CN101880662B - Microsatellite marker locus primer of ciconia boyciana and genetic individual recognizing method - Google Patents
Microsatellite marker locus primer of ciconia boyciana and genetic individual recognizing method Download PDFInfo
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- CN101880662B CN101880662B CN2010102037869A CN201010203786A CN101880662B CN 101880662 B CN101880662 B CN 101880662B CN 2010102037869 A CN2010102037869 A CN 2010102037869A CN 201010203786 A CN201010203786 A CN 201010203786A CN 101880662 B CN101880662 B CN 101880662B
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- ciconia boyciana
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Abstract
The invention relates to a microsatellite marker locus primer of a ciconia boyciana and a genetic individual recognizing method for a ciconia boyciana by utilizing a microsatellite marker locus. The method comprises the following steps of: collecting a tissue of the ciconia boyciana and extracting genome DNA; introducing a molecular marker locus primer for carrying out the PCR (Polymerase Chain Reaction) amplification of the microsatellite locus; genotyping a microsatellite locus PCR amplification product; and analyzing an individual of the ciconia boyciana by utilizing a genotyping result. By the microsatellite marker locus with high precision, the individual recognition on the ciconia boyciana can be realized on the genetic level, and the genetic distance and the affinity relationship among ciconia boyciana individuals can also be judged. The invention is to be successfully applied to the breeding protection work and the scientific research of the ciconia boyciana which is an endangered animal.
Description
Technical field
The present invention relates to the animal molecular genetics field, particularly the exploitation of the microsatellite markers primer of ciconia boyciana and utilize microsatellite markers ciconia boyciana to be carried out to the method for genetics individual recognition.
Background technology
Ciconia boyciana (Ciconia boyciana) is the large-scale wader of Ciconiiformes (Ciconiiformes), Ciconiidae (Ciconiidae).Ciconia boyciana once was distributed widely in a plurality of countries and regions (Collar et a1. in East Asia in history, 2001), after the seventies in last century due to many factors such as habitat fragmentation, habitat loss, overhuntings, the ciconia boyciana wild stocks is in succession in the ground such as Japan, Korea, Korea S extinction (Wang Qishan, 1995; Murata, 2004).The global wild stocks quantity of current ciconia boyciana is 3000 left and right only, and (Wang Qishan, Yang Zhaofen, 1995) on a declining curve, very urgent to the protection of these species.Therefore in the red species register of IUCN, being put into (EN) in imminent danger species, is also CITES appendix I species.
From the middle and later periods eighties 20th century, the breeding of raising in cages has successively been carried out to ciconia boyciana in the Duo Jia zoological park, and China is the maximum country of ciconia boyciana artificial propagation quantity at present.Yet usually run into some problems in the long-term reproductive process of raising in cages, as the pedigree confusion of ciconia boyciana, the relationship between individuality is not clear etc., and this brings difficulty also to the breeding measures such as artificial pairing of ciconia boyciana.Not only to pay close attention to the protection recovery of habitat, the work such as artificial breeding of Rejuvenate species to the protection of endangered species, also will pay attention to the understanding (Frankham etal., 2002) to its genetic background, and then make scientific and reasonable child care countermeasure simultaneously.Owing to lacking efficient strong molecule marker, at present still very limited to the genetic research of ciconia boyciana, be badly in need of from the molecular genetics angle, ciconia boyciana being carried out the research of conservation biology.Wherein, by molecule marker, carry out discriminate individuals, realize individual recognition, thereby provide technical support just to become very crucial for the child care of these species.
Micro-satellite (Microsatell ite) be at present the animals on the brink of extinction conservation genetics learn the optimal molecule genetic marker of research (
1998), have that polymorphism is high, codominant inheritance, spread all over whole genome, select neutral, the advantage such as can increase, be easy to detect, reproducible, the aspect researchs (Weigend, 2001) such as genetic map construction, paternity test, individual recognition, population genetic diversity analysis of many animals have been widely used at present.But microsatellite molecular marker has the species specificity of height, need to build micro-satellite library to screen special microsatellite locus and to design corresponding pcr amplification primer.In this application, the applicant is by building the screening in Bing Duigai library, the micro-satellite of ciconia boyciana library, specific 8 microsatellite genetic markers of ciconia boyciana have been obtained, and design corresponding primer, and used these microsatellite genetic markers to realize the individual recognition on the molecular level in white stork in the Orient, thereby provide important technical support for the formulation of the pedigree analysis of ciconia boyciana, marriage plan and Analysis of Genetic Background etc.
Summary of the invention
For the deficiencies in the prior art, the invention provides the microsatellite markers primer of ciconia boyciana and utilize the microsatellite molecular marker site ciconia boyciana to be carried out to the method for genetics individual recognition.
The present invention is achieved by the following technical solutions:
The first purpose of the present invention is to provide the microsatellite markers primer of ciconia boyciana:
Cbo?102-F：5’-AGCAGCTAAATAACCAG-3’
Cbo?102-R：5’-AGAGATTGTCCCCGAGATAC-3’
Cbo?108-F：5’-CCCAGGTCACAAATTATACG-3’
Cbo?108-R：5’-GAGCCTCACAAAGTTCCCTG-3’
Cbo?109-F：5’-GTGGTGTAGTCCAGTTTATG-3’
Cbo?109-R：5’-ATAACACATGAATGACCTGG-3’
Cbo?121-F：5’-CCACAATGGCAATTTTTCAC-3’
Cbo?121-R：5’-GTTCTCCCAGAGGCTTGCTC-3’
Cbo?133-F：5’-GGACAAAAGGCGATTCTAGC-3’
Cbo?133-R：5’-TTGAGCCAAACATCCGACAC-3’
Cbo?151-F：5’-AATCTGGTCTTGGTCCTTTC-3’
Cbo?151-R：5’-GGTTTTACCCTCTGACACTG-3’
Cbo?168-F：5’-GGGTGCAGTTGAATTAGAC-3’
Cbo?168-R：5’-AATATTTTGGTTTGGTAAAC-3’
Cbo?235-F：5’-TGGCTAAACATCTCCAAAC-3’
Cbo?235-R：5’-TACAAGTAACGCAAGGGTAC-3’。
The index parameter of the microsatellite locus corresponding with described primer is as following table:
The second purpose of the present invention is to provide a kind of ciconia boyciana microsatellite molecular marker site that utilizes and ciconia boyciana is carried out to the method for genetics individual recognition, it is characterized in that: gather the ciconia boyciana tissue sample; Extract genomic dna; Introduce molecule marker as claimed in claim 1 or 2 site primer and carry out the microsatellite locus pcr amplification; Gene type to the microsatellite PCR amplified production; Utilize the ontoanalysis of gene type result to ciconia boyciana.
Beneficial effect of the present invention is: by the slight satellite genetic marker of high precision site, not only realized the genetics individual recognition to these species of ciconia boyciana, can also judge the distance of genetic distance and sibship between each ciconia boyciana individuality simultaneously.The present invention will successfully apply to the breeding among Protection and scientific research of these animals on the brink of extinction of ciconia boyciana, and many-sided its value that embody such as foundation, pairing seed selection, family's pedigree analysis, genetic map construction, paternity test, genetic diversity context analyzer of white stork genes of individuals identity card in the Orient.
Embodiment
The concrete steps of the micro-satellite molecule marking method of ciconia boyciana are as follows:
1, ciconia boyciana DNA extracting
Utilize muscle or blood tissues, extract the genomic dna of ciconia boyciana.The extracting of genomic dna adopts the cracking of SDS/ Proteinase K, phenol-chloroform extracting approach (Sambrook et al., 1999).
2, the structure of ciconia boyciana enriched microsatellite library
Utilize and extract genomic dna, adopt AFLP Rapid Isolation (Fast Isolation by AFLP of Sequences Containing repeats, FIASCO) build enriched microsatellite library, detailed process is as follows: get genomic dna 250ng left and right, use MseI digestion, be connected with the AFLP acceptor joint (5 '-TAC TCA GGA CTC AT-3 '/5 '-GAC GAT GAG TCC TGA G-3 ') of MseI simultaneously.After ten times of digestion product dilutions, with the primer (5 '-GAT GAG TCC TGA GTA AN-3 ' is called for short MseI-N) of AFLP receptor-specific, do pcr amplification.Amplified production is the dispersion plating that is greater than 200bp after 30 minutes at 1% agarose gel electrophoresis.
Use probe (AC)
12perhaps (AG)
12after cutting the connecting fluid pcr amplified fragment and hybridized with enzyme, use magnetic bead to carry out absorb-elute, can obtain the micro-satellite repetitive sequence of required strand.The DNA caught of take is template, and the MseI-N of take carries out the double-stranded DNA recovery as primer.The PCR reaction volume is 50 μ L, and reaction composition is: dNTP 5 μ L, Buffer 5 μ L, r Taq 0.5 μ L, MseI-N Primer 2 μ L, magnetic bead elutriant 5 μ L.The circulation of PCR is set to: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30sec; 60 ℃ of annealing 30sec; 72 ℃ are extended 45sec, carry out 5 circulations; Carry out other 92 ℃ of sex change 30sec; 60 ℃ of annealing 30sec; 72 ℃ of 30 circulations of extending 45sec, 72 ℃ are extended 10min.
The PCR product is through PCR cleaning agents box (Axygen) purifying, and by 2% agarose-EB gel detection, result is that the dispersion plating that is greater than 200bp is considered as successfully.After being recovered to product and pMD19-T carrier (Takara) is connected, two strands after purifying transforms in DH5-α competent cell (Takara).The competent cell transformed is coated on the Amp+LB flat board that adds 0.5mM IPTG and 0.5 μ g/mL X-Gal, under 37 ℃, cultivated 13h, obtain the micro-satellite library that comprises AC, two kinds of repetitions of AG.
3, the screening of positive colony, order-checking and design of primers
The full bacterium colony of picking white be placed in Amp+LB liquid nutrient medium enlarged culturing (37 ℃, 5h).Adopt Tri-Primer-PCR (Zane et al., 2002) to detect the positive recombinant that contains micro-satellite fragment.Select the probe (AC) of the lifeless matter element mark that M13 carrier universal primer (M13-47) and clone are corresponding in experiment
12/ (AG)
12reacted.If product two or more band occurs and can think that this mono-clonal contains aim sequence, otherwise thinks without aim sequence.Successfully check out 107 of the positive recombinants of aim sequence by the three-primer method, these recons are checked order.
The preliminary screening of 4, sequential analysis, design of primers and microsatellite locus
Check sequencing result, after removing the carrier sequence of M13, use TRF software (TandemRepeats Finder, Version3.2.1) to find the sequence (Benson, 1999) that wherein contains micro-satellite repeating unit.(Lalitha 2000 to use PRIMER5 software; Singh etal., 1998) design primer in the flanking sequence of repeating unit sequence upstream and downstream, the purpose fragment is set between 100-200bp.Select altogether 28 micro-satellites to repeat site as alternative mark.
5, the gene type data reads the acquisition with microsatellite locus
One-sided primer 5 ' end to 28 microsatellite locus just selecting carries out fluorescent mark (FAM/HEX/TAMRA), then use the DNA of 23 wild ciconia boyciana individualities to be screened above-mentioned microsatellite locus, process is as follows: at first treat the pcr amplification of bit selecting point, circulation is set to: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30sec, the 60sec that anneals at the Tm temperature, 72 ℃ are extended 60sec, react 30 circulations; 72 ℃ are extended 10min.Amplified production is used Tamara350 fluorescence molecule amount standard in ABI 377DNA sequenator, to use polyacrylamide gel to carry out gene type, and the result of genetic analysis is used Genescan 2.0 softwares to be read.The analysis of gene type result, remove following alternative site: without the site of gene type result; In arbitrary individuality, number of alleles is in plural site; In 23 wild ciconia boyciana, allelotrope is in the site below two.After screening, finally obtain 8 stable microsatellite locus, the primer sequence in site title and this site is as shown in the table:
6, realize the individual recognition to ciconia boyciana by micro-satellite gene type in 8 sites
Use above-mentioned 8 microsatellite locus, to the gene type result of 23 wild ciconia boyciana, as shown in the table.Distinguish analysis by individuality, in 23 individualities in table, allelotrope difference minimum be 4 allelotrope, account for whole 16 allelic 25%, the individuality of these 4 allelotrope differences is to being: No. 1 to No. 13, No. 7 to No. 19, No. 13 to No. 15, No. 17 to No. 23, No. 21 to No. 23.Allelotrope difference between other individuality is all at 4 more than allelotrope.In table, 23 wild ciconia boyciana individualities are all distinguished by these 8 micro-satellites, thereby have realized the individual recognition on the molecular genetics level.
<110 > University of Anhui
<120 > the microsatellite markers primer of ciconia boyciana and genetics individual discrimination method
<140>201010203786.9
<141>2010-06-21
<160>24
<170>PatentIn?version?3.3
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<223 > primer
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agcagctaaa?taaccag
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<223 > primer
<400>2
agagattgtc?cccgagatac
<210>3
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<223 > primer
<400>3
cccaggtcac?aaattatacg
<210>4
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<223 > primer
<400>4
gagcctcaca?aagttccctg
<210>5
<211>20
<212>DNA
<213 > artificial sequence
<220>
<223 > primer
<400>5
gtggtgtagt?ccagtttatg
<210>6
<211>20
<212>DNA
<213 > artificial sequence
<220>
<223 > primer
<400>6
ataacacatg?aatgacctgg
<210>7
<211>20
<212>DNA
<213 > artificial sequence
<220>
<223 > primer
<400>7
ccacaatggc?aatttttcac
<210>8
<211>20
<212>DNA
<213 > artificial sequence
<220>
<223 > primer
<400>8
gttctcccag?aggcttgctc
<210>9
<211>20
<212>DNA
<213 > artificial sequence
<220>
<223 > primer
<400>9
ggacaaaagg?cgattctagc
<210>10
<211>20
<212>DNA
<213 > artificial sequence
<220>
<223 > primer
<400>10
ttgagccaaa?catccgacac
<210>11
<211>20
<212>DNA
<213 > artificial sequence
<220>
<223 > primer
<400>11
aatctggtct?tggtcctttc
<210>12
<211>20
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<220>
<223 > primer
<400>12
ggttttaccc?tctgacactg
<210>13
<211>19
<212>DNA
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<223 > primer
<400>13
gggtgcagtt?gaattagac
<210>14
<211>20
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<223 > primer
<400>14
aatattttgg?tttggtaaac
<210>15
<211>19
<212>DNA
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<400>15
tggctaaaca?tctccaaac
<210>16
<211>20
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<223 > primer
<400>16
tacaagtaac?gcaagggtac
Claims (2)
1. the microsatellite markers primer of ciconia boyciana, is characterized in that described micro-satellite mark molecule marker site primer sequence is as follows, and described microsatellite markers combination of primers is used:
Cbo102-F：5’-AGCAGCTAAATAACCAG-3’
Cbo102-R：5’-AGAGATTGTCCCCGAGATAC-3’
Cbo108-F：5’-CCCAGGTCACAAATTATACG-3’
Cbo108-R：5’-GAGCCTCACAAAGTTCCCTG-3’
Cbo109-F：5’-GTGGTGTAGTCCAGTTTATG-3’
Cbo109-R：5’-ATAACACATGAATGACCTGG-3’
Cbo121-F：5’-CCACAATGGCAATTTTTCAC-3’
Cbo121-R：5’-GTTCTCCCAGAGGCTTGCTC-3’
Cbo133-F：5’-GGACAAAAGGCGATTCTAGC-3’
Cbo133-R：5’-TTGAGCCAAACATCCGACAC-3’
Cbo151-F：5’-AATCTGGTCTTGGTCCTTTC-3’
Cbo151-R：5’-GGTTTTACCCTCTGACACTG-3’
Cbo168-F：5’-GGGTGCAGTTGAATTAGAC-3’
Cbo168-R：5’-AATATTTTGGTTTGGTAAAC-3’
Cbo235-F：5’-TGGCTAAACATCTCCAAAC-3’
Cbo235-R：5’-TACAAGTAACGCAAGGGTAC-3’。
2. utilize ciconia boyciana microsatellite molecular marker site ciconia boyciana to be carried out to the method for genetics individual recognition, it is characterized in that: gather the ciconia boyciana tissue sample; Extract genomic dna; Introduce molecule marker as claimed in claim 1 site primer and carry out the microsatellite locus pcr amplification; Gene type to the microsatellite PCR amplified production; Utilize the ontoanalysis of gene type result to ciconia boyciana.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1370834A (en) * | 2001-02-15 | 2002-09-25 | 华中农业大学 | Hog microsatellite DNA mark suitable for use in classifying hog breeds |
| CN1880477A (en) * | 2006-05-01 | 2006-12-20 | 中国海洋大学 | Method for detecting micro-satellite marker LJ_liu of Lateolabrax japonicus |
| KR20090026748A (en) * | 2006-05-05 | 2009-03-13 | 쌘디스크 코포레이션 | Non-volatile memory with background data latch caching during read operations and methods therefor |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1370834A (en) * | 2001-02-15 | 2002-09-25 | 华中农业大学 | Hog microsatellite DNA mark suitable for use in classifying hog breeds |
| CN1880477A (en) * | 2006-05-01 | 2006-12-20 | 中国海洋大学 | Method for detecting micro-satellite marker LJ_liu of Lateolabrax japonicus |
| KR20090026748A (en) * | 2006-05-05 | 2009-03-13 | 쌘디스크 코포레이션 | Non-volatile memory with background data latch caching during read operations and methods therefor |
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