CN106834517A - For the SSR label primer group of harbor seal paternity test and its application - Google Patents
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Abstract
The invention discloses for the SSR label primer group of harbor seal paternity test and application, the present invention is successfully screened and synthesizes 15 pairs of fluorescence labeling microsatellite primer sets, the primer sets can be used for setting up harbor seal paternity test technology, and disclose the kit of used paternity test.That authentication method of the present invention has is accurate, efficiently, it is reliable the features such as, the artificial breeding and field that can instruct harbor seal release work, and carry out popularization and application in the management of harbor seal family and Germplasm Identification.
Description
Technical field
The invention belongs to marine mammal paternity test technical field, and in particular to for harbor seal paternity test
SSR primer sets and application.
Background technology
Harbor seal (Phoca largha) belongs to Carnivora (Carnivora) dog shape suborder (Caniformia) Phocidae
(Phocidae), harbor seal category (Phoca), is China second class protection animal, is distributed mainly on northern and western part of North Pacific
Marine site and its littoral and island, are distributed mainly on the Bohai Sea and the Huanghai Sea, occasionally in the South Sea in China.Harbor seal is uniquely can be in China
The pinnipeds that marine site is bred, and Bohai Liao Dong Bay icing area is most southern in the breeding of harbor seal 8 areas one.Spot sea
Leopard Wild population is threatened, and artificial propagation turns into the important means for protecting the species, with the holy sub- holding stock of tourism in Dalian
It is common successfully to breed spot since harbor seal artificial propagation work is carried out as a example by part Co., Ltd (hereinafter referred to as " Dalian is holy sub- ")
Sea dog 44, now has 8~10 individualities to be born under artificial condition every year, and up to the present F1 generation individuality has accounted for whole raising group
The 60% of body, many F1 generations have reached sexal maturity.Increase over time, many F1 generations will unavoidably participate in breeding, regret
Be that the patriarchy relation of these F1 generations is still not clear, similar situation also other with the presence of harbor seal aquarium.
Also known as paternity test, microsatellite paternity test is a kind of paternity test technology popular in recent years for paternity identification, its
It is widely applied in the genetic pedigree identification of mankind's forensic identification, Important Economic animal and animals on the brink of extinction.However, at present
The technology is detected in evidential attribute (Tursiops spp), Yangtze finless porpoise (Neophocaena in marine mammal
A small number of Cetaceans such as asiaeorientalisasiaeorientalis).In recent years, the gene of the Phocidae species such as harbor seal
Group, the excacation of transcript profile sequence achieve some breakthrough so as to the species microsatellite paternity test technology research and development and
Using there is provided opportunity.
At present, microsatellite molecular marker is not applied to also the paternity test of harbor seal and the report of genetic management.This
Invention is intended to the micro-satellite primers group using invention, and harbor seal paternity test skill is set up using fluorescence labeling capillary electrophoresis technique
Art, for harbor seal reproductive population genetic pedigree structure, each aquarium between harbor seal exchange, exchange foundation is provided, to close
That manages instructs harbor seal artificial propagation.
The content of the invention
The present invention is intended to provide for the SSR label primer group of harbor seal paternity test, the primer can be used for harbor seal parent
Son identification, containing 15 pairs of primers, its nucleotide sequence is as shown in SEQ ID NO.1~SEQ ID NO.30.
It is another object of the present invention to provide the SSR fluorescent dye primer groups for harbor seal paternity test should
With.The kit for harbor seal paternity test is prepared into using the primer sets, or harbor seal is entered using the primer sets
Row paternity test, or carry out family management using it.
Described kit includes whole/part of the nucleotide sequence as shown in SEQ ID NO.1~SEQ ID NO.30
Primer sets, and 10 × PCR buffer solutions, MgCl2, dNTPs, Ex Taq archaeal dna polymerases and distilled water.
Invention additionally discloses a kind of method of harbor seal paternity test, it includes following operating procedure：Extract parent to be identified
Sheet and filial generation sample DNA, to the forward primer of primer sets mentioned above, the fluorescent dye of flag F AM, HEX, TAM or ROX；
Recycle the whole/part primer sets after mark fluorescent dyestuff to expand the sample DNA for extracting, carry out Capillary Electrophoresis,
Genotyping is carried out according to electrophoresis peak figure result, Genotyping is carried out using GeneMapper softwares, analysis result output is existed
In Excel forms.Contained using Cervus software statistics number of alleles (Na), average expected volume heterozygosity (He), polymorphism information
Amount (PIC), the first elimination factor of accumulation (CE-1P), the second elimination factor of accumulation (CE-2P) and parents' accumulation elimination factor (CE-PP), root
The value and Delta value standards of the LOD calculated according to Parentage modules, are as a result shown as "+" (i.e. 80% confidence level
Under level) it is its possible parent；Result is shown as " * " (under i.e. 95% level of confidence) for its most probable parent.Specifically,
Parent child relationship decision method is listed in the embodiment of the present invention 3, the SSR molecular marker primer of present invention offer is effectively provided
Group, can be used for the management of harbor seal family and Germplasm Identification, so that rational expectation, SSR molecular marker primer sets of the present invention
And its application process, can effectively instruct the artificial propagation of harbor seal, the genetic diversity and germplasm money of protection China harbor seal
Source.
Brief description of the drawings
The width of accompanying drawing of the present invention 15, is the part sequencer map of the primer sets in paternity test of invention, it is shown that design primer
Correctness, be respectively：
Fig. 1 is primer PLMS10 sequencer maps, and female parent 59368 (275bp, 278bp), filial generation 87492 are followed successively by from left to right
(278bp), male parent 59347 (275bp, 278bp).
Fig. 2 is primer PLMS12 sequencer maps, is followed successively by 59368 (280bp) of female parent from left to right, filial generation 87492 (277bp,
280bp), male parent 59347 (277bp).
Fig. 3 is primer PLMS17 sequencer maps, is followed successively by 59368 (261bp) of female parent from left to right, filial generation 87492 (258bp,
261bp), male parent 59347 (258bp, 261bp).
Fig. 4 is primer PLMS24 sequencer maps, and female parent 59368 (223bp), filial generation 87492 are followed successively by from left to right
(223bp), male parent 59347 (223bp).
Fig. 5 is primer PLMS28 sequencer maps, and female parent 59368 (263bp), filial generation 87492 are followed successively by from left to right
(263bp), male parent 59347 (263bp).
Fig. 6 is primer PLMS44 sequencer maps, and female parent 59368 (226bp), filial generation 87492 are followed successively by from left to right
(226bp), male parent 59347 (226bp).
Fig. 7 is primer PLMS45 sequencer maps, and female parent 59368 (189bp), filial generation 87492 are followed successively by from left to right
(189bp), male parent 59347 (189bp).
Fig. 8 is primer PLMS68 sequencer maps, and female parent 59368 (250bp, 258bp), filial generation 87492 are followed successively by from left to right
(250bp, 258bp), male parent 59347 (247bp, 258bp).
Fig. 9 is primer PLMS55 sequencer maps, and female parent 59368 (211bp, 223bp), filial generation 87492 are followed successively by from left to right
(211bp), male parent 59347 (211bp).
Figure 10 is primer PLMS58 sequencer maps, and female parent 59368 (231bp), filial generation 87492 are followed successively by from left to right
(231bp), male parent 59347 (231bp).
Figure 11 is primer PLMS71 sequencer maps, and female parent 59368 (248bp), filial generation 87492 are followed successively by from left to right
(248bp, 295bp), male parent 59347 (248bp, 295bp).
Figure 12 is primer PLMS76 sequencer maps, and female parent 59368 (264bp, 280bp), filial generation 87492 are followed successively by from left to right
(264bp, 280bp), male parent 59347 (280bp).
Figure 13 is primer PLMS79 sequencer maps, and female parent 59368 (314bp, 318bp), filial generation 87492 are followed successively by from left to right
(314bp, 316bp), male parent 59347 (316bp).
Figure 14 is primer PLMS83 sequencer maps, and female parent 59368 (217bp), filial generation 87492 are followed successively by from left to right
(217bp), male parent 59347 (217bp).
Figure 15 is primer PLMS90 sequencer maps, and female parent 59368 (287bp), filial generation 87492 are followed successively by from left to right
(287bp), male parent 59347 (287bp).
Specific embodiment
Technical scheme of the present invention, if not otherwise specified, is the conventional scheme of this area.
Sample collection：Patent application people carried out blood sample in 2014 to 2016 to 53 harbor seals that Dalian sage Asia is fed
Collecting work, wherein the mother-child relationship (MCR) of 10 sea dog young babies to be identified has record to be, it is known that candidate's male parent to be identified
17.The present invention takes about 5ml with disposable syringe by animal doctor or trainer in harbor seal physical examination from tail vein blood vessel
Blood, loaded on (EDTA-K2 anti-freezings) in medical blood routine pipe, 4 DEG C of preservations are stored in -20 DEG C for a long time after taking back laboratory.DNA
Extract using the poba gene group DNA purification kits of Tiangeng company, specific steps reference operation handbook.
Embodiment 1
The primer sets screening of harbor seal paternity test microsatellite marker and fluorescent dye primer group are in harbor seal paternity test
In application, comprise the following steps：
(1) primer sets screening
Because the genomic information of the harbor seal announced is little, inventor is used by only transcript profile data
MISA softwares carry out the search of SSR sites to Unigenes sequences, and the standard of the search is dinucleotides, trinucleotide, four cores
The minimum number of repetition of thuja acid, pentanucleotide, Hexanucleotide is respectively 14,9,6,4,4, by containing EST-SSR sequence profit
With 5.0 Software for Design micro-satellite primers of Primer Premier 195 pairs, design of primers principle is as follows：Primer length：18-
21bp, primer sequence G/C content：33-72%, primer pair Tm values between 53-61.5 DEG C (upstream and downstream primer Tm value differences 5 DEG C it
It is interior), amplified fragments size is between 185-315bp, it is to avoid hairpin structure, dimer, mispairing and primer dimer etc. occurs and draws
Thing secondary structure occurs.Primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..First with 20 for selecting at random
The DNA of harbor seal carries out primer amplification, and primary dcreening operation is carried out with 8% polyacrylamide gel electrophoresis, according to electrophoresis result, excludes without expansion
Volume increase thing, without polymorphism, band is weak, be unable to the primer of repeat amplification protcol after, the preliminary primer sets for obtaining amplification stabilization and polymorphism；
Using the random 2 harbor seal individualities of the primer pair of preliminary screening enter performing PCR expand, and by PCR primer send raw work bioengineering (on
Sea) limited company's cloning and sequencing.
Preliminary screening the is obtained sense primer of primer sets simultaneously is with different fluorescent dye FAM, HEX, TAM and ROX
Enter row stochastic mark, primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
(2) primer sets the selection result
The PCR reaction systems of primer preliminary screening are 25 μ l, wherein template DNA 50ng, mix dNTP 0.2mM, up and down
Trip primer each 200nM, 1 × PCR buffer, 1.5mM MgCl2, Ex Taq (Takara companies) 1U.Bar is reacted with following PCR
Part is expanded：95 DEG C of predegeneration 5min；94 DEG C of denaturation 30s, annealing 30s (table 3), 72 DEG C of extension 30s, totally 35 circulations；Last 72
DEG C extend 10min.Screening separation is carried out using 8% polyacrylamide gel electrophoresis, is repeated by screening and comparing, there are 25 positions
Point obtains clear, accurate, consistent allelic information.
The PCR reaction systems and reaction condition of fluorescent primer as shown in table 1 below, table 2, through capillary electrophoresis detection after, and
With reference to cloning and sequencing result, finally obtain 15 pairs of polymorphisms it is high and can stablize the microsatellite marker of amplification (SEQ ID NO.1~
Shown in SEQID NO.30).
The fluorescent primer reaction system of table 1
| Template (10ng/ μ l) | 1μl |
| Primers F (10mmol/ μ l) | 0.5μl |
| Primer R (10mmol/ μ l) | 0.5μl |
| dNTP 10mM | 0.5μl |
| 10×PCR Buffer | 2.5μl |
| 2.0μl | |
| Ex Taq enzymes (5U/ μ l) | 0.2 |
| Water (μ l) | 17.8 |
The fluorescent primer reaction condition of table 2
Embodiment 2
(1) microsatellite genotyping
To Dalian Sheng Ya harbor seals colony (the 53 individualities) DNA for extracting, the 15 pairs of fluorescence labelings obtained using screening are drawn
Thing group is expanded, and PCR primer carries out 3730XL sequenators (American AB I companies) capillary after being detected by 1% Ago-Gel
Electrophoresis detection, Genotyping is carried out using GeneMapper softwares, by analysis result output in Excel forms.
(2) feature of each microsatellite locus
The Capillary Electrophoresis peak figure of 53 the 15 of individuality pairs of fluorescent dye primers is carried out into base using GeneMapper softwares
Because of parting, by genotyping result output in Excel forms, and the softwares of Cervus 3.0 are imported, as a result as shown in table 3.It is each micro- to defend
Star gene seat allele number is 2~8, and average number of alleles is 4.1, and the average expected volume (He) of gene heterozygosity is
0.3669, the average value of polymorphism information content (PIC) is 0.3206.
The sequence of each microsatellite of table 3, annealing temperature, Gene frequency distribution and paternity test elimination factor
Embodiment 3
(1) parent child relationship judges
With the softwares of Cervus 3.0, count each microsatellite locus number of alleles (Na), average expected volume heterozygosity (He),
Polymorphism information content (PIC).And the parameters such as the elimination factor of each locus are calculated on this basis.
In the present invention, there are two kinds of situations in the probability of exclusion of father's identification：
Situation one：All suspicious individual genotype have all been listed, but mother-subrelation is unclear.In the case,
The probability of exclusion E of each microsatellite locusxAccording to the algorithm of formula below Jamieson (1965,1994), PiAnd PjIt is every
The gene frequency of individual seat allele:
Ex=∑ Pi 2(1-Pi)2-∑(PiPj)[4-3(Pi+Pj)]
Situation two：In mother, it is known that in the case that father is unknown, each site is in mother, it is known that the unknown feelings of father
Under condition, the probability of exclusion Ex of each site father identification is calculated according to the algorithm of the formula below such as Gaber：
Ex=∑ Pi 2(1-Pi)2+∑2(PiPj)(1-Pi-Pj)2
The joint elimination factor E in multiple sites is calculated according to the method for the formula below such as Jamieson, and i is the microsatellite for using
Number of loci：
E=1- (1-Ex1)(1-Ex2)(1-Ex3)…(1-Exi)
In the present invention, mother is, it is known that father is unknown.Judge parent child relationship using the softwares of Cervus 3.0：According to
The genotype of suspicious father calculates its LOD value (the natural logrithm value of likelihood ratio).Judge parent child relationship according to LOD scoring height：
Seek Δ value：
N >=2, Δ=LOD1-LOD2
N=1, Δ=LOD1
N=0, Δ value is without definition.
In formula, LOD1And LOD2It is two LOD values of harbor seal of most likely true father.
To ensure that the micro-satellite primers group used by the present invention can be simultaneously domestic other aquarium harbor seal paternity tests
There is provided and instruct, when the elimination factor of each microsatellite locus is calculated, counted according to the data of all harbor seals for collecting.
(2) qualification result：
According to each microsatellite locus allele situation of harbor seal for collecting, the genotype of each sample is obtained, calculated
The elimination factor of all sites (table 3), is computed, and the first elimination factor of accumulation (CE-1P) is 0.8214, accumulates the second elimination factor
(CE-2P) and parents' accumulation elimination factor (CE-PP) is respectively 0.9713,0.9977, because the sea dog mother that need to be identified is
Know, show that microsatellite marker used can be effective for the father of harbor seal identification.
Simulation (Simulation) module of Cervus3.0 softwares is run, simulation candidate parent 17 is circulated 10000 times
(filial generation is set to 10000), calculates a known parent and does not know the Δ distribution of parent, in runs software respectively
Parentage modules detect parent child relationship.Experimental population in this programme is the harbor seal fed, 10 sea dog childrens to be identified
Young mother-child relationship (MCR) has record to be, it is known that candidate's male parent to be identified has 17, is calculated according to Parentage modules
LOD value and Delta value standards, qualification result understand：Under 80% level of confidence, all 10 sea dogs to be identified
All have found most like father, wherein having the confidence level of 9 more than 95%, their affiliation refers to table 4.Can from table
Going out the filial generation sea dog that numbering is 59361 and 20946 has a common father：59331, they are brother and sister's relation；And male parent sea dog
59347 have 4 offsprings, are respectively：59365th, 87400,87492 and 59351.
The paternity identification result of table 4
Note：" * " represents that parent child relationship is extremely notable in table, and confidence level is more than 95%；"+" represents that parent child relationship is more significant, puts
Reliability is more than 80%.
Claims (5)
1. the SSR primer sets of harbor seal paternity test are used for, containing 15 pairs of primers, its nucleotide sequence such as SEQ ID NO.1~
Shown in SEQ ID NO.30.
2. application of the primer sets described in claim 1 in harbor seal paternity test.
3. application of the primer sets described in claim 1 in harbor seal paternity test kit is prepared.
4. application according to claim 3, it is characterised in that：Described kit includes whole/portion described in claim 1
Divide primer sets, 10 × PCR buffer solutions, MgCl2, dNTPs, Ex Taq archaeal dna polymerases and distilled water.
5. the method for harbor seal paternity test, it is characterised in that including following operating procedure：Extract parent to be identified and filial generation sample
Product DNA；To the forward primer of the primer sets described in claim 1, the fluorescent dye of flag F AM, HEX, TAM or ROX；Recycle
Whole/part primer sets after mark fluorescent dyestuff enter performing PCR amplification to the sample DNA for extracting, and carry out Capillary Electrophoresis, foundation
Electrophoresis peak figure result carries out Genotyping, uses Cervus software statistics number of alleles (Na), average expected volume heterozygosity
(He), polymorphism information content (PIC), the first elimination factor of accumulation (CE-1P), the second elimination factor of accumulation (CE-2P) and parents' accumulation
Elimination factor (CE-PP), the value and Delta value standards of the LOD calculated according to Parentage modules, in 80% confidence
Under degree level, "+" is as a result shown as its possible parent；Under 95% level of confidence, as a result it is shown as " * " and most may be used for it
Can parent.
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| CN201710179132.9A CN106834517B (en) | 2017-03-23 | 2017-03-23 | SSR (simple sequence repeat) labeled primer group for parent-child identification of parva and application of SSR labeled primer group |
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|---|---|---|---|
| CN201710179132.9A CN106834517B (en) | 2017-03-23 | 2017-03-23 | SSR (simple sequence repeat) labeled primer group for parent-child identification of parva and application of SSR labeled primer group |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103866004A (en) * | 2014-01-24 | 2014-06-18 | 中国水产科学研究院 | Molecular marker method for identifying fugu rubripes parentage as well as microsatellite and kit used for molecular marker method |
| CN105177143A (en) * | 2015-09-16 | 2015-12-23 | 中国科学院南海海洋研究所 | Method for identifying hippocampus kelloggi family by virtue of microsatelite |
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2017
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| CN103866004A (en) * | 2014-01-24 | 2014-06-18 | 中国水产科学研究院 | Molecular marker method for identifying fugu rubripes parentage as well as microsatellite and kit used for molecular marker method |
| CN105177143A (en) * | 2015-09-16 | 2015-12-23 | 中国科学院南海海洋研究所 | Method for identifying hippocampus kelloggi family by virtue of microsatelite |
| CN105331732A (en) * | 2015-12-04 | 2016-02-17 | 南京师范大学 | Method for identifying odontobulis mpotamophila family |
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| Title |
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| JOSEPH I. HOFFMAN ET AL.: ""GENETIC ANALYSIS OF TWINNING IN ANTARCTIC FUR SEALS (ARCTOCEPHALUS GAZELLA)"", 《JOURNAL OF MAMMALOGY》 * |
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