title: New Primers Targeting Full-Length Ciliate 18S rRNA Genes and Evaluation of Dietary Effect on Rumen Ciliate Diversity in Dairy Cows.
abstract: Analysis of the full-length 18S rRNA gene sequences of rumen ciliates is more reliable for taxonomical classification and diversity assessment than the analysis of partial hypervariable regions only. The objective of this study was to develop new oligonucleotide primers targeting the full-length 18S rRNA genes of rumen ciliates, and to evaluate the effect of different sources of dietary fiber (corn stover or a mixture of alfalfa hay and corn silage) and protein (mixed rapeseed, cottonseed, and/or soybean meals) on rumen ciliate diversity in dairy cows. Primers were designed based on a total of 137 previously reported ciliate 18S rRNA gene sequences. The 3'-terminal sequences of the newly designed primers, P.1747r_2, P.324f, and P.1651r, demonstrated >99% base coverage. Primer pair D (P.324f and P.1747r_2) was selected for the cloning and sequencing of ciliate 18S rRNA genes because it produced a 1423-bp amplicon, and did not amply the sequences of other eukaryotic species, such as yeast. The optimal species-level cutoff value for distinguishing between the operational taxonomic units of different ciliate species was 0.015. The phylogenetic analysis of full-length ciliate 18S rRNA gene sequences showed that distinct ciliate profiles were induced by the different sources of dietary fiber and protein. Dasytricha and Entodinium were the predominant genera in the ruminal fluid of dairy cattle, and Dasytricha was significantly more abundant in cows fed with corn stover than in cows fed with alfalfa hay and corn silage.
authors: State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuanxilu, Haidian, Beijing, 100193, People's Republic of China.
authors: Jun
authors: J
authors: Zhang
authors: State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuanxilu, Haidian, Beijing, 100193, People's Republic of China.
authors: Shengguo
authors: S
authors: Zhao
authors: State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuanxilu, Haidian, Beijing, 100193, People's Republic of China.
authors: Yangdong
authors: Y
authors: Zhang
authors: State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuanxilu, Haidian, Beijing, 100193, People's Republic of China.
authors: Peng
authors: P
authors: Sun
authors: State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuanxilu, Haidian, Beijing, 100193, People's Republic of China. budengpan@126.com.|CAAS-ICRAF Joint Laboratory on Agroforestry and Sustainable Animal Husbandry (ASAH), World Agroforestry Centre, East and Central Asia, Beijing, 100081, People's Republic of China. budengpan@126.com.|Synergetic Innovation Center of Food Safety and Nutrition, Harbin, 150030, People's Republic of China. budengpan@126.com.
authors: Dengpan
authors: D
authors: Bu
authors: State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, No. 2 Yuanmingyuanxilu, Haidian, Beijing, 100193, People's Republic of China. jiaqiwang@vip.163.com.
authors: Jiaqi
authors: J
authors: Wang
mesh_terms: D000818:Animals; D002417:Cattle; D016798:Ciliophora / Q000145:classification* / Q000235:genetics*; D016000:Cluster Analysis; D017931:DNA Primers; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D018744:DNA, Plant / Q000737:chemistry / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004032:Diet; D020459:Genes, rRNA; D014644:Genetic Variation; D008969:Molecular Sequence Data; D010802:Phylogeny; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D012417:Rumen / Q000469:parasitology*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D018744:DNA, Plant; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S