Variation in the methanogen community structure in the clone libraries based on different mcrA gene primer pairs

Four different mcrA gene primers were used in this study (Table 3.1). The modified primer MCRmf and MCRr (M-P4) was validated using pure cultures of methanogens (Methanospirillum hungatei DSM 864, Methanosarcina barkeri CM1,

Methanobacterium formicicum BRM9 and Methanobrevibacter ruminantium DSM 1093). When this modified primer pair was used to amplify the rumen samples, unidentified sequences which are partially similar to mrtA genes (74% similarity of

mrtA sequence of Methanosphaera stadtmanae) were obtained. These sequences were highly similar to each other and distantly clustered with mrtA sequences of

Methanosphaera stadtmanae and Methanobacterium formicicum (Figure 3.14). Occasionally a few mcrA sequences were also obtained with this primer pair (Table 3.3). Due to the dominance of these unidentifiable sequences, the modified primer pair M-P4 was omitted from the comparison study.

Table 3.4. The rumen samples used to validate the primer pair MCRmf and MCRr and the groups of sequences amplified

Rumen samples Unidentified sequences Methanimicrococcus species. Methanomicrobium species Total Sheep (S4) Winter pasture 68 (98.5%) 0 1(1.5%) 69 Cow (C2) Silage 13 (92.9%) 1 (7.1%) 0 14

86 The different methanogen groups identified in the clone libraries constructed by three different published mcrA primer pairs are shown in Figure 3.3.

Figure 3.3. The percentages of different methanogen groups identified by three different

mcrA gene primer pairs (M-P1 to M-P3) from the rumen samples obtained from a cow (C2) fed with lucerne silage and barley meal. The total number of clones in each primer pair is given at the top of each bar within parentheses.

All of the five methanogen groups identified by 16S rRNA gene primers were identified by the mcrA primers. The percentage contribution of different methanogen groups in the different clone libraries (different primer pairs) varied considerably (P = 1.15 × 10-28 in χ2-test for differences). A group of sequences amplified by primer pair M-P1 did not have any cultured relatives, but there were many clones in GenBank with similarity to these sequences. These sequences were assumed to be from the RCC group (RCC-like). Further studies were performed to confirm that these sequences are from the RCC group (Chapter 5). Only the primer pair M-P1 amplified RCC-like sequences and the percentage contribution of this group was comparable to the RCC group contribution in 16S rRNA gene clone libraries. The primer pair M-P1 amplified all five groups, which were amplified by 16S rRNA gene primers. Interestingly, the other two primers failed to amplify one or more groups of methanogens. The primer pair M-P2 amplified only three groups of methanogens. However, the primer pair M-P3 amplified five groups of methanogens and included a group of sequences which were

87 only partially similar to the mrtA gene. There were no clone sequences in GenBank with similarity to these unidentified sequences. These sequences clustered together and were highly similar to each other. In the clone library constructed with primer pair M-P3, the sequences contributed 13% of the total sequences. The clone library of M-P4 consisted exclusively from these sequences apart from one sequence from Methanimicrococcus

spp. (Table 3.3).

The contribution of different methanogen groups in different mcrA gene clone libraries varied highly. The only methanogen group amplified by all four mcrA gene primer pairs (including the modified primer pair M-P4) was Methanimicrococcus

species. However, its contribution to each clone library used in the comparison study varied from 5.8% to 71.9%. mcrA gene sequences belonging to the Mbb. ruminantium

group (3.6 - 55.1%) and Mbb. gottschalkii group (12.5 - 36.7%) were also amplified by all three primer pairs (M-P1, M-P2 and M-P3). However, the percentage contribution of these groups in different clone libraries varied highly. The percentage contribution of different methanogens groups in the clone library with primer pair M-P1 was comparable to most of the 16S rRNA gene clone libraries. However, the clone library created by M-P2 was dominated by sequences from the Mbb. ruminantium and Mbb. gottschalkii groups. In contrast, the clone library created by primer pair M-P3 had these two groups at a very low percentage and this clone library was dominated by

Methanimicrococcus sequences. Interestingly, one sequence which belongs to

Methanomicrobium was amplified by M-P3. The sequences of Methanomicrobium

species were not amplified by any other mcrA or 16S rRNA gene primer pairs. In addition, all of the sequences obtained from the Methanosphaera spp. group were found to be belong to mrtA gene sequences which was only amplified by primer pair M-P1 (25.5%).

The diversity indices (Shannon and Simpson‟s diversity indices) of mcrA gene clone libraries from three different published mcrA gene primer pairs are shown in Figure 3.4. Both indices were high for the clone library of primer pair M-P1 and low for the clone library of primer pair M-P3. Primer pair M-P3 amplified four groups of methanogens but was dominated by Methanimicrococcus species. According to the Shannon diversity t test, the clone library from M-P1 was different (P < 0.05) from

88 clone libraries of M-P2 and M-P3. Only the clone library of M-P1 was comparable to the clone libraries constructed with the 16S rRNA gene (P1, P2, P4, P5 and P6).

Figure 3.4. Diversity of the methanogen community in the rumen sample detected by three mcrA gene primer pairs. Shannon diversity index and Simpson‟s diversity index were calculated from contribution of this group in clone libraries.