An Integrated Transcriptome-Wide Analysis of Cave and Surface Dwelling Astyanax mexicanus

Abstract

Numerous organisms around the globe have successfully adapted to subterranean environments. A powerful system in which to study cave adaptation is the freshwater characin fish, Astyanax mexicanus. Prior studies in this system have established a genetic basis for the evolution of numerous regressive traits, most notably vision and pigmentation reduction. However, identification of the precise genetic alterations that underlie these morphological changes has been delayed by limited genetic and genomic resources. To address this, we performed a transcriptome analysis of cave and surface dwelling Astyanax morphs using Roche/454 pyrosequencing technology. Through this approach, we obtained 576,197 Pachón cavefish-specific reads and 438,978 surface fish-specific reads. Using this dataset, we assembled transcriptomes of cave and surface fish separately, as well as an integrated transcriptome that combined 1,499,568 reads from both morphotypes. The integrated assembly was the most successful approach, yielding 22,596 high quality contiguous sequences comprising a total transcriptome length of 21,363,556 bp. Sequence identities were obtained through exhaustive blast searches, revealing an adult transcriptome represented by highly diverse Gene Ontology (GO) terms. Our dataset facilitated rapid identification of sequence polymorphisms between morphotypes. These data, along with positional information collected from the Danio rerio genome, revealed several syntenic regions between Astyanax and Danio. We demonstrated the utility of this positional information through a QTL analysis of albinism in a surface x Pachón cave F2 pedigree, using 65 polymorphic markers identified from our integrated assembly. We also adapted our dataset for an RNA-seq study, revealing many genes responsible for visual system maintenance in surface fish, whose expression was not detected in adult Pachón cavefish. Conversely, several metabolism-related genes expressed in cavefish were not detected in surface fish. This resource will enable powerful genetic and genomic analyses in the future that will better clarify the heritable genetic changes governing adaptation to the cave environment.

RNA-Seq Technology and Its Application in Fish Transcriptomics

“…In this study, normal whitefish was found to overexpress the genes related to protein synthesis, while dwarf fish overexpress the genes associated with immunity, DNA replication and repair, and energy metabolism, and the correlation between RNA-seq results and a previous microarray data was positive. Transcriptomic analyses using RNA-seq were similarly employed to study the genetic bases for the phenotypic differentiation between siscowet and lean lake trout (Salvelinus namaycush) (Goetz et al, 2010), for the divergent pigment patterns in marine and freshwater sticklebacks (Gasterosteus aculeatus) (Greenwood et al, 2012), for the adaptation of Mexican tetra (Astyanax mexicanus) to the cave environment (Gross et al, 2013), and for the homologous relationship between zebrafish swimbladder and mammalian lung (Zheng et al, 2011). In these transcriptomic studies, many differentially expressed genes were detected, laying the ground for future functional genomic 106 QIAN ET AL.…”

The developmental transcriptome of contrasting Arctic charr (Salvelinus alpinus) morphs

“…For example microarrays were used to compare gene expression in anadromous and resident populations of brown trout ( Salmo trutta ), revealing that life history was a better predictor of gene expression in the liver than relatedness 9 . The newer technique, RNA-sequencing (RNA-seq) has been applied to species such as the Mexican cavefish ( Astyanax mexicanus ), cod ( Gadus morhua ) brook charr ( Salvelinus fontinalis ) and Atlantic salmon ( Salmo salar ) 10– 15 , addressing questions concerning evolution, molecular genetics, development and aquaculture. RNA-seq was used to study salinity tolerance in Arctic charr, linking expression and quantitative trait loci 16 .…”

Alterations in Mc1r gene expression are associated with regressive pigmentation in Astyanax cavefish

“…RNA-sequencing was performed in triplicate for 10 hpf – 72 hpf embryonic stages and in duplicate for juveniles using Illumina HiSeq 2500 Technology (TruSeq v.2 kit) at the Cincinnati Children’s Hospital Core Sequencing Facility (Cincinnati, OH). Sequencing reads (from fastq-formatted files) were aligned to a previously published comprehensive transcriptome template [Gross et al 2013] inclusive of the Mc1r sequence. Gene expression levels were calculated using the QSeq module of the ArrayStar software program (DNAStar v.11.0; Madison, WI) using an RPKM normalization strategy [Mortazavi et al 2008; Gross et al 2013].…”

“…Sequencing reads (from fastq-formatted files) were aligned to a previously published comprehensive transcriptome template [Gross et al 2013] inclusive of the Mc1r sequence. Gene expression levels were calculated using the QSeq module of the ArrayStar software program (DNAStar v.11.0; Madison, WI) using an RPKM normalization strategy [Mortazavi et al 2008; Gross et al 2013]. Our transcriptome template was generated using SeqMan NGen software (DNAStar v.11.0; Madison, WI) optimized for use with our 50-bp, unpaired read sets.…”