Transcriptome analysis of the plateau fish (Triplophysa dalaica): Implications for adaptation to hypoxia in fishes

Highlights

• We sequenced the transcriptome of a plateau fish (Triplophysa dalaica).

• We identified 61 positively selected genes in Triplophysa dalaica, of which 13 may be involved in hypoxia response.

• We provided lots of SSR and SNP markers for Triplophysa dalaica.

Abstract

Triplophysa dalaica, endemic species of Qinghai-Tibetan Plateau, is informative for understanding the genetic basis of adaptation to hypoxic conditions of high altitude habitats. Here, a comprehensive gene repertoire for this plateau fish was generated using the Illumina deep paired-end high-throughput sequencing technology. De novo assembly yielded 145, 256 unigenes with an average length of 1632 bp. Blast searches against GenBank non-redundant database annotated 74,594 (51.4%) unigenes encoding for 30,047 gene descriptions in T. dalaica. Functional annotation and classification of assembled sequences were performed using Gene Ontology (GO), clusters of euKaryotic Orthologous Groups (KOG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. After comparison with other fish transcriptomes, including silver carp (Hypophthalmichthys molitrix) and mud loach (Misgurnus anguillicaudatus), 2621 high-quality orthologous gene alignments were constructed among these species. 61 (2.3%) of the genes were identified as having undergone positive selection in the T. dalaica lineage. Within the positively selected genes, 13 genes were involved in hypoxia response, of which 11 were listed in HypoxiaDB. Furthermore, duplicated hif-α (hif-1αA/B and hif-2αA/B), EGLN1 and PPARA candidate genes involved in adaptation to hypoxia were identified in T. dalaica transcriptome. Branch-site model in PAML validated that hif-1αB and hif-2αA genes have undergone positive selection in T.dalaica. Finally, 37,501 simple sequence repeats (SSRs) and 19,497 high-quality single nucleotide polymorphisms (SNPs) were identified in T. dalaica. The identified SSR and SNP markers will facilitate the genetic structure, population geography and ecological studies of Triplophysa fishes.

Introduction

The genus Triplophysa is one member of the subfamily Nemacheilinae (Cypriniformes: Balitoridae) (Nelson, 2006), which is a strongly diverged fish group, with 137 valid species reported in the Fishbase (Froese and Pauly, 2014). Triplophysa fishes, also known as Plateau-dwelling loaches, are mainly distributed at high altitude rivers and lakes on the Qinghai-Tibetan Plateau (QTP) and its peripheral regions (Zhu, 1989, Prokofiev, 2001, He et al., 2011). The elevation in this area ranges from 700 to 5000 m, and the high altitudes are accompanied by hypoxia and low temperatures (Wu and Wu, 1992). As representatively endemic fishes of the QTP, Triplophysa dalaica (Kessler, 1876) (Cypriniformes: Balitoridae: Nemacheilinae) is adapted to the cold and hypoxic conditions of high-altitude habitats.

Recent studies of adaptation to high-altitude hypoxia mainly focused on endothermic vertebrates, including Ground tit, yak, Tibetan Mastiffs, Tibetan wild boar, and Tibetans (Qiu et al., 2012, Cai et al., 2013, Li et al., 2013, Qu et al., 2013, Li et al., 2014). EGLN1 (1egl 9 homolog 1), PPARA (peroxisome proliferator-activated receptor-a genes) and EPAS1, also known as HIF2α (endothelial PAS domain 1) were significantly associated with hypoxia adaptation (Beall et al., 2010, Bigham et al., 2010, Simonson et al., 2010, Storz, 2010, Yi et al., 2010). However, few studies have been performed on poikilothermic species. By comparing the two transcriptomes of Rana chensinensis and R. kukunoris which inhabit respective low- and high-elevation habitats, candidate genes were identified that may be involved in high-elevation adaption in poikilothermic species (Yang et al., 2012). Guan et al. (2014) concluded HIF-1αB may be the most important regulator in the adaptation of schizothoracine fish to the extreme environment of the Tibetan Plateau. In fact, as an important transcriptional factor, hypoxia-inducible factor (HIF) has evolved specialized roles in both low temperature acclimation and hypoxia response (Rissanen et al., 2006, Kaelin and Ratcliffe, 2008). For example, a study on a poikilothermic vertebrate, crucian carp (Carassius carassius) revealed that hypoxia-inducible factor-1 (HIF-1) could have a role in low temperature acclimation (Rissanen et al., 2006). Generally speaking, HIF is mainly regulated at the posttranscriptional level (Kallio et al., 1997), however, transcriptional control may be central for the regulation of HIFs in fishes. For example, in grass carp (Ctenopharyngodon idella), two distinct HIF-α isoforms (gcHIF-1alpha and gcHIF-4alpha) were differentially regulated at the transcriptional level in response to hypoxic stress in different organs (Law et al., 2006). Similar studies were performed in Atlantic croaker (Micropogonias undulatus), Chinese sucker (Myxocyprinus asiaticus) and Wuchang bream (Megalobrama amblycephala) and also showed that HIF-α isoforms were regulated in hypoxic conditions at the transcriptional level and potentially useful molecular indicators of environmental hypoxia exposure (Chan et al., 2005, Rahman and Thomas, 2007, Chen et al., 2012). Nevertheless, the contribution of HIF and the genetics basis to high-altitude adaptation in Triplophysa fishes were yet to be investigated. On the other hand, as an iconic symbol species of Qinghai-Tibetan Plateau, only a few genetic markers of Triplophysa fishes are available including partially mitochondrial DNA fragments, few nuclear genes and microsatellites (Tang et al., 2006, Hou et al., 2012, Liu et al., 2012, Wang et al., 2012, Yao et al., 2012, Li et al., 2013, Tang et al., 2013). The limited genetic resources, especially the scarcity of HIF information in Triplophysa fishes have greatly hindered understanding the genetic basis of adaptation to high-altitude environment.

With respect to non-model organisms, transcriptome sequencing is an effective way to obtain large amounts of coding sequences and identify large numbers of molecular makers (Wang et al., 2009, Sánchez et al., 2011, Wang et al., 2011, Fu and He, 2012, Ramayo-Caldas et al., 2012). Therefore, we attempt to reveal the genetic basis about how Triplophysa fishes cope with high-altitude environment using transcriptome data.

It has been proved that de novo assembly of high-throughput sequences from single tissue usually results in relative short sequences in some animal models, including rainbow trout (Oncorhynchus mykiss) transcriptome researches (Salem et al., 2010, Le Cam et al., 2012). In this study, we adopted paired-end Illumina deep sequencing to characterize the T. dalaica transcriptome from five mixed tissues (heart, liver, brain, spleen and kidney). The primary goal of this study was to generate a large amount of T. dalaica transcriptomic reads from multiple tissues, and provide a wealth of sequence data to serve as a reference transcriptome for future studies. The second goal is to detect candidate genes that may be involved in adaptation to high altitude hypoxia. The last one is to gather and apply a large number of genetic markers (microsatellites and SNPs) to the study of the genetic diversity, geographical distribution, expansion and origins of plateau-dwelling loaches.