Genetic Divergence of Thai Indigenous Pigs from Three Distinct Geographic Regions Revealed by Microsatellite Marker Analysis.

Thai indigenous pigs (TIPs) are important genetic resources. Crosses with exotic pig breeds and wild boars may cause genetic losses. To date, the physical characteristics of TIPs have been inconsistent. The classification of TIPs by genetic information is needed to pursue an appropriate conservation program. In this study, the genetic diversity, cluster analysis, and phylogenetic relationship of TIPs were investigated using twenty-nine pig microsatellite markers. Blood samples were collected from TIPs from three regions of Thailand: north (NT, n = 118), northeast (NE, n = 61), and south (ST, n = 75). The mean total number of distinct alleles and the effective number of alleles per locus were 11.851 and 5.497, respectively. The mean observed heterozygosity (Ho) and mean expected heterozygosity (He) were 0.562 and 0.837, respectively. The F values of the microsatellite loci were positive under Hardy-Weinberg Equilibrium at p < 0.001, with overall mean values of Fis, Fit, and Fst of 0.247, 0.281, and 0.046, respectively. A total of 5, 5, and 17 private alleles were found at frequencies greater than 0.050 in the NT, NE, and ST pigs, respectively. Three optimal clusters (K = 3) were proposed within the TIP populations. Pigs from the NT and NE regions were mixed in two clusters, while members of the ST region were clearly separated. The phylogenetic tree confirmed that the pigs from NT and NE were each divided into two subgroups, while the pigs from ST were clustered into one group. A microsatellite analysis revealed the high genetic diversity of the TIP populations and confirmed the genetic divergence of the TIPs from the European and Chinese breeds. A genetic admixture of the TIP with the local wild boars was detected.

Isolation of a gene encoding a cellulolytic enzyme from swamp buffalo rumen metagenomes and its cloning and expression in Escherichia coli.

Ruminants are capable of hydrolyzing lignocellulosic residues to absorbable sugars by virtue of the microbial communities residing in their rumen. However, large sections of such microbial communities are not yet culturable using conventional laboratory techniques. Therefore in the present study, the metagenomic DNA of swamp buffalo (Bubalus bubalis) rumen contents was explored using culture-independent techniques. The consensus regions of glycosyl hydrolase 5 (GH5) family of cellulases were used as primers for PCR amplification. A full-length metagenomic cellulase gene, Umcel5B29, with a complete open reading frame (ORF) of 1611 bp was identified. The similarity search analysis revealed that Umcel5B29 is closely related to the cellulases (73% to 98% similarity) of ruminal unculturable microorganisms, indicating its phylogenetic origin. Further analysis indicated that Umcel5B29 does not contain a carbohydrate binding module (CBM). Subsequently, Umcel5B29 was overexpressed in Escherichia coli. The recombinant enzyme worked optimally at pH 5.5 and 45°C, a condition similar to the buffalo's rumen. However, the enzyme retained more than 70% of its maximal activity after incubation at pH 4-7 and more than 50% maximal activity after incubation at 30-60°C for 30 min. These characteristics render Umcel5B29 as a potential candidate for the bio-stoning process of denim.

Identification and characterization of a cellulase-encoding gene from the buffalo rumen metagenomic library.

Microorganisms residing in the rumens of cattle represent a rich source of lignocellulose-degrading enzymes, since their diet consists of plant-based materials that are high in cellulose and hemicellulose. In this study, a metagenomic library was constructed from buffalo rumen contents using pCC1FOS fosmid vector. Ninety-three clones from the pooled library of approximately 10,000 clones showed degrading activity against AZCL-HE-Cellulose, whereas four other clones showed activity against AZCL-Xylan. Contig analysis of pyrosequencing data derived from the selected strongly positive clones revealed 15 ORFs that were closely related to lignocellulose-degrading enzymes belonging to several glycosyl hydrolase families. Glycosyl hydrolase family 5 (GHF5) was the most abundant glycosyl hydrolase found, and a majority of the GHF5s in our metagenomes were closely related to several ruminal bacteria, especially ones from other buffalo rumen metagenomes. Characterization of BT-01, a selected clone with highest cellulase activity from the primary plate screening assay, revealed a cellulase encoding gene with optimal working conditions at pH 5.5 at 50 °C. Along with its stability over acidic pH, the capability efficiently to hydrolyze cellulose in feed for broiler chickens, as exhibited in an in vitro digestibility test, suggests that BT-01 has potential application as a feed supplement.