Comparative Genomics Analysis Between Vestibuliferida And Entodiniomorphida In Rumen Ciliates

The rumen of ruminants uses its microbial system to efficiently utilize plant material,and this mode of digestion has contributed to the evolutionary success of ruminants in terms of species diversity,abundance,and geographic distribution.At present,the genomes of rumen microorganisms have been analyzed successively,which helps us to greatly understand the operation of rumen microbial system.Rumen ciliates account for about 50% of the microbial biomass in the entire rumen and are an important component of the rumen microbiome,yet their genomic evolutionary characteristics remain unclear.This study based on 52 high-quality genomes of 19 species in 13 genera from two orders newly assembled in our laboratory and transcriptome.With analysis of comparative genomics,the feature of rumen ciliates genome evolution got resolved.The results of the study are as follows:1.In this study,23 transcriptome data and e RF1 gene of rumen ciliates in Vestibuliferida and Entodiniomorphida were analyzed,and it was found that they are different from the stop codons of model ciliates.Both orders use UAA,UAG,and UGA as the stop codons.2.By pairwise alignment of 52 genomes,64,189 gene families were constructed,of which40,898 gene families were shared among species and 23,291 were species specific.Among the gene families shared among species,14,871 were shared among orders,6,091 were unique to Vestibuliferida,and 19,936 were unique to Entodiniomorphida.A large number of unique gene families make the gene family number of Entodiniomorphida higher than that of Vestibuliferida.3.Molecular clock analysis with the help of 113 single-copy genes and their phylogenetic tree further refined the species divergence time of rumen ciliates.The analysis of gene family expansion and contraction found that the gene families of Entodiniomorphida and Vestibuliferida showed different patterns: 2,784 gene family expansions occurred in the recent common ancestor(RCA)of Entodiniomorphida while 957 gene family expansions were detected,and 7 of 13 ancestral nodes are inclined to the expansion of gene families.1,113 gene family contractions occurred in the RCA of Vestibuliferida while 53 gene family expansions were detected,and 3 of 4 ancestral nodes are prone to gene family contractions.More expansions of gene family in Entodiniomorphida than Vestibuliferida may cause more gene counts.4.Through gene tree inference,a total 232,765 gene duplication events were detected during the evolution of rumen ciliates.There were 5,248 gene duplication events in the RCA of Entodiniomorphida,2,321 in the RCA of Diplodiniinae and Ophryoscolecinae,430 in the RCA of Vestibuliferida,6,290 in the RCA of Isotrichinae.It suggests that gene duplication events are important for the evolution of rumen ciliates.5.The selection pressure between gene families in Isotrichinae which was selected as background and gene families in Dasytrichinae,Entodiniinae,Diplodiniinae,Ophryoscolecinae were compared.The result showed that,compared with the Isotrichinae,there were 1,893,2,914,3,037,3,071 gene families with significant differences in Dasytrichinae,Entodiniinae,Diplodiniinae and Ophryoscolecinae,respectively,of which 939,2,338,2,212,and 2,247 are under intensified selection,and 954,576,825,824 are under relaxed selection.More gene families in Entodiniomorphida were subjected to stronger purifying selection than those in Vestibuliferida.6.According to result of gene annotation and functional domain annotation,it was found that the Vestibuliferida-specific gene families and gene families with a higher proportion of gene in Vestibuliferida compared to Entodiniomorphida are mainly related to environmental sensing while Entodiniomorphida-specific gene families and gene families with a higher proportion of gene in Entodiniomorphida compared to Vestibuliferida are mainly related to carbohydrate metabolism,membrane structure,vesicular transport,and antioxidant properties.In conclusion,with the help of comparative genomics,this study constructed the unique evolutionary history of rumen ciliates and demonstrated the genetic differences and evolutionary mechanisms of rumen ciliates,which provided a theoretical basis for follow-up research.