CN110484540B - Duck uaa gene and application thereof in duck MHC haplotype breeding - Google Patents

Duck uaa gene and application thereof in duck MHC haplotype breeding Download PDF

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CN110484540B
CN110484540B CN201910866354.7A CN201910866354A CN110484540B CN 110484540 B CN110484540 B CN 110484540B CN 201910866354 A CN201910866354 A CN 201910866354A CN 110484540 B CN110484540 B CN 110484540B
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uaa
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韩凌霞
王兴童
陈洪岩
张伟
于海波
周宇
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Harbin Veterinary Research Institute of CAAS
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Abstract

The invention discloses a duck uaa gene and application thereof in MHC haplotype breeding. The invention separates and obtains the uaa CDS sequences of 4 duck TAP haplotypes, and the nucleotide sequences are respectively shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4. The invention further determines that the genes of TAP1, TAP2 and uaa of the duck are closely linked through sequencing and bioinformatics analysis, namely the TAP haplotype can represent the MHC haplotype. The separated duck uaa CDS sequence can be used as a molecular marker to be applied to breeding of duck lines which are respectively homozygous for MHCI, tap1 and tap2 or directly applied to breeding of haplotypes in all duck MHC regions.

Description

Duck uaa gene and application thereof in duck MHC haplotype breeding
Technical Field
The invention relates to application of duck uaa genes and duck tap1, tap2 and uaa genes in MHC (major histocompatibility complex) haplotype variety breeding, in particular to application in breeding epidemic disease sensitive/resistant duck pure line varieties, belonging to the field of duck uaa genes and application thereof in variety breeding.
Background
China is a big duck breeding country, and the water poultry breeding amount is the first place in the world. According to the national waterfowl genetic improvement plan (2020-2035) published by the rural part of agriculture in China, the slaughtering amount of Chinese meat ducks in 2018 reaches 35 hundred million, which accounts for about 80% of the total slaughtering amount in the world. The egg-laying duck stock quantity of the adult is more than 2 hundred million, and accounts for more than 90% of the egg-laying duck stock quantity in the world. The yield of the duck eggs accounts for nearly 20 percent of the total yield of the poultry eggs. Ducks are important model animals in the field of life science research. There are 247 reports of pathogenic microorganism infection studies conducted in poultry, which were recorded by the National Center for Biotechnology Information (NCBI) in 2009 to 2014, involving various pathogens such as pathogenic or genetically engineered viruses, bacteria, epizoons or protozoa (wangxingtong, koreania, research progress of alternative methods in veterinary vaccines [ J ] experimental animal science, 2018,35 (1), 67-71.). In production, the duck embryo is a raw material and a verification material for producing various biological products for waterfowls specified in the veterinary drug dictionary (2010 edition) of the people's republic of China, and the number of duck eggs and the duck embryo for inspection and test of national biological product enterprises and scientific research institutes reaches hundreds of thousands. Although the duck breeding industry has an important socioeconomic status in China, various pathogenic infectious diseases cause serious harm to the quality of duck flocks, and a plurality of clinical cases are reported in the first half of 2019.
The haplotype (haplotype) is genetically a combination of alleles at multiple loci on the same chromosome, each closely arranged, and inherited together with little or no crossover or recombination. Major Histocompatibility Complex (MHC) is a group of closely linked, highly polymorphic, major histocompatibility antigen-encoding gene clusters that are widely present in vertebrate genomes, forming stable haplotypes. Animals of different species, including humans, all have multiple MHC haplotypes and are all typed with polymorphisms in MHC class I classical genes (Kaufman J, milne S, gobel TW, walker BA, jacob JP, auffray C, zoorob R, beck S (1999) The chicken B locus is a minimal essential male histocompatibility complex. Nature 401. MHC haplotypes not only influence the sensitivity to a variety of diseases (Hippophaea, korea, hanjialin, histocompatibility Complex in laboratory chickens, china journal of comparative medicine, 2010, 20 (2): 1-5.Kaufman J. Generalists and specialties. There are hundreds of MHC haplotype chicken lines that have been bred internationally (Fulton J E, lund A R, mcCarron A M, et al. MHC variable in reliability grades of chicken [ J ]. Poultry Science,2016,95 (2): 393.), but MHC haplotype duck lines have not been reported so far. The key bottleneck for restricting the breeding of duck MHC haplotype strains is that the artificial domestication time of domestic ducks is short, the ducks in a duck group have extremely rich genetic diversity, the MHC region is particularly complex, the biological characteristics related to the MHC I molecule allelic gene type are lack of research, and the suitable haplotype can not be determined for breeding.
The MHC I genes of ducks have 5 copies of genes such as UAA, UBA, UCA, UDA and UEA, the nucleotide homology is 87.4%, the two copies of the antigen transport related body genes TAP1 and TAP2 are positioned at the upstream of the UAA gene, and the UAA is predominantly Expressed (Moon D A, veniamin S M, parks-Dely J A, magor K E. The MHC of the Duck (Anas platyrnchos) continuous differential Expressed Class I genes. The Journal of Immunology,2005, 175. The glycoprotein encoded by TAP1 and TAP2 forms a heterodimer, and transports endogenous antigen peptides to MHC class I molecules, thereby stimulating cellular immune response.
The HBK ducks are standard Shaoxing Ma duck groups which are successfully bred in China at the earliest time, namely the duck groups are bred for 8 generations continuously in a positive pressure barrier environment, 9 specific pathogenic microorganisms and antibodies thereof are eliminated, and the quality of the HBK ducks meets the group standards of 'T/CALAS 18-2017 experimental animal SPF duck microbiology monitoring rule', 'T/CALAS 37-2017 experimental animal SPF duck genetics quality monitoring' and 'T/AS 17-2017 experimental animal CAL duck compound feed' published by the Chinese experimental animal society and the general technical requirements of 'DB 23/T2057.6-2017 experimental animal duck breeding isolators' in the Heilongjiang province standard. According to the group genetic structure analysis of F2, F3, F5 and F6 generations, the microsatellite DNA locus deviating from Hardy-Weinberg equilibrium is reduced after 6 generations of closed breeding, the group genetic diversity marked by the allele factor is reduced, and the inbreeding degree is in an increasing trend. Further analyzing the polymorphism of the short tandem repeat sequence of the Tap gene of the HBK duck group, and continuously breeding and selecting the detected 4 homozygous genotype individuals as breeding ducks. Respectively carrying out the genetic diversity analysis of a short tandem repeat sequence, a peptide binding region sequence of a Tap2 gene and a Tap2 whole genome sequence in 4 continuous generations, and finally breeding 4 haplotypes which are respectively named as Hvri: HB1, hvri: HB2, hvri: HB3 and Hvri: HB4 (Wangxingtong, benxing, 20319; hui, chenhong rock, hanlingxia, cultivation of MHC haplotype pathogen-free ducks, laboratory animal science, 2018,35 (4): 14-20).
In vivo challenge experiments show that 4 kinds of tap haplotype ducks have sensitivity differences on avian influenza vaccines (Zhao Li, yang Chao Na, xin Jiang, wen Hui Qiang, xian jin Xiong, chen Hong rock, aged orchid, han Lingxi, influence of different MHC haplotype of ducks on the immune effect of the avian influenza recombinant vaccine [ J ]. Experimental animal science, 2014,31 (3): 30-32.), duck plague (Wang Xintong, meng, 20319; hui Huiyi, chen rock, han Lingxi, cultivation of MHC monoploid ducks without specific pathogens, experimental animal science, 2018,35 (4): 14-20.).
If the MHC class I molecular genotype related to specific biology is identified on the basis and the relevance between the MHC class I molecular genotype and the tap haplotype is determined, the method has important application value for breeding duck MHC haplotypes with excellent growth performance, epidemic disease sensitivity or resistance and the like.
Disclosure of Invention
One of the purposes of the invention is to provide a CDS sequence of duck uaa gene;
the second purpose of the invention is to apply the CDS sequence of the duck uaa gene to the breeding of MHCI, tap1 and tap2 which respectively reach homozygous duck strains or the breeding of haplotypes in all duck MHC regions;
the above object of the present invention is achieved by the following technical solutions:
the invention takes tap haplotype breed ducks as the basis, and obtains the UAA CDS sequence of 4 kinds of ducks for the first time by measuring the UAA CDS region through reverse transcription-PCR; the nucleotide sequence of the UAA CDS sequence of the duck TAP haplotype B1 is shown in SEQ ID NO.1, the nucleotide sequence of the UAA CDS sequence of the duck TAP haplotype B2 is shown in SEQ ID NO.2, the nucleotide sequence of the UAA CDS sequence of the duck TAP haplotype B3 is shown in SEQ ID NO.3, and the nucleotide sequence of the UAA CDS sequence of the duck TAP haplotype B4 is shown in SEQ ID NO. 4.
According to the invention, the close linkage between the duck TAP1-TAP2-uaa genes is determined through bioinformatics analysis and comparison, namely the TAP haplotype simultaneously represents the individual MHC haplotype. The comparison result shows that in each strain (B1, B2, B3 and B4) homozygous for tap2, the uaa gene of each individual is completely homozygous, polymorphism exists among different strains, and polymorphism is completely linked with polymorphism of tap2, so that the uaa gene (MHC class I molecule heavy chain gene) can be used for directly breeding MHC haplotype duck strains without screening individuals homozygous for the tap2 gene and then indirectly breeding MHC haplotype.
Therefore, the CDS sequence of the duck uaa gene is further applied to breeding duck MHC haplotype varieties, and the breeding method comprises the following steps:
(1) Designing a primer pair for amplifying a tap gene by taking the tap gene of the duck as a target gene; designing a primer pair for amplifying the uaa gene by taking the duck uaa gene as a target gene;
(2) Preparing cDNA of a duck strain sample to be detected; respectively establishing a PCR amplification system by using the prepared cDNA as a template and the primer pair designed in the step (1) to amplify the CDS sequence of the tap gene or the uaa gene in the sample; sequencing the amplified product and performing bioinformatics analysis and alignment with the UAA CDS sequence; if the amplified UAA gene sequence is completely homologous with any UAA CDS sequence, other copy genes of MHCI, tap1 and tap2 of the detected duck individual are respectively homozygous; or if the amplified gene sequence is completely homologous with the UAA CDS sequence, the detected MHC core region of the duck individual except tap1, tap2 and UAA also achieves continuous homozygosis; the continuous homozygosity refers to dense single nucleotide site homozygosity in a section of region with unknown functions.
Wherein the nucleotide sequences of the primer pair for amplifying the TAP gene in the step (1) are shown in SEQ ID NO.5 and SEQ ID NO. 6; the nucleotide sequences of the primer pair for amplifying the UAA gene are shown as SEQ ID NO.7 and SEQ ID NO. 8.
The PCR reaction procedure for amplifying the TAP gene in step (2) is as follows: 95 ℃ for 5min,94 ℃ for 45s,64 ℃ for 60s,35 cycles, 72 ℃ for 10min.
The PCR reaction procedure for amplifying the UAA gene was: 2min at 94 ℃,50S at 94 ℃,50s at 68 ℃,40 cycles, and 10min extension at 72 ℃.
According to the invention, a duck UAA CDS sequence is obtained through separation, and the duck TAP1-TAP2-UAA genes are determined to be closely linked, namely the genotype of MHC class I molecules is consistent with the TAP haplotype and also represents the MHC haplotype; the molecular marker determined by the invention can be applied to breeding of duck strains respectively reaching homozygous MHCI, tap1 and tap 2.
The present invention relates to the definition of terms
Duck Major Histocompatibility Complex (MHC): is a group of closely linked highly polymorphic gene clusters, the haplotype of which is closely related to the disease resistance and the immune response level of a host, wherein an antigen presentation associated Transporter (TAP) gene is adjacent to an MHCI gene.
Haplotype (haplotype): the term "haploid genotype" is short, and genetically refers to a combination of alleles at a plurality of loci that are inherited together on the same chromosome, i.e., a genotype consisting of several closely linked genes that determine the same trait.
CDS (Coding sequence): a protein coding region.
Drawings
FIG. 1 shows the result of PCR amplification of CDS region of duck uaa gene.
Fig. 2 alignment of mega. Aln of duck tap2 gene sequence sequencing results.
FIG. 3 shows the alignment result of mega.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 cloning of UAA CDS sequence of TAP haplotype Duck lines (B1 line, B2 line, B3 line and B4 line)
1. Sample collection preparation and experimental design
5% sodium citrate solution is prepared aseptically, and 4 TAP haplotype duck individuals are collected in the wing vein for anticoagulation. Using AxyPrep TM Extraction of Duck peripheral blood Total RNA, prime Script Kit TM II1st Strand cDNA Synthesis Kit is reversely transcribed into cDNA, the sample quality is measured by a Nano Drop ND-1000 concentration measuring instrument according to the product instruction, and the concentration and the purity accord with OD 260 /OD 280 And (c) = 1.8-2.0, and the product is stored at-20 ℃.
2, PCR amplification:
designing a pair of CDS region primers for amplifying duck UAA:
Du-C-UAA-F：
5’-CCCATGGATGGGACCGGGACGGGCGGGGGCCCT-3’
Du-C-UAA-R：
5’-GCGTCGACTTAGACACTGGGGTTGCTCCCTGC-3’
the PCR reaction system is as follows: 2x TaqMix 25uL, du-C-UAA-F (10. Mu. Mol/ml) 1.5uL, du-C-UAA-R (10. Mu. Mol/ml) 1.5uL, cDNA template 4uL, ddH2O 18uL.
The PCR reaction program is: 2min at 94 ℃,30S at 76 ℃,30S at 72 ℃, 1min at 72 ℃,35 cycles, and extension for 10min at 72 ℃.
3. Sequence analysis:
the amplification results are shown in FIG. 1, and each strain obtains a single amplification product, and the molecular weight of each strain accords with a theoretical value. The 4 PCR amplification products are subjected to bidirectional detection by upstream and downstream primers, and are analyzed by MEGA software to obtain sequences shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 respectively.
Test example 1 detection of UAA CDS region homology test of tap2 haplotype duck by using UAA gene
B1, the number of the tested individuals is: a4675, a4679, a5090, a5091, a5092;
b2, the number of the tested individuals is: b3307, B4602, B4607, B4608;
b3, the number of the tested individuals is: c3304, C3321, C3341, C4629, C4636;
b4, the number of the detected individuals is: d4634, D4637, D4642, D4646 and D4652.
The sequence of the TAP2 gene is detected by using a primer pair D-TAP2-dF/D-TAP 2-dR.
D-TAP2-dF：5’-ATGGAGTTGCTGCCCACCTTGCGCCTG-3’,
D-TAP2-dR：5’-TGAAACCCATCAGGCACCATCCCAGGT-3’。
DNA template 4ul, primers 1.5ul each, 2 XTap PCR Starmix 25ul, ddH 2 O 18ul。
The PCR reaction program is: 95 ℃ for 5min,94 ℃ for 45s,64 ℃ for 60s,35 cycles, 72 ℃ for 10min. Total RNA of duck peripheral blood is extracted by using AxyPrepTM Multisource Total RNA Miniprep Kit, and Prime script II1st Strand cDNA Synthesis Kit is reversely transcribed into cDNA.
The UAA CDS region was amplified using primers Du-C-UAA-F/Du-C-UAA-R:
Du-C-UAA-F：
5’-CCCATGGATGGGACCGGGACGGGCGGGGGCCCT-3’
Du-C-UAA-R
5’-GCGTCGACTTAGACACTGGGGTTGCTCCCTGC-3’
the PCR reaction system is as follows: 2xTaqMix 25uL, du-C-UAA-F (10. Mu. Mol/ml) 1.5uL, du-C-UAA-R (10. Mu. Mol/ml) 1.5uL, cDNA template 4uL, ddH2O 18uL.
The PCR reaction program is: 2min at 94 ℃,30S at 94 ℃,50s at 68 ℃,40 cycles, and 10min extension at 72 ℃.
The PCR sample is identified by 1% agarose gel electrophoresis, has strong specificity and correct molecular weight, and is sent to Kuumei company for direct sequencing. The upstream and downstream primers are used for bidirectional detection respectively, and sequence splicing is completed by a company. The analysis was performed using MEGA ClustalW bioinformatics software,
the results of the mega. Aln alignment of the sequencing results are shown in fig. 2 (tap 2) and fig. 3 (uaa).
From the sequence alignment results of fig. 2 and 3, it can be determined that: the duck tap1-tap2-uaa genes are closely linked, namely, the haplotype determined according to tap2 also represents MHC haplotype. Therefore, the separated duck UAA CDS sequence can be used as a molecular marker for breeding duck lines respectively homozygous for MHCI, tap1 and tap2, or for breeding all duck MHC region haplotypes.
The results show that in each strain (B1, B2, B3 and B4) homozygous for tap2, the uaa gene of each individual is completely homozygous, polymorphism exists among different strains, and the polymorphism is completely linked with the polymorphism of tap2, so that the uaa gene (MHC class I molecule heavy chain gene) can be used for directly breeding MHC haplotype duck strains without screening individuals homozygous for tap2 gene and then indirectly breeding MHC haplotype.
Sequence listing
<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center of animal health and epidemiology)
<120> duck UAA gene and application thereof in duck MHC haplotype breeding
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agcccggggg tgccgcagta cgtggctgtg ggatacgtgg atggggaagc cttcacatac 180
tatgacagtg agaccaggag gacggagccc cgggtggact ggattgcggc ccatacggat 240
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aacctggaca cactgcggga gcgctacaac cagagcaggg ggtcttacac agtacaggtc 360
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ggggcacaaa tcaccaagag gaagtgggag gaggatggga ctgttgctga gcgcaggaag 540
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gatctggaca cgatgcggga gcgctacaac cagagcaggg ggtctcacat attgcagcgc 360
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gtgctggaga ggagagagcg cccagaggtc cgagtgtcgg ggatggaggc cgacaagatc 660
ctgaccttgt cctgccgtgc tcacggcttc tacccgcgac ccatctccat cagctggctg 720
aaggatggca tggtccagga gcaggagacc cagaggggga gcaccgtgcc caacagtgac 780
ggcacctacc atatctgggc aaccatcgat gtggtgccag gggacaagga caagtaccag 840
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agcccggggg tgccacgttt cgtgtccgtg gggtacgtgg acgggcacct catcgaccac 180
tatgatagcg agacccagag gacggagccc cgggcagact ggtttgcggc caatacggat 240
cagcagtact gggacaggca gactgagatc tcacgaggtg ctgagcagat tttccgcttg 300
gacctggaga cgctgcggga gcgctacaac cagagcaggg gctctcacac atggcagctc 360
atgtatggct gtgacctcct cgaggatggt agtactagag ggtttcgcca gtatggctac 420
gaaggaagag acttcgttgc ctttgacaaa gacacgctga cgttcactgc agcggacgct 480
ggggcacaaa tcaccaagag gaagtgggag caggaaggga ctgatgctga gcgctggaag 540
ttctacctgg agaacacctg cattgagggg ttgaggaaat acgtgagcta tgggaaggac 600
gtgctggaga ggagagagcg tcccgaggtc caagtgtcgg ggatggaggc cgacaagatc 660
ctgaccttgt cctgccgtgc tcacggcttc tacccgcgac ccatctccat cagctggctg 720
aaggatggca tggtccagga gcaggagacc aagaggggca gcaccgtgcc caacagtgac 780
ggcacctacc atatctgggc caccatcgat gtcctgccgg gagagaggga caagtatcag 840
tgccgcgtgg agcatgccag cctgccccag cctggcctct tctcatggga gccacagtcc 900
aacctgatcc ccatcgtggt ggtggtggct gttgctgtcg tggctgtcat cgctgccctg 960
gctggatttg ctgtctggaa gaggaagcag gggaagaagg agaagggcta caacgttgcg 1020
ccaggcagcg aagggggatc caacagctcg aacgcaggga gcaaccccag tgtctaa 1077
<210> 5
<211> 27
<212> DNA
<213> Artifical sequence
<400> 5
atggagttgc tgcccacctt gcgcctg 27
<210> 6
<211> 27
<212> DNA
<213> Artifical sequence
<400> 6
tgaaacccat caggcaccat cccaggt 27
<210> 7
<211> 33
<212> DNA
<213> Artifical sequence
<400> 7
cccatggatg ggaccgggac gggcgggggc cct 33
<210> 8
<211> 32
<212> DNA
<213> Artifical sequence
<400> 8
gcgtcgactt agacactggg gttgctccct gc 32

Claims (6)

1. The UAA CDS sequence of the duck TAP haplotype B1 line is characterized in that the nucleotide sequence is shown in SEQ ID NO. 1.
2. The UAA CDS sequence of the duck TAP haplotype B2 line is characterized in that the nucleotide sequence is shown in SEQ ID NO. 2.
3. The UAA CDS sequence of the duck TAP haplotype B3 line is characterized in that the nucleotide sequence is shown in SEQ ID NO. 3.
4. The UAA CDS sequence of the duck TAP haplotype B4 line is characterized in that the nucleotide sequence is shown in SEQ ID NO. 4.
5. Use of the UAA CDS sequence of any one of claims 1-4 for breeding MHC i, tap1 and tap2 to homozygous duck lines or whole duck MHC region haplotypes, respectively; the method comprises the following steps:
(1) Designing a primer pair for amplifying TAP gene by taking a TAP gene of a duck as a target gene; designing a primer pair for amplifying the UAA CDS sequence by using the UAA CDS sequence as the target gene in any one of claims 1-4;
(2) Preparing cDNA of a duck strain sample to be detected; respectively establishing a PCR amplification system by using the prepared cDNA as a template and using the primer pair designed in the step (1) to amplify a tap gene or a UAA CDS sequence in a sample;
(3) Sequencing the amplified product and performing bioinformatics analysis and alignment with the UAA CDS sequence of claims 1-4; if the amplified UAA gene sequence is completely homologous to the UAA CDS sequence of any one of claims 1-4, then the detected MHCI other copy genes, tap1 and tap2 of the duck individual are all homozygous, respectively; or if the amplified UAA gene sequence is completely homologous to the UAA CDS sequence of any one of claims 1-4, then the MHC core regions of the duck individual other than tap1, tap2 and UAA are detected as being contiguously homozygous;
the continuous homozygosis refers to dense single nucleotide site homozygosis in a section of region with unknown functions;
the nucleotide sequences of the primer pair for amplifying the tap gene in the step (1) are shown in SEQ ID NO.5 and SEQ ID NO. 6;
the nucleotide sequences of the primer pair for amplifying the UAA CDS sequence in the step (1) are shown as SEQ ID NO.7 and SEQ ID NO. 8.
6. The use according to claim 5, wherein the PCR reaction procedure for amplifying the tap gene in step (2) is: 95 ℃ for 5min,94 ℃ for 45s,64 ℃ for 60s,35 cycles, 72 ℃ for 10min; the PCR reaction program for amplification of UAA CDS sequence was: 94. 5min at 94 ℃ for 30s,68 ℃ for 50s,40 cycles, and an extension of 10min at 72 ℃.
CN201910866354.7A 2019-09-12 2019-09-12 Duck uaa gene and application thereof in duck MHC haplotype breeding Expired - Fee Related CN110484540B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643900A (en) * 2012-03-19 2012-08-22 上海市农业科学院 Single nucleotide polymorphism detection method of goose peroxisome proliferator-activated receptor (PPAR) gamma gene and application of single nucleotide polymorphism detection method
CN103966231A (en) * 2014-05-30 2014-08-06 湖南农业大学 Molecular cloning of GLP2R gene fragments associated with pork quality traits and application of GLP2R gene fragments

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2329364T3 (en) * 2003-01-17 2009-11-25 The Trustees Of Boston University HAPLOTIPOS ANALYSIS.
US20050123929A1 (en) * 2003-12-04 2005-06-09 Wisconsin Alumni Research Foundation Methods and compositions for genetically detecting improved milk production traits in cattle

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102643900A (en) * 2012-03-19 2012-08-22 上海市农业科学院 Single nucleotide polymorphism detection method of goose peroxisome proliferator-activated receptor (PPAR) gamma gene and application of single nucleotide polymorphism detection method
CN103966231A (en) * 2014-05-30 2014-08-06 湖南农业大学 Molecular cloning of GLP2R gene fragments associated with pork quality traits and application of GLP2R gene fragments

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
植物无融合生殖研究的现状及进展;莫饶等;《热带作物学报》;20000630;第23卷(第2期);全文 *

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