CN108048530A - Method based on est sequence exploitation EPICs primers - Google Patents
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- CN108048530A CN108048530A CN201810066162.3A CN201810066162A CN108048530A CN 108048530 A CN108048530 A CN 108048530A CN 201810066162 A CN201810066162 A CN 201810066162A CN 108048530 A CN108048530 A CN 108048530A
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Abstract
The invention discloses a kind of methods based on est sequence exploitation EPICs primers, include the following steps：(1) est sequence of searched targets biology or its nearly edge biology, the est sequence is compared with the genome sequence of NCBI；(2) if est sequence coverage rate in the genome of the NCBI is less than 25%, the length of single copy introne is filtered out in 400~1600bp, candidate gene of the similarity more than 85%；(3) primer is designed for the candidate gene, obtains the EPICs primers.The application method is easy to operate, only target organism genome sequence need to be compared with est sequence, picks out suitable molecular labeling, and the karyogene molecular labeling application of gained is extremely wide, is analyzed available for population genetic diversity.
Description
Technical field
The present invention relates to molecular genetic technique field, especially a kind of side based on est sequence exploitation EPICs primers
Method.
Background technology
The existing method on big mastacembelus aculeatus population analysis is mainly two methods of SSR and ISSR, and both approaches are being lost
Pass diversity, apply on phyloge- ography must compare it is more.SSR (Simple Sequence Repeats) marks are to send out in recent years
A kind of molecular marking technique based on specific primer PCR that exhibition is got up, also referred to as microsatellite DNA
(MicrosatelliteDNA), be it is a kind of by several nucleotide (generally 1~6) for repeat unit group into up to tens
The tandem repetitive sequence of a nucleotide.Due to the usually relatively conservative single-copy sequence of the sequence of each SSR both sides.
ISSR marks are a kind of new molecular labelings to grow up on the basis of simply repetition sequence YU (SSR), by adding
The Zietkiewicz etc. (1994) of University of Montreal of putting on airs is proposed.The technology is using the microsatellite DNA being anchored as primer, in SSR
The 3 ' of sequence or 5 ' ends plus 2.4 random nucleotides of anchor, in PCR reactions, anchor primer can cause specific site to be annealed, and lead
DNA segment carries out PCR amplification between the less big repetitive sequence in the interval of cause and anchor primer complementation.The Inter.SSR expanded
Multiple bands in region can be able to by polyacrylamide gel electrophoresis or agarose gel electrophoresis differentiate (Zhou Yanqing,
2005).It combines the advantages of RAPD labelling techniques and HSSR labelling techniques, is capable of providing more DNA informations.Show oneself
It is widely used in Medicinal Plant Germplasm Resources identification, evolves and examined with Genetic relationship, genetic diversity and In-door air pollution
The research of survey, genetic mapping, the assignment of genes gene mapping, molecular mark etc..
But above two method cannot meet the group higher to differentiation degree traced to the source (divergaence time, be distributed lattice
Office) and prospect (distributed area reconstruction, following dynamic prediction) research.It traces to the source in the group for carrying out higher to differentiation degree
, it is necessary to using karyogene mark during the research of (divergaence time, Distribution Pattern) and prospect (distributed area is rebuild, following dynamic prediction)
Note.
The content of the invention
Based on this, one kind is provided and can easily be opened it is an object of the invention to overcome above-mentioned the deficiencies in the prior art part
The method for sending out karyogene.
To achieve the above object, the technical solution taken of the present invention is：Based on EST (Expressed Sequence Tag,
EST) sequence exploitation EPICs primers method, include the following steps：
(1) est sequence of searched targets biology or its nearly edge biology, by the est sequence and the genome sequence of NCBI
It is compared；
(2) if est sequence coverage rate in the genome of the NCBI is less than 25%, single copy introne is filtered out
Length in 400~1600bp, candidate gene of the similarity more than 85%；
(3) primer is designed for the candidate gene, obtains the EPICs primers；
Wherein, the parameter of the primer includes：G/C content is 40%~60%, and annealing temperature is 55~65 DEG C, and length is
18~27bp.
It should be noted that " extron primer-include subchannel " molecular labeling (Exon-primed intron-
Crossing markers, EPICs), it is a kind of anonymous karyogene mark, principle is designed in anonymous exon 1 in primer pair
Between introne expanded so that the mark had not only had the conserved property of extron, but also with the fast spy of introne evolutionary rate
Point；Est sequence is compared with genome sequence, when having in genome two sections or more (i.e. consistent) all similar to est sequence
Sequence, the wherein coverage rate of one or several aligned sequences length are less than 20%, it is ensured that are single-copy sequences；It is if big
In or equal to 20%, exist may not be comparison same gene, and simply other similar genes.Wherein, coverage rate is general
Refer to the accounting of the length of similar sequence lengths and est sequence in NCBI genomes.In this application, coverage rate refers to NCBI genes
In group, the ratio of the other gene order length similar to est sequence and est sequence length is less than 20%.Karyogene (Gene,
Mendelian factor) refer to the DNA (i.e. karyogene is the DNA for having hereditary effect) for carrying hereditary information, also referred to as
Gene.
Preferably, the method further includes step 4)：The gene of target organism described in the EPICs primer pairs of step (3)
Group DNA carries out PCR amplification, if the DNA sequence dna length amplified unanimously proves the EPICs primer accurate and effectives with expected,
Molecular labeling can be used as, for population analysis.
Preferably, the other coverage rates in the genome of the NCBI of est sequence described in step (2) are interior less than 20%
Length containing son is 750~850bp, and similarity is more than 90%.It should be noted that contrast conting is likely to occur in genome
The length range of introne is drawn as 400-1600bp, is qualification,
800bp is optimal, is reference according to previous primer development length.Est sequence and candidate gene sequence in genome
Similarity (i.e. consistent degree) is greater than or equal to 85%, and the versatility of the primer of design is more preferable, and similarity is bigger, and conservative performance is got over
Good, the versatility of primer is higher.
Preferably, G/C content is 40%~60% in the step 3), and annealing temperature is 58~63 DEG C, length 20~
24bp。
Preferably, primer uses primer Software for Design in the step (3).
Preferably, in the step (3) primer use primer5.0 Software for Design, the primer be scored at 90 points with
On.It is highly preferred that the primer is scored at 95 points or more.
Preferably, the est sequence of swamp eel is retrieved in the step (1), the target organism is big mastacembelus aculeatus.
Preferably, the EPICs primers of the candidate gene such as SEQ ID NO.1 and 2, SEQ ID NO.3 and 4, SEQ ID
NO.5 and 6, SEQ ID NO.7 and 8, SEQ ID NO.9 and 10, SEQ ID NO.11 and 12 or the institutes of SEQ ID NO.13 and 14
Show.It is highly preferred that the EPICs primer sequences of candidate gene are SEQ ID NO.1 and 2.
As another aspect of the present invention, the present invention provides application of the above-mentioned method in population analysis.
Preferably, the population analysis is population genetic diversity analysis, group structure is analyzed, phylogeny reconstruction in kind
Analysis, the research of pedigree Geographical Analysis, Niche Model or the estimation of non-mode biological evolution selection pressure.
In conclusion beneficial effects of the present invention are：
1) method of the exploitation EPICs primers of the application is easy to operate, only need to be by target organism genome sequence and EST sequences
Row are compared, and pick out suitable molecular labeling, have EST suitable for certain biology or its sibling species
Biology；
2) the karyogene molecular labeling application obtained by the present processes is extremely wide, available for population genetic diversity, group
Structural analysis, phylogeny reconstruction in kind, pedigree geography and Niche Model research, non-mode biological evolution selection pressure
Estimation etc.；
3) the present processes are suitable for a variety of biologies, as long as certain biology or its sibling species have EST,
Suitable karyogene molecular labeling can be developed.
Description of the drawings
Fig. 1 is the comparison result schematic diagram of est sequence and genome；
Fig. 2 is EPICs marker development principle schematics；
Fig. 3 is the diversity schematic diagram of primer EPIC-1, wherein, mean is average value, and population is group.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
Embodiment 1
A kind of embodiment of the method based on est sequence exploitation EPICs primers of the present invention, includes the following steps：
(1) acquisition of est sequence：Log in ncbi database (https://www.ncbi.nlm.nih.gov/), retrieval
The est sequence of Monopterus albus (swamp eel)；
(2) genome sequence of the est sequence of swamp eel and NCBI is compared into (as shown in Figure 1), finds coverage rate and be less than
20% candidate gene singly copied, also, the sequence similarity of candidate gene and est sequence is greater than or equal to 85% (such as Fig. 2
It is shown), the length of intron in candidate gene is in 400~1600bp；
(3) for the candidate gene design primer of big mastacembelus aculeatus, the design standard of primer includes：G/C content for 40%~
60%, annealing temperature is 55~65 DEG C, and length is 18~27bp, is designed using primer5.0, score should be at 90 points or more；It
It is synthesized afterwards by primer Synesis Company；
(4) primer of synthesis is sieved with the genomic DNA of each 3 tail of big mastacembelus aculeatus of three Longyan, Baise, white sand groups
Choosing.
Wherein, general archaeal dna polymerase and buffer solution are used in PCR amplification system.PCR reaction conditions are pre- for 94 DEG C
It is denatured 4min；94 DEG C of denaturation 30s, anneal 45s, 72 DEG C of extension 60s, 30 Xun Huans；72 DEG C of extension 10min.
(5) 1% Ago-Gels, 5ul reactant point samples, 120V electrophoresis 20min, ultraviolet gel imager observation are taken pictures,
The PCR product for having clear band send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced.
(6) according to sequencing result, show that 7 pairs of primers of table 1 can be used as the EPICs primers (molecular labeling) of big mastacembelus aculeatus
In population analysis.The sequence length that 7 pairs of primer amplifications go out be 445~1482bp, genetic distance 0.250~1.222.On as a result,
It can be 7 DNA sequence dnas that the corresponding primer in EPIC-1,2,3,4,5,6 or 7 is obtained by PCR amplification in table 1 to state candidate gene
In one or more.
The filter information of 1 karyogene EPICs molecular labelings of table
The genetic diversity index of the primer EPIC-1 of 2 embodiment 1 of embodiment
Test method：Big mastacembelus aculeatus for primer EPIC-1 analyses has 9 groups totally 237 tail, wherein Fujian Longyan (LY) 30
Tail, Guangdong Wuhua (WH) 30 tail, Zengcheng (ZC) 30 tail, Chaozhou (CZ) 30 tail, 25 tail of Renhua (RH), Enping City (EP) 20 tail, Baise
(BS) 30 tail, 20 tail of Honghe, Yunnan (HH), 22 tail of Hainan white sand (HNBS).PCR amplification is carried out, system and reaction condition are referring to reality
Apply example 1.
Result of the test：As shown in table 2.
2 primer EPIC-1 genetic diversity indexs of table
Note：NA=Number of alleles (number of alleles), NE=Effective number of allele
(effective number of allele), I=Shannon's Information Index (shannon index), Ho=Observed
Heterozygosity (observation heterozygosity), HE=Expected heterozygosity (expectation heterozygosity).
Based on primer EPIC-1, for 694bp, (694bp includes the individual DNA sequence dna length amplified of 9 groups 267
Intron sequences and introne front-end and back-end partial exon sequences), the genetic diversity index such as table 2 of primer.By more
From the point of view of sample index comprehensive, Enping City (He=0.079, I=1.111) genetic diversity height (Fig. 3).It is used with the inventor of application
The method of ISSR marks compares and Enping City (I=0.233) genetic diversity is high, as a result basically identical.
In the present embodiment, in order to compare quality of the tri- kinds of marks of EPICs, SSR and ISSR for Swarm Evolution analysis, for
Big mastacembelus aculeatus is respectively adopted tri- kinds of marks of EPICs, SSR and ISSR and has carried out corresponding experiment, obtains the result such as the following table 3.SSR is marked
Remember that polymorphism is high, in Individual identification, affiliation and gene flow etc. are advantageous, and with reference to table 3, three kinds of methods are compared
It understands, ISSR marks use more convenient (primer is general) compared to EPICs；If group that will be larger to differentiation group
Evolutionary analysis is carried out, answers EPICs mark of the prioritizing selection based on est sequence.
The comparison of tri- kinds of marks of table 3 EPICs, SSR and ISSR
Note：- representing no data, nDNA refers to the EPICs based on est sequence.
It is carried out it can be seen from the above results using EST (EST) sequence of swamp eel and the genome of NCBI
It compares, the karyogene EPICs primers developed may be advantageously employed in the genetic diversity Journal of Sex Research of big mastacembelus aculeatus different groups.
When that from universal primer, can not screen the karyogene for being suitble to be studied in oneself kind, this research method can be fast
Speed finds the karyogene studied in suitable kind, is a kind of new development approach.By comparing ESTs and genome sequence in NCBI
Row identify extron, design primer amplification introne, and the primer developed can be widely used in kind, even inter-species.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And scope.
SEQUENCE LISTING
<110>Guangzhou University
<120>Method based on est sequence exploitation EPICs primers
<130> 2018
<160> 14
<170> PatentIn version 3.5
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Claims (10)
1. the method based on est sequence exploitation EPICs primers, which is characterized in that include the following steps：
(1) est sequence of searched targets biology or its nearly edge biology, the genome sequence of the est sequence and NCBI are carried out
It compares；
(2) if est sequence coverage rate in the genome of the NCBI is less than 25%, the length of single copy introne is filtered out
Degree is in 400~1600bp, candidate gene of the similarity more than 85%；
(3) primer is designed for the candidate gene, obtains the EPICs primers；
Wherein, the parameter of the primer includes：G/C content is 40%~60%, and annealing temperature is 55~65 DEG C, length for 18~
27bp。
2. according to the method described in claim 1, it is characterized in that, est sequence is in the target organism base described in step (2)
Because coverage rate is less than 20% in group, the length of introne is 750~850bp, and similarity is more than 90%.
3. according to the method described in claim 1, it is characterized in that, in the step (3) G/C content be 40%~60%, annealing
Temperature is 58~63 DEG C, and length is in 20~24bp.
4. according to the method described in claim 1, it is characterized in that, primer uses primer Software for Design in the step (3).
5. according to the method described in claim 4, it is characterized in that, primer is set using primer5.0 softwares in the step (3)
Meter, the primer are scored at 90 points or more.
6. according to the method described in claim 5, it is characterized in that, the primer is scored at 95 points or more.
7. according to the method described in claim 1, it is characterized in that, the est sequence of swamp eel, NCBI are retrieved in the step (1)
Genome be the genome being sequenced in NCBI, the target organism is big mastacembelus aculeatus.
8. the method according to the description of claim 7 is characterized in that EPICs primers of the candidate gene such as SEQ ID NO.1
With 2, SEQ ID NO.3 and 4, SEQ ID NO.5 and 6, SEQ ID NO.7 and 8, SEQ ID NO.9 and 10, SEQ ID NO.11
Shown in 12 or SEQ ID NO.13 and 14.
9. application of the claim 1~8 any one of them method in population analysis.
10. application according to claim 9, which is characterized in that the population analysis is population genetic diversity analysis, group
Phylogeny reconstruction analysis, the research of pedigree Geographical Analysis, Niche Model or non-mode biological evolution in fluid-structure analysis, kind
Select the estimation of pressure.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111477275A (en) * | 2020-04-02 | 2020-07-31 | 上海之江生物科技股份有限公司 | Method and device for identifying multi-copy area in microorganism target fragment and application |
| CN116064507A (en) * | 2022-12-13 | 2023-05-05 | 广州大学 | Specific DNA fragment for sex identification of large thorn loach, genetic sex marking primer and application |
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|---|---|---|---|---|
| CN104928367A (en) * | 2015-06-26 | 2015-09-23 | 中国海洋大学 | Shellfish distant hybridization offspring genetic identification method |
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2018
- 2018-01-23 CN CN201810066162.3A patent/CN108048530B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928367A (en) * | 2015-06-26 | 2015-09-23 | 中国海洋大学 | Shellfish distant hybridization offspring genetic identification method |
Non-Patent Citations (2)
| Title |
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| CHENHONG LI ET AL: "Exon-primed intron-crossing (EPIC) markers for non-model teleost fishes", 《BMC EVOL BIOL》 * |
| KONRAD LOHSE ET AL.: "Developing EPIC markers for chalcidoid Hymenoptera from EST and genomic data", 《MOLECULAR ECOLOGY RESOURCES》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111477275A (en) * | 2020-04-02 | 2020-07-31 | 上海之江生物科技股份有限公司 | Method and device for identifying multi-copy area in microorganism target fragment and application |
| CN116064507A (en) * | 2022-12-13 | 2023-05-05 | 广州大学 | Specific DNA fragment for sex identification of large thorn loach, genetic sex marking primer and application |
| CN116064507B (en) * | 2022-12-13 | 2023-07-18 | 广州大学 | Specific DNA fragment for sex identification of large thorn loach, genetic sex marking primer and application |
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