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Development of Microsatellite Markers in Mogurnda Adspersa Using

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Development of Microsatellite Markers in Mogurnda Adspersa Using Mogurnda adspersa microsatellite markers: multiplexing and multi-tailed primer tagging Author Real, Kathryn M, Schmidt, Daniel J, Hughes, Jane M Published 2009 Journal Title Conservation Genetics Resources DOI https://doi.org/10.1007/s12686-009-9095-7 Copyright Statement © 2009 Springer Netherlands. This is the author-manuscript version of this paper. Reproduced in accordance with the copyright policy of the publisher. The original publication is available at www.springerlink.com Downloaded from http://hdl.handle.net/10072/30628 Griffith Research Online https://research-repository.griffith.edu.au 1 Mogurnda adspersa microsatellite markers: multiplexing and multi-tailed 2 primer tagging. 3 4 Kathryn M. Real, Daniel J. Schmidt, Jane M. Hughes 5 Australian Rivers Institute 6 Griffith University, Nathan, 4111, Queensland, Australia 7 8 Abstract 9 10 A set of twelve microsatellite DNA loci were developed for the threatened Australian 11 freshwater fish Mogurnda adspersa (Eleotridae). Primers were tailed with one of four 20- 12 mer oligonucleotides for use in four-colour fluorescent detection and optimised for multiplex 13 PCR. The loci were used to genotype individuals from two populations in the Pioneer River 14 catchment of central Queensland, eastern Australia. Number of alleles per locus ranged from 15 2 to 33 and per locus heterozygosity ranged from 0.06 to 0.81. Successful cross-species 16 amplification of all loci was achieved in the congener M. mogurnda. These markers will be 17 used to estimate effective population size and to examine the relationship between flow 18 regime and population demographic parameters. 19 20 Keywords: freshwater fish, purple-spotted gudgeon, Eleotridae, M13 tailing, multiplex PCR. 21 22 The Purple-spotted gudgeon (Mogurnda adspersa) is an eleotrid freshwater fish endemic to 23 Australia. It is widely distributed throughout river catchments in East Coast Queensland, 24 Northern NSW, Murray-Darling Basin, Victoria and South Australia (Pusey et al. 2004). 25 Various authorities have listed the species as vulnerable or endangered due to substantial 26 declines in distribution and abundance in the southern and western parts of its range (Pusey et 27 al. 2004). Population viability of M. adspersa is potentially influenced by water resource 28 development in coastal catchments of Queensland where the species is currently common and 29 widespread. Highly polymorphic genetic markers are required to assess genetic diversity and 30 population structure of this species in relation to landscape variables including flow regime. 31 Twelve microsatellite loci were designed to be condensed into two multiplex combinations of 32 six loci for analysis on an ABI 3130 Genetic Analyser (Applied Biosystems). 33 34 The microsatellite library was constructed using DNA pooled from four individuals collected 35 in the Pioneer River, Queensland. Genomic DNA was extracted from tail muscle (Doyle and 36 Doyle 1987) and microsatellite fragments isolated using methods modified from Glenn and 37 Schable (2005). DNA (~750ng) was digested using the restriction enzyme DpnII, followed 38 by ligation with a nonphosphorylated Sau linker. The linker was made by annealing 39 equimolar amounts of Sau-L-A (5'-GCGGTACCCGGGAAGCTTGG–3') and Sau-L-B (5'- 40 GATCCCAAGCT-TCCCGGGTACCGC-3') oligonucleotides. Nine biotinylated oligo 41 probes (dCA13, dTA15, dAAC6, dACG6, dAGC6, dAAGT8, dAAAG8, dACAT8, dAAAT8) 42 were hybridised to the linker-ligated DNA and selectively maintained using Streptavidin 43 MagneSphere Paramagnetic Particles (Promega). This enrichment was repeated, then the 44 library was ligated into pGEM-4Z cloning vector (Promega) and electroporated into One Shot 45 TOP10 electrocompetent Escherichia coli cells (Invitrogen). Colonies found to possess DNA 46 fragment inserts approximately 200-500bp in size were sequenced then edited and aligned 47 using SEQUENCHER V4.9 (Gene Codes Corporation). Of the 96 colonies sequenced, 62 were 48 found to contain microsatellite repeats. 49 50 Repeat-containing sequences ranging in size between 150-380bp were selected for multiplex 51 PCR primer design (Markoulatos et al 2002; Heegariu et al 1997). Primers were designed 52 using PrimerBlast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/), and multiplex 53 combinations were checked for compatibility using AutoDimer (Vallone and Butler 2004). 54 Nineteen primer sets were tested and 12 were found to be variable within the two populations 55 examined. 56 57 As an alternative to universal M13 tailing of primers, we designed four unique 20-mer 58 oligonucleotide tails using the pGEM-4Z cloning vector sequence (GenBank accession: 59 X65305) as template (Table 2). These oligo tails were added to the 5' end of forward primers 60 to enable incorporation of corresponding fluorescently labelled tagging primers to different 61 loci in PCR reactions (Schuelke 2000). Three primers are used for the amplification of each 62 locus in this system: the tailed forward primer, reverse primer and fluorescently labelled tag 63 which corresponds to the tail sequence of the forward primer. By designing multiple tails, we 64 combine the economic benefits of universal M13 tailing with the ability to perform multiplex 65 reactions incorporating several fluorescent labels (Missiaggia and Grattapaglia 2006). Cost 66 of the present primer set was reduced by 37.75% compared with direct labelling, while 67 savings in money and time over universal M13 labelling depend on the reduction in PCR 68 reactions that can be achieved by multiplexing. Seven multiplex PCR combinations have 69 been optimised including 2MOG01+2MOG03, 2MOG02+2MOG10, 2MOG04+2MOG084, 70 4MOG02+4MOG03, 2MOG09+3MOG06, 2MOG08+2MOG10, 2MOG06+2MOG03 and future 71 additions to these will further increase throughput. 72 73 Amplification reactions were 7 µl in volume containing 1× reaction buffer, 1.5mM MgCl2, 74 0.1µM tailed forward primer, 0.4µM reverse primer, 0.4µM fluorescent tag, 0.2mM dNTP 75 and 0.2U Redtaq (Astral Scientific) polymerase and 7-100ng of total genomic DNA. The 76 PCR thermocycler conditions are as follows: 94oC (5mins) then 35 cycles at 94oC 77 (30sec)/60oC (30sec)/74oC (30sec) and a final extension of 72oC for 40mins. 78 79 Primers for the twelve microsatellite loci are described in Table 1 and the corresponding 80 labelled tagging primers are shown in Table 2. The tagging primers were labelled with 6- 81 FAM, VIC, NED and PET from the G5 fluorescent dye set (Applied Biosystems) allowing 82 two multiplex combinations of 6 loci : 2MOG01, 2MOG09, 3MOG03, 3MOG06, 4MOG02, 83 4MOG03 AND 2MOG02, 2MOG03, 2MOG04, 2MOG06, 2MOG08, 2MOG10 run on an ABI 84 3130 Genetic Analyser. Genotypes were scored using GENEMAPPER V4.0 (Applied 85 Biosystems). Data sets were corrected with MICRO-CHECKER (Oosterhout et al. 2004) and 86 found to have no null alleles. Allele number per locus varied from 2 to 33 (Table 1). Data 87 collected from two populations (Finch Hatton Ck. n = 31 and Bakers Ck. n = 16) were 88 analysed using GENEPOP V4.0 (Raymond and Rousset 1995) to calculate observed and 89 expected heterozygosity, test for deviations from Hardy-Weinberg predictions for each locus, 90 and to test for linkage disequilibrium between pairs of loci within each population. Expected 91 heterozygosity ranged from 0.06 – 0.81 (Table 1). No significant deviations from expected 92 Hardy-Weinberg proportions were observed for any locus. In both populations there were 93 four significant cases of genotypic disequilibrium from a total of 66 pairwise tests although 94 different pairs of loci were involved in each population, so significant linkage between loci is 95 not evident in our data. 96 97 The described microsatellite primers were screened on eight Mogurnda mogurnda individuals 98 collected from five sites in the Daly River including the upper Katherine (Northern 99 Territory). All loci amplified one or two alleles per individual except 4MOG03 which 100 requires further optimisation for use on this species (Table 1). 101 102 Acknowledgements: 103 104 This work is part of a collaborative project between Griffith University and Queensland 105 Department of Environment and Resource Management (DERM). We thank Steve Smith for 106 refining the protocol. Bernie Cockayne and Kate Engeldow collected fish samples, Ben Cook 107 provided M. mogurnda samples and Jake Butwell constructed laboratory equipment. 108 Australian Animal Ethics Committee approval number for this project is ENV/08/09/AEC. 109 Funding was provided by DERM as part of its Environmental Flows Assessment Program. 110 111 References 112 113 Doyle J.J. and Doyle J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh 114 leaf tissue. Phytochemistry Bulletin 19: 11-15. 115 116 Glenn T.C. and Schable N.A. (2005) Isolating microsatellite DNA loci. Methods in 117 Enzymology 395:202-222. 118 119 Heegariu O., Heerema N.A., Dlouhy S.R.,Vance G.H. and Vogt P.H. (1997) Multiplex PCR: 120 Critical parameters and step-by-step protocol. BioTechniques 23:504-511. 121 122 Markoulatos P., Siafakas N. and Moncany M. (2002) Multiplex Polymerase Chain Reaction: 123 A practical approach. Journal of Clinical Laboratory Analysis 16:47-51I. 124 125 Missiaggia A. and Grattapaglia D. (2006) Plant microsatellite genotyping with 4-color 126 fluorescent detection using multiple-tailed primers. Genetics and Molecular Research 5, 72- 127 78. 128 129 Oosterhout, C., Hutchinson, W., Wills, D. and Shipley, P. (2004) MICRO-CHECKER: 130 software for identifying and correcting genotyping errors in microsatellite data. Molecular 131 Ecology Notes 4: 535 - 538. 132 133 Pusey B., Kennard, M. and Arthington A. (2004) Freshwater fishes of North-Eastern 134 Australia. CSIRO Publishing: Melbourne. 135 136 Raymond M., Rousset F. (1995) GENEPOP (Version 1.2): a population 137 genetics software for exact tests and ecumenicism. Journal 138 of Heredity, 86, 248–249. 139 140 Schuelke M. (2000) An economic method for the fluorescent labelling of PCR fragments. 141 Nature Biotechnology 18: 233-234. 142 143 Vallone P.M. and Butler J.M. (2004) AutoDimer: a screening tool for primer-dimer and 144 hairpin structures.
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