CN104313146A - Method for developing genome simple sequence repeats (SSR) molecular marker - Google Patents
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Abstract
The invention discloses a method for developing genome simple sequence repeats (SSR) molecular marker. The method comprises the steps of taking two plant samples, respectively establishing banks for DNA of the samples, thereby obtaining two corresponding libraries; respectively sequencing the libraries by an Illumina sequencing technology, then carrying out micropackaging, seeking in Contigs obtained through micropackage so as to obtain the SSR of the samples, and screening the common SSR in the two samples, wherein the common SSR has the same chromosomal location and the same repetitive unit; and carrying out polymorphism screening on the two samples according to the common SSR, and judging whether the two samples are in porlymorphism according to the difference of times of the repetition of the SSR of the two samples. The method not only meets the requirement of seeking a lot of SSR molecular markers, but also has low cost; due to the advantages of high polymorphism, dominant heredity, wide distribution and few template DNA, the SSR molecular markers can be widely used in genetic diversity analysis, genetic mapping, quantitative trait loci (QTL) location, molecular marker assisted breeding and the like.
Description
Technical field
The invention belongs to biology field, particularly relate to a kind of technology about the SSR molecular marker exploitation in gene order-checking library.
Background technology
DNA molecular marker refers to the DNA fragment specific that can reflect certain species diversity in genome between biont or population.Compare with biochemical marker three class Genetic Markers with the morphological markers of routine, cytological marker, DNA molecular marker directly has following advantage: (1) is present in vegeto-animal any growth phase with the form of DNA, any organizing all can detect, not by season, environmental limit, there is not expression whether problem; (2) quantity is extremely many, and genome mutation is extremely abundant, and the quantity of mark is almost unlimited, and polymorphism is high, utilizes a large amount of primer, probe can complete the analysis of covering gene group; (3) manyly codominance is labeled as, very convenient to the selection of the proterties of recessiveness, the genotype of genotype and the heterozygosis of isozygotying can be identified, complete genetic information is provided.Due to more than, DNA molecular marker develops into current most widely used molecule marker.
SSR is the abbreviation of Simple Sequence Repeats, i.e. simple sequence repeats, also known as microsatellite sequence, is the s-generation molecule marker be based upon on PCR basis that development in recent years is got up.SSR is constantly repeated to form by 1-6 Nucleotide, and same class SSR can be distributed on whole genomic different positions, and on each seat, the number of repeating unit there are differences, and thus causes the polymorphism on each seat.Micro-satellite is stochastic distribution in prokaryotic organism and eukaryotic gene group, and this mark is codominance, meet Mendelian inheritance pattern, easy handling, there is high reproducibility and reliability, demonstrate the advantages such as stronger polymorphism, make it have important application in research fields such as genetic linkage maps structure, the assignment of genes gene mapping and assisted selection, pedigree and evolutionary relationship are studied, germplasm identification, genetic diversities, therefore SSR is considered to one of molecule marker best at present.
The development strategy of current SSR can be divided into two large classes, and first the first kind utilizes existing database developing SSR.In the databases such as Genebank, the genome sequence containing many species and est sequence information, just can search plain software conveniently by SSR by these sequences and obtain SSR.But for the huge species that the earth exists, in current database, much species gene group DNA information and est sequence thereof are also very limited or do not have, and this makes the suitability of first kind method be not very extensive at all.Equations of The Second Kind is by setting up DNA library, and then to library order-checking, carry out SSR exploitation, this kind of strategy is applicable to the species of all unknown gene group DNA or est sequence, mainly comprises following three kinds of modes:
(1) conventional Sanger method, comprises the steps: DNA extraction, SSR enrichment, builds TA clone library, plasmid extraction, Sanger order-checking, design of primers, screening polymorphic site;
(2) two generations sequencing (needing enrichment), comprise the steps: DNA extraction, SSR sequence enrichment, build two generation sequencing library, two generations order-checking, assembling, find SSR, design of primers, screening polymorphic site;
(3) two generations sequencing (not needing enrichment), comprise the steps: DNA extraction, build two generation sequencing library, two generations order-checking, assembling, find SSR, design of primers, screening polymorphic site.
Sanger method is individual chip sequencing, build a large amount of TA clone libraries, so expend time in very much and manpower, therefore can not show a candle to latter two and uses the convenient of the method for two generations order-checking.SSR classifies according to repeating unit base, and single base can be divided into repeat (totally 4
1kind), two bases repeat (totally 4
2kind), three bases repeat (totally 4
3kind), four bases repeat (totally 4
4kind) etc., only calculate SSR species number that is single, double, three bases repetitions and just reach 4
1+ 4
2+ 4
3=84 kinds more than, and the strategy of enrichment SSR is undertaken by probe hybridization, and the kind of probe can not design so much, and often design several, so just, several SSR can only be obtained, therefore, the library less than not enrichment SSR all far away on the kind of enrichment SSR and number, table 1 list occur in article by two generation sequencing developing SSR utilization power.Consider, use two generation sequencing, and do not do SSR enrichment, the method for directly building storehouse order-checking developing SSR is the most excellent.It is exactly 454 sequencing technologies and Illumina sequencing technologies that current two generations order-checking uses maximum, and what 454 sequencing technologies adopted is phosphoric acid sequencing, synthesis limit, the limit sequencing technologies of reversible cessation method that what Illumina sequencing technologies adopted is; Relative to 454 sequencing technologies, the price that Illumina sequencing technologies can be cheaper obtains genome sequence; But, at present develop genome SSR by two generations sequencing (not needing enrichment), utilization be 454 sequencing technologies, and for Illumina sequencing technologies, have not been reported exploitation genome SSR, only have exploitation transcript SSR, as shown in table 1.This is because utilize 454 sequencing technologies, it is 500bp that available Reads number is on average read long, directly can carry out SSR searching in these Reads.
Table 1 current two generation sequencing developing SSR molecule marker situation
Illumina sequencing technologies is by being attached to optically transparent surface by the random fragments of genomic dna, these DNA segments are by extending and bridge amplification, define the Flowcell with hundreds of millions of Cluster, each Cluster has the same DNA template of about 1000 copies, then carries out the order-checking of synthesis limit, limit by the fluorescently-labeled base of difference that 4 kinds of ends are closed.This novel method ensure that pinpoint accuracy and a real base connect the order-checking of a base, eliminates the specific fault of sequence aspect, the homopolymer that can check order and tumor-necrosis factor glycoproteins.This technology avoids at substantial human and material resources as conventional sequencing technology and carries out the loaded down with trivial details operation such as fragment clone, conversion, plasmid extraction.
In addition, after SSR develops, no matter carry out population genetic diversity assessment or carry out genetic linkage map structure, what first will carry out is get the prescreen that some individuals carries out the SSR of polymorphism, and then those SSR with polymorphism are extracted out separately, pcr amplification operation is carried out to colony or offspring.Prescreen is generally the method adopting polyacrylamide gel electrophoresis or capillary electrophoresis, but polyacrylamide gel electrophoresis is a kind of experimental technique of more difficult operation, very time-consuming, and is easy to when primer specificity is bad produce error of artificially reading tape; In addition, although capillary electrophoresis is convenient and tolerance range is very high, expense is also very high.So, based on the imperfection of current method, invent new
The method of SSR molecular marker exploitation.
Documents
Zhang Yucui, Yang Weihua, Kuang Meng, Xu Hongxia, Wang Yanqin, Zhou great Yun, Feng Xinai, Su Chang: SSR molecular marker progress and the application on cotton thereof. Cotton association 2011 years nd Annual Meeting compilations 2011.
Schnable?PS,Ware?D,Fulton?RS,Stein?JC,Wei?F,Pasternak?S,Liang?C,Zhang?J,Fulton?L,Graves?TA:The?B73?maize?genome:complexity,diversity,and?dynamics.Science2009,326:1112-1115.
Ekblom?R,Galindo?J:Applications?of?next?generation?sequencing?in?molecular?ecology?of?non-model?organisms.Heredity?2010,107:1-15.
Zalapa?JE,Cuevas?H,Zhu?H,Steffan?S,Senalik?D,Zeldin?E,McCown?B,Harbut?R,Simon?P:Using?next-generation?sequencing?approaches?to?isolate?simple?sequence?repeat(SSR)loci?in?the?plant?sciences.American?journal?of?botany?2012,99:193-208.
Summary of the invention
The object of the present invention is to provide a kind of method developing genome SSR molecular marker, to reduce order-checking cost, to improve prescreen efficiency, reduce manpower and financial resources.
In order to solve above technical problem, the concrete technical scheme of employing of the present invention is as follows:
Develop a method for genome SSR molecular marker, it is characterized in that: get two plant samples and sample A and sample B, respectively DNA is carried out to sample A and sample B and build storehouse, obtain two corresponding library A and library B, Illumina technology is adopted to check order to library A and library B respectively, then micro-assembling is carried out, the SSR of sample A and the SSR of sample B is found to obtain again from the Contigs obtained after micro-assembling, filter out sample A and sample B and have SSR and the identical and SSR that repeating unit is identical of chromosome position, total SSR described in utilization carries out polymorphism screening to sample A and sample B, with different the judging whether of SSR multiplicity of sample B, there is polymorphism according to sample A, thus directly carried out pre-screen steps in the aspect of data analysis, directly obtain the SSR of polymorphism, and do not need to carry out loaded down with trivial details experimental implementation again.
The determination methods of described polymorphism is as follows: when multiplicity is inconsistent in two samples for same repeating unit, then think that this SSR molecular marker has polymorphism, just can carry out design of primers and verify its polymorphism; Such as, AG repeating unit is repeated 10 times in sample A, AGAGAGAGAGAGAGAGAGAG, and in sample B, be repeated 11 times, AGAGAGAGAGAGAGAGAGAGAG, then think that this SSR molecular marker has polymorphism.
Although not as 454 sequencing technologies directly can find SSR like that in lower machine data, SSR number in Contigs after assembling is also fewer than 454 sequencing technologies strategies, but the requirement of the evaluation genetic diversity after also can meeting completely, structure genetic linkage map.A sample can obtain the data volume of 1G by Illumina sequencing technologies, cost is reduced to about 1/10th of 454 sequencing technologies, has both met the requirement finding a large amount of SSR, can reach low cost again.
In addition, because the data volume of 1G certainly exists bias concerning the larger species of some genome, survey two samples and just in time can supplement bias.
The present invention has beneficial effect
(1) the present invention is directly checked order to the genome of sample by Illumina sequencing technologies, reduces order-checking cost relative to 454 sequencing technologies;
(2) the present invention is directly screened SSR by bioinformatics method, does not need loaded down with trivial details experimental procedure to screen, has both met the requirement finding a large amount of SSR molecular marker, improve the efficiency of prescreen, reduce manpower and financial resources.
Accompanying drawing explanation
Fig. 1 is method flow diagram of the present invention;
Fig. 2 is the DNA extraction detection figure of the present invention's sample used;
Fig. 3 is that the agarose gel electrophoresis of sample used in the present invention detects PCR result figure.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Material: choose Rhizome of Giant Elephantsear and Colocasia gigantea as test sample, this sample in GeneBank except several chloroplast gene, without any genome and transcript profile.Get the wild Rhizome of Giant Elephantsear of two strains, a strain is taken from Chinese yunnan and is economized Xishuangbanna, and Vietnam is taken from a strain.Schema is shown in Fig. 1.
Method:
(1) DNA extraction: cut fresh and tender blade 0.4g CTAB method extract DNA, rear agarose gel electrophoresis Detection and Extraction effect, as shown in Figure 2, after namely master tape sample that is clear, that do not have disperse can be used for build storehouse operate in:
(2) build storehouse assembling: respectively get 1 μ g to two samples, DNA interrupts the fragment into 250bp, after carry out small segment and build storehouse.Check order with Hiseq PE151, lower machine data amount 1G.Use Cope and Soap denovo to assemble sequencing result, afterwards the Contig sequence filter of below 100bp is fallen.
(3) SSR is found: use MISA (http://pgrc.ipk-gatersleben.de/misa/) to find SSR marker in the Contig assembled, optimum configurations is as follows: mono-nucleotide repeats>10 nucleotides; Di-nucleotide repeats>12 nucleotides; Tri-nucleotide repeats>15 nucleotides; Tetra-nucleotide repeats>16nucleotides; Penta-nucleotide repeats>20 nucleotides and hexa-nucleotide repeats>24nucleotides.After finding out the Reads containing SSR, find out with Soap the SSR number that namely identical Reads have, and calculate two samples get union after total SSR Reads, as shown in table 2.
Table 2 is assembled and SSR number statistical
(3) SSR that can design primer is found: in the Reads with SSR site, picking SSR positional distance Reads two end is greater than the Reads of 50bp.Primer3 software is used to design primer.SSR statistics is as shown in table 3.
Table 3 can design the SSR number of primer
(4) statistics has the SSR of polymorphism: can design in the Reads of primer in total SSR site, (namely position on chromosome, SSR site is identical to find the SSR site in two samples with polymorphism, unit repetition number is different), in 987 Reads, obtain 621 SSR there is polymorphism.
(5) validity of primer: in 621 SSR, gets 100 SSR at random, carries out design of primers with Primer3 software.After sending Invitrogen company to carry out primer synthesis, get a strain Rhizome of Giant Elephantsear at random and carry out verifying (choosing be Vietnam's sample).Detect PCR result with agarose gel electrophoresis after PCR reaction, partial results is as Fig. 3; Result shows, and 86 SSR (86%) all can produce purpose fragment.
(6) polymorphism checking: choose 86 couples of SSR that amplification is in good condition, PCR reaction is carried out to an other strain Rhizome of Giant Elephantsear (Xishuangbanna), then the PCR result polyacrylamide gel electrophoresis of two strains is detected polymorphism.Result shows, 86 couples of SSR all demonstrate polymorphism in two samples, and the result of information analysis is completely the same.
From final analytical results, use Illumina sequencing technologies, the data volume of 1G surveyed by each sample, finally have found the Reads number that can design primer and reaches 4009, a kind of really efficient and cheap method of the scheme of explanation; Polyacrylamide gel electrophoresis result display, the polymorphism obtained by bioinformatic analysis and experimental result completely the same, the scheme demonstrated substantially increases the efficiency of prescreen really.In sum, the present invention is a kind of good method of SSR of cheap efficient exploitation unknown gene group species.
Claims (2)
1. develop a method for genome SSR molecular marker, it is characterized in that: get two plant samples and sample A and sample B, respectively DNA is carried out to sample A and sample B and build storehouse, obtain two corresponding library A and library B, Illumina sequencing technologies is adopted to check order to library A and library B respectively, then micro-assembling is carried out, the SSR of sample A and the SSR of sample B is found to obtain again from the Contigs obtained after micro-assembling, filter out sample A and sample B and have SSR and the identical and SSR that repeating unit is identical of chromosome position, total SSR described in utilization carries out polymorphism screening to sample A and sample B, with different the judging whether of SSR multiplicity of sample B, there is polymorphism according to sample A, thus directly carry out pre-screen steps in the aspect of data analysis, directly obtain the SSR of polymorphism, and do not need to carry out loaded down with trivial details experimental implementation again.
2. develop the method for genome SSR molecular marker for one kind, it is characterized in that the determination methods of described polymorphism is as follows: when multiplicity is inconsistent in two samples for same repeating unit, then think that this SSR molecular marker has polymorphism, just can carry out design of primers and verify its polymorphism; Such as, AG repeating unit is repeated 10 times in sample A, AGAGAGAGAGAGAGAGAGAG, and in sample B, be repeated 11 times, AGAGAGAGAGAGAGAGAGAGAG, and we think that this SSR molecular marker has polymorphism.
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Cited By (8)
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| CN104762395A (en) * | 2015-04-10 | 2015-07-08 | 西北农林科技大学 | Molecular marker screening method for qualitative trait genes with complementary action |
| CN105969862A (en) * | 2016-05-18 | 2016-09-28 | 中国科学院西北高原生物研究所 | Method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers |
| CN106282330A (en) * | 2015-12-02 | 2017-01-04 | 香港中文大学深圳研究院 | A kind of method developing Caulis et Folium Ammopiptanthi Mongolici Plant Genome simple repeated sequence molecular marker |
| CN108034696A (en) * | 2018-02-02 | 2018-05-15 | 中南大学 | A kind of method based on transcript profile sequencing SSR primers development |
| CN108441538A (en) * | 2018-04-17 | 2018-08-24 | 南昌大学 | The method for developing polymorphic micro-satellite molecular labeling based on multisample high-flux sequence |
| CN109536614A (en) * | 2018-10-22 | 2019-03-29 | 南昌大学 | Ascaris suum full-length genome microsatellite molecular marker and its primer and application |
| CN110195123A (en) * | 2019-06-20 | 2019-09-03 | 杭州师范大学 | Ku Zhi cpSSR labeled primer and its application are developed based on Chloroplast gene sequence |
| CN111540408A (en) * | 2020-05-12 | 2020-08-14 | 西藏自治区农牧科学院水产科学研究所 | Method for screening whole genome polymorphism SSR molecular marker |
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2014
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| CN104762395B (en) * | 2015-04-10 | 2017-12-12 | 西北农林科技大学 | For the method for screening molecular markers of complementation qualitative trait gene |
| CN104762395A (en) * | 2015-04-10 | 2015-07-08 | 西北农林科技大学 | Molecular marker screening method for qualitative trait genes with complementary action |
| CN106282330B (en) * | 2015-12-02 | 2019-09-06 | 香港中文大学深圳研究院 | A method of exploitation Ammopiptanthus mongolicus Plant Genome simple repeated sequence molecular labeling |
| CN106282330A (en) * | 2015-12-02 | 2017-01-04 | 香港中文大学深圳研究院 | A kind of method developing Caulis et Folium Ammopiptanthi Mongolici Plant Genome simple repeated sequence molecular marker |
| CN105969862A (en) * | 2016-05-18 | 2016-09-28 | 中国科学院西北高原生物研究所 | Method for designing, amplifying and sequencing twelve pairs of floccularia luteovirens microsatellite primers |
| CN108034696B (en) * | 2018-02-02 | 2020-07-28 | 中南大学 | Method for developing SSR primers based on transcriptome sequencing |
| CN108034696A (en) * | 2018-02-02 | 2018-05-15 | 中南大学 | A kind of method based on transcript profile sequencing SSR primers development |
| CN108441538A (en) * | 2018-04-17 | 2018-08-24 | 南昌大学 | The method for developing polymorphic micro-satellite molecular labeling based on multisample high-flux sequence |
| CN109536614A (en) * | 2018-10-22 | 2019-03-29 | 南昌大学 | Ascaris suum full-length genome microsatellite molecular marker and its primer and application |
| CN109536614B (en) * | 2018-10-22 | 2021-06-22 | 南昌大学 | Whole genome microsatellite molecular marker of ascaris suum, primer and application thereof |
| CN110195123A (en) * | 2019-06-20 | 2019-09-03 | 杭州师范大学 | Ku Zhi cpSSR labeled primer and its application are developed based on Chloroplast gene sequence |
| CN111540408A (en) * | 2020-05-12 | 2020-08-14 | 西藏自治区农牧科学院水产科学研究所 | Method for screening whole genome polymorphism SSR molecular marker |
| CN111540408B (en) * | 2020-05-12 | 2023-06-02 | 西藏自治区农牧科学院水产科学研究所 | Screening method of genome-wide polymorphism SSR molecular markers |
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