RSC Advances View Article Online PAPER View Journal | View Issue De novo assembly of Schizothorax waltoni transcriptome to identify immune-related genes Cite this: RSC Adv.,2018,8, 13945 and microsatellite markers† Hua Ye,ab Zhengshi Zhang,ab Chaowei Zhou,ab Chengke Zhu,ab Yuejing Yang,ab Mengbin Xiang,ab Xinghua Zhou,ab Jian Zhou*c and Hui Luo *ab Schizothorax waltoni (S. waltoni) is one kind of the subfamily Schizothoracinae and an indigenous economic tetraploid fish to Tibet in China. It is rated as a vulnerable species in the Red List of China's Vertebrates, owing to overexploitation and biological invasion. S. waltoni plays an important role in ecology and local fishery economy, but little information is known about genetic diversity, local adaptation, immune system and so on. Functional gene identification and molecular marker development are the first and essential step for the following biological function and genetics studies. For this purpose, the transcriptome from pooled tissues of three adult S. waltoni was sequenced and Creative Commons Attribution-NonCommercial 3.0 Unported Licence. analyzed. Using paired-end reads from the Illumina Hiseq4000 platform, 83 103 transcripts with an N50 length of 2337 bp were assembled, which could be further clustered into 66 975 unigenes with an N50 length of 2087 bp. The majority of the unigenes (58 934, 87.99%) were successfully annotated by 7 public databases, and 15 KEGG pathways of immune-related genes were identified for the following functional research. Furthermore, 19 497 putative simple sequence repeats (SSRs) of 1–6 bp unit length were detected from 14 690 unigenes (21.93%) with an average distribution density of 1 : 3.28 kb. We identified 3590 unigenes (5.36%) containing more than one SSR, providing abundant potential Received 21st January 2018 polymorphic markers in functional genes. This is the first reported high-throughput transcriptome Accepted 9th April 2018 analysis of S. waltoni, and it would provide valuable genetic resources for the functional genes involved This article is licensed under a DOI: 10.1039/c8ra00619a in multiple biological processes, including the immune system, genetic conservation, and molecular rsc.li/rsc-advances marker-assisted breeding of S. waltoni. Open Access Article. Published on 16 April 2018. Downloaded 9/28/2021 1:03:04 AM. Introduction shes on the QTP.4 There are 15 genera and over 100 species belonging to schizothoracine in the world, and about 76 species The Qinghai-Tibetan Plateau (QTP), 2.5 million km2 with an and subspecies belong to 11 genera on the QTP and the adjacent average altitude reaching 4000 m, is the highest and the largest, areas in China.5,6 The schizothoracine shes are characterized and also one of the youngest plateaus in the world. Known as by slow growth rates, late maturity, low fecundity, long-lived, the World's Roof, QTP has become a global hotspot for research and restricted distributions.5 Those characteristics of the on biodiversity, phylogeny, adaptation, and evolution.1–3 schizothoracine shes make them more affected by habitat – Accompanied by the upli of QTP, the environment had modication, intense exploitation, and biological invasions.7 9 undergone tremendous changes. Extreme environment, such as Therefore, the schizothoracine shes can be used as the hypoxia, chilliness, high radiation, has almost certainly affected paragon models to study high altitude adaptation, historical the component fauna of the plateau and the adjacent areas.3 and contemporary environmental changes, and evolutionary The schizothoracine shes are well adapted to the harsh biology.10,11 conditions, and became the predominant group of endemic Schizothorax waltoni, an indigenous economic tetraploid sh to Tibet in China, is one kind of the subfamily Schizothoracinae and only distributed in the middle reaches of the Yarlung 12 aCollege of Animal Science, Southwest University, Chongqing 402460, China. E-mail: Tsangpo River. It is rated as a vulnerable species in the Red List [email protected] of China's Vertebrates.13 Owing to overexploitation and biological bKey Laboratory of Freshwater Fish Reproduction and Development (Ministry of invasion, the population and catched individual size of S. wal- Education), Key Laboratory of Aquatic Science of Chongqing, 400175, China toni have been declining rapidly in recent years.14 Although S. cFisheries Research Institute, Sichuan Academy of Agricultural Sciences, Chengdu, waltoni plays an important role in ecology and local shery 611731, China. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: economy, little information is known about genetic diversity, 10.1039/c8ra00619a local adaptation, immune system and so on. Existing related This journal is © The Royal Society of Chemistry 2018 RSC Adv.,2018,8, 13945–13953 | 13945 View Article Online RSC Advances Paper studies of S. waltoni were mainly focused on population unigenes with an average length of 956 bp (ranging from 224 to recording, morphology, phylogeny, mitogenome, and several 29 395 bp) (Table 1). The N50 lengths of transcripts and unig- fundamental aspects of biology.5,9,12,15–17 In order to protect enes were 2337 and 2087 bp, respectively, which is similar to germplasm resources of S. waltoni, the research on articial that of Gymnodiptychus dybowskii (N50 of 2407 bp) and Schizo- culture and breeding has been carried out in Tibet. However, thorax pseudaksaiensis (N50 of 2283 bp),24 and Gymnodiptychus the shery industry is threatened by the multiple infectious pachycheilus (N50 of 2322 bp),10 and longer than that of Gym- pathogens, since S. waltoni is particularly vulnerable to patho- nocypris przewalskii (N50 of 1495 bp),25 G. przewalskii (N50 of genic microorganism and environmental pollutants under 1836 bp),26 shorter than that of Schizothorax prenanti (N50 of articial culture conditions. Prerequisite condition to preven- 2539 bp).27 Irrespective of difference between organisms, several tion and treatment of diseases is understanding the functions factors can affect the N50 length of transcripts and unigenes, of genes and pathways involved in the immune system. Mean- including sequencing technique, read number, assembly so- while, microsatellite markers, the versatile and popular genetic ware, and parameters. Among these unigenes, 42 890 (51.6%) marker with applications in conservation biology, population unigenes were no more than 500 bp in length, 28 037 (33.7%) genetics, and evolutionary biology, can be used for the studies unigenes exceeded 1000 bp and 14 968 (18.0%) unigenes were of marker-assisted selection in the improvement of economic longer than 2000 bp (Fig. 1). These proportions were also traits.18,19 The large-scale development of microsatellite markers similar to that of S. prenanti.27 The detailed frequency distri- is necessary to carry out the research of marker-assisted selec- bution of the unigenes length is shown in Fig. 1. tion, conservation biology, and population genetics. However, functional gene identication and marker development of S. waltoni still remain poorly explored.14,20 Functional annotation With the advent and development of next generation Functional annotation of S. waltoni transcriptome was carried sequencing (NGS) technology, it is a cost- and time-efficiency out by searching against several public nucleotide and protein Creative Commons Attribution-NonCommercial 3.0 Unported Licence. method of transcriptome sequences to identify genes and databases. As a result, 57 301 (85.6%), 35 510 (53.0%), and develop genetic markers.21–23 In this study, we used the Illumina 28 877 (43.1%) unigenes showed signicant similarities (E- À Hiseq 4000 platform to analyze the pooled tissues tran- value < 10 5) to the NCBI nucleotide (NT), protein (NR), and scriptome of S. waltoni. The major objective of this study was to Swiss-prot databases, respectively (Table 2). A total of 58 934 obtain a comprehensive S. waltoni transcriptome information (88.0%) unigenes showed homologous matches in at least one from the multiple tissues, and provide an abundant resource for database (Table 2). The annotation ratio of assembled unigenes the following functional studies. Genes involved in immune higher than previously reported in other sh, such as Hypo- system were annotated and emphasized since the general rthodus septemfasciatus (41.5%),28 G. przewalskii (77.8%),25 Cyp- interest of immune response of sh species, including S. wal- rinus carpio (81.1%)29 and Ctenopharyngodon idella (82.8%),30 This article is licensed under a toni. A large number of microsatellite markers were also slightly lower than that of S. prenanti (94.4%).27 The high detected and analyzed for the conservation genetics studies and percentage of annotation ratio might be due to many long marker-assisted selection breeding in S. waltoni. sequences obtained from transcriptome data and higher Open Access Article. Published on 16 April 2018. Downloaded 9/28/2021 1:03:04 AM. average length of unigenes, as well as the abundant sequence Results and discussion databases.31,32 The E-value distribution of unigenes which could be correctly annotated in the NR database showed that 28.1% of de novo Transcriptome sequencing and assembly the unigenes had perfect matches, 30.3% of the unigenes Aer sequencing using Illumina Hiseq 4000 platform, three showed signicant homology to the previously stored À cDNA libraries of S. waltoni generated 155 932 320 raw reads sequences (less than 1 Â 10 45), and 41.6% of the unigenes À À with a read length of 150 bp. Aer read quality evaluation, low showed homology ranging from 1 Â 10 45 to 1 Â 10 5 (Fig. 2A). quality trimming and length ltering, 147 852 684 clean reads The similarity distribution of the top Blast hits for each were le. As a result for the trinity assembly, we obtained 83 103 sequence ranged from 17–100%. Among the similarity distri- transcripts ranging from 224 to 29 395 bp with average length of bution, 16.5% of the unigenes had the similarity of 60–80%, 1139 bp (Table 1).