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Isolating Microsatellite Markers in the Marine Sponge Cinachyrella Alloclada for Use in Community and Population Genetics Studies

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Isolating Microsatellite Markers in the Marine Sponge Cinachyrella Alloclada for Use in Community and Population Genetics Studies Isolating microsatellite markers in the marine sponge Cinachyrella alloclada for use in community and population genetics studies A thesis submitted to the University of Manchester for the degree of MPhil Environmental Biology in the Faculty of Life Sciences Sarah Miriam Griffiths 2013 1 Contents List of figures and tables.......................................................................................................... 3 Abstract................................................................................................................................... 4 Declaration................................................................................................................................ 5 Copyright statement.................................................................................................................. 5 Acknowledgements................................................................................................................... 6 Abbreviations............................................................................................................................ 7 Introduction............................................................................................................................. 8 Marine sponges.............................................................................................................. 9 Genetic markers............................................................................................................. 10 Allozymes................................................................................................... 10 mtDNA........................................................................................................ 10 AFLP........................................................................................................... 11 Nuclear DNA.............................................................................................. 11 SNP............................................................................................................ 12 Microsatellites............................................................................................. 12 Microsatellite development............................................................................................. 12 Optimizing Seq-to-SSR approaches.......................................................... 14 Aims and objectives....................................................................................................... 16 Methods and Results............................................................................................................. 17 Sample collection and DNA extraction........................................................................... 17 Library prep and sequencing.......................................................................................... 17 Quality filtering................................................................................................................ 17 Microsatellite identification and primer design................................................................ 19 PCR amplification of selected loci.................................................................................. 20 Discussion.............................................................................................................................. 23 References.............................................................................................................................. 25 Word count – 11,118 2 List of figures and tables Figure 1: Comparison of per base sequence quality scores of the left (L) and right (R) reads in the raw reads and filtered reads ........................................................................................................... 18 Figure 2: Sequence length distributions over all sequences in the left hand (L) and right hand (R) reads after quality trimming by TRIMMOMATIC.............................................................................. 19 Figure 3: Agarose gel electrophoresis of PCR products for eight Florida Bay C. alloclada individuals (FB 1 – 8). Size standard = Hyperladder IV (Bioline). Typical results for a) a ‘successful’ locus (Call 13) b) ‘unsuccessful’ loci due to i) Amplification failure (Call 11) ii) Non-specific priming (Call 10)........................................................................... 21 Table 1: Success rates in obtaining amplifiable, polymorphic microsatellite loci from the loci screened in studies using NGS approaches to microsatellite development ....................................15 Table 2: Number of microsatellite loci [and number of PALs] found by PAL_FINDER in the raw reads vs. the filtered reads ............................................................................................................. 20 Table 3: Primer sequences and repeat motifs for successfully amplified microsatellite loci ...........22 3 Abstract Molecular genetics is commonly used to address ecological questions and aid in ecosystem management and conservation. The level of genetic variation, and its distribution among populations, has ecologically important consequences, such as for associated community biodiversity and ecological resilience. Microsatellites are popular and useful molecular markers with many applications, but must be developed a priori for every species, which traditionally has been a costly and time-consuming process. Recently, next-generation sequencing approaches have been successfully used to isolate microsatellite markers for a lower cost and much more quickly than traditional methods offer. Here, we develop an existing method that uses Illumina paired-end sequencing, to isolate microsatellites for the marine sponge Cinachyrella alloclada. We use longer read lengths than previously used (2 x 250bp) and filter sequence data by quality in order to increase the number of amplifiable loci located and the chances of successful amplification. Out of 35 loci tested, 13 were successfully amplified in PCR reactions. These loci can go on to be used in population genetics and community genetics studies for C. alloclada, aiding understanding of its ecology. In addition, this method of microsatellite isolation offers a faster and cheaper way of obtaining molecular markers than traditional approaches, and uses quality filtering and longer read lengths to enhance the capture of amplifiable loci. This method may therefore be used for other species that would benefit from the availability of microsatellite markers, including other understudied marine sponges. 4 Declaration No portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institute of learning. Copyright statement i. The author of this thesis (including any appendices and/or schedules to this thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. ii. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. iii. The ownership of certain Copyright, patents, designs, trade marks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. iv. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=487), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on Presentation of Theses 5 Acknowledgements This project was only possible with the support of my supervisor, Professor Richard Preziosi, who provided invaluable assistance throughout the whole project. I also thank Nathan Truelove, who helped greatly with the project and provided much support with the sequencing and bioinformatics stages of the project especially. Thanks also to the Preziosi lab group for valuable feedback and assistance, and especially Rachael Antwis for the tea and moral support. I also thank the Walton lab for use of their lab space and equipment, and Dr. Petri Kampainnen for always being willing to share advice and ideas over a coffee. The expertise and knowledge of Andy Hayes and Stacey Holden at the Genomic Technologies Core Facility, and Peter Briggs and Ian Donaldson at the Bioinformatics Core Facility (both at the University of Manchester) have been an essential part of this project. Many thanks also to all of their colleagues
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