Identification and characterization of known and novel microRNAs in three tissues of Chinese giant salamander base on deep sequencing approach.

MicroRNAs (miRNAs) are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although animal miRNAs have been extensively studied in model systems, less is known in other animal with limited genome sequence data, including Chinese giant salamander (Andrias davidianus). The identification of the full repertoire of miRNAs expressed in the liver, spleen and muscle of A. davidianus would significantly increase our understanding for physiological function of A. davidianus, in this ancient and endangered urodele amphibian. In this study, three independent small RNA libraries were constructed from the liver, spleen and muscle of A. davidianus. The libraries were subjected to high-throughput sequencing by using the Illumina deep sequencing. As a result, a total of 12,831,239, 13,592,195 and 9,887,531 raw reads representing 2,240,771, 1,363,266 and 1,964,252 clean reads per library were obtained separately. Through bioinformatics analysis, we identified total of 553 known miRNAs and 44 putative novel miRNAs in our small RNA dataset from liver, spleen and muscle tissues. Five known miRNAs (gga-miR-10a-5p, pma-miR-29d-5p, aca-miR-338-3p, hsa-miR-455-3p and ssa-miR-2184-5p_R-1) and three novel miRNAs (PC-5p-891_1763, PC-5p-32538_50 and PC-3p-33645_48) showed different expression in eight different tissues as revealed by stem-loop qPCR analysis. This study characterized the miRNA of A. davidianus for the first time, which provides an opportunity for further understanding of miRNA regulation function in A. davidianus ranked as living fossils.

Rediscovery of Protohynobius puxiongensis (Caudata: Hynobiidae) and its phylogenetic position based on complete mitochondrial genomes.

The mysterious Asian hynobiid salamander, Protohynobius puxiongensis, was described based on a single specimen collected in 1965 and never found again since then. Because the specimen had an internasal bone, Pr. puxiongensis was thought to retain a primitive character lost by a common ancestor of all other hynobiid salamanders, and it was thus considered to be not only a new genus, but also a new subfamily. This conclusion bothered herpetologists for decades because it was based on only one specimen and one character without other living specimens being rediscovered. After years of field effort, we rediscovered living individuals of Pr. puxiongensis at its type locality. All characters observed in rediscovered specimens are identical to the original description of the holotype except the internasal bone, implying that the internasal bone observed in the holotype may be just an individual variation. To examine the phylogenetic position of Pr. puxiongensis, we sequenced complete mitochondrial genomes for this species, together with two Pseudohynobius species. By combining 18 published hynobiid mitochondrial genomes and our new sequences, we reconstructed a comprehensive phylogenetic relationship of Hynobiidae at the genus level. Our results indicate that Pr. puxiongensis is deeply nested within the hynobiid phylogeny. It is the sister group of the Pseudohynobius species, and the validity of subfamily Protohynobiinae is not supported.

A new species of the genus Batrachuperus (Urodela: Hynobiidae) from Southwestern China.

A new stream salamander species, Batrachuperus daochengensis sp. nov., from southwestern China, is described herein based on morphological and molecular evidence. Molecular phylogeny derived from the mitochondrial gene together with previous nuclear data revealed that B. daochengensis sp. nov. is sister to B. yenyuanensis. The new species differs from all other species of the genus by the following combination of characters: brown horny epidermis on tips of fingers and toes absent; tubercles on palms and soles absent; costal grooves 12; dorsal brown, mottled with blackish spots; fingers 2-3-4-1 in order of decreasing length; tips of longest digits of fore- and hindlimbs largely separated by one to two costal spaces when adpressed towards each other along sides of body. The new species is currently known in the central and southern Shaluli Mountains in southwestern China.

Transcriptome analysis of the Chinese giant salamander (Andrias davidianus) using RNA-sequencing.

The Chinese giant salamander (Andrias davidianus) is an economically important animal on academic value. However, the genomic information of this species has been less studied. In our study, the transcripts of A. davidianus were obtained by RNA-seq to conduct a transcriptomic analysis. In total 132,912 unigenes were generated with an average length of 690 bp and N50 of 1263 bp by de novo assembly using Trinity software. Using a sequence similarity search against the nine public databases (CDD, KOG, NR, NT, PFAM, Swiss-prot, TrEMBL, GO and KEGG databases), a total of 24,049, 18,406, 36,711, 15,858, 20,500, 27,515, 36,705, 28,879 and 10,958 unigenes were annotated in databases, respectively. Of these, 6323 unigenes were annotated in all database and 39,672 unigenes were annotated in at least one database. Blasted with KEGG pathway, 10,958 unigenes were annotated, and it was divided into 343 categories according to different pathways. In addition, we also identified 29,790 SSRs. This study provided a valuable resource for understanding transcriptomic information of A. davidianus and laid a foundation for further research on functional gene cloning, genomics, genetic diversity analysis and molecular marker exploitation in A. davidianus.

Characterization of conserved and novel miRNAs using deep sequencing and prediction of miRNA targets in Crucian carp (Carassius auratus).

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs of -22 nucleotides that can base pair with their target mRNAs, which represses their translation or induces their degradation in various biological processes. However, little is known about identification of miRNAs and their target genes in C. auratus. In the present study, a small RNA library from pooled tissue of C. auratus was constructed and sequenced using the deep sequencing. A total of 320 conserved miRNAs (belonging to 105 families) as well as 11 potentially novel miRNAs were identified. Stem-loop qRT-PCR analysis confirmed that both conserved and novel miRNAs were expressed in C. auratus, and some of them were preferentially expressed in certain tissues. Subsequently, a total of 1668 potential target genes were predicted for these identified miRNAs and GO analysis showed that most of the targets were involved in lots of physiological actions. This study represents a first large-scale identification and characterization of C. auratus miRNAs and their potential target genes. Taken together, our results add new information to existing data on C. auratus miRNAs and should be useful for investigating the biological functions of miRNAs in fishes and other aquatic species.

Sequencing and de novo transcriptome assembly of the Chinese giant salamander (Andrias davidianus).

Next-generation technologies for determination of genomics and transcriptomics composition have a wide range of applications. Andrias davidianus, has become an endangered amphibian species of salamander endemic in China. However, there is a lack of the molecular information. In this study, we obtained the RNA-Seq data from a pool of A. davidianus tissue including spleen, liver, muscle, kidney, skin, testis, gut and heart using Illumina HiSeq 2500 platform. A total of 15,398,997,600 bp were obtained, corresponding to 102,659,984 raw reads. A total of 102,659,984 reads were filtered after removing low-quality reads and trimming the adapter sequences. The Trinity program was used to de novo assemble 132,912 unigenes with an average length of 690 bp and N50 of 1263 bp. Unigenes were annotated through number of databases. These transcriptomic data of A. davidianus should open the door to molecular evolution studies based on the entire transcriptome or targeted genes of interest to sequence. The raw data in this study can be available in NCBI SRA database with accession number of SRP099564.

A comparative study of the hyobranchial apparatus in Hynobiidae (Amphibia: Urodela).

The morphology of the adult hyobranchial apparatus has played an important role in understanding the systematics and evolution of urodeles, but the hyobranchial apparatus of hynobiid salamanders has received little attention so far. In this study, the hyobranchial apparatus of eight hynobiid salamanders (Hynobius leechii, Onychodactylus zhangyapingi, Ranodon sibiricus, Batrachuperus pinchonii, Salamandrella keyserlingii, Liua shihi, Pachyhynobius shangchengensis and Pseudohynobius flavomaculatus) is described and compared based on the clearing and double-staining method. The basic elements of the hyobranchial apparatus of the eight species are similar, including one basibranchial, cornua, one pair of radial loops, one pair of ceratohyals, one pair of hypobranchials II, one pair of ceratobranchials II, one urohyal (absent in O. zhangyapingi), one pair of the complex of hypobranchial I and ceratobranchial I (separated in certain species). Although the hyobranchial apparatus is similar among hynobiid salamanders and shows a unique morphological pattern, there are also certain species-specific distinctions that may be used for specific or generic diagnosis. The results of an ancestral state reconstruction of five traits showed that the ossified basibranchial, the presence of a separated hypobranchial I and ceratobranchial I, the absence of a urohyal, the ossified hypobranchial I and the partially ossified ceratohyal are derived traits. The state shown by the traits of each species is consistent with the phylogenetic position of each species. Compared with other Urodela, the hyobranchial apparatus of this group shows certain distinctive features that may represent the diagnostic characters of the family Hynobiidae. The partially ossified ceratohyal is correlated with the habitat and represents an ecological adaptation.