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Genetic analyses of population structure of Bhutanesecattle reproduced by traditional cattle breeding systems

Tashi DORJI BTN - 10801

Chief,Ministry of Agriculture and Forests,Diary Development Division, Department of Livestock

Japanese Advisor : Hideyuki MANNENProfessor, Kobe University

The indigenous cattle are themost important livestock geneticresources for food, animal energyand household income in Bhutan.The native breeds have uniquedisease resistance and adaptationsto the local environment andprovide basic raw material for breedimprovement for the Bhutanesefarmers. Nevertheless, increase indemand for domestic livestockproducts favors crossbreeding oflow producing indigenous cattlewith the specialized exotic breeds.As a consequence, the domesticindigenous animal resources ofBhutan are threatened by geneticerosion. An effective managementof farm animal genetic resourcesrequires comprehensive knowledgeof the breed characteristics,including molecular data on withinand between breed genetic diversity.However, cattle populations in Bhutanare poorly characterized anddescribed. Therefore, the objectivesof this study were to:

• Describe the traditional cattle breeding systems and their folk taxonomy and • Understand the genetic constitution of cattle population using different

genetic tools (milk protein polymorphism, mtDNA, microsatellite markers)and interpret its implications on native cattle conservation programme in

Photo-1: Laboratory work with co-supervisor professor YoshiKawamoto at the Laboratory of Primate Research Institute,Inuyama, Kyoto University

Photo-2: Field survey with principal Supervisor ProfessorHideyuki Mannen (Kobe University) at 4000m, AMSL , Paro,Bhutan

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Bhutan. In this study, we have documented the rich folk nomenclatures that exist for

different bovines and its categories in Bhutan. Although, these terms continue to beused today among farmers in rural communities throughout Bhutan, for theyounger generation, this knowledge is fast disappearing. The documentation of thisfolk taxonomy provides better understanding of the less-known local breeds and inpreserving the precious branch of such indigenous knowledge for future reference.Further, this study contributes to standardization of the use of folk terminologiesfor cattle types and its categories in Bhutan. Specifically, we propose to use theterm Nublang-Thrabum (Nublang for male; Thrabum for female) for the indigenouscattle breed of Bhutan. By using this term, we recognize the long tradition ofnaming indigenous breeds, which has its own logical reasons that describe itsorigin; Nub-meaning its origin from western Bhutan or Thra-diverse colour patternsobserved in Thrabum.

Livestock farming in Bhutan is typically related to low input subsistenceproduction systems and cattle herders inhabit marginal areas characterized byextreme weather patterns ofunreliable rainfall or situated in thehigh altitudes. As such, the traditionalbreeding strategies have resulted inanimals that survive and reproducein hostile environments. Thisindigenous animal breedingknowledge, particularly involvingMithun (Bos frontalis) andNublang-Thrabum provides arepository of information for thescientifically undocumented breedsand traits. However, this traditionalMithun-Thrabum hybridization hasnever been verif ied by genomicanalysis. In this research, weinvestigated milk protein variationin cattle (Bos indicus), Mithun (Bosfrontalis), yak (Bos grunniens) andtheir hybrid populations in Bhutanto estimate genetic variability,conduct genetic characterizationand assess the possibility of geneflow between Mithun and cattle.Isoelectric focusing of 372 milk

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Photo-3: During field survey in Paro, Western Bhutan withprincipal Supervisor Professor Hideyuki Mannen (KobeUniversity)

Photo-4: During field survey in Zhemgang, central Bhutan withCo-Supervisor Professor Yoshi Kawamoto (Kyoto University)

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samples from 11 populations detected four molecular types of β-lactoglobulin (A,B, E and M), five molecular types of αS1-casein (A, B, C, E and X) and threemolecular types of k-casein (A, B and X). Mithun and yak shared alleles but werefound to exhibit different allele frequencies for the proteins studied. The degree ofgenetic variability within populations was measured by average heterozygosity andranged from 24–40% in cattle, 26% for yak and 33% for Mithun. Typically, thedetection of a Mithun specific molecular type M for β-lactoglobulin provideduseful tool to quantify Mithun genes into the village Thrabum populations. Also,the variant αS1-CnX previously identified in the Nepalese and Dali cattle fromwestern Yunnan, China was found in the Bhutanese Thrabum and Indian Jaba butabsent in the Yak and Mithun populations. Similarly, k-CnX, a specific variant ofYak occurred not only in Yak but also in Mithun with appreciable frequency.Further investigation of these markers could provide useful tool to study the originand trace gene flow in the Asian cattle populations.

Furthermore, to determine genetic diversity and relationship of Bhutaneseindigenous cattle, we analyzed mtDNA D-loop sequence of cattle from threepopulations of Bhutanese native cattle (Thrabum) and two Bhutanese populationseach of Tibetan (Goleng) and Indian (Jaba), and together with a Nepalese cattlepopulation. Although Bhutanese native cattle morphologically reveal them as Bosindicus with the prominent hump, the three Thrabum populations included bothBos indicus and Bos taurus types of mtDNA haplotypes. Almost all of Bhutaneseindigenous populations included both haplotypes assigned to either I1 or I2 cladeof Bos indicus with adequate frequencies, while Bos taurus type of haplogroups ofT1, T3 and/or T4 clades were observed in all the five Bhutanese indigenouspopulations. The result indicated that indigenous cattle of Bhutan are admixture ofBos taurus and Bos indicus, and that ancient taurine had contributed in theformation of Bhutanese native cattle.

Although Mithun has been used in Bhutan for centuries, not much is knownabout its origin and phylogeny. In order to characterize Mithun (Bos frontalis) andincrease our understanding of its origin, we sequenced the 16S rRNA gene inmDNA. We compared Mithun with Yak, European cattle, Bhutanese zebu andIndian zebu. Sequencing revealed low nucleotide diversity within the Mithunpopulation and their phylogenetic proximity to Gaur. A close relationship betweenBhutanese Mithun and Gaur was confirmed by an additional comparison with wildGaur specimens from three locations in Bhutan. Direct domestication of Mithunfrom Gaur was supported, while maternal contribution from the cattle lineageduring domestication was not supported.

Likewise, we analyzed the genetic diversity and relationship of nativeNublang-Thrabum populations of Bhutan using 20 microsatellite markers. A totalof 120 Nublang-Thrabum cattle were sampled and were grouped according to theirgeographical locations in four populations. Each included 30 individuals named

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Nublang-Thrabum West, Nublang-Thrabum South, Nublang-Thrabum Central andNublang-Thrabum East. Also, 30 samples of Indian Jaba (Bos indicus), TibetanGoleng (Bos taurus), Nepal Hill (Bos indicus), Holstein Friesian and Mithun (Bosfrontalis) were included. The mean number of alleles per loci (MNA) and observedheterozygosity (Ho) were higher in the Nublang-Thrabum populations (MNA7.2±0.3 – 8.9±0.5 and Ho 0.67±0.04 – 0.73±0.03) compared to Holstein Friesian(MNA 5.8±0.5 and Ho 0.57±0.04). The smallest coeff icient of geneticdifferentiation and genetic distance (FST 0.015 and DA 0.073) was obtained betweenNublang-Thrabum West and Central. The Nublang-Thrabum East was differentfrom other Nublang-Thrabum populations being closer to the Indian Jaba (FST 0.024and DA 0.084). A high bootstrap value of 96% supported placement of Nublang-Thrabum South, Nublang-Thrabum Central and Nublang-Thrabum West on thesame cluster.

In conclusion, the study contributed to better understanding of the traditionalbreeding practices and genetic constitution of cattle genetic resources in Bhutan. Inparticular, we have established that Bhutan has two Nublang-Thrabum populationsat the genetic level. Nublang-Thrabum West, South and Central are geneticallysimilar. The Nublang-Thrabum East is genetically apart from other Nublang-Thrabum but closer to Jaba. We suggest that Nublang-Thrabum conservationprogram in Bhutan should focus on the former population as it has received lessgenetic influence from other cattle breeds. Future research is required to betterunderstand the origin of Mithun and develop genetic methods to evaluate its hybridpopulations. Additional information including data on population size andstructure, geographical distribution, their specific merits and socio-economic valuewithin the local context will facilitate effective management of animal geneticresources in Bhutan.

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Photo-5: During field survey in Zhemgang, central Bhutan with Co-Supervisor Professor Yoshi Kawamoto (Kyoto University) andprofessor Tetsyu Inamura (Aichi Prefecture University, Japan).

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