Conservation Genet Resour (2012) 4:689–693 DOI 10.1007/s12686-012-9623-8
TECHNICAL NOTE
Development of twenty-three novel microsatellite markers for the seagrass, Zostera muelleri from Australia Craig D. H. Sherman • Annalise M. Stanley Michael J. Keough • Michael G. Gardner • Peter I. Macreadie
•
Received: 19 February 2012 / Accepted: 20 February 2012 / Published online: 1 March 2012 ! Springer Science+Business Media B.V. 2012
Abstract Seagrasses are one of the most productive and economically important habitats in the coastal zone, but they are disappearing at an alarming rate, with more than half the world’s seagrass area lost since the 1990s. They now face serious threat from climate change, and there is much current speculation over whether they will survive the coming decades. The future of seagrasses depends on their ability to recover and adapt to environmental change—i.e. their ‘resilience’. Key to this, is understanding the role that genetic diversity plays in the resilience of this highly clonal group of species. To investigate population structure, genetic diversity, mating system (sexual versus asexual reproduction) and patterns of connectivity, we isolated and characterised 23 microsatellite loci using next
C. D. H. Sherman (&) ! A. M. Stanley School of Life and Environmental Sciences, Centre of Integrative Ecology, Deakin University, Pigdons Road, Waurn Ponds, VIC 3217, Australia e-mail:
[email protected] M. J. Keough Department of Zoology, The University of Melbourne, Parkville, VIC 3052, Australia M. G. Gardner School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia M. G. Gardner School of Earth and Environmental Science, Australian Centre for Evolutionary Biology and Biodiversity, University of Adelaide, Adelaide, Australia P. I. Macreadie School of the Environment, University of Technology, Sydney, NSW 2007, Australia
generation sequencing for the Australian seagrass species, Zostera muelleri (syn. Z. capricorni), which is regarded as a globally significant congeneric species. Loci were tested for levels of variation based on eight individuals sampled from Lake Macquarie, New South Wales, Australia. We detected high to moderate levels of genetic variation across most loci with a mean allelic richness of 3.64 and unbiased expected hetrozygosity of 0.562. We found no evidence for linkage disequilibrium between any loci and only three loci (ZosNSW25, ZosNSW2, and ZosNSW47) showed significant deviations from Hardy–Weinberg expectations. All individuals displayed a unique multi-locus genotype and the combined probability of identity across all loci was low (PID = 1.87 9 10-12) indicating a high level of power in detecting unique genotypes. These 23 markers will provide an important tool for future population genetic assessments in this important keystone species. Keywords Seagrass ! Dispersal ! Genetic structure ! Mating system ! Gene flow ! Sexual ! Asexual ! Clonal ! Recruitment ! Life history ! Zostera capricorni Seagrasses are highly productive group of plant species that are distributed throughout most of the world’s coastlines. They provide many important ecosystem services, such as: shoreline stablisation (Bos et al. 2007), nutrient cycling (worth US$19,002 ha-1 year-1; Costanza et al. 1997), habitat provision (for fish, bird, and invertebrate species; Heck et al. 2003; Hughes et al. 2009), and carbon sequestration (McLeod et al. 2011). However, seagrasses are currently facing a global crisis (Orth et al. 2006); 29% of the world’s seagrasses have disappeared (Waycott et al. 2009), and 14% of all seagrass species (8 of *60 species) are at risk of extinction (Orth et al. 2006).
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Zostera species are among of the most important and widely distributed seagrass species and have become model organisms for a range of ecological, demongraphic and genetic studies. In Australia, Z. muelleri (syn. Z. capricorni) is widely distributed throughout the temperate and tropical waters of the south and east coast (inc. Queensland, New South Wales, Victoria, South Australia, and Western Australia), however, the taxonomic status of Zostera in Australia remains contentious (Tomlinson and Posluzny 2001, Les et al. 2002, Jacobs et al. 2006, Jacobs and Les 2009). Zostera muelleri is considered conspecific with Z. capricorni, however, the lack of reliable morphological characters and no evidence of genetic divergence across a number of key genetic loci has led to the recommendation for the taxonomic merger of Z. capricorni and Z. muelleri into a single species, with the retention of the name Z. muelleri as the original and final species name (Les et al. 2002, Jacobs et al. 2006). Here we developed a set of microsatellite markers to examine key population parameters; including: genetic structure, diversity, clonal structure and patterns of connectivity in Z. muelleri from Lake Macquarie, New South Wales, Australia. Approximately 10 lg of genomic DNA was isolated from fresh leaf tissue from a single individual using DNeasy plant kits (QIAGEN), following the manufacturers instructions. We developed microsatellites following the methodology of Gardner et al. (2011). Briefly, we sequenced one-sixteenth of a plate using the GS-FLX 454 platform (Roche, Germany), providing 76,598 sequenced reads. A total of 165 unique sequence reads possessing microsatellite motifs were identified using the software QDD v 1.3 (Megle´cz et al. 2010). Primer pairs were designed using Primer3 for 48 of the fragments (Rozen and Skaletsky 2000). Multiplexes consisting of four loci were designed where forward primers had a fluorescent dye associated tag added (FAM-GCCTCCCTCGCGCCA; NED-GCCTTGCCAGCCCGC; VIC-CAGGACCAGGCTA CCGTG; PET-CGGAGAGCCGAGAGGTG) (Blacket et al. 2012). To test loci for levels of polymorphism, we screened eight individuals collected from Lake Macquarie, New South Wales, Australia. DNA extraction followed the method reported above. Polymerase chain reactions (PCR) were conducted in 11 lL volumes containing; 10 ng of genomic DNA; 5 lL PCR Master Mix (Qiagen, USA) and 4 lL primer multiplex (0.26 lM of each forward primer and fluorescent dye, 0.13 lM of reverse primer). PCR products were amplified using a touchdown programme; initial hot start at 94"C for 15 min; five cycles of 94"C for 45 s, 65"C for 45 s, 72"C for 45 s; five cycles of 94"C for 45 s, 60"C for 45 s, 72"C for 45 s; 10 cycles of 94"C for 45 s, 57"C for 45 s, 72"C for 45 s: 20 cycles
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Conservation Genet Resour (2012) 4:689–693
of 94"C for 45 s, 55"C for 45 s, 72"C for 45 s; final elongation at 72"C for 15 min. PCR amplicons were electrophoresed using an ABI 3130xl Genetic Analyzer, incorporating LIZ 500 (-250) size standard (Applied Biosystems). Alleles were scored using GeneMapper, v3.7 (Applied Biosystems). Population genetic statistics were calculated with GENEPOP on the web (Raymond and Rousset 1995) and the program GENALEX (V6.41) (Peakall and Smouse 2006). We tested the statistical power of the marker system to identify different clones by calculating the probability of identity, PID, for increasing locus combinations (Waits et al. 2001) using the program GENALEX (V6.41) (Peakall and Smouse 2006). This identification estimator calculates the probability that two individuals drawn at random from a population will have the same genotype at multiple loci and is used to assess the statistical confidence of the marker system for individual identification. PID was calculated for each locus and then multiplied across loci to give an overall PID (Waits et al. 2001). Twenty-three microsatellite primer pairs (Table 1) out of 48 amplified clearly and consistently; of these 20 were found to be polymorphic within the populations sampled. Overall we detected moderate levels of polymorphism across loci with the number of alleles per locus ranging from 2 to 10 and a mean of 3.64 alleles per locus. Estimates of unbiased expected heterozygosity ranged from 0.233 to 0.912, with a mean of 0.562, while observed heretozygosity varied from 0.00 to 1.00, with a mean of 0.412 (Table 1). Five loci showed significant deviations from Hardy– Weinberg equilibrium, however only three of these (ZosNSW25, ZosNSW29 and ZosNSW47) remained significant after corrections for multiple comparisons. Tests for linkage disequilibrium found no significant linkage between any of the loci after corrections for multiple tests. We report details of three monomorphic primer pairs (ZosNSW27, ZosNSW35 and ZosNSW44), as these loci may prove useful for population genetic studies of other widely geographically isolated populations despite the lack of variability in the sampled population. All individuals displayed a unique multi-locus genotype and the combined probability of identity across all loci was very low (PID = 1.87 9 10-12), suggesting that these loci have a high level of power in detecting unique genotypes in this highly clonal species. The markers developed here will be a powerful tool for assessing patterns of population connectivity, levels of genetic diversity and an understanding of the relative importance of asexual versus sexual reproduction to population persistence in this ecologically important species.
JQ085968
JQ085969
JQ085970
JQ085971
JQ085972
JQ085973
JQ085974
JQ085975
JQ085976
JQ085977
JQ085978
JQ085979
JQ085980
JQ085981
JQ085982
JQ085983
ZosNSW13
ZosNSW15
ZosNSW17
ZosNSW18
ZosNSW19
ZosNSW20
ZosNSW23
ZosNSW25
ZosNSW27
ZosNSW28
ZosNSW29
ZosNSW34
ZosNSW35
ZosNSW36
ZosNSW38
ZosNSW40
JQ085984
JQ085967
ZosNSW02
ZosNSW42
Genbank Acc. No.
Locus
R: TCCTGCAGTTGAGAATCCTG
F: AGGAGAGCCAACCAAGGAC
R: TGCTAACGAATTGTGACGGG
F: TGCAATTAGCGCAAGAGTACG
R: CGAAGACGATTCACCACCTG
F: TCATGGTCGTGCTTTAACATCC
R: TGATTCACATGGCTTCAATGTTC
F: GCGCATAAATTAGAATCTGCGG
F: TGCATTCACGTCATTAGAATCCG R: ACGGCCGGAAGGTCTTG
R: GGCGTGGAGTTAAAGAGTCG
F: GCTCGCTCCAAACACCTTG
R: ACATGTTTGGAGGCTTTGC
F: GCTCCACTCAAACTACCTATCG
R: TGGGAATTTGGGATTCGACC
F: TGGACAAGCTGCAGAAACG
R: GGTTGCTGATGGCGAGAAC
F: CCTGGCCCATATCTCACGG
R: ATGCATAGAGAATCCTGAGGTC
F: TCAGGCCCAGTTAGATACGG
R: TCTCGGACCATCTTTTCGAG
F: CACCACCGTATCAACTCCCT
R: ATTGTTTGGGCAGTTGATCG
F: ACTACCGGGAATTTCATCCC
F: ATCATTCCTGCTCCGACGTA R: CGCACCATTCCCTATTCACT
R: TCAATTAAGTGGCTCCAGGC
F: GGGAAGACAGCGTTGAAGAG
R: TGCAAAATTATGTATGTTCTGGA
F: CAGTACTCATGTGCTATCCTGTATTTG
R: GGGAGAAGTTGTTGGAAGAGG
F: GAACCTTTTCACTTACCAAACCA
R: CGTATTTCAATTGGTTGATCG
F: GAGGGGAATATGCATCATGG
R: AGCTTCAAGGGCACAATGAT
F: TATTGCCGATCCCTCTTGTC
Primer sequence (50 –30 )
AT
AT
CT
AT
AT
AG
AT
AT
CT
AT
AAGACG
AGAT
AATG
ACGT
AT
AT
AT
AT
Motif
Table 1 Microsatellite loci developed for the Australian seagrass Zostera muelleri (syn. Z. capricorni)
VIC
PET
PET
FAM
VIC
VIC
VIC
NED
PET
FAM
NED
PET
NED
FAM
NED
VIC
PET
PET
Fluorescent tag
409–411
403–405
357–375
64–88
313–313
267–269
223–227
195–199
186–186
156–164
146–165
200–232
164–188
120–128
183–187
220–224
200–208
150–174
Size rangea
8
8
8
5
8
8
8
8
8
8
7
7
7
7
7
7
6
8
N
2
2
3
3
1
2
3
2
1
3
2
8
5
3
3
3
2
3
A
0.000
0.000
0.750
0.200
0.000
0.500
0.000
0.500
0.000
0.000
0.429
1.000
0.429
0.429
0.429
0.571
0.500
0.375
HO
0.400
0.233
0.700
0.644
0.000
0.400
0.700
0.400
0.000
0.633
0.363
0.912
0.593
0.385
0.692
0.538
0.409
0.575
UHE
0.461
0.634
0.193
0.265
–
0.461
0.193
0.461
–
0.248
0.497
0.042
0.232
0.445
0.203
0.323
0.461
0.270
PID
ns
ns
ns
ns
N/A
ns
**
ns
N/A
*
ns
ns
ns
ns
ns
ns
ns
ns
HWE
Conservation Genet Resour (2012) 4:689–693 691
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Product size range including the associated fluorescent dye tag (FAM-GCCTCCCTCGCGCCA; NED-GCCTTGCCAGCCCGC; VIC-CAGGACCAGGCTACCGTG; PET-CGGAGAGCCGAGAGGTG) (Blacket et al. 2012)
N number of individuals screened, A number of alleles, HO observed hetrozygosity, UHE unbiased expected hetrozygosity
Significant departures from Hardy–Weinberg equilibrium (* P \ 0.05, ** P \ 0.001, after sequential Bonferroni correction)
a
** 0.248 0.000 3 8 306–324 NED F: CCTACCAACCAATGCTGGC JQ085989 ZosNSW47
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R: GAGGTTGTGGTTTGGACCG
AATT
GAGT R: ACCTGCTCAGTGGTAACGC
JQ085988 ZosNSW46
F: GATTGTTTCCAAATTCCAATAGAGG
ATCT R: TGACGTGTGTTGCGATAAGG
R: ACCCACCCAGAAGATATCGC
JQ085987 ZosNSW45
F: GACACCGGAATGATGGAACC
0.633
ns 0.039 1.000 10 8 FAM
258–314
8 238–258 NED
NED AAGTT JQ085986 ZosNSW44
F: AGTGGTTGAGTTTCCACTTCAC
0.900
ns 0.161 4
0.625
0.725
N/A – 280–280
8
1
0.000
0.000
ns 0.461 0.400 0.500 2 8 212–242 NED AAGTT F: ACTTGTTAAAGCTCACCTGCC JQ085985 ZosNSW43
R: TGTCTAGCCAAATGGCCTTC
Genbank Acc. No. Locus
Table 1 continued
Primer sequence (50 –30 )
Motif
Fluorescent tag
Size rangea
N
A
HO
UHE
HWE
Conservation Genet Resour (2012) 4:689–693
PID
692
Acknowledgments We would like to thank A. Miller and A. Fitch for technical assistance. This work was support by funding provided by the Paddy Palin Foundation and Humane Society International to PM, and funding from the Centre for Integrative Ecology, Deakin University to CS.
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