Journal of Genetics (2020)99:67 Ó Indian Academy of Sciences https://doi.org/10.1007/s12041-020-01221-x (0123456789().,-volV)(0123456789().,-volV) RESEARCH ARTICLE Sequencing and characterization of complete mitogenome DNA of Rasbora tornieri (Cypriniformes: Cyprinidae: Rasbora) and its evolutionary significance HUNG HUI CHUNG1* , CYNTHIA KERANTI ANAK KAMAR1, LEONARD WHYE KIT LIM1*, JILL SELA ROJA1, YUNSHI LIAO2, TOMMY TSAN-YUK LAM2 and YEE LING CHONG3 1Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Sarawak, Malaysia 2School of Public Health, The University of Hong Kong, Sassoon Road, Pokfulam, Hong Kong 3Department of Science and Environmental Studies, The Education University of Hong Kong, Tai Po Campus, NT, Hong Kong *For correspondence. E-mail: Hung Hui Chung, [email protected]; Leonard Whye Kit Lim, [email protected]. Received 8 October 2019; revised 12 January 2020; accepted 27 April 2020 Abstract. The yellowtail rasbora (Rasbora tornieri) is a miniature ray-finned fish categorized under the genus Rasbora in the family of Cyprinidae. In this study, a complete mitogenome sequence of R. tornieri was sequenced using four primers targeting two halves of the mitogenome with overlapping flanking regions. The size of mitogenome was 16,573 bp, housing 22 transfer RNA genes, 13 protein-coding genes, two ribosomal RNA genes and a putative control region. Identical gene organization was detected between this species and other members of Rasbora genus. The heavy strand encompassed 28 genes while the light strand accommodated the other nine genes. Most protein-coding genes execute ATG as start codon, excluding COI and ND3 genes, which utilized GTG instead. The central conserved sequence blocks (CSB-E, CSB-F and CSB-D), variable sequence blocks (CSB-1, CSB-3 and CSB-2) as well as the terminal associated sequence (TAS) were conserved within the control region. The maximum likelihood phylogenetic family tree revealed the divergence of R. tornieri from the basal region of the Rasbora clade, where its evolutionary relationships with other Rasbora members are poorly resolved as indicated by the low bootstrap values. This work acts as window for further population genetics and molecular evolution studies of Rasbora genus in future. Keywords. mitogenome; gene arrangement; light strand origin; phylogeny; Rasbora tornieri. Introduction (where the yellowtail rasbora resides), Acheilognathinae, Cyprinninae, Cultrinae, Squaliobarbinae, Gobioninae, The yellowtail rasbora (Rasbora tornieri) is categorized Tincinae and Xenocypridinae (Liao et al. 2010). The under the genus Rasbora within the family Cyprinidae. As Rasbora group is well-known as ‘catch-all’ group due to its name suggests, this freshwater ray-finned fish is uniquely the taxonomical complications known to exist since their different from other Rasbora fishes in terms of the striking discovery contributed by their closely resembled mor- yellow colouration with broad black margins at its caudal fin phological characters (Brittan 1954; Kottelat and Vid- (Kottelat et al. 1993). With regard to length, the maximum thayanon 1993; Siebert and Guiry 1996;Kottelat2005; growth of this fish species can reach up to 17 cm, and they Liao et al. 2010;Tanget al. 2010). The most widely feed on exogenous insects, often spawn in ponds and rivers known and accepted Rasbora characterization is of Brittan (Kottelat et al. 1993). It is commonly found in canals and (1954), where the Rasbora sensu lato concept is applied, streams of the lowlands and are distributed across Indonesia, categorizing them into eight species complexes: Indochina as well as Malaysia (Kottelat et al. 1993). pauciperforata, einthovenii, sumatrana-elegans, lateris- Cyprinidae family accommodates 11 subfamilies, triata, argyrotaenia, trisfasciata, caudimaculata and namely Barbinae, Leuciscinae, Labeoninae, Danioninae daniconius (Brittan 1954). 67 Page 2 of 7 Hung Hui Chung et al. The research advancement of the Rasbora genus is in its Table 1. Primers utilized in the amplification of the R. tornieri infancy stages rendering the occurrence of cryptic diversity mitogenome. and taxonomical impediments (Liao et al. 2010; Kusuma et al. 2016). With the recent research limelight shining on R. Primer Tm Amplification name Primer sequence (°C) length (bp) sarawakensis and R. caverii (Wijeyaratne and Pathiratne 2006; Lim et al. 2018), the main stage is now open to SF1 GTGCTTCCTCTACACCAC 55.3 8923 encompass more Rasbora species, especially with the help of SR1 TGATGTTGAGAAGGCTAC the next-generation sequencing. Thus, a total of 18 Rasbora LF1 CCTATCTTACCGAGAAAG 48.6 9990 mitogenome has been sequenced including the one in this LR1 GAGGCCTTCCCATCTAGA study, R. tornieri, as well as R. myersi, R. heteromorpha, R. sarawakensis, R. borapetensis, R. lateristriata, R. hobel- mani, R. aprotaenia, R. argyrotaenia, R. pauciperforata, R. maculata, R. vaterifloris, R. espei, R. steineri, R. The polymerase chain reaction (PCR) was executed uti- cephalotaenia, R. sumatrana, R. daniconius and R. trilineata lizing the T-100 Thermal Cycler (BioRad, USA) in a 20 lL (Miya 2009; Tang et al. 2010; Chang et al. 2012;Hoet al. total reaction volume containing 2.5 mM dNTP, 10 lM 2014; Zhang et al. 2014; Kusuma and Kumazawa 2015; forward and reverse primer each, 2.5 U high-fidelity Taq Kusuma et al. 2017; Chung et al. 2020b). With the addition polymerase, 2.0 lL 10x PCR buffer (with Mg2?), 14.6 lL of the yellowtail rasbora, the discovered Rasbora mito nuclease-free water and 25 ng genomic DNA extract. The genome had just crossed the 20% milestone, taking up thermal cycler parameters are defined as follow: one cycle of 20.7% of the total 87 Rasbora fish species discovered to date predenaturation at 94°C for 2 min, followed by 35 cycles of (Eschmeyer 2015). The mitogenome has the great potential denaturation, annealing and extension at 94°C (30 s), pri- to bridge molecular evolution studies to population genetics, mer-specific temperature (30 s) and 72°C (5 min) respec- thus providing the link between genotypes and phenotypes. tively and a final extension cycle at 72°C for 5 min. The Therefore, this study aimed to unveil the mitogenome con- agarose gel electrophoresis was run following the PCR tents of R. tornieri as well as its evolutionary significance reaction to size separate the PCR products before visualizing across the other 17 Rasbora genus counterparts. them under UV light. High purity and quality amplicons were subjected to Illumina HiSeq 400 system pair-ended sequencing reactions with short reads. Quality checks were Materials and methods done on all the sequencing reads prior to adaptor trimming employing cutadapt (Martin 2011). The assembly was con- Sampling and mitogenome extraction from total tissue ducted using de novo assembler SPAdes v. 3.12.0 (Banke- vich et al. 2012). The yellowtail R. tornieri fishes were captured from Kano- wit River, Sarawak, Malaysia (2°01036.700 N, 110°24041.300 E) with the permit granted by the Sarawak Forestry Mitogenome characterization and gene analysis Department (permit number: NCCD.94047(Jld13)-178). The fishes were Tricane humanely euthanized using the protocols The construction and visualization of the mitogenome map established by the Animal Ethics Committee, Universiti of R. tornieri was done using MitoFish (Iwasaki et al. 2013) Malaysia Sarawak (reference number: UNIMAS/TNC(PI)- with gene annotations. All protein-coding genes were clo- 04.01/06-09(17)). The mitogenome extraction was con- sely inspected via translation of nucleotide sequences into ducted using the muscle tissues from the total tissue of R. amino acid sequences to ensure full functionalities whereby tornieri via CTAB method as followed by Chung (2018). the truncated and premature stop codons were edited using The remaining parts of the fish body was preserved in 95% MEGA 7.0 (Kumar et al. 2016). The tRNA-scan SE v. 2.0 ethanol for long-term storage. (Lowe and Chan 2016) was exercised to detect anti-codons of tRNA genes via default search mode. The L-strand origin (OL) was predicted and visualized in terms of its secondary PCR and sequencing structure via RNAstructure 6.0 (Reuter and Mathews 2010) with considerations of sequence homology. NCBI Bankit The primers targeting the desired mitogenome regions were was utilized to deposit the entire mitogenome sequences into designed based on conserved motifs across four closely the GenBank database. related Rasbora species (R. aprotaenia, R. agryrotaenia, R. trilineata and R. sumatrana) (table 1). The two primer pairs must cover the two halves of the mitogenome with Phylogeny 2000-bp flanking overlapping regions to ensure good sequencing reads. The removal of these overlapping regions A total of 18 Rasbora mitogenomes (inclusive of R. tornieri) will reveal the complete mitogenome sequences. were selected for the building of phylogenetic family tree in Complete mitogenome of R. tornieri Page 3 of 7 67 Table 2. Features of the complete R. tornieri mitogenome. Position Codon Length Anti- Intergenic Gene Start End(bp) codon Start Stopa nucleotideb Strand tRNAPhe 1 69 69 GAA 0 H 12S rRNA 70 1025 956 0 H tRNAVal 1026 1096 71 TAC 0 H 16S rRNA 1097 2784 1688 0 H tRNALeu (UAA) 2785 2859 75 TAA ? 1H ND1 2861 3835 975 ATG TAA ? 4H tRNAIle 3840 3911 72 GAT - 2H tRNAGln 3980 3910 71 TTG 1 L tRNAMet 3982 4050 69 CAT 0 H ND2 4051 5095 1045 ATG T– 0 H tRNATrp 5096 5167 72 TCA ? 2H tRNAAla 5237 5170 68 TGC ? 1L tRNAAsn 5311 5239 73 GTT ? 37 L tRNACys 5414 5349 66 GCA ? 1L tRNATyr 5485 5416 70 GTA ? 1L COI 5487 7037 1551