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A Revisit to a Low-Cost Method for the Isolation Of bioRxiv preprint doi: https://doi.org/10.1101/384651; this version posted August 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 A revisit to a low-cost method for the isolation of microsatellite 2 markers: the case of the endangered Malayan tapir (Tapirus indicus) 3 4 Qi Luan Lim1, Nurul Adilah Ismail1, Ramitha Arumugam1, Wei Lun Ng2, Christina Seok 5 Yien Yong1, Ahmad Ismail1, Jeffrine J. Rovie-Ryan3,4, Norsyamimi Rosli3, Geetha Annavi1* 6 7 1Department of Biology, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, 8 Malaysia 9 2China-ASEAN College of Marine Sciences, Xiamen University Malaysia 1 10 3National Wildlife Forensic Laboratory (NWFL), Ex-Situ Conservation Division, 11 Department of Wildlife and National Parks (DWNP), Kuala Lumpur, Malaysia. 12 4Institute of Tropical Biodiversity and Sustainable Development, Universiti Malaysia 13 Terengganu, Kuala Terengganu, Terengganu, Malaysia. 14 15 * Corresponding Author: Geetha Annavi 16 Email: [email protected] (GA) bioRxiv preprint doi: https://doi.org/10.1101/384651; this version posted August 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 17 Abstract 18 19 There are many approaches to develop microsatellite markers. We revisited an easy and rapid 20 Polymerase Chain Reaction (PCR)-cloning-sequencing method to design microsatellite 21 markers for Tapirus indicus. Using six random amplified microsatellite (RAM) markers, this 22 study had rapidly generated 45 unique genomic sequences containing microsatellites. After 23 screening 15 terminal and seven intermediate microsatellite loci, we shortlisted five and 24 seven which were amplified either by single- or multiplex PCR using the economical three- 2 25 primer PCR method. Genotyping attempts were made with ten Tapirus indicus individuals 26 using three of the terminal microsatellite loci and all seven intermediate loci. However, none 27 of the terminal microsatellite loci were considered useful for population genotyping studies, 28 while the seven intermediate loci showed good amplification but were monomorphic in the 29 ten samples. Despite successful detection of amplified loci, we would like to highlight that, 30 researchers who are interested in this alternative method for isolation of microsatellite loci to 31 be cautious and be aware of the limitations and downfalls reported herein that could render 32 these loci unsuitable for population genotyping. 33 34 Introduction 35 36 Microsatellites, also known as short tandem repeats (STRs) or short sequence repeats (SSRs), 37 are stretches of DNA consisting of tandemly repeated 1-6 nucleotides occurring at high 38 frequency in the nuclear genomes of most organisms, with the length of a microsatellite locus 39 typically in 5-40 repeats [1]. Slippage event during DNA replication is generally considered 40 as the main mechanism for the expansion and contraction of microsatellites [2]. Mutation 41 rates of the slippage events elevate from background rates when the repeat numbers in bioRxiv preprint doi: https://doi.org/10.1101/384651; this version posted August 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 42 microsatellites are large [3]. Because of the hypermutability, microsatellite markers have 43 been shown to be highly polymorphic [4]. They are widely used for molecular genetic studies 44 including fingerprinting, parental or kinship analysis, population genetic structure and 45 biological resources conservation [2,5,6]. 46 47 There are various methods to develop microsatellite markers [7,8]. These methods have been 48 improved over decades and are well described. An extensive review of microsatellite 49 isolation methods has been published [7]. Some of these methods are still sufficient and 50 successfully used in recent microsatellite marker development projects [9,10]. 3 51 52 One of the conventional microsatellite isolation methods employs conversion of a random 53 amplified microsatellite (RAM) marker, which is a multi-locus marker system, to one or more 54 single-locus microsatellite marker. RAM was first described by Zietkiewicz et al. [11] and 55 later further improved by Fisher et al. [12] to anchor the RAM primers consistently at the 5’ 56 ends of two adjacent microsatellites. PCR amplification then produces amplicons that contain 57 microsatellites at both ends of sequence. Extra microsatellite may also be present between 58 the two termini of microsatellites. Next, specific primers can be designed for both the 59 terminal and intermediate microsatellites. This technique has been used in the rapid 60 generation of microsatellite markers in earlier studies [13–15]. 61 62 T. indicus, or commonly known as Malayan Tapir or Asian Tapir, is an odd-toed ungulate 63 (Order Perissodactyla) that occurs solely in Southeast Asia and also the only Old World 64 extant member of Tapiridae family [16]. Its current population distribution includes regions 65 of Myanmar, Thailand, Peninsular Malaysia, and Sumatra with not more than 2500 mature 66 individuals worldwide, and it has been listed as ‘Endangered’ by the International Union for 67 Conservation of Nature (IUCN) Red List [17,18]. The population genetic structure of this 68 species in Peninsular Malaysia remains poorly understood, and this may potentially hamper bioRxiv preprint doi: https://doi.org/10.1101/384651; this version posted August 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 69 conservation efforts (e.g. breeding management) in the nation. Genetic marker such as 70 microsatellite has been used to aid conservation management in other tapir species [19], but 71 microsatellites have not been developed or published for this spectacular species yet. 72 73 We followed the same technique as described in [20] with minor modifications and 74 genotyped a few tapir individuals using microsatellite markers isolated from RAM marker. 75 To our knowledge, our work was the first study to analyse microsatellites isolated with RAM 76 markers on capillary electrophoresis system. We reported the potential of combining RAM 77 or specific anchor markers and other techniques namely multiplex PCR and three-primer- 4 78 method for genotyping. We also discussed the advantages and downfalls when using RAM 79 markers in isolating microsatellites. Even though we did not detect any polymorphism in the 80 genotyped samples through this protocol, we hope the reporting of the process and limitations 81 that we had encountered would help other researchers with similar interest on this method to 82 be cautious in the generation and interpretation of the data obtained. 83 84 Materials and Methods 85 86 Sample collection and DNA extraction 87 88 Whole blood samples were collected for microsatellite isolation from three T. indicus 89 individuals kept in enclosures at the National Zoo of Malaysia or Zoo Negara (3°12'N, 90 101°45'E) and the Sungai Dusun Wildlife Reserve (3°40'N, 101°21'E) by the respective 91 veterinary officers at the study sites and transported on ice to the laboratory at Universiti 92 Putra Malaysia. All the sampling procedures were approved by the Institutional Animal Care 93 and Use Committee, Universiti Putra Malaysia (ethical approval ref.: UPM/IACUC/AUP- bioRxiv preprint doi: https://doi.org/10.1101/384651; this version posted August 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 94 R033/2016). For genotyping, blood samples on FTA® cards (Whatman, UK) and tissue 95 samples of ten putatively unrelated individuals were provided by the Department of Wildlife 96 and National Park (PERHILITAN), Malaysia. Genomic DNA (gDNA) was extracted from 97 these samples using the QIAamp® DNA Mini Kit (Qiagen, Germany) following the 98 manufacturer’s spin protocol. The gDNA was quantified by QuantusTM Fluorometer using 99 ONE dsDNA dye (Promega, USA) and ranged from 0.2 to 100 ng/μL. 100 101 Development of microsatellite markers 5 102 103 Six 5’-anchoring RAM primers selected from published literature (Table 1) were used for 104 amplification. These RAM primers were selected based on the length of 5’-anchors 105 (minimum 7 nucleotides). Single-primer PCR reaction mixtures contained final 106 concentrations of approximately 10 ng of template DNA, 1× MyTaq Red Mix (Bioline, 107 Germany), and 1.0 μM RAM primer, in a total volume of 25 μL. Two types of PCR reaction 108 profiles were used: general and touchdown (PCR condition I in Table 2). Details on the PCR 109 reaction profile and Ta used for each RAM primer are listed in Table 1. PCR amplification 110 products were visualised on 2% agarose gel containing RedSafeTM Nucleic Acid Staining 111 Solution. The remaining PCR products were purified using the Wizard® PCR Clean-Up 112 System (Promega, USA) or the QIAquick® Gel Extraction Kit (Qiagen, Germany), following 113 manufacturer’s instructions. The purified DNA amplicons were ligated and cloned. 114 115 116 117 118 bioRxiv preprint doi: https://doi.org/10.1101/384651; this version posted August 3, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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