| Chapter I:Investigation of Inhibition locus and inheritance of Two-end black in Tongcheng pigsCoat color is an important trait for breed standard. Two classic color loci, Inhibition and Extension, have been mapped and fully characterized in modern commercial European pigs. Extension locus encodes the MC1R gene, which is responsible for the exclusive synthesis of eumelanin or pheomelanin, as called "switch gene" in pigment system. Locating in pig chromosome 8, KIT gene controls the Inhibition locus. So far, at least four copy number variations and one splicing mutation have been found in KIT gene. The combination of these variations forms different haplotypic effect on inhibiting the normal migration of melanoblasts, resulting in completely white color of Large white and Landrace pigs, patch color of Pietrain pigs and belt color of Hampshire pigs. In contrast, the genetic basis of white color is still barely known in Chinese domestic pigs. As a typical breed in two-end black population, Tongcheng pigs is a famous breed in central China. In this study, we constructed two color segregating populations, including one backcross population from Tongcheng and Duroc pigs and one F2 population from Tongcheng and Large white pigs. Based on the distribution of each colour phenotypes, we proposed the inhibition mode of two-end black color. Moreover, we detected the four reported segmental duplications as well as the splicing mutation of KIT in Tongcheng pigs, and perform the linkage analysis between genotype/phenotype in two resource populations. In this study, we descripted the genetic rule of two-end black color, and discussed the relationship between this trait and Inhibition locus, which provides the fundamental materials for mapping the causative genes for two-end black color. The main results of this study:(1) In the backcross population from Tongcheng and Duroc pigs, all Fl individuals were solid black with few white coat in the legs, which confirmed that two-end black is a recessive white color comparing to the solid color. In backcross generation,39 individuals were found with "intermedia" color. This non-parental phenotype demonstrates the two-end black color are controlled by multiple genes;(2) In Tongcheng pigs, we did not find the four kinds of duplication in KIT gene, and the RT-PCR experiment also revealed splicing mutation is absent in this breed. Thus, we concluded Tongcheng pigs did not carry the dominant white allele in KIT gene. Compared to Duroc pig, we found 8 synonymous and 4 non-synonymous substitutions in the coding sequence of KIT gene in Tongcheng pigs. Using the c.551C>T as genetic marker, we performed the linkage analysis between KIT and color phenotype in the backcross population, and elucidated that two-end black color is controlled by the genetic locus independent from KIT gene.(3) In the F2 population from Tongcheng and Large white, we performed the breakpoint amplification of DUP1 to trace the dominant white allele from Large white pigs. As a result, all complete white and patch white color individuals carried the dominant white alleles, and the ratio between these individual and other color type pigs is consistent with the Mendelian single gene segregation. This results reveals that the Inhibition locus is epistatic to the two-end black color.Based on two color segregating populations, this study demonstrated the two-end black is a recessive trait that controlled by multiple genes, and Inhibition locus is epistatic to the two-end black color.Chapter II:Genome-wide analysis of selection regions in Tongcheng pigs by next generation sequencingPig domestication is occurred-9000 years ago. During this process, the coat color, body size and behavior were dramatically changed in domesticated pigs, and these phenotypic changes were usually caused by artificial selection on the favorable trait. However, due to the independent domestication, huge phenotypic difference were observed between Chinese and European domestic pigs. Thus, analyzing genomic regions that under artificial selection could uncover the genetic basis underlying the morphologic characteristics of Chinese domestic pigs, which could further contribute to the molecular pig breeding. In this study, we chose Tongcheng pigs as the research object, and performed the whole-genome analysis by using next generation sequencing. Combined with data of individually sequenced pigs, we screened the genomic intervals with strong selection signature, and discuss the biologic function of gene embedding in the selective regions.(1) After comparing genome sequence between Tongcheng pigs and Chinese wild boars, we identified 7,416,043 SNPs with high quality. These SNPs were annotated and further validated by the EST sequences. As a result, we found 6 putative non-sense substitution in the whole genome of Tongcheng pigs, but none of them were fully fixed in this breed, which confirmed the gene inactivation did not contribute to the domestication of Tongcheng pigs. Moreover, according to the sequence data from 5 outgroup species, we analysed the synonymous/non-synonymous substitutions in Tongcheng and Chinese wild boar populations. The ratio between two substitutions is not statistically different between two populations, which is consistent to the fact that Tongcheng pigs did not subjected to strong selection pressures during the recent history.(2) After scanning with 150 Kb sliding window, we analyzed the distributions of Hp and Fst among the whole genome, and set 3% left tail of Hp and 3% right tail of Fst as the thresholds. As a result, we identified 196 regions with both Hp and Fst thresholds reached, indicating these regions were under strong selection pressure during domestication. We performed the functional analysis of 570 encoding gene harboring in the selective regions, and found these gene were participating in various biological processes, including lipid metabolism, melanocyte development and neural regulation. This processes are highly consistent with phenotype changes in Tongcheng pigs, which confirmed the reliability of the selection analysis.(3) Specially, a region with extreme heterozygosity (Hp=0.012) is found in the 64.7-101.2 Mb of chromosome X in Tongcheng pigs. Pairwise comparison of haplotype indicated 5 Chinese indigenous breeds:Meishan, Jinhua, Neijiang and Ya’nan pigs were also fix in this region with identical haplotype of Tongcheng pigs. In contrast, the region is also homozygous in Large white, Landrace and Duroc pigs but with a distinct haplotype. In addition, a 12 Mb heterozygous region (Hp=0.47) was also observed in Tongcheng pigs spanning 51.45-63.53 Mb of chromosome X.(4) Two candidate genes, EDNRB and MITF,separately locating in chrll: 54.60-54.75 Mb and chr13:56.4 Mb-56.7 Mb are highly related to two-end black color for their functional association. To elucidate the relationship between two genetic loci and white color variation in Chinese domesticated pigs, we employed the 60K SNP chip data of 118 Chinese indigenous pigs, including 5 white spotting breeds, Ningxiang, Wuzhishan, Tongcheng, Luchuan and Bama pigs, as well as one solid black breed, Laiwu pigs, and searched the signatures of homozygousity in both EDNRB and MITF regions. As a result, the homozygosity dramatically drop in all white spotting breeds at both loci, but not in solid black Laiwu pigs. Scanning the sequence of EDNRB and MITF reveals no convincing candidate mutation in coding region, indicating regulatory mutation of these two gene could contribute for the disruption of normal migration of melanoblasts.(5) In the selective region detected from Tongcheng pigs, we also observed the ESR1 gene, which is significantly associated with liter size as previous report. In this gene, a synonymous substitution c.669T>C shows high allele difference between Tongcheng and wild boars populations. We called the genotype of 669 from all individually sequenced pigs, and found the variant C allele increased in frequency in all tested Chinese domestic population. In addition, other three selective regions from chromosome 3,13,14 are also functionally associated with reproductive traits, which harbors PRM1, PRM2, TNP2, GPR14 and JMJD1C genes. In these three loci, Chinese domestic breeds show high haplotype similarity with Tongcheng pigs, which is further confirmed by phylogenetic tree analysis. These findings suggest that these three loci potentially have been under artificial selection before the breed creation, and generally affect the reproductive traits in nowadays Chinese domestic breeds.Using the next generation sequencing, this study screened the genomic intervals under strong artificial selection by Hp and Fst methods. Functional analysis of genes embedding in the selective regions revealed these genes were highly associated with the phenotypic characteristics of Tongcheng pigs. In the further experiment, we confirmed 6 selective regions affected coat color and reproduction traits in Chinese indigenous breeds. |
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