Genetic diversity, differentiation and relationship of Nepalese sheep genetic resources: using DNA markers

Abstract

Four distinct indigenous domestic sheep breeds - Bhyanglung in the alpine region, Baruwal in the high hills, Kage in the mid hills and Lampuchhre in the low lands - are distributed in Nepal, which borders India in the south and China in the north. In this study, we unraveled the origin, genetic diversity, relationship and differentiation of the Nepalese sheep populations, compared with sheep from Asia using mtDNA control region and SRY 5’ promoter region and SRYM18 microsatellite marker of Y-chromosome. In addition, we extended our objectives by using genome-wide SNPs markers (Illumina SNP50K ovine Beadchip) to: (i) investigate the phylogenic relationship between these four Nepalese sheep and previously studied sheep to estimate the origin of the Nepalese sheep; (ii) compare the population genomic structure of the high-altitude sheep with those of lowland sheep to identify molecular mechanism underlying the high-altitude adaptation in these breeds; and (iii) identify if high-altitude adaptation-related genes occurred among different sheep breeds and to determine if adaptation had a single or multiple origins. Analyses of mitochondrial DNA (mtDNA) and nuclear genome revealed high diversity among these breeds. The mtDNA analyses showed that all haplotypes in these breeds belonged to three haplogroups (A to C). Among the four breeds, three residing in mid to high hills, had all three haplogroups while Lampuchhre sheep from lowlands carried only haplogroups A and B. This study revealed southwestern route of gene flow in sheep probably originating from China to India via Nepal. In addition, we provide evidences that the genetic diversity and structure in mtDNA genome among Nepalese sheep were shaped not only by the extensive gene flow to the high-altitude sheep (specifically Bhyanglung) from China but also by the continuous exchanges between sheep found in mid hills and lowlands in Nepal and India. This phenomenon can be explained by considering occurrence of gene flow through ancient trading route as well as the current movement of sheep from/to the geographic vicinities in India and China. Heteroplasmy due to length polymorphism with tandem repeats in mtDNAs within individual has been scarcely studied in domestic animals. Interestingly, in the present study, we identified intra-individual length variation in the control region of mtDNAs in Nepalese sheep by molecular cloning and sequencing techniques. We observed one to four tandem repeats of a 75/76-bp nucleotide sequences in the mtDNA control region in 45% of the total Nepalese sheep sampled in contrast to the Chinese sheep, indicating that the heteroplasmy is specific to Nepalese sheep. The high rate of heteroplasmy in Nepalese sheep could be a result of the mtDNA mutation and independent segregation at intra-individual level or a strand slippage and mispairing during the replication. Very few studies have been done to separate out the male-specific contribution specifically during breed development in domestic animals, specifically in the Asian region. We had investigated the ancestral origin and genetic diversity in four Nepalese sheep populations along with wild sheep, Argali (O. ammon byth) sampled in the northwestern China using Ychromosome microsatellites and SNPs variation in SRY 5’ promoter gene. In the present study, most of the Nepalese sheep breeds exhibited paternally monomorphic except Kage breed which exhibits three allelic variants (141 bp, 143 bp and 147 bp) in SRYM18 microsatellite region. In this present study, we have identified haplotype HY-20 (A-oY1/147-SRYM18) for the first time in domestic sheep. More than 90% of Nepalese sheep shared the haplotype which corresponds with defined haplotype HY-6. This haplotype includes mouflons and high percentage of domestic sheep. Argali, wild species found in northwestern China and also in Nepal, showed the paternal monomorphism. Nepalese sheep and Argali (speculated ancestor population) clustered separately revealed that there is no introgression from any of the extant wild populations to today’s sheep population. Low polymorphism in Y-chromosome in Nepalese male sheep might be due to an exaggerated reduction in male effective population size; however, the reason for monomorphism in case of Argali is not clear. In order to draw any rigid conclusion, further research including more sample size in domestic and wild sheep with inclusion of different Ychromosome markers is warranted. Domestic animals are perfect materials for the detection of signatures of selection since they have been adapted to different kinds of environment and developed genotypes and phenotypes accordingly over the time of domestication and through the subsequent selection resulting from the condition of their habitat such as high-altitude environment. Due to the unique geography of Nepal, containing eight of the world’s ten highest mountains in the north and lowland Terai plains in the south, Nepalese sheep are one of the best domestic animals for the study of high-altitude adaptation. Population structure analyses from our study also revealed that one highland breed (Bhyanglung) has similar genetic background as the Tibetan sheep, while another highland breed (Baruwal) shows a distinctly different population structure. The two lowland breeds (Kage and Lampuchhre) were found to be mixed with the highland sheep and Indian lowland breeds. To understand the genetic basis of adaptation to high altitude in farm animals, by comparing the breeds from different altitudes we revealed strong signals of population differentiation at the four loci of hypoxia-related genes. Out of these, XXX-7 is the most promising one with epithelium specific expression and an important role in early lung organogenesis and the morphogenesis of epithelium. Furthermore, we identified one novel upstream regulatory novel mutation specific to the high-altitude sheep. To the best of our knowledge, our study is the first one to characterize genetic polymorphism of valuable Nepalese sheep genetic resources which have very unique properties of adaptation to high-altitude environment. We have also provided important information on the genetic diversity of the Nepalese sheep and potential mechanisms for the adaptation to hypoxia caused by high-altitude. This study further opens a new avenue to study on the identified genes for the functional mechanism in the adaptive processes. This thesis has documented the information on phylogeny and population diversity within and between Nepalese indigenous breeds taking into account three different directions, viz. mtDNA, Y-chromosome DNA and nuclear DNA, and further undergone meta-analysis with GenBank dataset of different sheep of the region. It has shown the genetic structure and high levels of biodiversity within and among sheep populations. Importantly, it provides the genetic basis for the highland sheep for adapting to the hypoxic environment. These are useful baseline scientific information on which any policy on holistic conservation decision related to sheep in the regions should be based.