Study On Genetic Background And Molecular Marker Of Tibet Mini-pigs

BackgroundTibet hog is one kind of smaller mini-pig in the world,which mainly live in semi-agricultural and semi-pastoral areas(average elevation:2500～4300m) in Qinghai-Tibet plateau located in southwest China.Tibet hogs are distributed mainly in Tibet autonomous region and Yunnan,Sichuan,and Gansu.It is the only pig breed that can adapt to the high altitude climate.Because of long-term geographical isolation and environmental difference,the differentiation of Tibet hog in different regions(such as Sichuan Tibet hog and Yunnan Tibet hog) appear.Sichuan Tibet hog diverses more from the law of Hardy-Weiberg than Yunnan Tibet hog do.Sichuan Tibet hog is smaller than Yunnan Tibet hog.Although some research work has been done on Tibet hog(mostly on Sichuan Tibet hog and Yunnan Tibet hog),little work has been done on Tibet hog from Tibet autonomous region.To develop a laboratory pig strain,50 indigenous Tibet pigs(25 males and 25 females) were moved from Tibet autonomous region to Guangzhou city in 2004,which were raised in the base of Tibet mini-pig breeding in Zengcheng,Guanzhou city.These pigs were bred for the purpose of laboratory animalization and were named as Tibet mini-pig.The number of Tibet mini-pigs are about 600 now.Objectives1.To analyze the sequence of mitochondrial DNA D-loop region and Cyt b gene of Tibet mini-pigs,to investigate genetic differentiation and the impact on partial blood parameters and to explore their cytoplasmic DNA markers used to identify them.To find the phylogenetic relationship between Tibet mini-pigs and several other breeds of Chinese domesticated pigs.2.To investigate the population structure feature of HAL locus,and to detect allozygote individuals of HAL genotype in different Tibet mini-pig,and to guide the breeding of laboratory animal Tibet mini-pigs in abstracto.Methods1.Collecting blood from 120 Tibet mini-pigs randomly and genome DNA was extracted from the blood with Genome DNA Extraction Kit.Primer were designed. The fragment of Mitochondrial DNA D-loop region in 120 Tibet mini-pigs,7 Bama miniature pigs and 23 Guizhou xiang pigs and 17 WZS pigs were amplified and sequenced,followed by being multiple alignment compared with each other using ClustalW software and MEGA3.0 software,then variation sites and haplotype were made.Established genetic relationship tree between Tibet mini-pig and other China domestic pigs with CLUSTALW software.Additionally,blood physiological and biochemical parameters of 58 Tibet mini-pigs(8 months old) were measured which included WBC,RBC,HGB,PLT,LYM,MONO,NEU,EOS,BASO,HCT, MCV,MCH,MCHC,RDW,ALT,AST,AST/ALT,TP,AKP,GLU,BUN, CREA,CHOL,TG and ALB.After classified,these index were compared.The software SPSS13.0 and Independent samples t-test were used to make statistics analysis,and all the data was denoted as mean±standard deviation((?)±SD).2.Making research on genome DNA from Tibet mini-pig(representing haplotype of D-loop sequence),some Bama miniature pigs,Guizhou xiang pigs and WZS pigs.Then designing Primer and amplifing the sequence of Cyt b gene from Tibet mini-pig,Bama miniature pigs,Guizhou xiang pigs and WZS pigs.After sequencing, the base sequence and amino acid sequence were compared and analysed using MEGA3.0 software,then phylogenetic tree and evolution position of Tibet mini-pig were established. 3.PCR-RFLP methods were used in the research of HAL polymorphism. Genome samples from 36 Duroc and 20 Changbai as control group,HAL fragment were amplified using specific primer,then the fragment was digested with Hha I and the result was verified.Through reading genotype,genotypic frequency data of the group were obtained.The genotype can be classified to 3 kinds by electrophoresis analysis,of which HAL~NHAL~N represented two electrophoretic bands of 493bp and 166bp,HAL~NHAL~n represented three electrophoretic bands of 659bp,493bp and 166bp respectively,HAL~nHAL~n represented one electrophoretic band of 659bp.Results1.Previous research showed the length heterogeneity(15～29 10 bp repeat motif) in the tandem repeat sequence of mtDNA D-loop in pigs and all of the motifs is GTACACGTGC,a perfect repeat,but the data from this study demonstrated that there are not only perfect repeats(i.e,type A) but also some imperfect repeats(type B),that is to say,moiety Chinese Tibet mini-pigs contain an alternate array in three motifs:GTACACGTGC,GTACACATGC and GTACACGTAC.By performing pedigree analysis,array types were inherited from mother to offspring and had no relation with father's types.340 bp sequence at 3' end of the Tibet mini-pigs D-loop which is very conservative particularly exhibited high homology as in the same region in domestic pigs.704 bp sequence at 5' end of the Tibet mini-pigs D-loop had 20 polymorphic sites which deduced 26 haplotypes.The three transform sites(305,500 and 691) nearly corresponded to type A and B of tandem repeat sequence.All type B are t,a,a(100%);the bases in three mutation site of type A are all c,g,g(87%);the rest mutation account 13%.The mutation sites in two haplotype of type A is similar to type B.Compared with Tibet mini-pigs,Bama miniature pigs,Guizhou xiang pigs and WZS pigs had 4,4 and 3 haplotypes,respectively,with only one type of tandem repeat sequence,that is type A.Although many studies were performed to investigate the repeat arrays of D-loop previously,little is known about the function of the repeat sequence.In this study,among these blood physiological and biochemical parameters described above,only RBC of Tibet mini-pigs(8 months old) was found to have significant differences between type A and B(P＜0.01),which demostrated tandem repeat sequence has influence on the fuction of blood cells.2.Tibet mini-pigs have close relation with domestic pigs in southwest China,by using phylogenetic tree building by haplotypes deduced from polymorphic sites of 5' terminal flanking region.3.There are 16 mutation between domestic pig in China and pig from Europe. Besides there was a significant difference in two nucleotide site:a T-C switch in in site 420 and a G-A switch in in site 883,with a proportion about 50%respectively. Compare with pigs from Europe,Tibet mini-pig is different in three amino acids site. In site 295,most of type A Tibet mini-pig and pig from Europe are all Valine,while type B Tibet mini-pig is Isoleucine in this site.Bama miniature pigs,Guizhou xiang pigs and WZS pigs is coincide with type B and a small fraction of type A Tibet mini-pig in site 295 which is isoleucine.Our research showed that Bama miniature pigs,Guizhou xiang pigs and WZS pigs had very close blood relationship with some of Tibet mini-pigs.The research further proved there is differentiation among Tibet mini-pig group.4.The HAL gene type of Tibet mini-pig are all HAL~NHAL~N,no stress sensitive gene is discovered.The HAL genotype frequency of Tibet mini-pig colony is HAL~NHAL~N 100%,HAL~NHAL~n and HAL~nHAL~n 0.4 heterozygote in samples from Duroc pig and 1 heterozygote in samples from Long-white pigs were detected.There is no allozygote in all these samples.As a result,a conclusion is drawed that there is no stress sensitive recessive gene and Tibet mini-pig population have higher ability to defense the press in Tibet mini-pig groups.Conclusions1.Based on the variety of tandem repeat motif and the variety sites of the entire sequence of the hypervariable mtDNA D-loop region,Tibetan mini-pig can be divided into two types:A and B.Types A and B Tibet mini-pig had significant differences in RBC count(type A＜type B).There is a closely genetic relationships between a piece of Tibet mini-pig(type) A and Chinese domestic pigs.Type A,type B and the three base pair transition site(site 305,site 500 and site 691) can be uesd as the united genetic marker of Tibet mini-pigs together.Through artificial selection, classifying type A and type B as two groups,eliminating some of the mutative individuals in type A(13%),reinforcing the genetic marker,we can breed two closed colony of Tibet mini-pigs.All type A is c,g,g and all type B is t,a,a.2.By performing pedigree analysis,array types(A and B) were inherited from mother to offspring and had no relationships with father's types.Because all of the reported domestic pigs in China are type A,we infer that some of Tibet mini-pigs(A) may be the common origin of Chinese domestic pigs.3.Two variable sites in Cyt b gene of Tibet mini-pigs were discovered,including a T-C transform in site 420 and a G-A transform in site 883,which corresponded with type A and type B Tibet mini-pig.Compare with pigs from Europe,Tibet mini-pig is different in three amino acids site.In site 295,type A Tibet mini-pig and pig from Europe are all Valine,while type B Tibet mini-pig is Isoleucine in this site.Bama miniature pigs,Guizhou xiang pigs and WZS pigs is coincide with type B Tibet mini-pig in site 295.It was confirmed again that there is differentiation in the Tibet mini-pig.4.The HAL genotype frequency of Tibet mini-pig is very low,which demostrate the Tibet mini-pig have a low stress sensibility.So Tibet mini-pig is fit to breed as medical laboratory animal.