CN104611440A - Method for carrying out parentage identification and paternity identification by utilizing multiple haliotis discus hannai Ino fluorescence-labeled microsatellite - Google Patents
Method for carrying out parentage identification and paternity identification by utilizing multiple haliotis discus hannai Ino fluorescence-labeled microsatellite Download PDFInfo
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Abstract
The invention belongs to the field of marine biotechnology, and particularly relates to a method for carrying out parentage identification and paternity identification by utilizing a multiple haliotis discus hannai Ino fluorescence-labeled microsatellite. Multiple PCR reaction is adopted by the fluorophore-labeled haliotis discus hannai Ino microsatellite, so that the microsatellite can be applied to distinguishment of different parentages of haliotis discus hannai Ino or haliotis discus hannai Ino paternity identification. Two combinations disclosed by the invention both integrate original nine microsatellite PCR amplification and detection to one time, the detection cost is lowed, and the work efficiency is improved. An ABI 3730xl full-automatic DNA sequenator is used for performing microsatellite classification, classification error caused by denaturing polyacrylamide gel electrophoresis separation and sensitive silver staining is avoided, and the accuracy of paternity identification is improved. The data analysis in studying of haliotis discus hannai Ino genetics is more standardized and normalized by the method established in the invention, and data communication and sharing among different researchers are facilitated.
Description
Technical field
The invention belongs to field of marine biotechnology, a kind ofly specifically utilize multiple haliotis discus hannai Ino fluorescence labeling microsatellite to carry out the method for parentage identification and paternity identification.
Background technology
Haliotis discus hannai Ino (Haliotis discus hannai Ino) is the mollusk that a class has Important Economic and is worth, and because of its delicious meat, nutritious, becomes one of important sea farming object of China already.Along with improving constantly of living standards of the people, the demand of haliotis discus hannai Ino increases day by day, and therefore all kinds of Biotechnology in Genetic Breeding is also carried out in succession in these species.Setting up the more stable family of good character is one of current main task, but because the growth cycle of haliotis discus hannai Ino is longer, breeding process frequent operation cause breeding with cultivating process in be subject to the pollution of external source family, therefore need to carry out paternity identification to the haliotis discus hannai Ino individuality of breeding process, specify the genealogical relationship of each individuality.
At present, generally approve microsatellite marker in the world, particularly multiple microsatellite PCR reaction system is the preferred embodiment solving this problem.The good news is, at long oyster (Taris et al., 2005), American oyster (Wang et al., 2010), sinamia (Zhu et al., 2010), atlantic salmon (Olafsson et al., 2010) etc. successfully construct multiple microsatellite PCR system in marine fishery animal, these systems just can carry out the researchs such as paternity identification, pedigree analysis, genetic construction analysis efficiently, quickly by one group or a small amount of several groups of PCR reaction systems.
Though haliotis discus hannai Ino micro-satellite multiplex PCR system construction appears in the newspapers (Nie Hongtao etc., 2013), each combination of this system only has 3 microsatellite markers, and utilize 6% denaturing polyacrylamide gel electrophoresis to be separated, the colour developing of silver dye, therefore, efficiency is lower, be subject to subjective impact.For increasing work efficiency, reduce subjective judgement error, suitable microsatellite marker is selected from haliotis discus hannai Ino genetic map 18 linkage groups, different fluorophors is marked to each microsatellite marker, build multiple microsatellite PCR system, can high-throughput, carry out the qualification of parental right relation at low cost.The foundation of this system also will make the paternity identification of haliotis discus hannai Ino and pedigree analysis quick at a high speed, and analyzes stdn more and standardization to research haliotis discus hannai Ino data on genetics, is beneficial to the data exchange between different investigator and shares.
Summary of the invention
The object of the invention provides a kind of and utilizes multiple haliotis discus hannai Ino fluorescence labeling microsatellite to carry out the method for parentage identification and paternity identification.
For achieving the above object, the present invention adopts technical scheme to be:
With the micro-satellite of the haliotis discus hannai Ino of mark fluorescent group, adopt multi-PRC reaction, and then high-throughput ground is used for differentiation or the haliotis discus hannai Ino paternity identification of the different family of haliotis discus hannai Ino.
Being marked on by fluorophor obtains on microsatellite marker from the linkage group of haliotis discus hannai Ino genetic map, build two groups of multiple microsatellite PCR systems, the PCR sample obtained utilizes the full-automatic DNA sequencer of ABI 3730xl to carry out micro-satellite somatotype, ROX fluorescence molecule is as interior mark, GeneMarkerV1.75 software is utilized to obtain micro-satellite typing data, and according to the Mendel's law of segregation of microsatellite marker, data are passed through PAPA (Package for the Analysis of Parental Allocation) software, process amorphs site and detect the somatotype error that may exist, and then high-throughput ground is used for differentiation or the haliotis discus hannai Ino paternity identification of the different family of haliotis discus hannai Ino.
The micro-satellite of haliotis discus hannai Ino of described two group echo fluorophors is respectively:
Combination one is:
Haliotis discus hannai Ino microsatellite marker HD12
Forward primer (5 '-3 '): FAM-TGGATCTGCTGTTTGTCAC,
Reverse primer (5 '-3 '): GGCTAATGTCCACTACTTTC;
Haliotis discus hannai Ino microsatellite marker CHh157
Forward primer (5 '-3 '): FAM-ATAATAAACAGCATTGGCGGACG,
Reverse primer (5 '-3 '): CCACCCCTACTCCTAAATATTTTGG;
Haliotis discus hannai Ino microsatellite marker CHh104
Forward primer (5 '-3 '): FAM-TGGCAATGTGGATTTTGTTGATG,
Reverse primer (5 '-3 '): GCTTCGATAAGCATTCTACATGGAG;
Haliotis discus hannai Ino microsatellite marker CHh012
Forward primer (5 '-3 '): HEX-GCAATCTAACCTATTCAGCCACC,
Reverse primer (5 '-3 '): GAACATAAGCAACATCACCATCA;
Haliotis discus hannai Ino microsatellite marker Awb039
Forward primer (5 '-3 '): HEX-TCGTTGTTCAAAGATAAAGATGTGA,
Reverse primer (5 '-3 '): CTTACGTCCAGTGTTATGCGGTCTC;
Haliotis discus hannai Ino microsatellite marker CHd533
Forward primer (5 '-3 '): HEX-TCGTATCTCAGTAGGCTGGCAT,
Reverse primer (5 '-3 '): TTGTTCATTCTTGATTATTATCGTG;
Haliotis discus hannai Ino microsatellite marker KHdh86
Forward primer (5 '-3 '): TAM-AGTTGTTGAACCTTTCTGTTG,
Reverse primer (5 '-3 '): GAAAACTTCACCCACTACTTG;
Haliotis discus hannai Ino microsatellite marker CHh139
Forward primer (5 '-3 '): TAM-TTTGCTAAAGCTGCGACATCGTATC,
Reverse primer (5 '-3 '): CTTCTGTATTTCATGGTGTCCGTGG;
Haliotis discus hannai Ino microsatellite marker CHh001
Forward primer (5 '-3 '): TAM-GAAGCAAAACCAGATGCCAAGGAGG,
Reverse primer (5 '-3 '): TTCAAGGTAGCAGCAACAGCAACGAC;
Combination two is:
Haliotis discus hannai Ino microsatellite marker CHh011
Forward primer (5 '-3 '): FAM-GATCTTTATTTGGCAAACTAA,
Reverse primer (5 '-3 '): TGAAACCTGATACTGGTAAGC;
Haliotis discus hannai Ino microsatellite marker Eab630
Forward primer (5 '-3 '): FAM-GTACTTGTTGTTACCTTGGCATTC,
Reverse primer (5 '-3 '): ACGTAGACTTACGATAGTCATAGC;
Haliotis discus hannai Ino microsatellite marker CHh165
Forward primer (5 '-3 '): FAM-CGTACCCCCTGTTGTGGTAGTAGAT,
Reverse primer (5 '-3 '): TGTAGTGTTATACCAGTAGACGGCG;
Haliotis discus hannai Ino microsatellite marker CHh107
Forward primer (5 '-3 '): HEX-GGTCCTCTGTGGTCTGAAGCAAC,
Reverse primer (5 '-3 '): TCAAGCATATCATACTTTGTACTACGCAC;
Haliotis discus hannai Ino microsatellite marker CHh036
Forward primer (5 '-3 '): HEX-GCTTAGTCAGACCTGAAGGATACC,
Reverse primer (5 '-3 '): GTTCTACTCACTGTAAAACAATCAATG;
Haliotis discus hannai Ino microsatellite marker CHh193
Forward primer (5 '-3 '): HEX-TAACACCTGCGTCGTCTTCGTC,
Reverse primer (5 '-3 '): TATCACAATTATTCCACAACCTTATGT;
Haliotis discus hannai Ino microsatellite marker Tr001
Forward primer (5 '-3 '): TAM-ATGTGAATGCACGTCACTATG,
Reverse primer (5 '-3 '): ATAACTGTCTTAGCACTAGGATTG;
Haliotis discus hannai Ino microsatellite marker CHh170
Forward primer (5 '-3 '): TAM-GGTATCCCAAAACATTTTCTGAGCA,
Reverse primer (5 '-3 '): GTTAGTGACCCCAAAAGTTCATCCA;
Haliotis discus hannai Ino microsatellite marker CHh175
Forward primer (5 '-3 '): TAM-TTTACTGGATGTGAAACAGTGACAC,
Reverse primer (5 '-3 '): ATTGACTAGGAAGATGAGAAGCTGA.
Described multi-PRC reaction condition is
taq DNA Master Mix (ABI) 7.5ul, Primer Mix 1ul, GC Enchaner 1.5ul, template DNA (40-80ng/ul) 1ul, distilled water polishing is to 15ul.PCR reaction conditions is 94 DEG C of sex change 4min, then carries out 13 circulation (94 DEG C of sex change 30s; Annealing temperature is down to 53 DEG C from 59 DEG C, and every 1 cycle annealing temperature reduces by 0.5 DEG C, and annealing time is 45s; 72 DEG C extend 45s), and then carry out 22 circulation (94 DEG C of sex change 30s; 53 DEG C of annealing 45s; 72 DEG C extend 45s), finally, 72 DEG C extend 10min, 4 DEG C of preservations.
Its concrete steps are as follows:
(1) genomic dna of haliotis discus hannai Ino parents and offspring individual is extracted;
(2) according to the feature such as amplified production length, temperature of reaction of microsatellite marker in haliotis discus hannai Ino genetic linkage maps 18 linkage groups, pick out 18 microsatellite markers, mark three kinds of fluorophors (being respectively FAM, HEX and TAM).
(3) Auele Specific Primer and the mark fluorescent group of two groups of microsatellite markers is obtained.
(4) haliotis discus hannai Ino micro-satellite multi-PRC reaction condition is optimized.
(5) PCR primer utilizes the full-automatic DNA sequencer of ABI 3730xl to carry out micro-satellite somatotype, and ROX fluorescence molecule is as interior mark.GeneMarker V1.75 software is utilized to read micro-satellite typing data.
(6) according to the Mendel's law of segregation of microsatellite marker, data are input in EXCEL, utilize the grand function computational analysis in software, and aid in Manual analysis, this step can process amorphs site and detect the somatotype error that may exist.
(7) data that previous step is handled well are brought in PAPA (Package for the Analysis ofParental Allocation) software, automatically calculate, identify the parents of each offspring individual, i.e. paternity identification.
Advantage of the present invention: original 9 microsatellite PCRs amplification and detection are all incorporated into 1 time by two combinations of the present invention, reduce testing cost, improve working efficiency.Utilize the full-automatic DNA sequencer of ABI 3730xl to carry out micro-satellite somatotype, evaded denaturing polyacrylamide gel electrophoresis separation, silver dye develops the color the somatotype error brought, and improves the accuracy of paternity identification.Foundation of the present invention also will make research haliotis discus hannai Ino data on genetics analyze stdn more and standardization, be beneficial to the data exchange between different investigator and share.
Accompanying drawing explanation
Fig. 1 is the filial generation parental right efficiency diagram that in embodiment of the present invention, two multi-PRC reaction system combinations are used for identifying haliotis discus hannai Ino 10 ♀ × 10 ♂ mating combination.Along with the increase of each combination microsatellite marker number, its paternity identification efficiency is higher.When use the 1st group of multi-PRC reaction system, 758 offspring individuals (99.7%) identify its parents, and when use the 2nd group of multi-PRC reaction system, 757 offspring individuals (99.6%) identify its parents.
Embodiment
Embodiment
Build haliotis discus hannai Ino 10 ♀ × 10 ♂ mating combination, extract the DNA of 20 parents and 760 offspring individuals.Adopt 2 groups of different multi-PRC reaction system amplifications respectively.
Be specially, reaction conditions is:
taq DNA Master Mix (ABI) 7.5ul, Primer Mix 1ul (each primer concentration is in table 1), GC Enchaner 1.5ul, template DNA (40-80ng/ul) 1ul, distilled water polishing is to 15ul.PCR reaction conditions is 94 DEG C of sex change 4min, then carries out 13 circulation (94 DEG C of sex change 30s; Annealing temperature is down to 53 DEG C from 59 DEG C, and every 1 cycle annealing temperature reduces by 0.5 DEG C, and annealing time is 45s; 72 DEG C extend 45s), and then carry out 22 circulation (94 DEG C of sex change 30s; 53 DEG C of annealing 45s; 72 DEG C extend 45s), finally, 72 DEG C extend 10min, 4 DEG C of preservations.
Two groups of PCR samples of above-mentioned acquisition utilize the full-automatic DNA sequencer of ABI 3730xl to carry out micro-satellite somatotype respectively, and ROX fluorescence molecule is as interior mark.GeneMarker V1.75 software is utilized to read micro-satellite typing data.According to the Mendel's law of segregation of microsatellite marker, data are input in EXCEL, utilize the grand function computational analysis in software, and aid in Manual analysis, this step can process amorphs site and detect the somatotype error that may exist.The data that previous step is handled well are brought in PAPA (Package for the Analysis of Parental Allocation) software, automatically calculate, identify the parents of each offspring individual, i.e. paternity identification.
When use the 1st group of multi-PRC reaction system, 758 offspring individuals (99.7%) identify its parents, and when use the 2nd group of multi-PRC reaction system, 757 offspring individuals (99.6%) identify its parents (Fig. 1).If when using this two groups of multi-PRC reaction systems, then the equal successful identification of all offspring individuals is to its parents.
Each primer information in table 1 haliotis discus hannai Ino micro-satellite multi-PRC reaction system
Reference:
OLAFSSON,K.,HJORLEIFSDOTTIR,S.,PAMPOULIE,C.,HREGGVIDSSON,G.O.&GUDJONSSON,S.2010.Novel set of multiplexassays(SalPrint15)for efficient analysis of 15 microsatellite loci ofcontemporary samples of the Atlantic salmon(Salmo salar).Molecular EcologyResources,10,533-537.
TARIS,N.,BARON,S.,SHARBEL,T.F.,SAUVAGE,C.&BOUDRY,P.2005.A combined microsatellite multiplexing and boiling DNA extractionmethod for high-throughput parentage analyses in the Pacific oyster(Crassostrea gigas).Aquaculture Research,36,516-518.
WANG,Y.,WANG,X.X.,WANG,A.M.&GUO,X.M.2010.A16-microsatellite multiplex assay for parentage assignment in the eastern oyster(Crassostrea virginica Gmelin).Aquaculture,308,S28-S33.
ZHU,Z.Y.,WANG,C.M.,LO,L.C.,LIN,G.,FENG,F.,TAN,J.,CHOU,R.,LIM,H.S.,ORBAN,L.&YUE,G.H.2010.A standard panel ofmicrosatellites for Asian seabass(Lates calcarifer).Animal Genetics,41,208-212.
Nie Hongtao, Li Qi, & hole makes cutting edge of a knife or a sword. haliotis discus hannai Ino micro-satellite multiplex PCR system construction and the application in Parentage determination thereof. and aquatic product journal, 37 (2), 207-215.
Claims (4)
1. utilize multiple haliotis discus hannai Ino fluorescence labeling microsatellite to carry out a method for parentage identification and paternity identification, it is characterized in that:
With the micro-satellite of the haliotis discus hannai Ino of mark fluorescent group, adopt multi-PRC reaction, and then high-throughput ground is used for differentiation or the haliotis discus hannai Ino paternity identification of the different family of haliotis discus hannai Ino.
2. utilize multiple haliotis discus hannai Ino fluorescence labeling microsatellite to carry out the method for parentage identification and paternity identification by according to claim 1, it is characterized in that: fluorophor is marked on and obtains on microsatellite marker from the linkage group of haliotis discus hannai Ino genetic map, build two groups of multiple microsatellite PCR systems, the PCR sample obtained utilizes the full-automatic DNA sequencer of ABI 3730xl to carry out micro-satellite somatotype, ROX fluorescence molecule is as interior mark, GeneMarker V1.75 software is utilized to obtain micro-satellite typing data, and according to the Mendel's law of segregation of microsatellite marker, data are passed through PAPA (Package for the Analysis ofParental Allocation) software, process amorphs site and detect the somatotype error that may exist, and then high-throughput ground is used for differentiation or the haliotis discus hannai Ino paternity identification of the different family of haliotis discus hannai Ino.
3., by utilizing multiple haliotis discus hannai Ino fluorescence labeling microsatellite to carry out the method for parentage identification and paternity identification described in claim 1 or 2, it is characterized in that:
The micro-satellite of haliotis discus hannai Ino of described two group echo fluorophors is respectively:
Combination one is:
Haliotis discus hannai Ino microsatellite marker HD12
Forward primer (5 '-3 '): FAM-TGGATCTGCTGTTTGTCAC,
Reverse primer (5 '-3 '): GGCTAATGTCCACTACTTTC;
Haliotis discus hannai Ino microsatellite marker CHh157
Forward primer (5 '-3 '): FAM-ATAATAAACAGCATTGGCGGACG,
Reverse primer (5 '-3 '): CCACCCCTACTCCTAAATATTTTGG;
Haliotis discus hannai Ino microsatellite marker CHh104
Forward primer (5 '-3 '): FAM-TGGCAATGTGGATTTTGTTGATG,
Reverse primer (5 '-3 '): GCTTCGATAAGCATTCTACATGGAG;
Haliotis discus hannai Ino microsatellite marker CHh012
Forward primer (5 '-3 '): HEX-GCAATCTAACCTATTCAGCCACC,
Reverse primer (5 '-3 '): GAACATAAGCAACATCACCATCA;
Haliotis discus hannai Ino microsatellite marker Awb039
Forward primer (5 '-3 '): HEX-TCGTTGTTCAAAGATAAAGATGTGA,
Reverse primer (5 '-3 '): CTTACGTCCAGTGTTATGCGGTCTC;
Haliotis discus hannai Ino microsatellite marker CHd533
Forward primer (5 '-3 '): HEX-TCGTATCTCAGTAGGCTGGCAT,
Reverse primer (5 '-3 '): TTGTTCATTCTTGATTATTATCGTG;
Haliotis discus hannai Ino microsatellite marker KHdh86
Forward primer (5 '-3 '): TAM-AGTTGTTGAACCTTTCTGTTG,
Reverse primer (5 '-3 '): GAAAACTTCACCCACTACTTG;
Haliotis discus hannai Ino microsatellite marker CHh139
Forward primer (5 '-3 '): TAM-TTTGCTAAAGCTGCGACATCGTATC,
Reverse primer (5 '-3 '): CTTCTGTATTTCATGGTGTCCGTGG;
Haliotis discus hannai Ino microsatellite marker CHh001
Forward primer (5 '-3 '): TAM-GAAGCAAAACCAGATGCCAAGGAGG,
Reverse primer (5 '-3 '): TTCAAGGTAGCAGCAACAGCAACGAC;
Combination two is:
Haliotis discus hannai Ino microsatellite marker CHh011
Forward primer (5 '-3 '): FAM-GATCTTTATTTGGCAAACTAA,
Reverse primer (5 '-3 '): TGAAACCTGATACTGGTAAGC;
Haliotis discus hannai Ino microsatellite marker Eab630
Forward primer (5 '-3 '): FAM-GTACTTGTTGTTACCTTGGCATTC,
Reverse primer (5 '-3 '): ACGTAGACTTACGATAGTCATAGC;
Haliotis discus hannai Ino microsatellite marker CHh165
Forward primer (5 '-3 '): FAM-CGTACCCCCTGTTGTGGTAGTAGAT,
Reverse primer (5 '-3 '): TGTAGTGTTATACCAGTAGACGGCG;
Haliotis discus hannai Ino microsatellite marker CHh107
Forward primer (5 '-3 '): HEX-GGTCCTCTGTGGTCTGAAGCAAC,
Reverse primer (5 '-3 '): TCAAGCATATCATACTTTGTACTACGCAC;
Haliotis discus hannai Ino microsatellite marker CHh036
Forward primer (5 '-3 '): HEX-GCTTAGTCAGACCTGAAGGATACC,
Reverse primer (5 '-3 '): GTTCTACTCACTGTAAAACAATCAATG;
Haliotis discus hannai Ino microsatellite marker CHh193
Forward primer (5 '-3 '): HEX-TAACACCTGCGTCGTCTTCGTC,
Reverse primer (5 '-3 '): TATCACAATTATTCCACAACCTTATGT;
Haliotis discus hannai Ino microsatellite marker Tr001
Forward primer (5 '-3 '): TAM-ATGTGAATGCACGTCACTATG,
Reverse primer (5 '-3 '): ATAACTGTCTTAGCACTAGGATTG;
Haliotis discus hannai Ino microsatellite marker CHh170
Forward primer (5 '-3 '): TAM-GGTATCCCAAAACATTTTCTGAGCA,
Reverse primer (5 '-3 '): GTTAGTGACCCCAAAAGTTCATCCA;
Haliotis discus hannai Ino microsatellite marker CHh175
Forward primer (5 '-3 '): TAM-TTTACTGGATGTGAAACAGTGACAC,
Reverse primer (5 '-3 '): ATTGACTAGGAAGATGAGAAGCTGA.
4., by utilizing multiple haliotis discus hannai Ino fluorescence labeling microsatellite to carry out the method for parentage identification and paternity identification described in claim 1 or 2, it is characterized in that:
Described multi-PRC reaction condition is
taq DNA Master Mix (ABI) 7.5ul, Primer Mix 1ul, GC Enchaner 1.5ul, template DNA (40-80ng/ul) 1ul, distilled water polishing is to 15ul.PCR reaction conditions is 94 DEG C of sex change 4min, then carries out 13 circulation (94 DEG C of sex change 30s; Annealing temperature is down to 53 DEG C from 59 DEG C, and every 1 cycle annealing temperature reduces by 0.5 DEG C, and annealing time is 45s; 72 DEG C extend 45s), and then carry out 22 circulation (94 DEG C of sex change 30s; 53 DEG C of annealing 45s; 72 DEG C extend 45s), finally, 72 DEG C extend 10min, 4 DEG C of preservations.
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| CN105200051A (en) * | 2015-10-29 | 2015-12-30 | 中国海洋大学 | SNP (single nucleotide polymorphism) marker for distinguishing Chinese population and Japan population of haliotis discus hannai |
| CN109680077A (en) * | 2019-01-25 | 2019-04-26 | 福建出入境检验检疫局检验检疫技术中心 | A kind of method of fluorescence quantitative PCR detection identification haliotis discus hannai Ino |
| CN109680077B (en) * | 2019-01-25 | 2022-03-22 | 福建出入境检验检疫局检验检疫技术中心 | Method for detecting and identifying Haliotis discus hannai by fluorescent quantitative PCR (polymerase chain reaction) |
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