Combining next‐generation sequencing with microarray for transcriptome analysis in rainbow trout gonads

Abstract

Microarray technology is a powerful tool for studying genome-wide gene expression. As the genome of many fish has not yet been determined, however, cDNA microarrays can only be designed from limited expressed sequence tag data. In this study, we designed a microarray based on the sequencing data (337,466 reads) obtained by next-generation sequencing of RNA extracted from rainbow trout (Oncorhynchus mykiss) embryonic genital ridge, testis, and ovary. These data (307,264 reads) were assembled into 28,668 contigs; 3,298 reads could not be assembled and 26,904 reads were unique sequences that did not cluster with other reads. Based on this information, 55,928 microarray probes were designed for a microarray, which was validated by hybridization experiments with RNA extracted from type A spermatogonia (A-SG) and testicular somatic cells. Expression of known spermatogonial markers was confirmed to be higher in A-SG than in testicular somatic cells whereas supporting-cell markers were expressed at higher levels in testicular somatic cells. This microarray analysis revealed that 8,068 transcripts showed at least fourfold higher signal in A-SG than testicular somatic cells. Fourteen of 17 randomly selected transcripts were expressed at significantly higher-levels in A-SG than somatic cells, by quantitative RT-PCR. In addition, three transcripts analyzed with in situ hybridization showed A-SG-specific signals in immature trout testis, with one of them exhibiting a heterogeneous expression pattern in A-SG. The rainbow trout gonad microarray developed in this study therefore appears to be a useful tool to understand gametogenesis in rainbow trout.

Expression patterns of gdnf and gfrα1 in rainbow trout testis

“…This expression pattern resembles that of mice. However, we recently discovered that tubulin alpha chain homolog is expressed in a subpopulation of ASG, but not in the entire population, in rainbow trout (Hayashi et al, 2012). Furthermore, in rainbow trout germ cell transplantation, only 4.6×10 -2 % of transplanted ASG are successfully incorporated into recipient gonads and act as SSCs (Okutsu et al, 2006a).…”

“…At this age, germ cells were mostly ASG. Cell sorting was performed as previously described (Hayashi et al, 2012). For DsRed detection, a 488 nm sapphire laser and 575 nm band-pass filter were used.…”

“…After the cell sorting, GFP+ cells, DsRed+ cells, GFP-and DsRed-cells, and unsorted cells were subjected to conventional RT-PCR. Extraction of total RNA was performed using 6×10 4 cells of each sample as previously described (Hayashi et al, 2012).…”

“…First, there exist two transgenic rainbow trout strains: pvasa-Gfp and pinhibin-DsRed. In pvasa-Gfp rainbow trout, spermatogonia are labeled by green fluorescence protein (GFP) under the control of the vasa-gene regulatory region (Yoshizaki et al, 2000b;Yano et al, 2008), which enables enrichment of ASG, including SSCs, according to the intensity of green fluorescence (Okutsu et al, 2006a;Hayashi et al, 2012).…”

Germ Cell-Specific Excision of loxP-Flanked Transgenes in Rainbow Trout Oncorhynchus mykiss1

“…The development of various experimental tools, including germ cell transplantation [21][22][23][24], in vitro germ cell culture [25][26][27], germ cell purification or enrichment using a centrifugal elutriation system [28] or a flow-cytometer [29][30][31], long-term storage of germ cells by cryopreservation [32,33], and comprehensive transcriptomic analysis of germ cells [34], have resulted in significant advances in germ cell biology of salmonid species [35,36]. Nevertheless, the full range of genetic tools available for functional studies of some model species is not available for salmonid species.…”