Molecular diversity of rumen Archaea from Jinnan cattle

Abstract:

Molecular diversities of rumen archaea of Jinnan (South Shanxi Province, China) cattle was analyzed and compared by 16S rRNA gene sequencing from three clone libraries generated using three different archaea-specific primer sets, respectively. DNA from rumen of 4 Jinnan cattle was extracted, and methanogen 16S rRNA gene was amplified using archaea-specific primer sets. Three clone libraries were generated by using vector pGEM-T and cloning into E.coli JM109. One hundred clones were randomly picked up for each library and RFLP was analyzed for each clone to obtain OTUs. Sequences from each OTU were analyzed and compared with available sequences in GenBank. The first library, generated with primers Arch f364/1386, produced four groups of sequences, affiliated with 4 Methanobrevibacter strains, 1Y (61% of clones), SM9 (23% of clones), NT7 (14% of clones), and AK-87(2% of clones). The second library, generated with primers 1 Af/1100Ar, two groups of sequences, one affiliated with Methanobacterium aarhusense(72% of clones) and the other with Methanosphaera stadtmanae DSM 3091 (28% of clones). The third library, generated with primers Met86F/Met1340R, produced a high degree of diversity. It included the sequence groups found in the first and the second libraries, as well as sequences affiliated with the Methanomicrobium mobile (2% of clones) and uncultured euryarchaeote sequences (7% of clones). The phylogenetic analysis indicated that archaea found in the three libraries were clustered in Methanobrevibacter, Methanobacterium, Methanosphaera, Methanomicrobium, and unidentified euryarchaeote of the Euryarcharota. There were 25 unidentified sequences belonged to Euryarchaeota. This suggests the existence of novel methanogens in the rumen of Jinnan cattle.