Sample collection and genomic DNA isolation
Habitat, distribution, management practices, utility, performance and socio-economic status of the farmers rearing ‘Bhangor’ buffalo was collected through preset questionnaires during the pilot survey conducted in Tripura state. A volume of 10 ml blood sample was collected from 76 animals from several villages located in the remote area of the districts West Tripura, Khowai, Dhalai, Unakoti, Gomati, and North Tripura. Blood samples were collected under aseptic conditions through jugular vein puncture in sterile polypropylene tubes containing anticoagulant i.e., ethylene diamine tetra acetate (EDTA). Genomic DNA was isolated following standard phenol-chloroform extraction method . The genomic DNA samples were evaluated for their purity, quality and concentration using agarose gel documentation and NanoDrop spectrophotometer (absorbance ratio 260/280nm).
Selection of markers, PCR amplification and Microsatellite typing
Bhangor buffaloes were genotyped using a battery 24 recommended microsatellite markers/loci (www.fao.org) present on the bovine genome. The information about these microsatellite markers along with corresponding primer sequences annealing temperature and chromosomal location are depicted in Table 1. 5` end of forward primer of each microsatellite marker was labeled with one of the fluorescent dyes, viz., FAM (Blue), VIC (Green), NED (Yellow) or PET (Red) to assess the fragment length of genotyped PCR product with automated DNA sequencer (ABI 3100). However, only 15 microsatellite loci (CSSM33, BM1818, CSRM60, HEL13, ILSTS019, ILSTS025, ILSTS028, ILSTS029, ILSTS030, ILSTS033, ILSTS036, ILSTS056, ILSTS058, ILSTS061, ILSTS089 and ETH003) were found to be polymorphic for Bhangor population and hence were selected for studying genetic diversity. The markers used in this study were dinucleotide repeats which are more polymorphic than tri-nucleotides. The amplified products are clearly seen in the Agarose gel, and no non-specific amplification or PCR failure was observed.
PCR amplification was carried out in thermal cycler in a final reaction volume of 15 μl containing 10 pmol/μl of each primer, 10 mM of each dNTP, 1.5mM MgCl2 and 1.2U Taq polymerase (Invitrogen, California), after optimization of annealing temperature for each microsatellite locus. After multiplexing of different dye-labeled amplified markers, the pooled samples were run on ABI automated DNA sequencer along with internal control LIZ standard. The data was extracted using GENEMAPPER software documenting the allele sizes for each marker in each animal .
The GenAlEx software (version 6.503)  was employed to calculate different within-population diversity measures viz, mean number of alleles per locus (Na), effective number of alleles per locus (Ne), observed heterozygosity (Ho) and coefficient of genetic diversity (He) of Nei for microsatellite loci analyzed in Bhangor buffaloes. Within-population-inbreeding estimates (FIS) were calculated using FSTAT computer program (version 126.96.36.199) . Polymorphism information content (PIC) values were estimated through allele frequencies using the following equation: see equation 1 in the supplementary files.
Hardy–Weinberg equilibrium test was used to detect whether the present population has undergone any recent reduction in the effective population size or genetic bottleneck. Three different tests: sign test, standardized differences test and Wilcoxon sign-rank test under stepwise mutation model, the infinite allele model and the two-phase model of microsatellite evolution were used to compare the expected heterozygosity (HE) at Hardy–Weinberg equilibrium with the heterozygosity expected at mutation drift equilibrium (Heq). Bottleneck 1.2.01 software was used to conduct sign test, standardized differences test and Wilcoxon sign-rank test to detect whether buffalo population has undergone any recent reduction in the effective population size or genetic bottleneck . For genetic relationship studies between Bhangor buffalo with established riverine Murrah, Nilliravi, Manda, Chilika, Kalahandi and swamp population of Manipur and Assam, microsatellite data generated from same set of loci in the Lab were used.