PCR products were cloned with a pGEM-T Easy Vector System (Promega, San Luis Obispo, CA) according to the manufacturer’s instructions. Clones containing the correct insert were sequenced at Takara Bio (Yokkaichi, Japan). Clone nomenclature was as follows: for the alfalfa and orchardgrass hay-associated Ulixertinib Treponema libraries, clone names began with ALTC and OGTC, respectively, followed by the clone number. Clone names in the concentrate-associated Treponema library began with CTC followed by the clone number. All the sequences were deposited into the GenBank database with the accession numbers AB537568 through AB537880. A total of 313 16S rRNA gene sequences, obtained
from the three clone libraries and representative rumen Treponema sequences from the NCBI database, were included in the analysis. The sequences were automatically
aligned using clustal x ver.1.81 multiple sequence alignment software (Thompson et al., 1997). A neighbor-joining tree (Saito & Nei, 1987) with a Kimura-2 correction was constructed in mega v.3.1 software. (Tamura et al., 2007). In order to statistically evaluate the branching of the tree, bootstrap analysis (Felsenstein, 1985) was carried out with 1000 resamplings of the data. Sequences from the three rumen Treponema clone libraries were compared with 16S rRNA gene sequences in the GenBank database using the blast program (Altschul et al., 1990) to obtain similarity
values. Operational taxonomic units (OTUs) were defined based on a 97% sequence identity criterion (Stackebrandt find more & Goebel, 1994). Analysis of the diversity for the individual and combined libraries was carried out using the nonparametric estimator Chao1 (Chao, 1984) and the Shannon index (Shannon & Weaver, 1949) using fastgroupii software. (http://biome.sdsu.edu/fastgroup/fg_tools.htm) (Yu et al., 2006). The percentage of coverage of the clone libraries was calculated by Good’s method with the formula [1−(n/N)] × 100, where n is the number of singletons and N is the total number of sequences Ribonuclease T1 (Good, 1953). The statistical differences among the 16S rRNA gene clone libraries from the respective feeding conditions were compared using the web-based library shuffling (web-libshuff) program version 0.96 (http://libshuff.mib.uga.edu) (Henriksen, 2004) to determine whether a given pair of libraries was drawn from the same population. The significant difference level for comparison of the three libraries was defined as P=0.0085. The sequences were initially aligned by clustal x and genetic distances were generated in the dnadist program of the phylip package (v.3.67) (Felsenstein, 2007) using the Jukes–Cantor model before submitting to web-libshuff.