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. Biotechniques 372:226-31.
145 Primer Sequence (5’-3’) HO, HE Size F=Forward primer, R=Reverse primer, *=5’ tail Range Number o Locus Repeat TA ( C) 5’ Tail (Bp) of Alleles Finch Hatton Ck Bakers Ck 2MOG01 (GT)7 60 F: *TACTTGACTCCACGCGGCTT 1 200-218 8 0.57,0.55 0.56,0.65 R: GCAGAGGGAGGAGATGGAGAA
2MOG02 (GT)18 60 F: *AGCAGCGCTGGCTCAGAT 2 250-280 10 0.60,0.53 0.44,0.37 R: TTCATCGCTGTCTCCTGGTG
2MOG03 (GT)10 60 F: AAAGGGGGAAGAAAACGGC 2 260-176 9 0.37,0.39 0.75,0.54 R: TCCCTTCTCCTGCCCAAAGT
2MOG04 (GT)13A(TG)15 60 F: *AGGGTGAGGCTCCTGTGG 4 150-216 20 0.70,0.73 1.00,0.73 R: CATCGTCTGAAGTGCAGTGCT
2MOG06 (CA)11 60 F: *TGCTGCTGCGTGTGGAATTA 2 188-218 7 0.23,0.21 0.56,0.50 R: GGGGCACAACTCAATCTGGA
2MOG08 (GT)10 60 F: *AGCTTGACGCAAAGGCACTTA 3 230-265 7 0.33,0.33 0.50,0.57 R: CCCTGACACTGAACTAAACCTGC
2MOG09 (GT)15 60 F: *TCCCTCCTCTGGACTCGGAC 2 260-278 9 0.60,0.74 0.50,0.48 R: GTGACGGAGGTGTGCTCCC
2MOG10 (CA)11 60 F: *CACCACGCTGAGGAAGAAGC 3 160-210 13 0.50,0.49 0.06,0.06 R: TTCGCCGAGGTCAGAGGATA
3MOG03 (CAA)21 60 F: *CGTCCTGGAGTCTAACAGAGCA 4 150-265 29 0.93,0.84 0.63,0.54 R: CCCACCAGCCTCACGTCTAT
3MOG06 (GTT)3GT(GTT)4 60 F: *TGACCTTCTCGCACTCTCCC 2 160-195 6 0.33,0.28 0.31,0.42 R: CAGTTCCTCCGCTCACCG
4MOG02 (CATA)24 60 F: *GACGGGCAGGTGATTCCATA 3 290-388 23 0.77,0.77 0.69,0.73 R: CCAAGGCAGTGTCAAAAGCC
4MOG03 (TGTC)5(TGTA)12 60 F: *CACATCAAAAGTATAAGGATGCGTG 3 160-200 10 0.70,0.64 0.94,0.73 R: AAAGCCCTCTGAGGACCAGC
Table 1: Primers attributes for the twelve Mogurnda adspersa microsatellite loci. Forward priming sequences presented do not include the additional 5’ tagging sequence modification. Successful multiplexing pairs include 4MOG02/4 MOG03, 2MOG09/3MOG06 2MOG08/2MOG10 2MOG06/2 MOG03 2 MOG01/2MOG03, 2MOG02/2MOG10, 2MOG04/2MOG08. The microsatellite loci were designed to be run in two combinations of 6 primers: 2MOG01 2MOG09 3MOG03 3MOG06 4MOG02 4MOG03 2MOG02 2MOG03 2MOG04 2MOG06 2MOG08 2MOG10 5’ Tail ID Dye Sequence (5’ – 3’) Tail 1 FAM CTCTTCGCTATTACGCCAGC Tail 2 VIC GCTGCAAGGCGATTAAGTTG Tail 3 NED CGGCCAGTGAATTGGATTTA Tail 4 PET TAGAGTCGACCTGCAGGCAT Table 2: Tags fluorescently labelled from G5 dye set for ABI Genetic Analysers that correspond to the 20bp sequence added to the 5’ end of the forward primer.
Doyle, J.J., and Doyle, J.L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry Bulletin 19: 11-15.
Glenn T.C. and Schable N.A. (2005) Isolating microsatellite DNA loci. Methods in Enzymology 395:202-222.
Heegariu O., Heerema N.A., Dlouhy S.R.,Vance G.H. and Vogt P.H. (1997) Multiplex PCR: Critical parameters and step-by-step protocol. BioTechniques 23:504-511.
Markoulatos P., Siafakas N. and Moncany M. (2002) Multiplex Polymerase Chain Reaction: A practical approach. Journal of Clinical Laboratory Analysis 16:47-51I.
Oosterhout, C., Hutchinson, W., Wills, D. and Shipley, P. (2004) MICRO- CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Molecular Ecology Notes 4: 535 - 538.
Pusey B., Kennard, M. and Arthington A. (2004) Freshwater fishes of North-Eastern Australia. CSIRO Publishing: Melbourne.
Raymond M., Rousset F. (1995) GENEPOP (Version 1.2): a population genetics software for exact tests and ecumenicism. Journal of Heredity, 86, 248–249.
Schuelke M. (2000) An economic method for the fluorescent labelling of PCR Fragments. Nature Biotechnology 18: 233-234.
Vallone P.M. and Butler J.M. (2004) AutoDimer: a screening tool for primer-dimer and hairpin structures. Biotechniques 372:226-31.