Isolation and characterization of 12 microsatellite markers for the vulnerable subshrub Comesperma polygaloides (Polygalaceae) Collin W. Ahrens & Elizabeth A. James
Conservation Genetics Resources ISSN 1877-7252 Conservation Genet Resour DOI 10.1007/s12686-014-0258-9
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Author's personal copy Conservation Genet Resour DOI 10.1007/s12686-014-0258-9
MICROSATELLITE LETTERS
Isolation and characterization of 12 microsatellite markers for the vulnerable subshrub Comesperma polygaloides (Polygalaceae) Collin W. Ahrens • Elizabeth A. James
Received: 22 May 2014 / Accepted: 24 June 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract We isolated 12 new polymorphic microsatellite markers from Comesperma polygaloides, a rare and vulnerable subshrub from southeastern Australia. 454 pyrosequencing was used to identify 81,804 possible primer pairs. Of 96 pairs, 32 amplified a product of expected size in the target species. Subsequently, 12 loci were screened in five populations of C. polygaloides and one population each of C. ericinum and C. calymega. All 12 loci were polymorphic and showed a diploid pattern. Observed and expected heterozygosity were calculated (0.04–0.71 and 0.04–0.76, respectively). Alleles per locus ranged from 2 to 14 (mean = 4.9). Cross species amplification was successful for three primer pairs. These loci will improve understanding of genetic structure and enhance long-term viability of a species under state protection. Keywords Small milkwort Microsatellites 454 GSFLX Grassland Vulnerable Comesperma polygaloides (F. Muell.) is a perennial subshrub listed as vulnerable in Victoria, Australia and its disjunct distribution is limited to southern Western Australia and southeastern Australia. It is considered ‘‘at risk’’ due to continued depletion of its grassland habitat and competition with introduced species. Information about the genetics and ecology of this species is lacking but seed
Electronic supplementary material The online version of this article (doi:10.1007/s12686-014-0258-9) contains supplementary material, which is available to authorized users. C. W. Ahrens (&) E. A. James Royal Botanic Gardens Melbourne, National Herbarium of Victoria, Birdwood Avenue, Private Bag 2000, South Yarra, VIC 3141, Australia e-mail:
[email protected]
collection and propagation of the species is underway to support reintroduction efforts and long-term germplasm storage. Genetic markers have been developed herein to aid the genetic investigation of this vulnerable species. Genomic DNA was isolated from 300 mg of silica-dried leaf tissue from five pooled C. polygaloides individuals using a DNeasy Plant Maxi Kit (QIAGEN, Hilden, Germany) at AGRF (Australian Genomic Research Facility, Adelaide, South Australia). The genomic DNA library was prepared according to Roche’s Rapid Library Preparation Method Manual and quantified by PicoGreen fluorimetry (Quant-iT PicoGreen dsDNA Assay Kit; Invitrogen, USA). DNA was nebulized and cleaned with a QIAGEN MiniElute purification kit. Fragments underwent end repair and subsequent barcoded adapter ligation with the 454 Adapter-A and Adapter-B (Lib-L). Small fragments were removed using Agencourt AMPure Beads (Beckman Coulter, Australia). Final library quality was assessed using the Agilent Bioanalyzer (Agilent Technologies, USA) and quantitative PCR (qPCR) using KAPA reagents (KAPA Biosystems, USA). DNA was sequenced with a Roche GSFLX machine for a 1/8 coverage (454 Life Sciences, USA). The program QDD was used to identify tandem repeats and to design microsatellite primers (Meglecz et al. 2009). Of 81,804 possible microsatellite loci isolated, 96 loci were selected for screening based on penalty score (lowest score indicates ‘‘best’’ primer pair at the locus), DNA fragment size (100–400 bp), and number of motif repeats. Primer pairs were manufactured by IDT (Integrated DNA Technologies, USA) and screened for amplification and variability in seven C. polygaloides individuals using Blacket et al.’s (2012) three-primer approach to enable efficient simultaneous amplification of loci in multiplex PCRs using four fluorochromes. PCRs were conducted in 10-lL reactions consisting of 20 ng of DNA, 5 lL Master Mix from
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Author's personal copy Conservation Genet Resour Table 1 Genetic properties of 12 new microsatellite markers within and among 5 popopulations of C. polygaloides Locus
Shelford
Derrinallum
Cressy
Hamilton Hwy
Mt. Mercer CR
Total
(n = 19)
(n = 19)
(n = 19)
(n = 18)
(n = 12)
(n = 87)
A
Ho
He
A
Ho
He
A
Ho
He
A
Ho
He
A
Ho
He
A
Ho
He
cp011
2
0.32
0.27
4
0.26
0.28
2
0.05
0.05
2
0.56
0.40
3
0.25
0.23
5
0.29
0.24
cp022
4
0.56
0.44
2
0.26
0.23
3
0.89
0.52
3
0.83
0.57
3
1.00
0.57
4
0.71
0.47
cp027 cp028
3 4
0.53 0.68
0.56 0.57
3 3
0.37 0.32
0.48 0.34
3 3
0.74 0.61
0.51 0.45
3 2
0.28 0.56
0.25 0.48
3 3
0.75 0.42
0.50 0.45
3 5
0.53 0.52
0.46 0.46
cp029
2
0.21
0.19
1
0.00
0.00
1
0.00
0.00
1
0.00
0.00
1
0.00
0.00
2
0.04
0.04
cp042
4
0.58
0.46
5
0.63
0.61
2
0.53
0.50
3
0.61
0.64
3
0.08
0.29
6
0.49
0.50
cp043
3
0.63
0.54
3
0.53
0.48
3
0.89
0.52
2
0.61
0.42
2
0.08
0.08
3
0.55
0.41
cp046
2
0.50
0.38
3
0.42
0.43
2
0.11
0.10
2
0.44
0.50
2
0.50
0.38
3
0.40
0.36
cp060
2
0.11
0.10
2
0.05
0.05
2
0.58
0.45
2
0.33
0.28
1
0.00
0.00
2
0.22
0.18
cp062
10
0.35
0.84
11
0.65
0.88
5
0.26
0.46
7
0.94
0.79
8
1.00
0.83
14
0.64
0.76
cp072
4
0.47
0.64
5
0.22
0.60
4
0.50
0.62
3
0.35
0.58
4
0.91
0.68
6
0.49
0.62
cp093
4
0.56
0.58
6
0.68
0.71
3
0.18
0.30
3
0.50
0.41
4
0.73
0.63
6
0.53
0.52
A = number of alleles. Ho = observed heterozygosity. He = expected heterozygosity. Bold numbers indicate significant departure from HWE adjusted for Bonferonni correction
the Type-It Microsatellite PCR Kit (QIAGEN), 0.1 lM of each forward primer, 0.2 lM of each reverse primer, and 0.1 lM of each fluorescently tagged universal primer (FAM, VIC, NED, PET). PCRs were run under the following conditions: 5 min denaturation at 95 °C, followed by 28 cycles of 95 °C for 30 s, annealing at 60 °C for 1 min 30 s and 72 °C for 30 s, and a final extension at 60 °C for 30 min. Of the 96 primer pairs screened, 12 had distinct polymorphic banding patterns and are developed further. Repeat motifs included three dinucleotides, five trinucleotides, and four tetranucleotides (Online Resource 1). Markers were tested for locus variability and marker consistency within and among five populations (Table 1). After PCR, capillary electrophoresis was used to determine fragment size on an ABI 3130xl (Applied Biosystems, USA) by Macrogen (Seoul, South Korea) with GeneScan 500 LIZ (Applied Biosystems) used as the internal size standard. Alleles were scored with the microsatellite plugin in Geneious software (version 7.0.2). Each locus was checked for evidence of null alleles, scoring error, and allele drop out using MICRO-CHECKER 2.2.3 (Van Oosterhout et al. 2004). No loci showed significant null allele frequencies. The number of alleles per locus (A = 2–14), observed heterozygosity (Ho = 0.04–0.64), and expected heterozygosity (He = 0.04–0.76) were calculated for each locus in FSTAT version 2.9.3 (Goudet 1995). Deviation from HWE was tested using GENEPOP 4.2 (Raymond and Rousset 1995) with P values adjusted using
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the sequential Bonferroni method. We checked all pairs of loci for linkage disequilibrium in GENEPOP and none were significant. The microsatellite markers cp022 and cp028 amplified polymorphic patterns in C. ericinum and cp060 amplified polymorphic patterns in C. calymega. Acknowledgments The authors acknowledge the Royal Botanic Gardens Melbourne; the study was funded by Hansons Construction Materials Pty Ltd.
References Blacket MJ, Robin C, Good RT, Lee SF, Miller AD (2012) Universal primers for fluorescent labelling of PCR fragments: an efficient and cost-effective approach to genotyping by fluorescence. Mol Ecol Resour 12:456–463 Goudet J (1995) FSTAT (Version 2.9.3): a computer program to calculate F-statistics. J Hered 86:485–486 Meglecz E, Costedoat C, Dubut V, Gilles A, Malausa T, Pech N, Martin J-F (2009) QDD: a user-friendly program to select microsatellite markers and design primers from large sequencing projects. Bioinformatics 26:403–404 Raymond M, Rousset F (1995) GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J Hered 86:248–249 Van Oosterhout C, Hutchinson WF, Wills DP, Shipley P (2004) MICRO-CHECKER: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4:535–538
