Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.
Rumen ciliate protozoa represent important functional members of the rumen environment, as most have some cellulolytic or amylolytic abilities (1-3). Most studies of rumen ciliate protozoa are performed using microscopy and traditional culturing techniques (2, 4-10), quantitative PCR (11, 12), denaturing gradient gel electrophoresis (13), and full-length 18S rRNA clone libraries (13,14). A few studies of rumen ciliate protozoa use highthroughput sequencing, although primer selection remains a problem, as some studies use universal eukaryotic primers, primers which target only one ciliate protozoon signature region, or primers which produce long amplicons that are unsuitable for current high-throughput technology (15)(16)(17)(18)(19)(20).18S rRNA genes range from 1.5 kb to more than 4.5 kb (21), and in rumen ciliate protozoa, they are generally 1.5 kb to 1.8 kb in length. Like the 16S rRNA genes of prokaryotes, the 18S rRNA genes of eukaryotes have nine hypervariable regions (V1 to V9) which can be used for genus/species identification. Four gut ciliate signature regions exist within rumen protozoal 18S rRNA genes which represent areas of high variability that can improve identification down to the species level (22, 23). Signature region 1 occurs between bp 440 and 460 (within V3), signature region 2 occurs between bp 590 and 620 (between V3 and V4), signature region 3 occurs between bp 1220 and 1260 (within V6), and signature region 4 occurs between bp 1560 and 1580 (after V8) (Fig. 1). Additionally, rumen ciliate protozoa have a slightly different 18S rRNA secondary structure from nonrumen ciliates, in that rumen protozoa are missing helix E23-5 from the V4 region and other hel...