Molecular Diversity Of Methanogens In The Rumen Of Jinnan Cattle And Goat

The ruminal methanogen population has become a focus of increasing scientific attention for the negative effect of methanogensis on dietary energy and environment. The knowledge of composition and diversity of ruminal methanogens is the foundation to control the methanogens and, finally, the methane emissions from rumen. However, up to now, the available information of ruminant gastrointestinal methanogens all come from overseas research mainly on ruminal methanogens of sheep and cow. Therefore, in the present work, the 16S rRNA gene clone libraries were constructed to investigate the diversity of methanogens in the rumen of Jinnan cattle and goat. And by comparing the results of clones, the distribution characteristics of methanogenic archaea in the rumen and caecum were revealed. This dissertation was described in the following four sections.1 Primers screening for molecular diversity analysis of rumen methanogensMolecular diversity of rumen methanogens of Jinnan cattle was analyzed and compared by 16S rRNA gene sequencing from the three clone libraries generated using two different archaea-specific and one methanogen-specific primer sets, respectively. One hundred clones were randomly picked up for each library. The first library, which was generated with primers Arch f364/1386, revealed four groups of sequences, affiliated with four Methanobrevibacter strains,1Y (61%of clones), SM9 (23%of clones), NT7 (14%of clones), and AK-87(2%of clones). The second library, which was generated with primers 1 Af/1100Ar, inculded two groups of sequences, one affiliated with Methanobacterium aarhusense(12%of clones) and the other with Methanosphaera stadtmanae DSM 3091 (28%of clones). The third library, which was generated with primers Met86F/Met1340R, revealed a high degree of diversity. It includes the four Methanobrevibacter found in the first library (the percentage of them were 47%,26%, 11%and 3%, respectively) and the sequence groups found in the second library (M. aarhusense 1%, M. stadtmanae 3%) as well as sequences affiliated with the Methanomicrobium mobile (2%of clones) and Aciduliprofundum boonei (7%of clones). The sequences, found with primers Met86F/Metl340R, had greater diversity than other libraries, were clustered in Methanobrevibacter, Methanobacterium, Methanosphaera, Methanomicrobium, and unidentified euryarchaeote of the Euryarcharota. Then, Met86F/Met1340R was more suitable for molecular diversity analysis of ruminant gastrointestinal methanogens. There were 25 unidentified sequences belonged to Euryarchaeota. This suggests the existence of novel methanogens in the rumen of Jinnan cattle.2 Distribution characteristics of methanogenic archaea in the rumen of Jinnan cattleThree 16S rRNA gene libraries were constructed from liquid-associated methanogen (LAM), solid-associated methanogen (SAM), and epithelium-associated methanogens (EAM) in the rumen of Jinnan cattle using methanogen-specific primers Met86Fand Met1340R. The clone numbers were 268,135 and 267 in LAM, SAM and EAM library, respectively. In the three libraries, the dominant methanogen sequences,86.94%of clones in the LAM library,77.78%in the SAM library and 76.12%in the EAM library, were all most related to the genus Methanobrevibacter. And Methanobrevibacter strains 1Y and SM9, M. alcaliphilum-like, M. stadtmanae-like and A. boonei-like clones existed in all libraries. But M. mobile, M. aarhusense-like, M. stadtmanii-like and Methanobrevibacter sp. AbM4-like clones were only found in the LAM library, M. ruminantium only in the SAM library, Methanobrevibacter sp. SM9-like clones were only found in the EAM library, and Methanobrevibacter sp. AK-87-like clones were found in the LAM and SAM libraries.In LAM library,12.30%of the clones were unidentified euryarchaeota,23.69%in SAM library and 24.71%in EAM library. And in those unidentified sequences, five phylotypes (E17, E18, E19, L25 and S14) were novel sequences. These suggest that there was apparent difference in methanogen populations in different part of the rumen of Jinnan cattle.3 Comparison of the methanogenic archaea diversity between rumen and caecum of Jinnan cattleTwo methanogenic 16S rRNA gene libraries were constructed from rumen and caecum of Jinnan cattle to reveal the methanogen population compositions in different part of gastrointestinal tract using methanogen-specific primers Met86F/Met1340R. Two hundred clones were randomly picked up for each library. The rumen library revealed 12 groups of sequence, affiliated respectively M. alcaliphilum (1%), M. aarhusense (2%), M. stadtmanae (3%), M. stadtmanii (1%), M. mobile (2%), A. boonei (11.5%), and Methanobrevibacter strains ruminantium (0.5%), SM9 (26%),1Y(44.5%), AK-87 (6%), NT7 (2%) and AbM4 (0.5%).The caecum library could be divided into 7 groups of sequences and these sequences were related M. alcaliphilum (5%), M. aarhusense (1%), M. stadtmanae (4%), Methanobrevibacter strains SM9 (54.5%),1Y (27%), AK-87 (6.5%), NT7 (2%), respectively. The clones that related to M. mobile, Methanobrevibacter sp.1Y, M. stadtmanii, Methanobrevibacter sp. AbM4 and A. boonei were found in the rumen, but no in the caecum. But in the rumen library, no Methanobrevibacter sp. SM9-like clone was found that existed in caecum. The results show the difference between rumen and caecum methanogenic community of Jinnan cattle.4 Molecular diversity analysis of rumen methanogens from goatMolecular diversity of rumen methanogens of goat was analyzed and compared by 16S rRNA gene sequencing from the two clone libraries generated using methanogen-specific primers Met86F/Met1340R and archaea universal primers w017/ w002, respectively. One hundred archaeal clones were randomly picked up for each library. The first library, which was generated with primers Met86F/Met1340R, revealed nine groups of sequences, affiliated with M. stadtmanae (1%), M. aarhusense (2%), A. boonei (3%) and six Methanobrevibacter strains, AK-87 (62%), ZA-10 (19%), OCP (7%), SM9 (2%),30Y (2%) and 1Y (2%). The second library, which was generated with primers w017/ w002, included eight groups of sequences, affiliated with Methanosphaera stadtmanii (47%), Methanobrevibacter sp. SM9(1%), Methanobacterium sp. DSM 11106 (21%), M. stadtmanae DSM 3091 (4%), Methanobacterium alcaliphilum (3%), Methanobacterium sp. OM15 (1%), Picrophilus oshimae (4%) and A. boonei (19%). And the unidentified euryarchaeota sequences accounted for 97%of the archaea clones in the second library, while uncultured bacteria existed.