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Characterization and Expression of Xiphophorus Maculatus Microsatellite Msb069 Full Sequence in Subgenus Poecilia

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Characterization and Expression of Xiphophorus Maculatus Microsatellite Msb069 Full Sequence in Subgenus Poecilia Journal of Genetics, Vol. 97, No. 4, September 2018, pp. 817–824 © Indian Academy of Sciences https://doi.org/10.1007/s12041-018-0968-y RESEARCH ARTICLE Characterization and expression of Xiphophorus maculatus microsatellite Msb069 full sequence in subgenus Poecilia KEONG BUN POH∗ , ZARIF MUAZ ROSLAN, ROSMILAH MISNAN and SOM CIT SINANG Faculty of Science and Mathematics, Department of Biology, Universiti Pendidikan Sultan Idris, 35900, Tanjong Malim, Perak, Malaysia *For correspondence. E-mail: [email protected]. Received 21 April 2017; revised 26 October 2017; accepted 21 November 2017; published online 3 August 2018 Abstract. Msb069 primer pairs encompassed region is believed to be associated with a quantitative trait loci (QTL) of dorsal fin length in subgenus Poecilia. However, detailed investigation on Msb069 which originated from Xiphophorus on subgenus Poecilia remains unexplored. In this study, full sequence of Msb069 was characterized by sequencing bioinformatics analysis and gene expression. The sequence analysis of Msb069 primer pairs encompassed region on three species of Poecilia revealed higher number of microsatellite tandem repeats in Poecilia latipinna (ATG16) compared to P. sphenops (ATG13–14). There is no notable pattern of ATG tandem repeats discovered in the hybrids. The full sequence of Msb069 is 734 bp in length and showed a 233 bp conserved region between Xiphophorus and Poecilia. BLAST search performed on this sequence revealed no significant similarities. Nonquantitative RT-PCR exhibited the presence of Msb069 transcripts in three different tissues in subgenus Poecilia. Meanwhile, quantitative RT- PCR expression on two different tissues showed relatively higher expression of Msb069 transcript in P. latipinna dorsal fin tissues in both male and female fishes, suggesting a repressive function of this transcript with respect to dorsal fin length. However the exact gene expression event of Msb069 is still unknown and requires further investigation. Keywords. microsatellite; Msb069 full sequence; quantitative trait loci, dorsal fin length; Poecilia; Xiphophorus maculatus. Introduction In the subgenus Poecilia, dorsal fin is used for court- ing during mating process. This is especially for male Xiphophorus maculatus microsatellite Msb069 full P.latipinna, where the enlarged dorsal fin is fanned against sequence is a microsatellite containing sequence which potential females to induce copulation. Dorsal fin length has ATG tandem repeats and was isolated from DNA in P. latipinna is sexually dimorphic. Enlarged dorsal fin is of X. maculatus (platy fish). The Msb069 full sequence only present in the males but this feature is absent in the was 734-bp long and the Msb069 primer pairs encom- females (Ptacek 2002; Ptacek and Breden 1998). Besides passed region was mapped in Xiphophorus linkage map the importance of dorsal fin in courting, this feature has positioned at linkage group 14 (Walter et al. 2004). This diverse roles and functions. For instance, dorsal fin plays sequence was reported to be associated with a quantita- an important role in stabilizing the fish during forward tive trait loci (QTL). Briefly, the QTL study was conducted swimming and also withstanding lateral water forces that in subgenus Poecilia utilizing X. maculatus microsatellite might cause the fish to turn. In some fish species, dorsal markers designed by Walter et al. (2004) who had suc- fin has spines which serve as a defence against predators cessfully identified the Msb069 primer pairs encompassed (Kalish-Achrai et al. 2017). region to be linked with dorsal fin length, which showed Despite these, developmental process and genetic events high association (LOD > 3.0) (Keong et al. 2014). behind the varying length of dorsal fin remained vague and only a few researches were conducted. For example, Keong Bun Poh and Zarif Muaz Roslan contributed equally to this a developmental study on frog embryo had shown that work. dorsal fin emerged from migration of cells from neural 817 818 Keong Bun Poh et al. crest (NC) cells and somite located in the dorsomedial PCR amplification conditions and reverse region into the core of dorsal fin. In addition, a calcium transcription-polymerase chain reaction (RT-PCR) regulated gene, Wnt11-R is also expressed in migratory cells during dorsal fin development (Garriock and Krieg PCR and nonquantitative two-step RT-PCR reactions 2007). Another related yet different study focussing on were carried out by using 2× PCR master mix (1st Base, genetic analysis of mutated gene effect was also conducted Science Park 2, Singapore) in a 20 μL reaction volume on zebra fish. The study discovered that adult zebra fish comprising of 1× PCR master mix, 0.4 μM primer and which inherited stein und bein (sub) was found to be lacking 0.1 μg of DNA or cDNA. In two-step RT-PCR, the unpaired dorsal fin in homozygous individuals. Moreover, RNA was reverse transcribed to cDNA by using Quanti- stein und bein (sub) mutant fish also exhibited severely Nova Reverse Transcription Kit (Qiagen) prior to PCR. reduced pelvic fin (van Eeden et al. 1996). To the best The PCR amplification protocol was as follows: 30 cycles ◦ of our knowledge, genetic investigation on dorsal fin is of 1 min predenaturing step at 95 C, followed by 1 min of ◦ ◦ quite limited. Two most recent research related to dor- denaturing at 95 C, 1 min of annealing at 54 C, 1 min of ◦ ◦ sal fin in Actinopterygii or ray-finned fish were found, extension at 72 C and a final extension at 72 C for 5 min. but mainly focussed on development pattern (Richter and Morit 2017) and structure, composition, mechanics and growth of spines in dorsal fin (Kalish-Achrai et al. 2017). Electrophoresis and visualization Despite the important contribution of the dorsal fin in social activities (e.g. courting) and swimming performance, Amplified PCR products were electrophoresed in 2% × . μ / detailed data related to the QTL effect of Msb069 on dor- agarose gel (1 TBE buffer, 0 5 g mL gel red). Stained sal fin length is not reported so far. Therefore, this study gel was visualized under UV light using Imager Sys- was conducted (i) to characterize Xiphophorus Msb069 full tem (SmartView Pro Imager System,Taoyuan, Taiwan). sequence and (ii) to investigate the gene expression events GeneRuler 100 bp (Fermentas, Waltham, USA) and 1 kbp of this QTL in subgenus Poecilia. (Fermentas) were used as molecular size standard. Analysis of different loci in X. maculatus microsatellite Msb069 Materials and methods full sequence in Poecilia Experimental animals To test whether other regions within Msb069 DNA full sequence were conserved between Xiphophorus and Poe- The Poecilia samples which comprised of P.latipinna (sail- cilia, three primer pairs flanking the same tandemly fin molly), P. sphenops (shortfin molly) and hybrids (dal- repeated region (ATG) but with different product size were mation molly) were sampled from local breeding aquaria. designed (table 1;figure1). The three primer pairs namely; All fish samples were sexually mature adults with average Msb069_687bp, Msb069_450bp and Msb069_233bp were length of 2.50±1.23 cm SL. Wild samples of Poecilia are tested for amplifications on representative DNA samples. not available locally as this fish species is not native to The representative samples include two P. latipinna (one this country. Most of the existing fish stocks are artificially male and one female), two P. sphenops and two hybrids. breed in captivity. Analysis of Msb069 sequences among species of Poecilia Genomic DNA extraction A Msb069 primer pairs designed by Walter et al. (2004) and was downloaded from Xiphophorus Microsatellite DNA was extracted from freshly sampled fin clips Loci Database, (http://www.xiphophorus.org/microsats/ ∼ ( 10 mg) using an extraction kit (Qiagen, Hilden, Ger- microsat.htm) or alternatively, full sequence information many). Extraction procedure was conducted in accordance could be retrieved from EMBL/GeneBank Data Libraries with manufacturer’s instructions. under accession no. AY258838.1. The Msb069 primer pairs is flanking a tandemly repeat region of ATG (table 1; figure 1). This primer pairs were tested for PCR amplifi- Tissue specific RNA extraction cations on 30 DNA samples isolated from three different species of Poecilia. The fish samples include 10 P.latipinna From each fish, RNA was extracted from ∼10 mg of tis- (five male and five female), 10 P. sphenops and 10 hybrids. sues of dorsal fin, muscles and gonad (male and female). Positive amplified PCR products were send for direct These tissues were immediately frozen in liquid nitrogen sequencing to 1st Base Laboratory Sdn Bhd (Seri Kem- prior to sample disruption. The RNA was isolated by using bangan, Malaysia). Sequence alignment was performed by RNeasy Mini kit (Qiagen). using online software, ClustalW. Characterization and expression of X. maculatus microsatellite Msb069 819 Identifications of open reading frame (ORF) and nonquantitative expression of Msb069 in cDNA of dorsal fin, muscle and gonad A search for putative ORF was conducted on X. macu- latus microsatellite Msb069 DNA full sequence by using accession no. Full sequence ORF Finder online graphical analysis tool. This search was conducted to identify any potential transcript encod- ing for protein residing within this locus. All statistical data wasexpressedasmeans± STDEV. Two-step nonquantitative RT-PCR was used to deter- mine the expression of Msb069 on tissue specific cDNA isolated from dorsal fin, muscle and gonad. The fish sam- ples include four P. latipinna (two male and two female), Marker size (bp) four P. sphenops and four hybrids. Prior to tissue extrac- tion, fishes were euthanized in crushed ice for 10 min. Dorsal fin, muscle and gonad were dissected out from the same fish and immediately crushed separately with liquid nitrogen before proceed with RNA isolation. A reference C) ◦ gene (beta actin; atcb1) were PCR on RNA samples to test ( m for any possible genomic contamination prior to cDNA T conversion (table 1). First strand cDNA conversion was Melting temp. carried out using Qiagen’s Quantinova Rev.
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