Conservation Genet Resour DOI 10.1007/s12686-010-9219-0
TECHNICAL NOTE
Isolation and characterization of microsatellite markers for the banded ironstone endemic Acacia karina (Leguminosae: Mimosaceae) and cross-species amplification with A. stanleyi and A. jibberdingensis Paul G. Nevill • Janet M. Anthony Siegfried L. Krauss
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Received: 24 February 2010 / Accepted: 26 February 2010 Ó Springer Science+Business Media B.V. 2010
Abstract Microsatellite markers were developed for the banded ironstone endemic shrub Acacia karina to examine genetic diversity, range-wide differentiation and mating system parameters. Nine loci were developed and in a sample of 20 individuals from one population the number of alleles ranged from 4 to 12 per locus and observed heterozygosities from 0.556 to 0.824. All loci were tested for cross-species amplification in two other south-west Australian Acacia species thought to be closely related to A. karina. Of these nine loci, eight were polymorphic in A. jibberdingensis and three in A. stanleyi. Keywords Acacia South-west Australia Microsatellites Cross-species amplification Banded ironstone
Acacia karina Maslin is a recently described, narrowrange, conservation priority-listed, species endemic to the Blue Hill Banded Ironstone Ranges of the Midwest region of south-west Western Australia (Maslin and Buscumb 2007). The Midwest region of WA has an exceptionally high diversity of Acacia species (Hnatiuk and Maslin 1988), many with a restricted distribution and high conservation value. Microsatellite markers were developed to examine genetic diversity, range-wide genetic differentiation and mating system parameters of A. karina. This P. G. Nevill (&) J. M. Anthony S. L. Krauss Botanic Gardens and Parks Authority, Kings Park and Botanic Garden, West Perth, WA 6005, Australia e-mail:
[email protected] P. G. Nevill S. L. Krauss School of Plant Biology, University of Western Australia, Nedlands, WA 6009, Australia
understanding is critical to identify and manage potential genetic impacts from proposed mining activities, and subsequently potential impacts on population, and species, viability. The utility of these markers for studies on other putatively closely related Acacia species from sect. Juliflorae was tested on A. stanleyi Maslin, and A. jibberdingensis Maiden and Blakely (Maslin and Buscumb 2007). Phyllodes were collected from A. karina plants in the Mt Karara area (477875 E 6771530 N). Genomic DNA was extracted from frozen (-80°C) phyllode material using the CTAB procedure as described in Butcher (1998). Microsatellite enriched libraries were developed for four different repeat motifs (CAn, GAn, AACn and ATGn) by Genetic Identification Services, California, USA (http://www.geneticid-services.com/). Briefly, genomic DNA was restricted with seven blunt-end cutting enzymes (Rsa1, HaeIII, BsrB1, PvuII, StuI, ScaI, EcoRV). Fragments in the size range of 300–750 bp were linker adapted with oligonucleoties that contained a HindIII site and then subjected to magnetic bead capture (CPG Inc.). Molecules were restricted with HindIII and ligated into the HindIII site of the pUC19 plasmid. Ligation products were introduced into E. coli strain DH5 alpha (ElectroMax, Invitrogen) by electroporation. Blue-white selection was used to identify recombinant clones for sequencing on an ABI 377 (Applied Biosystems) using Amersham’s DYEnamic Terminator Cycle Sequencing Kit (Amersham Biosciences). One hundred and 43 clones were sequenced, including 37 from the CA library, 35 from the GA library, 39 from the AAC library and 40 from the ATG library. Ninety-eight different microsatellite containing clones were identified from the four libraries and primers were designed for 71 sequences using DesignerPCR version 1.03 (Research Genetics Inc.) and synthesized for 23. These
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primer pairs were initially tested to verify amplification, determine the optimum annealing temperature and to establish size ranges for later polymerase chain reaction (PCR) multiplexing, using DNA from six individuals of A. karina, and also six individuals each of A. jibberdingensis and A. stanleyi collected in the Kalanie area (537315 E 6671343 N). For amplification of microsatelite loci, PCR was carried out in 12.5 ll reaction volumes containing 1.25 ll of 109 PCR buffer (Fisher Biotec), 2 mM of MgCl2 (Fisher Biotec), 0.2 mM of dNTP mix (Fisher Biotec), a primer concentration of 0.2 lM for each of forward and reverse primers, 20 ng of template DNA and 0.5 U of Taq DNA polymerase (Fisher Biotec). PCR was carried out in a Corbett Palm-Cycler (Corbett Life Science) using the following reaction conditions: initial denaturation for 3 min at 94°C, 35 cycles of 40 s at 94°C, 40 s at an annealing temperature of 57°C, and an extension of 30 s at 72°C; with a final extension of 4 min at 72°C. PCR products were separated on a 2% agarose gel and fragment sizes determined by comparison to a Low DNA Mass Ladder (Invitrogen). Twenty individuals of A. karina were genotyped using 15 loci from the original screening that were polymorphic and amplified reliably. Multiplexing was performed on three groups of primers using the QIAGEN Multiplex kit (QIAGEN) in 12.5 ll reactions as follows: 19 Multiplex PCR Master Mix, 2 lM each primer, dH2O, and 20 ng DNA. Forward primers were fluorescently labeled (see
Table 1). The multiplex PCR followed the manufacturers protocol and the following profile was used: an initial denaturation for 5 min at 95°C; 28 cycles of 30 s at 95°C, 1 min 30 s at an annealing temperature of 57°C, and an extension of 30 s at 72°C; with a final extension of 30 min at 60°C. Multiplex PCR products were diluted 1:40 in deionised formamide and separated by electrophoresis using a CEQ8800 (Beckman-Coulter). Fragment analysis and sizing was performed using CEQ 8800 Genetic Analysis System software version 9.0.25 (Beckman-Coulter). A. karina genotypes were analysed using GDA 1.1 (Lewis and Zaykin 2001) (available at http://hydrodictyon.eeb.uconn. edu/people/plewis/software.php). The number of alleles (A), and the observed (Ho) and expected (HE) heterozygosities were calculated for each locus. The loci were tested for deviation from Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium using Fisher’s exact tests in GDA 1.1 and applying sequential Bonferroni correction (Hochberg 1988). Four out of 15 loci did not amplify reliably and two others were not in HWE. There was no significant linkage disequilibria between all loci pairs (P \ 0.05) after sequential Boneferroni correction (Hochberg 1988). Primer sequences and characteristics of the nine polymorphic microsatellites that reliably amplified and were in HWE are shown in Table 1. In a sample of 20 plants from one population of A. karina, between four and twelve alleles per locus were found. Observed (Ho) and expected (HE)
Table 1 Characteristics of nine microsatellite loci isolated from Acacia karina Locus ID
Repeat motif
Primer sequences (5’–3’)
Dye label
Size range (bp)
No. alleles
HO
HE
Genbank Acc. no.
AkA103-F
(AC)13
ATT TGT GCT TGG TCT TGA AC
D4 (1)
155–175
11
0.556
0.708
GU903900
D3 (3)
144–198
11
0.722
0.747
GU903901
D3 (3)
269–297
12
0.824
0.691
GU903902
D2 (2)
276–316
12
0.706
0.851
GU903903
D2 (1)
251–279
9
0.765
0.863
GU903904
D3 (2)
196–223
6
0.667
0.818
GU903905
D3 (2)
111–120
4
0.778
0.728
GU903906
AkA103-R AkA112-F
TTG GTG CGA GTC TCA GTC (GT)19
AkA112-R AkB10-F
TTC TGC GTT GAA ATA ACC TG (CT)20
AkB10-R AkB108-F
(AG)14 (AG)13
ATC GTG TCT TAC CTG ATG AGT C AAG CAG CCA CTG TCA ATG
(GTT)9
AkC103-R AkC104-F
CCC TCA ACA TCA TTT TCT GG AAG CAA GTT TCT CAG CAA AGG
AkB114-R AkC103-F
TAA GGA GCG TCT TGT CAC AC CGA GCC TAA CAG ATT TGT AAA G
AkB108-R AkB114-F
GCT CTC TCC AAG CCT TCT T
TGG GGT AGG TTA ATG ATT GAT C CAG CCA CAA AGA CAT CAG TAA G
(CAA)8
AAA GCC TCC TTC TTC CAT TC
AkC108-F AkC108-R
(CAA)13(TAA)7
TGT GCG TGT GTG TGT AGT TG AGC ATT TAC ATG CAC TTT ATG C
D4 (3)
211–250
7
0.706
0.848
GU903907
AkD118-F
(TCA)8
GTT GAT GTT GAT TCC CAT TCA C
D4 (2)
185–200
4
0.615
0.757
GU903908
AkC104-R
AkD118-R
TCA GCC ATC ACA GGT TCA TA
AAG GAT GAT GAT TGT TCA GTC G
Shown are locus names, repeat motif, primer sequences, dye label and multiplex group, allele size range, number of alleles, observed and expected heterozygosities and GenBank accession number
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Conservation Genet Resour Table 2 Results of cross amplification of microsatellites developed for A. karina in A. stanleyi (n = 20) and A. jibberdingensis (n = 6) Locus ID
A. stanleyi
A. jibberdingensis
AkA103
a
5 (150–170)
AkA112
5 (142–150)
7 (140–188)
AkB10
a
4 (246–272)
AkB108
a
a
AkB114
3 (249–265)
5 (251–271)
AkC103
a
1 (201)
3 (117–126)
3 (108–120) 5 (213–258)
AkC104 AkC108 AkD118
a a
Acknowledgments We gratefully acknowledge funding from Karara Mining Ltd. Thanks to Genetic Identification Services, California, USA for construction of the library. Thanks also to David Coultas (Woodman Environmental Consulting) and Bruce Maslin (Western Australian Herbarium) for advice on sample collection. Phyllode material was collected under permit number SW012710 issued under the Western Australian Wildlife Conservation Act 1950.
3 (163–190)
Given are the number of alleles and their size range a
they are closely related, but distinct, species. In contrast, A. stanleyi is more distantly related to A. karina, based on the relatively poor cross species transferability of these markers, in contrast to the earlier suggestion that these species are most closely related (Maslin and Buscumb 2007).
References
No or poor amplification
heterozygosities ranged from 0.556 to 0.824, and from 0.690 to 0.863, respectively. Cross amplification was successful at eight loci in A. jibberdingensis and three in A. stanleyi (Table 2). In A. stanleyi, lower levels of diversity or poor/unreliable amplification were found for all loci. The microsatellite loci presented in this study will be useful in the examination of genetic diversity and mating systems in A. karina and A. jibberdingensis but not A. stanleyi. The cross-species transferability of simple sequence repeats from A. karina to A. jibberdingensis supports the suggestion that, based on morphological similarity,
Butcher R (1998) RFLP diversity in the nuclear genome of Acacia mangium. Heredity 8:205–213 Hnatiuk RJ, Maslin BR (1988) Phytogeography of Acacia in Australia in relation to climate and species-richness. Aust J Bot 36:361– 383 Hochberg Y (1988) A sharper Boneferroni procedure for multiple tests of significance. Biometrika 75:800–803 Lewis PO, Zaykin D (2001) Genetic data analysis: computer program for the analysis of allelic data. Version 1.0 Maslin BR, Buscumb C (2007) Two new Acacia species (Leguminosae: Mimosoideae) from banded ironstone ranges in the Midwest region of south-west Western Australia. Nuytsia 17:263–272
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