CN102226214A

CN102226214A - Duck parental testing method by multiple polymerase chain reactions (PCRs) and kit

Info

Publication number: CN102226214A
Application number: CN2011101161079A
Authority: CN
Inventors: 刘伟; 徐桂云; 杨宁; 詹凯; 李俊营
Original assignee: Institute of Animal Husbandry and Veterinary Medicine of Anhui Academy of Agricultural Sciences
Current assignee: Institute of Animal Husbandry and Veterinary Medicine of Anhui Academy of Agricultural Sciences
Priority date: 2011-05-06
Filing date: 2011-05-06
Publication date: 2011-10-26

Abstract

The invention discloses a duck parental testing method by multiple polymerase chain reactions (PCRs) and a kit. By the method, 20 short tandem repeat (STR) markers are subjected to combination of multiple PCR amplifications, the workload is greatly simplified. The method has the characteristics of high efficiency, quickness, accuracy and high flux, is particularly suitable for determination of genetic relation between parents and progeny in a duck breeding population performing mass natural random mating, can reduce workload and improve laying rate, fertility rate and hatching rate of breed ducks in breeding, and has better economical benefit.

Description

A kind of multiplex PCR carries out the method and the test kit of duck paternity test

Technical field

The invention belongs to biological technical field, be specifically related to carry out the technological method that the pcr amplification of STR mark of duck paternity test and detection and sibship are identified, be adapted at that duck is cultured and breeding in carry out Idioplasm identification and sibship evaluation.

Background technology

Paternity test (parentage identification) claim paternity identification, individual recognition etc. again, it is the theory and technology that utilizes biology, molecular genetics and medical science, from the form structure of filial generation and parental generation or the similar characteristics of aspects such as physiological function, physical basis of heredity, analyze hereditary feature, judge whether akin method (Qian Lindong etc., 2010) of parental generation and filial generation.The paternity test technology has important use to be worth in daily forensic medical examination and livestock industry are produced, and the paternity test method has multiple, and early stage have blood group method of testing, human leucocyte antigen identification method and a chromosomal polymorphism identification method etc.; Along with going deep into of molecule genetics research, the molecular genetic marker detection technique of several genes group level has been applied to paternity test, these dna molecular marker technology are the genetic markers based on genetic material inner nucleotide sequence variations between individuality, are the direct reflections of dna level genetic polymorphism.Dna molecular marker has plurality of advantages, and the dna molecular marker that wherein is suitable for most paternity test has: (Hill such as Mitochondrial DNA mark, dna fingerprint mark, moonlet variation tumor-necrosis factor glycoproteins mark, microsatellite marker STR Deng,

2008).

Microsatellite marker (microsatellite marker) claims simple repeated sequence (simple sequence repeats again, SSR), short series connection repeats (short tandem repeat, STR) etc., be widely used in the field such as structure, individual recognition, paternity test, the assignment of genes gene mapping, analysis of genetic diversity, medical diagnosis on disease of linkage map.Utilize microsatellite marker to carry out paternity test and on human medical jurisprudence, be widely used, a large amount of reports are also arranged in animals and plants.Utilize DNA microsatellite marker and SNP site that the animal population of unknown pedigree is carried out sibship and identify existing in the world successful Application.The microsatellite marker that has proposed in ISAG (ISAG, International Society of Animal Genetics, http://www.isag.org) in 1996 annual meeting to use in several domestic animal paternity tests such as ox, pig, horse, dog is advised.Open inferior equality nineteen ninety-five and separated the paternity test that 10 giant panda microsatellite markers can be used for giant panda, Zhang Yuguang etc. were applied to the paternity test of northeastern tiger in 2003 with microsatellite marker, had all obtained good effect, had very high accuracy; Ye Junhua etc. (2005) adopt multiplex PCR and genome scanning technology for detection the Ministry of Public Security directly under four tame police dog base import kind dogs and 7 kinds of accurate numerous dog 10 microsatellite DNA polymorphisms of totally 269 samples, the parentage exclusion probability that found that these 10 microsatellite locus is 99.7287%, the individual recognition rate is 99.999%, has set up police dog STR-DNA paternity test method; Ellegren etc. (1992) judge that with the microsatellite marker of 5 equines the parental right relation of horse can make elimination factor reach more than 98%, and 10 mark elimination factors reach 99.99%; Glowatzki-Mullis etc. (1995) have utilized 6 polymorphic STR marker detection on the coloured differently body gene frequency of 238 oxen, the parental right that has successfully solved 35 oxen can't determining with traditional blood group authentication method is confirmed problem; Ozkan etc. (2009) utilize 12 microsatellite markers that holstein cow colony has been carried out the paternity test analysis, comprising 9 sites of recommending by ISAG, the result shows that the eliminating probability of these 12 marks is at least 99.9%, and the parental right relation of most of (95.3%) has obtained affirmation; Stevanovic Deng(2009,2010)

Utilize 11 microsatellite markers to carry out paternity test respectively in different Simmental colonies, the result shows that its eliminating probability can reach 99.9%.

At present to be marked at the research of carrying out paternity test in the bird few for STR, is used for analysis of genetic diversity and genetic (Qu such as mobile more Deng,

2006; Berthouly Deng,

2008,2009); It is few to utilize the STR mark to carry out the research of paternity test in aquatic bird, and Reichart etc. (2010) utilize protein fingerprint technology and 10 microsatellite markers to identify sibship between ruddy duck.Above result of study shows that the microsatellite DNA mark research of duck has obtained impressive progress and good application has been arranged, and how further bringing into play bigger effect in the breeding work of duck is a field that is worth very much research.

China is maximum in the world meat duck producing country and country of consumption, and the proportion in aviculture is increasing.There are many local duck varieties with good hereditary property in China, but lack effectively development and use, the breeding work of duck is made slow progress, and scientific and technological content is lower, is necessary state of the art and quickening breeding progress by the breeding of molecular biological means service hoisting duck.

The pedigree record of setting up complete and accurate is the basis of duck breeding work, and for promoting China's duck breeding level, improving breeding efficiency has vital role and meaning.Utilize present method not changing that present kind of duck ground is raised scattered, to set up the pedigree record between two generation duck groups under the prerequisite of the raising pattern of pangamy accurately, and accurately fast, help accelerating the seed selection progress.

Summary of the invention

The present invention has developed and has a kind ofly utilized 20 STR marks to carrying out the technological method of paternity test between the duck parent of unknown sibship and filial generation, and the working method of this technology is provided.

The technical scheme of this method is as follows:

One aspect of the present invention relates to the method that a kind of multiplex PCR carries out the duck paternity test, it is characterized in that comprising the steps: parent and filial generation duck are extracted DNA, according to the synthetic fluorescent primer of following table;

Primer in the use table carries out the multiplex PCR amplification to the DNA that extracts, and carries out fluorescence electrophoresis and detect, and electrophoresis result analysis is obtained all individual genotype datas, and parent and filial generation are carried out the sibship analysis.

In a preferred embodiment of the present invention, it is characterized in that the described condition of according to the form below carries out pcr amplification:

In a preferred embodiment of the present invention, it is characterized in that described duck is Chaohu duck.

In a preferred embodiment of the present invention, it is characterized in that by software electrophoresis result and/or sibship being analyzed, described software is selected from GeneMaper and/or Cervus.

The present invention also relates to a kind of test kit of duck paternity test in addition on the one hand, it is characterized in that comprising primer and the DNA extraction reagent described in claim 1 table.

The present invention has the advantage of following several respects:

1. high-throughput: the present invention is by a large amount of research of groping, continue to optimize and improve the PCR reaction conditions, 20 STR marks are optimized combination, formed 7 multi-PRC reaction systems, workload has been reduced by 66.7%, saved a large amount of time, artificial and expense.

2. accuracy rate height: according to the Mendelian genetics theory, utilize 20 STR marks to carry out the duck paternity test, its non-father gets rid of probability and can reach more than 99.999%.Its rate of accuracy reached to 98.969% when in duck breeding group, carrying out this technical identification.

Description of drawings

Fig. 1 a, the Genemapper analytical results of 1b: embodiment 2.

Embodiment

Two generations Chaohu duck colony employing present method of 1 pair of known pedigree record of embodiment is carried out paternity test

At Chaohu duck seed farm according to existing pedigree record, 5 drakes of picked at random at first, find and these 5 ducks that drake carries out mating according to pedigree record then, totally 23, again according to pedigree record 65 of picked at random in the filial generation of these male and female ducks, totally 93 are individually adopted the present invention to confirm sibship between its individuality.

Above 93 individualities are carried out venous blood collection under the wing, extract genomic dna, it is standby to be diluted to 50ng/ μ l.According to the synthetic little satellite fluorescent primer of micro-satellite primers information in the table 1, and carry out the multiplex PCR amplification, carry out electrophoresis detection then.Electrophoresis result is analyzed with GeneMapperv3.0 software, obtain all individual genotype datas, carry out the sibship analysis with 2.0 couples of parents of software Cervus and filial generation, calculate its most probable parents' individuality of each filial generation, choose the highest drake of degree of confidence respectively and duck is confirmed as its parent according to analytical results, obtain 93 sibship records between the duck individuality at last.Result and the existing pedigree record of seed farm of using the present invention to analyze are contrasted, in 65 filial generation individualities, have 2 individualities carry out the parent when assert both results inconsistent, and be male parent or difference appears in a maternal side, under the right-on prerequisite of the hypothesis original pedigree record of breeding station, its accuracy rate of the present invention is 96.923%.

Embodiment 2 uses the present invention in two generation Chaohu duck colonies of unknown pedigree record

Carry out the application test of present method at Chaohu duck seed farm.

Choose reproductive performance good 40 age in week 150 of Chaohu duck kind ducks, all wear wing number, 20 drakes wherein, 130 ducks, this duck group focuses on and carries out jumpbogroup in the big colony house and raise random mating.Raise collection every day kind of an egg after 15 days, go into kind of egg storehouse and preserve, the kind egg of collecting altogether 4 days goes into to incubate.To duckling band wing number, obtain 318 healthy offspring individuals altogether during hatching.Gather whole parents and offspring individual blood sample, carry out the multiplex PCR amplification after extracting DNA, and carry out genotype detection, the genotype data of parent and filial generation is analyzed, set up the sibship record between two generation Chaohu ducks.Above laboratory work part method is with embodiment 1.

Multi-PRC reaction system following (20 μ l system):

※: n represents the microsatellite marker number that comprises in each multi-PRC reaction system

The PCR product detects: pcr amplification product after suitably diluting (advising 5～10 times of dilutions), is mixed the back with mark in the molecular weight and carry out electrophoresis on ABI 3730xl type dna sequencing instrument, operate according to the instrument service requirements.

Interpretation of result: electrophoresis result utilizes GeneMaper 3.0 softwares to handle, and obtains each individual STR marker genotypes data.The genotype data that each is individual utilizes software Cervus 2.0 to calculate, and analyzes the probability of sibship between individuality, gets rid of the individuality of non-parent, obtains the sibship between parent and filial generation at last, finishes paternity test.

The result has 314 individualities can all assert the parent at 318 offspring individuals, and other 4 individualities can be assert the side among the parents, and its accuracy rate can reach 98.742%.

Should be understood that, for those of ordinary skills, can be improved according to the above description or conversion, and all these improvement and conversion all should belong to the protection domain of claims of the present invention.

Sequence table

＜120〉Title: a kind of multiplex PCR carries out the method and the test kit of duck paternity test

<130>AppFileReference：201110116107.9

<140>CurrentAppNumber：

<141>CurrentFilingDate：___-_-__

--------

Sequence

＜213〉OrganismName: Husbandry ﹠ Veternity Research Inst. of Anhui Prov. Agriculture Science Academy

<400>PreSequenceString：

acagcttcag?cagacttaga 20

<212>Type：DNA

<211>Length：20

SequenceName：DL01-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gcagaaagtg?tattaaggaa?g 21

<212>Type：DNA

<211>Length：21

SequenceName：DL01-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tccacttggt?agaccttgag 20

<212>Type：DNA

<211>Length：20

SequenceName：DL02-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tgggattcag?tgagaagcct 20

<212>Type：DNA

<211>Length：20

SequenceName：DL02-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

ctgggtttgg?tggagcataa 20

<212>Type：DNA

<211>Length：20

SequenceName：DL03-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tactggctgc?ttcattgctg 20

<212>YType：DNA

<211>Length：20

SequenceName：DL03-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tgctatccac?ccaataagtg 20

<212>Type：DNA

<211>Length：20

SequenceName：DL04-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

caaagttagc?tggtatctgc 20

<212>Type：DNA

<211>Length：20

SequenceName：DL04-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tttaggtaaa?actgtgaatc?aa 22

<212>Type：DNA

<211>Length：22

SequenceName：DL05-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

atcaaagcag?ggagctaag 19

<212>Type：DNA

<211>Length：19

SequenceName：DL05-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

acgtcacatc?accccacag 19

<212>Type：DNA

<211>Length：19

SequenceName：DI06-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

ctttgcctct?ggtgaggttc 20

<212>Type：DNA

<211>Length：20

SequenceName：DL06-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gtgcctaacc?ctgatggatg 20

<212>Type：DNA

<211>Length：20

SequenceName：DL07-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

cttatcagat?ggggctcgga 20

<212>Type：DNA

<211>Length：20

SequenceName：DL07-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gataatggct?ggctccttga 20

<212>Type：DNA

<211>Length：20

SequenceName：DL08-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gaccacaaca?tcgtgcagag 20

<212>Type：DNA

<211>Length：20

SequenceName：DL08-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gggacatctc?ttggagcaaa 20

<212>Type：DNA

<211>Length：20

SequenceName：DL09-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

agtgaaagct?gctgctggat 20

<212>Type：DNA

<211>Length：20

SequenceName：DL09-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gaaagaatga?gttaaaaagg?ca 22

<212>Type：DNA

<211>Length：22

SequenceName：DL10-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tgaggcttaa?ttggtggaca 20

<212>Type：DNA

<211>Length：20

SequenceName：DL10-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

ttagtaaact?cttgccatct 20

<212>Type：DNA

<211>Length：20

SequenceName：DL11-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tgtagtttag?ttgctggata 20

<212>Type：DNA

<211>Length：20

SequenceName：DL11-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

ggacaagtgg?catgtgtcat 20

<212>Type：DNA

<211>Length：20

SequenceName：DL12-1

SequenceDescription：

Sequence

--------

<400>PreSequenceStrlng：

ggcttctgtg?ctcctcagat 20

<212>Type：DNA

<211>Length：20

SequenceName：DL12-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

agaaagctcc?tgtatgtgat 20

<212>Type：DNA

<211>Length：20

SequenceName：DL13-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

atgctggtgt?gagatttgaa 20

<212>Type：DNA

<211>Length：20

SequenceName：DL13-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gatgtgtctt?ttccaggtgc 20

<212>Type：DNA

<211>Length：20

SequenceName：DL14-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gcatcccatc?ttcaaagcag 20

<212>Type：DNA

<211>Length：20

SequenceName：DL14-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

cagcgtcccc?agaaacttga 20

<212>Type：DNA

<211>Length：20

SequenceName：DL15-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

ccctcgtatt?atccagtccg 20

<212>Type：DNA

<211>Length：20

SequenceName：DL15-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gctgcctgga?atataaccgc 20

<212>Type：DNA

<211>Length：20

SequenceName：DL16-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gcatttgcag?agtcaggacc 20

<212>Type：DNA

<211>Length：20

SequenceName：DL16-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gcaataaagg?cgtgagca 18

<212>Type：DNA

<211>Length：18

SequenceName：DL17-1

SequenceDescription：

Sequence

-------

<400>PreSequenceString：

atatcctctc?cctcgcat 18

<212>Type：DNA

<211>Length：18

SequenceName：DL17-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gaaaaaggca?gcacagcac 19

<212>Type：DNA

<211>Length：19

SequenceName：DL18-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gcaaagttga?ggcatgtaat?c 21

<212>Type：DNA

<211>Length：21

SequenceName：DL18-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tgacattaca?cacccaaac 19

<212>Type：DNA

<211>Length：19

SequenceName：DL19-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

caagggcagg?ggtaaggat 19

<212>Type：DNA

<211>Length：19

SequenceName：DL19-2

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

tgacctccct?gacgtttgc 19

<212>Type：DNA

<211>Length：19

SequenceName：DL20-1

SequenceDescription：

Sequence

--------

<400>PreSequenceString：

gtctgcgtgt?ccgtgtatg 19

<212>Type：DNA

<211>Length：19

SequenceName：DL20-2

SequenceDescription：

Claims

1. the method that multiplex PCR carries out the duck paternity test is characterized in that comprising the steps: parent and filial generation duck are extracted DNA, according to the synthetic fluorescent primer of following table;

2. the method for carrying out the duck paternity test according to the described a kind of multiplex PCR of claim 1 is characterized in that the described condition of according to the form below carries out pcr amplification:

3. duck paternity test method according to claim 1 and 2 is characterized in that described duck is Chaohu duck Or Beijing duck

4. according to any described duck paternity test method of claim 1-3, it is characterized in that by software electrophoresis result and/or sibship being analyzed, described software is selected from GeneMaper and/or Cervus.

5. the test kit of a duck paternity test is characterized in that comprising primer and the DNA extraction reagent described in claim 1 table.