www.nature.com/scientificreports OPEN High-density Genotyping reveals Genomic Characterization, Population Structure and Genetic Received: 24 November 2017 Accepted: 20 June 2018 Diversity of Indian Mithun (Bos Published: xx xx xxxx frontalis) Anupama Mukherjee1,3, Sabyasachi Mukherjee1, Rajan Dhakal 2, Moonmoon Mech1, Imsusosang Longkumer1, Nazrul Haque1, Kezhavituo Vupru1, Kobu Khate1, I. Yanger Jamir1, Pursenla Pongen1, Chandan Rajkhowa1, Abhijit Mitra1, Bernt Guldbrandtsen 2 & Goutam Sahana 2 The current study aimed at genomic characterization and improved understanding of genetic diversity of two Indian mithun populations (both farm, 48 animals and feld, 24 animals) using genome wide genotype data generated with Illumina BovineHD BeadChip. Eight additional populations of taurine cattle (Holstein and NDama), indicine cattle (Gir) and other evolutionarily closely related species (Bali cattle, Yak, Bison, Gaur and wild bufalo) were also included in this analysis (N = 137) for comparative purposes. Our results show that the genetic background of mithun populations was uniform with few possible signs of indicine admixture. In general, observed and expected heterozygosities were quite similar in these two populations. We also observed increased frequencies of small-sized runs of homozygosity (ROH) in the farm population compared to feld mithuns. On the other hand, longer ROH were more frequent in feld mithuns, which suggests recent founder efects and subsequent genetic drift due to close breeding in farmer herds. This represents the frst study providing genetic evidence about the population structure and genomic diversity of Indian mithun. The information generated will be utilized for devising suitable breeding and conservation programme for mithun, an endangered bovine species in India. Mithun (Bos frontalis), also known as gayal, a unique bovine species, has a limited geographical distribution primarily restricted to the North-Eastern Hilly (NEH) region of India, Myanmar, hilly provinces of Bangladesh, Bhutan and Yunan province of China1. Tough it is difcult to ascertain the actual population of Mithun in the world, India possesses 0.30 million mithun which constitutes ~97.57% of the world population (19th Livestock Census, 2012)2. Mithun has a very special socio-cultural status among the indigenous tribal population of NEH region of India. Having the very high value in the barter system, owning mithun is considered as a sign of prosperity. Mithun are traditionally raised as a meat animal in a free-range system in the sub-tropical rain forest with almost zero input. As a part of socio-cultural practices, mithuns are sacrifced during rituals and festivals to ofer feast3. Mithun meat is one of the most preferred sources of animal protein among local tribes. Compared to the meat from local cattle and bufalo, mithun meat is preferred due to both quality (better marbling, fner texture, and tenderness) and quantity (higher dressing %)4–6. Mithun have higher feed conversion efciency than local 1Animal Genetics and Breeding Lab., ICAR-National Research Centre on Mithun, Medziphema, Nagaland, 797106, India. 2Center for Quantitative Genetics and Genomics, Department of Molecular Biology and Genetics, Aarhus University, 8830, Tjele, Denmark. 3Present address: Dairy Cattle Breeding Division, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India. Anupama Mukherjee and Sabyasachi Mukherjee contributed equally to this work. Correspondence and requests for materials should be addressed to S.M. (email: sabayasachimukherje@ gmail.com) SCIENTIFIC REPORTS | (2018) 8:10316 | DOI:10.1038/s41598-018-28718-x 1 www.nature.com/scientificreports/ Sample size Location of collected Admixture PCA Sl. No. Name/Code used samples analysis analysis 1 Mithun – farm population (Bos frontalis)/Farm India 48 20 2 Mithun – feld population (Bos frontalis)/Field India 24 20 3 NDama (Bos taurus)/NDA Africa 23 20 4 Bali cattle (Bos javanicus)/BLI Indonesia (Bali) 20 20 5 Gir (Bos indicus)/ GIR Brazil 50 20 6 Holstein (Bos taurus)/HOL Denmark 20 20 7 Wild bufalo (Bubalus depressicornis)/OWB Africa 10 10 8 Yak (Bos grunniens)/OYK China 4 4 9 Bison (Bison bison)/OBB USA 4 4 10 Gaur (Bos gaurus)/OGR USA 6 6 Table 1. Species/breeds included in the ADMIXTURE and PCA analyses genotyped with Illumina BovineHD BeadChip (HD) or BovineSNP50 (50 k). cattle7. Te current population size of mithun in India is not large. However, considering its importance in wid- ening the biodiversity base and adaptation to a humid sub-tropical climate and a hilly topography, it could be exploited as an alternative means of livelihood as well as for improved food and nutrition security in its present habitat and in similar environments elsewhere. Genetic improvement of mithun, however, is impeded by the way mithun is presently reared. Under tradi- tional system of rearing, mithuns are let loose in the forest with minimal intervention. Recording of traits and pedigree information is almost non-existent. Te size of a typical mithun herds ranges from 50 to 100. Herds are generally served by a few dominant bulls. Te mithun population may therefore sufer from inbreeding. Under these circumstances, the genetic diversity of the mithun population needs to be described. Tis will assist ongoing mithun conservation and genetic improvement programme and for devising a suitable breeding policy. Among a wide range of molecular markers developed, single nucleotide polymorphisms (SNPs) are the most abundant, widely dispersed throughout genomes, and have variable distribution among species8. Te avail- ability of high-throughput SNP genotyping platforms makes it feasible to undertake high-resolution scans by using large numbers of SNP markers distributed across the whole genome. SNPs are useful in studying livestock genetic diversity and population structure8,9. Although a large number of SNPs have been identifed in bovine genome-sequencing projects, few of these have been validated outside Bos taurus, as for example in mithun10. Te Illumina BovineHD BeadChip (Illumina, San Diego, CA) with 777k SNPs was introduced and utilized for gen- otyping studies in various breeds of cattle, but has also been used in other members of the bovidae, for example, yak, gaur, bufalo and wild anoa10–16. Other bovine species including indigenous cattle, yak and gaur are also found in the North Eastern parts of India along with mithun. Forest cover and natural habitats of mithuns in the North Eastern Hill States of India have shrunk over the years. Tis raises concerns about possible introgression of local cattle with mithun, shar- ing the same habitat. Sporadic instances of crossing mithuns with cattle bulls by mithun owners in the feld to increase milk production have also been recorded17. Hence, we used the unsupervised clustering analysis (carried out with the ADMIXTURE sofware)18 to estimate individuals’ ancestries from SNP genotypes to assess the extent of admixture in mithun with other cattle species (Table 1). Terefore, the present study aimed to generate high-resolution information on the genomic diversity and pop- ulation structure of mithun using Illumina BovineHD BeadChip. To our knowledge, this is the frst study using BovineHD BeadChip in mithun. We further studied the evolutionary relationship of mithun with closely related bovine species to understand mithun phylogeny and origin. Results Population diversity parameters - Observed Ho, expected He, FIS and ROH. Observed (Ho) and expected (He) heterozygosities estimated in the Indian mithun population (farm and feld population) ranged from 0.25 to 0.17, and 0.25 to 0.18, respectively (Table 2). Te inbreeding coefcient estimates (FIS) based on the observed versus expected number of homozygous genotypes, were found to be 0.06 ± 0.02 and 0.02 ± 0.01 for the farm and feld animals (Table 2). Te mean FST estimate between farm and feld mithun populations was 0.03 ± 0.01. Runs of homozygosity. Runs of homozygosity (ROH) in the autosomes of 48 farm mithun and 24 feld mithun animals were determined using PLINK1.919 and consisted of 139,350 SNPs in each of the farm and feld data set afer quality control. Te total length of ROH per animal was averaged within population in six sized windows: 250 kb–1 Mb, 1 Mb–2 Mb, 2 Mb–4 Mb, 4 Mb–8 Mb, 8 Mb–16 Mb and >16 Mb. Te average total length of ROH per animal was 823.7, 242.1, 92.30, 47.03, 66.22 and 110.9 Mb in the farm and 735.8, 245.0, 121.3, 84.4, 86.0 and 87.4 Mb in the feld population for these six length categories. Summary statistics of ROH observed are outlined in Table 3 (farm) and 4 (feld). Average numbers of ROH per animal for these six categories were 1793.0, 182.8, 35.3, 8.9, 6.0 and 4.25 in farm and 1596.0, 181.9, 45.5 15.5, SCIENTIFIC REPORTS | (2018) 8:10316 | DOI:10.1038/s41598-018-28718-x 2 www.nature.com/scientificreports/ Population Location No. of animals Average Ho Average He Inbreeding Coefcient (FIS) Research- farm (Farm) Nagaland 48 (28 males and 20 females) 0.25 ± 0.08 0.25 ± 0.07 0.06 ± 0.02 Nagaland, Arunachal Farmers’ herds (Field) Pradesh, Manipur, 24 (11 males and 13 females) 0.17 ± 0.03 0.18 ± 0.03 0.02 ± 0.01 Mizoram Table 2. Geographic location, sample size (N), composition of mithuns and diversity parameters in mithun population. Average observed and expected heterozygosities are indicated as Ho and He, respectively. ROH length category (Mb) Statistics 250 kb-1 Mb 1–2 Mb 2–4 Mb 4–8 Mb 8–16 Mb >16 Mb Mean 823.7 242.1 92.30 47.03 66.22 110.9 SD 85.3 42.1 39.3 34.2 54.6 134.7 Length of ROH per animal (Mb) Min 610.7 137.8 25.31 4.52 8.2 16.7 Max 971.6 322.8 179.8 138.1 205.6 498.6 Mean 1793.0 182.8 35.3 8.9 6.04 4.25 SD 199.8 30.1 14.5 6.2 4.7 5.02 Number of ROH per animal Min 1313.0 108.0 10.0 1.0 1.0 1.00 Max 2125.0 240.0 69.0 26.0 18.0 19.00 Mean 0.335 0.098 0.038 0.019 0.027 0.045 SD 0.035 0.017 0.016 0.014 0.022 0.007 FROH Min 0.248 0.056 0.010 0.002 0.003 0.007 Max 0.395 0.131 0.073 0.056 0.084 0.202 Table 3.