A high quality assembly of the Nile Tilapia (Oreochromis niloticus) genome reveals the structure of two sex determination regions

Abstract

Background: Tilapias are the second most farmed fishes in the world and a sustainable source of food. Like many other fish, tilapias are sexually dimorphic and sex is a commercially important trait in these fish. In this study, we developed a significantly improved assembly of the tilapia genome using the latest genome sequencing methods and show how it improves the characterization of two sex determination regions in two tilapia species.

Results: A homozygous clonal XX female Nile tilapia (Oreochromis niloticus) was sequenced to 44X coverage using Pacific Biosciences (PacBio) SMRT sequencing. Dozens of candidate de novo assemblies were generated and an optimal assembly (contig NG50 of 3.3Mbp) was selected using principal component analysis of likelihood scores calculated from several paired-end sequencing libraries. Comparison of the new assembly to the previous O. niloticus genome assembly reveals that recently duplicated portions of the genome are now well represented. The overall number of genes in the new assembly increased by 27.3%, including a 67% increase in pseudogenes. The new tilapia genome assembly correctly represents two recent vasa gene duplication events that have been verified with BAC sequencing. At total of 146Mbp of additional transposable element sequence are now assembled, a large proportion of which are recent insertions. Large centromeric satellite repeats are assembled and annotated in cichlid fish for the first time. Finally, the new assembly identifies the long-range structure of both a ~9Mbp XY sex determination region on LG1 in O. niloticus, and a ~50Mbp WZ sex determination region on LG3 in the related species O. aureus.

Conclusions: This study highlights the use of long read sequencing to correctly assemble recent duplications and to characterize repeat-filled regions of the genome. The study serves as an example of the need for high quality genome assemblies and provides a framework for identifying sex determining genes in tilapia and related fish species.

Keywords: Aquaculture; Gene duplication; Genome assembly; Pacific Biosciences SMRT sequencing; Sex chromosome; Sex determination; Tilapia; Transposable elements.

Fig. 1

Assembly overview. Flowchart detailing the processing of the raw 44X PacBio sequencing reads, producing candidate assemblies, polishing, breaking, and final assembly anchoring. Metrics are provided at each step

Fig. 2

Repeat Landscape comparison. The percentage of both the Orenil1.1 and O_niloticus_UMD1 and assemblies that each TE family is represented at in particular substitution levels analogous to the age of TEs (Kimura substitution level – CpG adjusted)

Fig. 3

Vasa gene duplication. a The top row shows the vasa transcript (NCBI accession number AB032467.1) aligned to Orenil1.1 assembly scaffolds with gaps shown in solid red. b The middle row shows this same vasa transcript aligned to the separate BAC assemblies (NCBI accession numbers AB649031-AB649033). c The bottom row shows the vasa transcript aligned to O_niloticus_UMD1 LGs. For each row there are three alignments corresponding to the three copies of each vasa transcript

Fig. 4

Whole genome O. niloticus sex comparison. a F_ST comparison of XX female pool versus XY male pool on Orenil1.1. b Sex-patterned variants across Orenil1.1. c F_ST comparison of XX female pool versus XY male pool on O_niloticus_UMD1. d Sex-patterned variants across O_niloticus_UMD1

Fig. 5

LG1 O. niloticus sex comparison. a F_ST comparison of XX female pool versus XY male pool on LG1 of Orenil1.1. b Sex-patterned variants on LG1 of Orenil1.1. c F_ST comparison of XX female pool versus XY male pool on LG1 of O_niloticus_UMD1. Anchored contig boundaries are depicted with grey bars. d Sex-patterned variants on LG1 of O_niloticus_UMD1

Fig. 6

Whole genome O. aureus sex comparison. a F_ST comparison of ZW female pool versus ZZ male pool on Orenil1.1. b Sex-patterned variants across Orenil1.1. c F_ST comparison of ZW female pool versus ZZ male pool on O_niloticus_UMD1. d Sex-patterned variants across O_niloticus_UMD1

Fig. 7

LG3 O. aureus sex comparison. a F_ST comparison of ZW female pool versus ZZ male pool on LG3 of Orenil1.1. b Sex-patterned variants on LG3 of Orenil1.1. c F_ST comparison of ZW female pool versus ZZ male pool on LG3 of O_niloticus_UMD1. Anchored contig boundaries are depicted with grey bars. d Sex-patterned variants on LG3 of O_niloticus_UMD1