism of 0/1 matrix or AA/BB letter matrixes by the requirement of software.
Specifically, in identification method step (1) of the present invention, sample genomic dna extraction to be detected can take the prior art
Known method, for example, Chinese cabbage sample genomic dna extraction, reference Liu Shuantao etc. (Journal of Agricultural Biotechnology, 2014,22
(7):853~861) method.
In the preferred embodiment of the present invention, codominant marker of the present invention is one kind in SSR, InDels, SNP.
In a preferred embodiment of the invention, codominant marker marks for InDels.Selected in step (2) of the present invention
InDels labels can be that the prior art has disclosed report, such as the meeting of instrument pool, Chinese cabbage simple sequence repeats (SSR) and
The exploitation of insertion/deletion (InDel) label and General Use Analysis, Journal of Agricultural Biotechnology, 2012,20 (12):1398~
1406 or label newly developed, such as Zhang Zhigang, Journal of Agricultural Biotechnology, 2016,24 (4):510~518, the two article
Content is included in the application together.
For example, in marker development, sequence is resurveyed by carrying out full-length genome to Chinese cabbage sample, screens in sequencing data and inserts
Enter to lack difference in the site of more than 5bp, in both sides conserved regions design primer, progress PCR amplification and PAGE detections.
In the selection and screening criteria of specific InDels codominant markers, select InDels labels in sample variant
And it is merely able to amplify the primer combination of single band respectively, for carrying out PCR amplification and capillary electrophoresis detection.
Preferably, exploitation label material therefor be Japanese Chinese cabbage crossing kind be good for the spring selfing separation strain 06-247 with
The selfing separation strain He102 in two packet header, Chinese cabbage local varieties Henan.
In specific embodiment, testing result assignment of the single marking in specific sample is made of two letters, pure
Mould assembly is AA, GG, TT, CC, and heterozygous is assigned a value of AG, AT, AC, GT, GC, TC or CT, CG, CA, TG, TA, GA, in a collection of sample
Scoring criteria is consistent.
Specifically, sequence or relatively large segment assignment of the heterozygous assignment in a collection of sample are first, relatively small
Segment assignment is rear, it is possible to be that AG, AT, AC, GT, GC, TC or i.e. relatively small segment assignment on the contrary are first, relatively large
Segment assignment rear, i.e. CT, CG, CA, TG, TA, GA follow a sequence in a collection of sample.
In specific embodiment, one kind in DNAMAN, clusterW, MEGA of nucleic acid diversity analysis software or
It is several.
In specific embodiment, the present invention uses Relationship iden- tification of the DNAMAN and MEGA6.0 softwares to cenospecies, with
Differentiate the affiliation between new listing cenospecies and the kind sold；Using MEGA6.0 to Core Germplasms and cenospecies
Carry out UPGMA cluster analyses, not only analyze the genealogical relationship of cenospecies, but also be easier to judge the parents of certain cenospecies
Source.
In preferred embodiment, identification method of the present invention can be used for Relationship iden- tification between cabbage hybrid.
In preferred embodiment, identification method of the present invention carries out molecular labeling mirror for the cenospecies resource of collection
It is fixed, judge its affiliation with existing germplasm, find the material of affiliation relatively and carry out backcross transformation, with purposeful
Ground carries out germplasm innovation, avoids blindness.
The present invention achieves following advantageous effect：
Relative to Software of Fuzzy Clustering Analysis NYSYS, popgene etc., DNA compares software DNAMAN, MEGA etc. in molecular biosciences
Learn researcher in popularity rate it is higher, therefore by the testing result of the simple InDels codominant markers of banding pattern be converted to A, G, T,
Tetra- letters of C, four letters represent that band is descending successively, since Chinese cabbage is diploid crop, the detection of a label
As a result it is made of two letters, i.e., it is homozygous to be represented with AA, GG, TT or CC, otherwise sequence of the heterozygous in a collection of sample is
First, the relatively small segment of relatively large segment is rear, it is possible to be AG, AT, AC, GT, GC, TC or otherwise it is i.e. relatively small
First, the relatively large segment of segment rear, i.e. CT, CG, CA, TG, TA, GA follow a sequence in a collection of sample.Separately
Outside, selected label has lacks completely in part detection sample, if missing sample proportion is more than detection sample
This more than 60 percent eliminates the label, and missing band is represented with NN during less than 60 percent.The detection of one group echo
As a result it being arranged in certain sequence in a collection of sample, all label testing results of a certain material are exactly one section " DNA sequence dna ", this
The result of sample can be easily compared with DNAMAN and MEGA softwares, export intuitive comparison result, warp and NTSYS
Cluster result compares, basically identical.The assignment method eliminates the tab file preparation process of NTSYS Software of Fuzzy Clustering Analysis, behaviour
Make simple, it is important to which this method is suitble to analyze the relationship between cenospecies and pure lines, unlike NTSYS can only be analyzed between pure lines
Relationship, so convenient and practical.
Description of the drawings
The screening of Fig. 1 labels
The DNAMAN5.0 analysis results of 40 cenospecies of Fig. 2
6.0 analysis results of MEGA of 40 cenospecies of Fig. 3
The MEGA6.0 analysis results of 127 parts of resources of Fig. 4
2.0 cluster results of NTSYS of 127 parts of resources of Fig. 5
40 cenospecies of Fig. 6 and the affiliation of 127 parts of core resources
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.It is unless another
It indicates, all technical and scientific terms used herein has usual with general technical staff of the technical field of the invention
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singulative
It is also intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation and/or combination thereof.
1 material and method：
1.1 material
Exploitation label material therefor is selfing separation strain 06-247 and the Chinese cabbage that Japanese Chinese cabbage crossing kind is good for the spring
The selfing separation strain He102 in two packet header, local varieties Henan.40 cabbage hybrids are bought from market refers to 1,127 parts of table
Core resource is independently formulated for Chinese cabbage seminar of vegetable or flower research institute of Shandong Academy of Agricultural Sciences, refers to table 2.
1 40 cenospecies information of table
2 127 parts of Germplasm Resources Informations of table
1.2 material to be tested extracting genome DNAs
With reference to (Journal of Agricultural Biotechnology, 2014,22 (7) such as Liu Shuantao:853~861) method.
The exploitation of 1.3InDels labels and screening criteria
Marker development is shown in (Journal of Agricultural Biotechnology, 2016,24 (4) such as Zhang Zhigang:510~518) method, that is, pass through
Full-length genome is carried out to 06-247 and He102 and resurveys in sequence, screening sequencing data insertion and deletion difference in the site of more than 5bp,
In both sides conserved regions design primer, PCR amplification and PAGE detections are carried out.Those is selected to have the good primer of specificity of polymorphism
Combination, i.e., it is variant in He102 in 06-247 and be merely able to amplify the primer of single band respectively and combine, to cenospecies with
Core Germplasms carry out PCR amplification, capillary electrophoresis detection.
1.4 label assignment methods and assignment
All labels to be detected are arranged in certain sequence, the sequence marked in a collection of sample cannot be changed.Due to selected
Label specificity is good, and 1-2 bands, detection knot of the single polymorphism mark in all material can only be amplified in single sample
Fruit is assigned a value of A, G, T, C successively by target fragment is descending, if the single labelled target fragment in all detection samples
Size is more than four kinds, then eliminates this label.Since Chinese cabbage is diploid crop, then inspection of the single marking in specific sample
Surveying result will be made of two letters, and homozygous is AA, GG, TT, CC, otherwise sequence of the heterozygous assignment in a collection of sample is
First, the relatively small segment assignment of relatively large segment assignment is rear, it is possible to be AG, AT, AC, GT, GC, TC or on the contrary
First, the relatively large segment assignment of i.e. relatively small segment assignment is rear, i.e. CT, CG, CA, TG, TA, GA, in a collection of sample
Follow a sequence.It is lacked completely in part detection sample in addition, selected label has, if missing sample
Proportion is more than more than 60 the percent of detection sample, eliminates the label, and missing band is represented with NN.
The output of 1.5 results
According to the method described above, a sample is detected with X label, will obtain one section be made of 2X letter
" DNA " sequence, such sequential file can hold by identifications such as nucleic acid diversity analysis software such as DNAMAN, clusterW very much.
2 results
The label of 2.1 screenings
PAGE testing results during selection markers as shown in Figure 1, number is marker number in figure, and each label is two corresponding
Swimming lane, left side is using 06-247 as template, and right side is using He102 as template, and according to testing result, eliminating those does not have polymorphism mark
The label (such as No. 3, No. 4, No. 6) of (such as No. 9, No. 17), dominant marker's (such as No. 11) and amplified band complexity, only selects that
The label such as No. 1, No. 16 and No. 18 of single polymorphic bands can be amplified respectively in 06-247 and He102 a bit.Using this
Stringent screening technique selects 80 pairs of labels to carry out subsequent research, numbers from X1 to X80 altogether.
The identification of 2.2 pairs of cenospecies
With DNAMAN and MEGA6.0 softwares to the Relationship iden- tification results of 40 cenospecies as shown in Figures 2 and 3,
The not no related report about Relationship iden- tification between cenospecies in some documents.By above-mentioned qualification result, can hold very much
Easily differentiate the affiliation between new listing cenospecies and the kind sold, it is known that it is the substitute or liter of that kind
Grade version.By with old varietal check, the molecular labeling chain with Ameliorative character can be screened and for germplasm innovation.
The identification of 2.3 pairs of Core Germplasms
UPGMA cluster analyses are carried out to 127 parts of Core Germplasms with MEGA6.0, the results are shown in Figure 4, the result with
The analysis result (such as Fig. 5) of NTsys2.0 is basically identical.Have and have no that analyzing germ plasm resource with MEGA softwares mutually closes in document
The related report of system.
2.4 cenospecies genealogical relationships are identified
By the source of peculiar its parent of marker for judgment of a cenospecies, need to analyze the parent between cenospecies and germplasm
Edge relationship, not about the research of this respect in having document.Assignment method using the present invention can very easily divide
The genealogical relationship of cenospecies is analysed, if Fig. 6 is all 40 cenospecies and the relationship of 127 parts of germplasm, can therefrom be easier to sentence
Break and the parental origin of certain cenospecies.
It with the universal of cenospecies and promotes, farm variety is almost withered away, and causes to collect cenospecies also as germ plasm resource
A kind of important form collected carries out the cenospecies of collection to be selfed separation again after Agronomic characteristic and comprehensive resistance identification
Elite plant strain is improved by backcross transformation and has strain, is a kind of principal mode of current Chinese cabbage germplasm innovation.In order to avoid
Parents' homogeneity needs to carry out molecular markers for identification to the resource of collection in advance, judges its affiliation with existing germplasm, looks for
The material of affiliation relatively carries out backcross transformation, purposefully to carry out germplasm innovation, avoid blindness.
Claims (10)
1. the identification method of a kind of Chinese cabbage germplasm and kind genealogical relationship, which is characterized in that include the following steps：
(1) sample genomic dna extraction to be detected；
(2) selection of codominant marker and screening criteria：Primer is designed according to codominant marker and carries out amplification and PAGE detections, choosing
The good primer combination of specificity with polymorphism is selected, carries out PCR amplification and capillary electrophoresis detection；
(3) assignment is marked：All labels to be detected are arranged in order, the sequence consensus marked in a collection of sample；It is if single
The target fragment size in all detection samples is marked to be more than four kinds, then eliminates this label；Single polymorphism mark is in institute
There is the testing result in sample to be assigned a value of A, G, T, C successively by target fragment is descending；Single marking is in specific sample
Testing result assignment is made of two letters；For the complete degree of lacking of label selected in detection sample, missing sample institute accounting
Example is more than more than 60 the percent of detection sample, eliminates the label, and missing band is represented with NN；
(4) qualification result of germplasm and kind genealogical relationship exports：One section be made of A/T/G/C will be generated after label assignment
The label testing result of different samples to be detected with nucleic acid diversity analysis software is analysed and compared, drawn by " DNA " sequence
Homology tree obtains the affiliation between each sample to be detected.
2. identification method according to claim 1, which is characterized in that the codominant marker is in SSR, InDels, SNP
One kind.
3. identification method according to claim 1, which is characterized in that codominant marker marks for InDels.
4. identification method according to claim 3, which is characterized in that resurveyed by carrying out full-length genome to Chinese cabbage sample
Insertion and deletion difference in both sides conserved regions design primer, carries out PCR amplification in the site of more than 5bp in sequence, screening sequencing data
It is detected with PAGE.
5. identification method according to claim 4, which is characterized in that InDels marks variant and difference in selection sample
It is merely able to amplify the primer combination of single band, for carrying out PCR amplification and capillary electrophoresis detection.
6. according to claim 1-5 any one of them identification methods, which is characterized in that in step (3), single marking is specific
Testing result assignment in sample is made of two letters, homozygous for AA, GG, TT, CC, heterozygous be assigned a value of AG, AT, AC,
GT, GC, TC or CT, CG, CA, TG, TA, GA, scoring criteria is consistent in a collection of sample.
7. identification method according to claim 6, it is characterised in that sequence of the heterozygous assignment in a collection of sample or
First, the relatively small segment assignment of relatively large segment assignment is rear, i.e. AG, AT, AC, GT, GC, TC or otherwise i.e. opposite
First, the relatively large segment assignment of small segment assignment is rear, i.e. CT, CG, CA, TG, TA, GA.
8. according to claim 1-5 any one of them identification methods, which is characterized in that nucleic acid diversity analysis software is selected from
One or more of DNAMAN, clusterW, MEGA.
9. identification method according to claim 8, which is characterized in that using DNAMAN and MEGA6.0 softwares to cenospecies
Relationship iden- tification, to differentiate the affiliation between new listing cenospecies and the kind sold；Using using MEGA6.0
The genealogical relationship of cenospecies is analyzed in carry out UPGMA cluster analyses to Core Germplasms and cenospecies, judges the double of certain cenospecies
Close source.
10. according to claim 1-5 any one of them identification methods, which is characterized in that the identification method is used for Chinese cabbage
Relationship iden- tification between cenospecies.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109694919A (en) * | 2018-12-29 | 2019-04-30 | 山东省农业科学院蔬菜花卉研究所 | Four seed type cabbage hybrid Purity core primers and purity of hybrid InDel molecular markers for identification kit |
| CN109811075A (en) * | 2019-01-28 | 2019-05-28 | 北京市农林科学院 | A kind of method for identifying Chinese cabbage cultivar authenticity and its combination of dedicated SNP primer |
| CN111445953A (en) * | 2020-03-27 | 2020-07-24 | 武汉古奥基因科技有限公司 | Method for splitting tetraploid fish subgenome by using whole genome comparison |
| CN111793708A (en) * | 2019-10-11 | 2020-10-20 | 北京市农林科学院 | Method for identifying authenticity of Chinese cabbage variety and special SSR primer combination thereof |
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| CN109811075A (en) * | 2019-01-28 | 2019-05-28 | 北京市农林科学院 | A kind of method for identifying Chinese cabbage cultivar authenticity and its combination of dedicated SNP primer |
| CN109811075B (en) * | 2019-01-28 | 2020-11-13 | 北京市农林科学院 | Method for identifying authenticity of Chinese cabbage variety and special SNP primer combination thereof |
| CN111793708A (en) * | 2019-10-11 | 2020-10-20 | 北京市农林科学院 | Method for identifying authenticity of Chinese cabbage variety and special SSR primer combination thereof |
| CN111793708B (en) * | 2019-10-11 | 2021-04-27 | 北京市农林科学院 | Method for identifying authenticity of Chinese cabbage variety and special SSR primer combination thereof |
| CN111445953A (en) * | 2020-03-27 | 2020-07-24 | 武汉古奥基因科技有限公司 | Method for splitting tetraploid fish subgenome by using whole genome comparison |
| CN111445953B (en) * | 2020-03-27 | 2022-04-26 | 武汉古奥基因科技有限公司 | Method for splitting tetraploid fish subgenome by using whole genome comparison |
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