Cross-amplification of ungulate microsatellite markers in the endemic Indian antelope or blackbuck (Antilope cervicapra) for population monitoring and conservation genetics studies in south Asia.

The Indian antelope or blackbuck (Antilope cervicapra) is endemic to the Indian subcontinent, inhabiting scrublands and dry grasslands. Most of the blackbuck populations are small, isolated, and threatened by habitat fragmentation and degradation. Management of such disjunct populations requires genetic characterization, which is critical for assessing hazards of stochastic events and inbreeding. Addressing the scarcity of such information on the blackbuck, we describe a novel panel of microsatellite markers that could be used to monitor blackbuck demography and population genetic parameters using non-invasive faecal sampling. We screened microsatellites (n = 40) that had been reported to amplify in bovid and cervid species using faecal samples of the blackbuck collected from Kaimoor Wildlife Sanctuary, Uttar Pradesh, India and its vicinities. We selected 12 markers for amplification using faecal DNA extracts (n = 140) in three multiplex reactions. We observed a mean amplification success rate of 72.4% across loci (92.1-25.7%) with high allele diversity (mean number of alleles/locus = 8.67 ± 1.03). Mean genotyping error rates across the markers were low to moderate (allelic drop-out rate = 0.09; false allele rate = 0.11). The proportions of first- and second-order relatives in the study population were 0.69% and 6.21%, respectively. Based on amplification success, genotyping error rates and the probability of identity (PID), we suggest (i) a panel of five microsatellite markers (cumulative PID = 1.24 × 10-5) for individual identification and population monitoring and (ii) seven additional markers for conservation genetics studies. This study provides essential tools capable of augmenting blackbuck conservation strategies at the landscape level, integral to protecting the scrubland-grassland ecosystem.

Characterization of a partially folded intermediate of papain induced by fluorinated alcohols at low pH.

A systematic investigation of the effects of aqueous 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and 2,2,2-trifluoroethanol (TFE) on the structure of acid-unfolded papain (EC. 3.4.22.2) was made using circular dichroism (CD), intrinsic tryptophan fluorescence, and 1-anilino 8-sulfonic acid (ANS) binding. At pH 2, papain exhibits substantial secondary structure as beta-sheet and is relatively less denatured as compared to 6 M guanidine hydrochloride (GdnHCl) but loses the persistent tertiary structure of the native state. Addition of HFIP and TFE caused an induction of alpha-helical structure as evident from the increase in the mean residue ellipticity value at 208 and 222 nm. Induction was 20% more in HFIP than TFE. Interestingly, at 13% (v/v) HFIP and 30% (v/v) TFE a near-UV CD spectrum approaches the native-like spectral features. Tryptophan fluorescence studies indicate the change in the environment of the tryptophan residues on the addition of HFIP and TFE to acid-unfolded papain. Maximum ANS binding occurs at 13% (v/v) HFIP and 30% (v/v) TFE, suggesting a compact "molten globule"-like conformation with enhanced exposure of hydrophobic surface area. Acid-unfolded papain in presence of 13% (v/v) HFIP and 30% (v/v) TFE showed the recovery of enzymatic activity by 54 and 61%, respectively. Thermal stability of these states was assessed by changes in fluorescence emission maximum and absorbance at 292 nm. Temperature-induced unfolding of papain at pH 2 was non-cooperative and the transition curves were biphasic in nature. Temperature-induced unfolding of HFIP and TFE-induced state was weakly cooperative in comparison to cooperative transition of native.