title: Dasytricha dominance in Surti buffalo rumen revealed by 18S rRNA sequences and real-time PCR assay.
abstract: The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR-RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10(5) vs. 1.3 × 10(4)) in per ml ruminal fluid.
authors: Department of Animal Biotechnology, College of Veterinary Science and Animal Husbandry, Anand Agricultural University, Anand, Gujarat 388 001, India. kmsingh18@gmail.com
authors: K M
authors: KM
authors: Singh
authors: A K
authors: AK
authors: Tripathi
authors: P R
authors: PR
authors: Pandya
authors: D N
authors: DN
authors: Rank
authors: R K
authors: RK
authors: Kothari
authors: C G
authors: CG
authors: Joshi
mesh_terms: D000818:Animals; D044822:Biodiversity; D002020:Buffaloes; D016798:Ciliophora / Q000145:classification* / Q000235:genetics* / Q000254:growth & development; D016000:Cluster Analysis; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004032:Diet; D020459:Genes, rRNA; D054892:Metagenome; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D016053:RNA, Protozoan / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012417:Rumen / Q000469:parasitology*; D017422:Sequence Analysis, DNA
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D016053:RNA, Protozoan; D012337:RNA, Ribosomal, 18S