The present application is a U.S. National Phase of International Patent Application Serial No. PCT/CN2014/000937 entitled “METHOD FOR IDENTIFYING WUZHISHAN MINIATURE PIG INBRED LINE BY USING 145 SNP,” filed on Oct. 21, 2014. The entire contents of the above-referenced application are hereby incorporated by reference for all purposes.
Incorporated by reference in its entirety herein is a computer-readable nucleotide sequence listing submitted herewith and identified as follows: 24,964 bytes ASCII (Text) file named “Sequence_Listing_JEE17305PCTUS,” created Apr. 21, 2017.
The present invention relates to a method for identifying Wuzhishan miniature pig inbred line by using 145 SNPs.
During the period of 1989-2003, under the support from seven related programs including the 7th, 8th and 9th Five-Year key industrial R & D Programs of China's Ministry of Agriculture, and “Chinese National Programs for High Technology Research and Development Program (863 Program)” of China's Ministry of Science and Technology, key and major projects of National Natural Science Foundation of China etc., the research working on “the breeding, germplasm characteristics, as well as development and utilization of Wuzhishan inbred line” had made many breakthroughs, including but not limited to, taking a boar and a sow from Wuzhishan pigs (also referred as Wuzhishan miniature pigs) as line ancestors, continuously employing “offspring-parent”, “full sibs” mating, and other comprehensive measures, gradually overcoming the adverse effects that inbreeding resulted in very low breeding survival rate, which was less than 20% at that time, establishing F17 inbred line population and genealogy, monitoring the research process of inbred line breeding of inbred line varieties F7-F16 by using many advanced technical means, such as molecularly genetic means, DNA fingerprinting similarity factor, microsatellite etc., preliminarily revealing genetic regularity and characteristics of Wuzhishan miniature pig inbred line, and demonstrating the facticity and scientificity of the breeding process of this inbred line. Moreover, many studies on exploitation and utilization of Wuzhishan miniature pig inbred line had been completed, showing the incomparable superiority of this inbred line in serving as an ideal animal model for human disease, and consequently achieving a certain economic benefits and major social benefits. Two achievements of the researches—“Researches on germplasm characteristics as well as exploitation and utilization of Wuzhishan miniature pigs” (Achievements Appraisal of Academy of Agricultural Sciences 95, No. 075) and “Wuzhishan miniature pig inbred line breeding for experiments and molecule genetic basic research” passed the Science and Technology Achievement Appraisals hosted by China's Ministry of Agriculture in 1995 and 2005 respectively, and won the Third Prize of China's Ministry of Agriculture Science and Technology Progress Award in 1999.
Preliminary research achievements of miniature pig inbred breeding had great influences at home and abroad. For example, industrial specialists from the United States, Italy, Japan, West Germany, Korea, and other countries had proposed for introduction and cultivation, and or cooperative study. An institute of the United States proposed to purchase 50 boars with 5 million USD in 1996; Korea proposed to cooperatively set up factories in China by funding 5 million RMB in 2000 after its introduction and cultivation proposal was rejected. The main reason behind the influences is that the inbred line animals, as special genetic resources for animals, can rapidly obtain scientific data for life science researches with the same extent of sensitivity and accuracy as from analytical reagents in chemistry and precise instruments in physics. With their very high use value and research significance, more than 450 inbred lines of mice, rats, and small mammals etc, have been bred in the world at present, and widely used as animal models for studying difficult and complicated human diseases, solving difficult problems in bioscience, medical science, and pharmaceutics, and other research fields. Due to the various differences between pigs and those small animals in interspecific physiology, the breeding of inbred line pigs has been very difficult. It was verified by novelty searching (2013. 11) that there was no successful report in the world. Moreover, compared with rodents, pigs possesses greater similarity with human, and will play irreplaceable, unique roles in the researches for studying difficult and complicated human diseases and solving difficult problems in bioscience, medical science, and pharmaceutics. Inbred line pigs are rare, valuable genetic resources of large animals in the world. Previously, in 1960s, many departments of the United States and Europe carried out the breeding research of inbred line pigs, but failed with the highest inbreeding coefficient no more than 0.75. After a 15-year breeding research, China has obtained F17 with the special Chinese miniature pig resource, and the inbreeding coefficient is up to 0.965, and more encouragingly the high incidence stage of piglet death due to inbred breeding has been overcome. Therefore, the successful breeding of inbred line pigs will be likely achieved in China firstly. After a further work in current years, Wuzhishan miniature pig inbred lines F17 to F22 have been obtained.
Because there was no successful breeding report for inbred line pigs in the world yet, and either the standard of establishment or identification method of inbred line pigs does not exist, either, we strictly referred to and followed the breeding method of inbred line mouse or rat, that was, inbreeding more than 20 generations from two pigs in same ancestor and establishing complete genealogy, testing the genetic stability thereof, establishing identification method, establishing line standard, specially on interspecific differences from current Hainan Wuzhishan pigs. Only in this way, scientific and credible evidence can be provided to the industry and the world, and such novel innovation project of genetic resource can be accomplished, thereby to benefit human beings.
Once the miniature pig inbred line is successfully bred, China will possess the proprietary intellectual property rights and the international leading level of innovative genetic germplasm resource of pig, which will fill up research blank within this research field in the world, and enrich theory and practice of inbred line breeding for large animals. Meanwhile, as an ideal “animal models” for human beings, inbred line pig is an important foundation and innovation basis for the progress of biotechnological research, which will significantly promote collaborative development and innovation in bioscience, human medical science, and pharmacy, etc. in China, and play irreplaceable position for studying difficult and complicated human diseases, extending lives, so as to benefit human beings. Therefore, it possesses important practical and theoretical significance.
The purpose of the present invention is to provide a method for identifying Wuzhishan miniature pig inbred line by using 145 SNPs.
The present invention provides a method for auxiliarily identifying whether the pig to be tested is inbred line of Wuzhishan miniature pig, comprising the following steps:
Testing genotypes based on 145 SNP sites of the pig to be tested;
If all the standards of the following (1) to (145) are satisfied, the pig to be tested is a candidate for Wuzhishan miniature pig inbred line:
If any standard of the above is not satisfied, the pig to be tested is a candidate for non-Wuzhishan miniature pig inbred line.
The pig to be tested can be the Wuzhishan miniature pig inbred line or Hainan Wuzhishan pig.
The Wuzhishan miniature pig inbred line can belong to any one of F20 to F22 generations.
The Wuzhishan miniature pig inbred line can belong to F20 generation or generations later than F20 generation.
In this method, specific chips can be employed to test the genotype based on 145 SNP sites of the pig to be tested; the specific chips are nucleic acid chips in which single-stranded DNA molecules shown as Sequences 1-145 in the sequence listing are immobilized at different points, respectively.
The present invention is also used to protect a nucleic acid chip in which single-stranded DNA molecules shown as Sequences 1-145 in the sequence listing are immobilized at different points, respectively.
The present invention is also used to protect the use of the nucleic acid chip, which is the following (a) or (b): (a) auxiliarily identifying whether the pig to be tested is Wuzhishan miniature pig inbred line; or (b) auxiliarily identifying whether the pig population to be tested is Wuzhishan miniature pig inbred line population.
The pig to be tested can be the Wuzhishan miniature pig inbred line or Hainan Wuzhishan pig.
The Wuzhishan miniature pig inbred line can belong to any one of F20 to F22 generations.
The Wuzhishan miniature pig inbred line can belong to F20 generation or generations later than F20 generation.
The present invention also provides a method for auxiliarily identifying whether the pig population to be tested is Wuzhishan miniature pig inbred line population, comprising the following steps:
Randomly sampling from the pig population to be tested to obtain samples to be tested, then testing the genotypes based on 145 SNP sites of each of samples to be tested, and if all the samples to be tested satisfy all the standards of the following (1) to (145), the pig population to be tested is a candidate for Wuzhishan miniature pig inbred line population:
If more than one of the samples to be tested do not satisfy all the standards of the above (1) to (145), the pig population to be tested is a candidate for non-Wuzhishan miniature pig inbred line population.
In the method, samples to be tested with statistic significance are obtained by randomly sampling from the pig population to be tested.
In the method, more than 16 pig samples to be tested are obtained by randomly sampling from the pig population to be tested.
The pig to be tested can be Wuzhishan miniature pig inbred line or Hainan Wuzhishan pig.
The Wuzhishan miniature pig inbred line can belong to any one of F20 to F22 generations.
The Wuzhishan miniature pig inbred line can belong to F20 generation or generations later than F20 generation.
In the method, specific chips can be employed to test genotypes based on 145 SNP sites of the pig to be tested; the specific chips are nucleic acid chips in which single-stranded DNA molecules shown as Sequences 1-145 in the sequence listing are immobilized at different points, respectively.
The following examples are convenient for better understanding the present invention, but not to limit the present invention. Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the experimental materials used in the following examples are all commercially available from conventional biochemical reagent stores. The experimental animals include 48 Wuzhishan miniature pigs inbred line, of which 16 for F20 generation, 16 for F21 generation, and 16 for F22 generation, as well as 16 non-inbred line Hainan Wuzhishan pigs.
Wuzhishan Miniature Pig Inbred Line:
Li Kai, Feng Shutang, Mou Yulian, Yang Shulin, Han Jianlin, Liu Lan, Yuan Xinxu, Guo Yong, Study on Genetic Regulation of Microsatellite Loci Gene of Three Inbred Families of Wuzhishan Miniature Pig, Scientia Agricultura Sinica, 2012, 42 (5): 1751-1760.
Hainan Wuzhishan Pig:
Hou Guanyu, Wang Dongjin, Guan Song, Rong Guang, Huang Xianzhou, Detection of Genetic Quality of Small Population of Wuzhishan Pig, Acta Ecologiae Animalis Domastici, 2007, Volume 28, Issue 6: 44-47.
Example 1, the Establishment of the Method
The inventors of the present invention found that there are 145 SNP sites when genome researches are made on Wuzhishan miniature pig inbred line and non-Wuzhishan miniature pig inbred line, and Wuzhishan miniature pig inbred line and non-Wuzhishan miniature pig inbred line can be identified according to their genotypes. The genotypes of 145 SNP sites of Wuzhishan miniature pig inbred line are all homozygous types (genotypes described in Table 1). The genotypes of 145 SNP sites of non-Wuzhishan miniature pig inbred line are all heterozygous types.
In the 145 SNP sites, 12 SNP sites are located at first segment of chromosome 1, 16 SNP sites are located at 15th segment of chromosome 1, 14 SNP sites are located at 50th segment of chromosome 1, 16 SNP sites are located at 78th segment of chromosome 1, 15 SNP sites are located at second segment of chromosome 2, 14 SNP sites are located at 12th segment of chromosome 2, 13 SNP sites are located at 19th segment of chromosome 2, 20 SNP sites are located at fifth segment of chromosome 18, 13 SNP sites are located at 14th segment of chromosome 18, and 12 SNP sites are located at 20th segment of chromosome 18.
Based on 145 SNP sites, the inventors of the present invention designed 145 probes (the binding domain of probe with genomic DNA is adjacent to SNP site, and located upstream or downstream of SNP site, therefore, genotype of SNP site can be obtained via terminal extension).
As for names of 145 SNPs, their located chromosomes, their positions on the chromosomes, the genotypes of Wuzhishan miniature pig inbred line, source types of SNPs, nucleotide sequences of 145 probes, and source sequences of SNP on genomic DNA, see Table 1.
CAAGATGCTCAGACCC
CGACGCGAGACTCTCG
TTCTGTAGACCAAA
GA
AAACAAATTCCGACTT
GTGGAATTGTAAATATT
TGTCGATTC
[A/G]TCTTT
TCCCGTTTGCTTCTAA
GATACCTCCCTCCCAC
GCGCAGCCAACACCA
C
GGACAAAGGAAACCTG
TTTCCTACCGTTCCATT
ACGTCAGTGTG
[A/C]AA
AAAAAGAAAGCAGGCC
AAAAAAGAGTTCACAC
TCTACAAGGCCA
[T/C]G
TCCTTTGGTGCTGGAA
CTACAGAGAGTCTGTA
TTTTCTTCGATTCG
CCT
TACACAACGATGACAG
ACGCGACACAAGAAGG
ACTGAGTAAAGCAG
AA
AAGACTGTCGCAAGAC
TGTCGCAGTGTCGCAG
TGACAGCACCC
[A/G]GG
GAGCAGACAAATCAGC
CTTAAACACTGCTGAC
CTTAAACATCAG
[T/C]T
CCAGCAGCGTCTTTTT
ATTTGTATTAGTGGTGT
CTGCCAACAAG
[T/G]GG
GCATCTCCATCCGAGC
TTCTGCTTCCAAATCCA
TGTTCCTCCAGCC
CCG
GCTGCGGAAAATGGAT
TCGGTGTAAATGAATC
TTACCTGGAAGAG
ATG
CTTTAATATAGGCGTTC
CTAACCTGGATGTTTTT
GAGTTGAAC
[A/G]TAGG
TCTCAATACCTGGTCC
AAGAACTTCCATATGC
TGCAGGCATGGCC
AAA
TAGATAGACAGATAGTT
AGATAGCAGACAATAG
ATACAGAAA
[A/G]ATGG
TGTGATTGTGAAAGGT
GTTGATTTAATTCTTTT
GTGTATTCCCA
TCCTGA
TGGAGGAGAACTCTTT
TCATCTCAGCTAGAAA
TTCTTCTTGA
ACTTGAT
TTATGTGAGGGAGAGG
GAGCTGACATATCTTT
CCTACTGTATATC
ATAC
AGTTTGCTTATCTTAGG
AAGAAGTGAGAGTTTC
AAGTTCAGAATC
ATTTC
ATATGTCTATCAGTAAA
GAGTCAGCTAATACTTT
AAACTTTGCC
[T/C]GCC
ACAAATGTGCTACTTTT
ATATCTTTTTCCCATTT
TAAAACTTT
[A/G]CATTT
CATGGTATTTAAAAATT
GCTATAAGAGGAGTTA
CCATTGTGGCTC
AGCA
ATTCCTGTCTTGGGCA
GCAAAGCTCTTGAAAA
GGAAAAAGGAAG
GAGT
AGTATTTAAAAGGCAG
AAAAAAGGAAGGCTAC
AAAAAAGACCAA
GCTG
AGACATGGTTGTGGCT
TGACCTTGGAAAGATG
AGCTAAAGGTGC
[T/G]G
CATACTGAAAAGTGAT
GGAACATATCTTGAAA
CCAATTTGAC
[T/C]TAGT
GTGCTTCAGAGCTTTG
GTAATTTGGGCATCCT
TGTATCACCTT
[A/G]ATT
CACTCAAAGTACATATA
GAACTAACCATTTCTTA
TTACCTCCATTG
CTGCC
CTTCATAGTTAATAGCT
ATTAGAATCCTAGAGCT
GCATAGAGAA
[T/C]ATA
GTGGTGGCTGTGCCCT
GGGCTTTCAGAGGCTT
GTCTGGTGGACTGTT
C
TGATGCAGCAAGCTCT
GTGATGTAACAGGTAC
CCCACTCTGCTG
[A/G]C
CTCAGTCTGGACAGGG
CAGTGTGTTCTTTTTAT
GATAATGGGC
[A/G]CTG
TGGTTTTATGCCAAGA
GACTCCACAGTCAGCT
TAATAACCTTA
[A/G]GC
GCACATGGTTGCTTAG
AACAGTCCTCTCTTTT
GGGTCAGAACCATTT
C
CTGAGGGATTCTGGGA
TAGGAGAGACAAGGCA
GGCATTGGGAAGTCCA
GGCATTTTCCACAGAA
CAAAGTGACAGTTGCA
CTTATTGCTGCAGT
TTC
AACATGGATCTCTTCT
GTCCAATCTTTTCCCC
AAAGAACAAATG
AGAG
CACAAGGGGATATTCT
ATTGAATTAATGGCCCT
GGAAGGACAA
[A/C]AG
AGCCTTATGTTTTACTT
ACCTCGGAAGCATGAG
CCATGTGATA
[A/C]CTG
AAGTGGATGGTGTGGT
ATCACTGAACGTTGGC
TGTATTCAGAT
TGTCTA
ATCCTAATGCCAGTCAT
TCCAACTCACTCGTGG
TACAAATTGAT
GCAACT
CCAGGGCAACATCTTC
CATGAAGCTCATGTAA
CTTTAATGCCT
[T/C]GTC
TTCCCGTTCTCCCATTT
CTAATATAAGACAGGG
ATGAGGATAC
[A/C]NKK
CTGAGTTAGGTACTTG
GAGGGTGAAGGATAAA
AAAGATACAGA
[T/C]TA
TGCTCTTGAAAATCCC
AATTCAATTCAAACCC
AGCTGTTCTGAT
[A/C]A
GTCTTACTTTCAAACT
GATACAACAGTCCTCA
AATGCCAGATCA
GCTCC
TATTAAGCAACATCTAT
ATTTTAAATATATATAG
GGGGAGGG
TTTTCTGT
AGGGAGTCCCTGCTGT
GGCACAACCAGATTGG
CACAAACTCTA
GAGGC
TGCCCTGTAATATTTGG
AACAAACCTTATTTAGT
CA
GAGTGCTTCA
CTTGCCAAAGAATTAA
AATAAAGATAAGCAAT
GCTTTAAAAAA
[A/G]AT
TGTTTGCTTCTGATTAG
CCTGTGTGTATGTGTT
GGATGAATGAGC
AATT
TTCCCTGCCTAAAGCC
ACATAGCATCATCTTAG
GAAATAAACC
[T/C]GGG
ATGTATACGTAGATTCT
TATAACTGTCCAATGAA
CTAAGTGTC
[T/C]AACA
GTTTTCAGTCCACTGT
GAAATTTTAACCTTTTT
CGTATGTCTA
[A/G]CCA
CAGTGTGCAAATATAAT
ATGCAAGACCAAAAAA
A
[A/G]KWACATATTGAGT
TTCACTCTTGTCGATG
CAGCCAAGTGGCAGG
GAAGTCCTCAGC
[A/C]A
ACAGGTGGCTCTGGAG
GAAACACACTTGGTCT
GAGATATCACGG
[A/G]G
CACTGGGTAGCTGAGT
GACTTGTACAAACTCA
CCAACTCATTTTTTAT
C
ACCAATGAAAGATGAG
TCACCTTACTATCTTAC
ATATTGGCTTCC
TCTTC
GAACATTTCTTCATTAG
GGGCTCTACTGGGGAG
AAAGGACCCTTTG
ACA
AAATTACCACAAACTT
GGTGGCTTGTAACAAT
AGAACCGTGTT
[T/C]TC
TCTCATGCTCATCTTGA
GCCCTCGTATGTGAGG
CAAAGTGTGG
[T/C]GCC
TGGATTGGCTCTCACT
TAGCAAGTCAAACTAG
TTGTAATATT
ACTACAA
TATAAAACAGTATAGTA
TCTGCATATAACCTATA
CACGTCCTC
[T/C]TATAT
GTATCATCTGGGGATTT
CCATTTCCCAAGTCGG
CTGAGACTTGGA
GGGT
TCACTCAAATGAGAAC
AAAGGAGCAGGATGGG
TGGAGCTAGGAG
[A/G]C
TCAGGTCCTCATCCTAT
ATCTCCATTTCTCCCTT
CTGTGATGTGT
AAGGAT
CTTTTTATTTCTTTCAT
TAGTTCATGTCCTTCG
GCAGTTACTTT
ATGAGG
CACATGTACAGTTCTG
TGAGTCATCCTGGCTG
TGGCCAGTGAC
[T/C]GG
AATCCCAACCATGCCA
TGACCATTTTGTAGCT
CATTTCAACCT
[A/G]AA
TTGAAGTTTCCAGCTC
GCTGACCACTCTTTCT
TCACCTCCCTC
[A/G]CT
CATGGGAGCTTATGAT
CTACTTTTGGACATGTT
TGCTATTTTCTCTT
TCT
ACTTTAGGTTTGACCT
TTTGAGGAGCTGCCAA
ACTGTTCTCCACA
GTG
GCTGACTGGCTGTTAA
CCTGCGCACCTCGGCT
TTCCTTCCTATGCG
CTC
AATTCAGGCATCTCCAT
AGACTACCTTAGGTTG
GATAATTCCCTA
GAACA
GGAAAATGGTACTATA
AATAAAACCTAGATGAT
TAACTGTTTAA
AGGATA
AAGTCACAGGTACGGC
TGGGATCTGCTATTGC
GGTGCCTATGGCAT
AA
TCATGCCTGAATTACA
CAGCTGATAAATAGCA
GGGATTCAGAG
[A/G]AG
GCAGGATGTGATGAGG
AAGGAGCCAAGAAAAG
TAGACTGCAGAC
[A/G]G
TCACGCTTTACACCTG
TCCCTAGCTGCTCCAC
TGGAGGAGGTG
[A/C]C
ACAATACAAGAAAAGG
GGGAATCCGCAACTTG
GACATTGGCA
[A/C]AAG
GGTTTCTATTTGTCTCT
GAGAAGTCAAGTTCCC
TGTTAGCTAC
[T/C]CCCT
GACCAACAAGGAACTC
CCAGATTTTCCAGATT
GGAATATTCTATA
ACTA
GCAAAAGGATGTCTCC
GTAGATAAGACTTCAA
GGGAATATAGAGGGT
T
TTGCCAGGCTAGGGGT
CCAATCAGAGCTGATG
CCACCCAACTA
[T/C]GC
AACTACGACCACATCC
TCAGCACAGGCTGTGC
ACCCCCAGGAAAGAAC
AT
CTACTATAAG
TCTTCCTCCTGGAGCT
ATTGGAGATCTGTGCA
GCAGTTCTGATG
CTGG
GGAGACTGAGAGCTCT
CAGGATTATTGTATATT
TGCCCAAGAC
[T/C]CTAT
TTGCATTACCATTATTA
GTGATCAAAATAATCA
GCAATTTTA
ATTACAGT
AGTTAAATTCGTCCAC
CCAGTGCAGGTTTCAG
TATCTACCAAA
[T/C]AG
GAATGAAAGCCAATCA
GGGGAGGCCATGATTC
TAGAGTGGCTA
[A/G]AG
CCTCTATAGCTCCTTCC
AGTGAGTGTGTGAGAC
TGGCAAATAGGAT
GGA
CTACTGATGCAAACCT
TCAACTTATAACTAACC
AGTGACCTAAT
[A/G]GA
ATGTTAAGTGCAGGTG
AGAGAAGAGGATTTGG
GAAGAAAAATT
[A/C]AA
GTTCCCACAGTAGAAT
AGCTCTCCTGCCAACC
TCATGTCCATATT
CAGC
CTGGGGTTAATCTTCT
CCAGTACGTACACACA
ATATTGTTCATCC
ATTT
AAATGAACTTTTAAATA
AAATGGTATTTCCTACA
ATGAGTTCCA
[T/C]GAC
TGCTATGCCCGGGCAT
TGAAATTTCTTTAGAAA
CTAATGATAT
[T/C]CTTA
GCAAAGGCATTAGTAA
GAGTTTAAAAGGTTTG
TGCTACAAAGT
[T/G]AA
CCTCAGCTTACAGGCA
ATTGGGTAGATAAGGC
AAAATCTGCTA
CGATGA
GGAGGACACAGAGAA
ACATGCCTGTTAGATAT
TCAACCCATTGAT
GGCC
TGTTTAAGTGTTTGTAT
TATTGTACACTGTCATT
CAGCTGT
TCTCCTATAT
TATGGAACTTCCTGGG
TTAGGGGTCAAATCAG
AGCTACAGCTGC
TGGT
GGGCTTTTCCCTGTGT
TCTTGACTTTCCACTG
CCATCTCTCAC
[T/C]AC
CTGGAAGAGTCCCAGC
TCCGAGTTCGGGTGAC
AATGGGACCTGGGC
TC
TGGCCTGGCTGAAATG
ATAAGGGTGACTTCCT
ACCTTACTCTTA
[T/C]G
GCAACCTCTTCCTACT
CTAAACTCTAATAGCAC
TTTATGCAGA[T/C]TTTT
AAATCACTCAGGCTAG
CTGTGTCCCCAGGTAG
CAGCTCATTCC
TGAGC
GTGATGGATGGTCTTT
TCTGCGCTAATGGGGA
AGAGAGTGCACAGG
GC
GCTCTTTGTGTTGGTA
TTGGTACTTTCTCAATA
CAGTGGGTGAA
CTTCA
TCCGGCATTGCCACAG
CTGCAGCGTAGGTCAC
AACTGCGGCTTGAA
TC
CCTTAGCCAGAGACAT
AAACAGGCACAGAGCT
GTAGTTGCCCT
[T/C]CG
TCTCTGCAAGCTTCCT
GGTCACCCATCACTGC
TTGAGGACACA
[A/C]AA
TATGTAAGCATAAATTT
AAAGGCCCCTAGAGGA
AGACAATATAT
[A/G]TTT
AATATAACAACTTTAAT
GGTCCTCACTTAGCTT
TAGGATTCC
[A/G]AATG
CATAGCTTTGATCCCC
AGCTCCGTGCAGTGTG
TTACAGGATCTG
[T/G]C
TTCTAACCAAACTAGA
GTAGCAAAATTTGAGT
TCAAGTTGTAGT
[A/G]G
TCCAGTCATCACCTCA
GGAACAAGCTGTGTGT
TCTTGCCCACTCATG
AT
AGACATTTAGATTTTTC
CATATAGCTGGCTCCTA
AACTCAAAGGC
ACCTT
AGGGATCAGGACCTAA
GTAGGGCCATTCTGGA
CAAGTGGGCAAA
[T/C]G
CTAGAGTATAGGAGAG
AAAAATAGAAAAGAAA
ATAAGGATGTTTGA
ATC
ATTGAGCTCCTTGCCC
TCCTATCCCTCTATAGC
CGGCTGCCAGA
ACGAA
GATAGTCCAGCCAAGC
ATTGAAGGACGGAAGG
GGGTTAGGACA
[A/G]GA
AACCTCTGGGCCAGGA
AAGAGGATGAGTGACA
GGTGGCTTAGAGGCCC
C
AAGCAGTCGA
TTCAAAGAGAGAACAG
TAATCTTCTCCTTCAAA
GCCCTTTACTC
ACAATT
GGAAAGTGTAACCCAG
GTGTCCTCAAAGAGAA
GGGGGGAGATTG
[A/G]
TGCTGTAGTATCTTTCA
GCCATTACCTGGGAGC
AGGTTACTGAG
[T/C]CT
ATAAACAGATATATAGA
TGTAATCAAGCTTCCT
CCTCCACCAA
[T/C]GAA
TGACACATCAAACGCT
TTATCTACAGTCTGGC
CCACTTCCAGG
GAGAT
ACAGTGTCATGAGGAT
GTGGGTTCAATCCCTG
CCCTCACTCAGT
[A/G]G
CCCCATCTAGCTAGAC
TTGGCTGAGGACTCCC
TCCTAAAACAAAG
AATA
TGGGCTCCAAGAAAGA
ATGCAAGAGCCTTCAA
GGTTTCTAGAGG
CCCA
TTCTTCCCACCCTGCA
TGGCCAGAAGCAGGTC
ACATTTGATAAGAC
TTT
GGAAAGCACTGAGTAT
CAGCCATCCAGCCAAG
GTAGGTACAGGA
[T/C]G
ACACCATCCAATAAAC
ACTGACCACATCCTGA
CCAGAGCTAAG
[A/G]AC
GTAATTTAGTGCAGAG
TGCCAGAATCCCGAGT
AGCTGGGTTCC
[A/G]GT
CATTTTCCTCCTCCCGT
GGACACAATGAATGTG
CAGCTACACAC
[A/G]RA
TAAGTTAAAACAAGCT
GTGCCCAGAGGAACAT
ATAGTCTGGCTC
[T/C]T
AATTGTGTTTTCATCCC
TGAGTTTCCAACTGTT
CTATTTAAGCA
[A/G]AA
TGTCCTTCTCTTTCAA
GAATTTTCCCGGCTATT
TTCAGGATTA
TCCCTCC
TTACATGTGGGACTGA
GTCACTCGGCTGCACA
CCTGAAGCTAA
[T/C]GC
AAGTCTAATGGAATAG
GCTGTCTCAAAGTTTC
CCACCTCTAGATTT
ATA
TTTTCGTCAAAGCAGG
AGGCAAAGAGGAAATG
TCTGAAGTTGATTAA
TG
TTGGCTCTAGAATAAG
CTCCGGATTGTACAAA
TCTGACTCGGT
[T/C]GT
TTCGTTGCTGCTGAGC
CACAACAGGAATTCAT
ATTAAGAACAT
[T/C]TTT
In table 1, N represents A, T, C, or G. In table 1, underlines in source sequence label the regions to which probes correspond (containing two cases, the nucleotide sequence of probe is same as that of underline region, the nucleotide sequence of probe is reversely complementary to that of underline region). In table 1, when source type of SNP is [T/G], heterozygous type is TG; when source type of SNP is [A/G], heterozygous type is AG; when source type of SNP is [T/C], heterozygous type is TC; and when source type of SNP is [A/C], heterozygous type is AC.
Example 2. Identifying Wuzhishan Miniature Pig Inbred Line and Non-Wuzhishan Miniature Pig Inbred Line by Using 145 SNPs
1. Selecting experimental animals, collecting blood from ear veins, and extracting genomic DNA from blood samples;
2. Hybridizing the genomic DNA obtained in Step 1 with nucleic acid chips immobilized with 145 probes (these 145 probes are single-stranded DNA molecules shown as Sequences 1-145 in the sequence listing, respectively);
3. After accomplishing Step 2, terminal extensions were performed on each point in nucleic acid chips, so as to obtain the genotypes of 145 SNP sites in genomic DNA;
4. Whole genome sequencing was performed on genomic DNA obtained in Step 1 to obtain the genotypes of 145 SNP sites in genomic DNA.
The results demonstrated that the results of Step 3 are completely consistent with those of Step 4.
Genotypes of 145 SNP sites of 48 experimental animals of Wuzhishan miniature pig inbred line are all homozygous type (genotypes described in Table 1 of Example 1). Genotypes of 145 SNP sites of 16 experimental animals of Hainan Wuzhishan pig are all heterozygous type.
The present invention is of a significant value for the identification of germplasm resource of Wuzhishan miniature pig inbred line. The method of the present invention can be used for breeding of Wuzhishan miniature pig inbred line, preliminarily screening all the pigs in the pig population to be tested, weeding out non-Wuzhishan miniature pig inbred line, finding out candidate Wuzhishan miniature pig inbred line, and further confirming in combination with other methods. The present invention also can be applied to test whether the purchased Wuzhishan miniature pig inbred line is counterfeit.
Filing Document | Filing Date | Country | Kind |
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PCT/CN2014/000937 | 10/21/2014 | WO | 00 |
Publishing Document | Publishing Date | Country | Kind |
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WO2016/061711 | 4/28/2016 | WO | A |
Number | Date | Country |
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103923993 | Jul 2014 | CN |
103923994 | Jul 2014 | CN |
104293963 | Jan 2015 | CN |
Entry |
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ISA State Intellectual Property Office of the People's Republic of China, International Search Report Issued in Application No. PCT/CN2014/000937, dated Jul. 27, 2015, WIPO, 4 pages. |
Number | Date | Country | |
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20170327903 A1 | Nov 2017 | US |