Development of novel microsatellite markers for Cymodocea rotundata Ehrenberg (Cymodoceaceae), a pioneer seagrass species widely distributed in the Indo-Pacific Dan M. Arriesgado, Yuichi Nakajima, Yu Matsuki, Chunlan Lian, Satoshi Nagai, Motoshige Yasuike, Yoji Nakamura, Miguel D. Fortes, et al. Conservation Genetics Resources ISSN 1877-7252 Conservation Genet Resour DOI 10.1007/s12686-013-0025-3
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Author's personal copy Conservation Genet Resour DOI 10.1007/s12686-013-0025-3
MICROSATELLITE LETTERS
Development of novel microsatellite markers for Cymodocea rotundata Ehrenberg (Cymodoceaceae), a pioneer seagrass species widely distributed in the Indo-Pacific Dan M. Arriesgado • Yuichi Nakajima • Yu Matsuki • Chunlan Lian • Satoshi Nagai • Motoshige Yasuike • Yoji Nakamura • Miguel D. Fortes • Wilfredo H. Uy • Wilfredo L. Campos • Masahiro Nakaoka • Kazuo Nadaoka
Received: 22 July 2013 / Accepted: 12 August 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Genetic diversity and population genetic structure are key components of seagrass resilience and contribute to an understanding of its conservation and management. We isolated 29 polymorphic microsatellite (SSR) markers from a widely distributed pioneer seagrass, Cymodocea rotundata, by two methods; next generation sequencing and compound SSR marker isolation. Twentynine markers had 2–14 alleles per locus, and the observed and expected heterozygosity ranged from 0 to 0.688, and 0.113 to 0.770, respectively. These loci will facilitate investigation of the genetic diversity and population genetic connectivity and structure of C. rotundata. Keywords Clonal plant Simple sequence repeat Cymodocea rotundata Pioneer Seagrass
D. M. Arriesgado K. Nadaoka Department of Mechanical and Environmental Informatics, Graduate School of Information Science and Engineering, Tokyo Institute of Technology, 2-12-1 O-okayama, Meguro, Tokyo 152-8552, Japan D. M. Arriesgado Y. Nakajima Y. Matsuki C. Lian (&) Asian Natural Environmental Science Center, The University of Tokyo, 1-1-8 Midori-cho, Nishitokyo, Tokyo 188-0002, Japan e-mail:
[email protected] D. M. Arriesgado W. H. Uy Institute of Fisheries Research and Development, Mindanao State University, 9023 Naawan, Misamis Oriental, Philippines
The pioneer species, Cymodocea rotundata, is one of the most important and widespread seagrasses. It can colonize diverse environments, and often forms a fringing bed, being present in areas exposed to impact from coastal development and high anthropogenic activity (Terrados et al. 1998). C. rotundata is a clonal plant that can reproduce either sexually by seed production or asexually by vegetative propagation of rhizomes. This species is included in the Catalogue of Endangered Species, yet no specific conservation measures are currently in place, and the genetics of this species have not been investigated. In this study, microsatellite (SSR) markers of C. rotundata were developed in order to assess the genetic diversity, connectivity and population genetic structure throughout its distribution range. Genomic DNA was extracted from silica-gel dried leaves of C. rotundata collected from Rizal, Zamboanga
M. D. Fortes Marine Science Institute CS, University of the Philippines, Diliman, 1101 Quezon City, Philippines W. L. Campos Division of Biological Sciences, College of Arts & Sciences, University of the Philippines Visayas, 5023 Miag-ao, Iloilo, Philippines M. Nakaoka Akkeshi Marine Station, Field Science Center for Northern Biosphere, Hokkaido University, Aikappu, Akkeshi, Hokkaido 088-1113, Japan
S. Nagai M. Yasuike Y. Nakamura Harmful Algal Bloom Division, Toxic Phytoplankton Section, National Research Institute of Fisheries and Environment of Inland Sea, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan
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Author's personal copy Conservation Genet Resour Table 1 Characteristics of the twenty-nine microsatellite loci developed from C. rotundata Locus name
Repeat motif
Protocol
Size range (bp)
Primer sequences (50 –30 )
Cymrot001
(AT)12
NGS
110–114
F:U19-CTCATCAAGAACAAGAGCACA
Cymrot002
(TC)13
NGS
312–316
Na
Ho
He
FIS
Accession no.
2
0.042
0.353
0.882
AB845675
3
0.063
0.119
0.473
AB845676
5
0.563
0.77
0.269
AB845677
F:GGAAAACTTCAGCCAATACCAG R:U19-TGGTGCAGCCATAAGACTAAGA
4
0.531
0.615
0.136
AB845678
F:ACACACACACACACAGAGAG
8
0.688
0.728
0.056
AB845679
2
0.125
0.469
0.733
AB845680
3
0.375
0.654
0.427
AB845681
4
0.156
0.202
0.225
AB845682
2
0.032
0.481
0.933
AB845683
2
0.156
0.242
0.354
AB845684
R:ATGAAGTGTGACCTGCTACACG F:U19-ATGTCGTAGTCCTGGAGGATGT R:TCGATCATGGAACGAACAGAT Cymrot003
(AG)5
C
124–134
F:ACACACACACACACAGAGAG R:GTGCATCTAATATAACGCCCTAG
Cymrot004
(CA)15
NGS
220–228
Cymrot006
(AG)8
C
91–115
R:GCCTGATAAATGTCATTAATTCTT Cymrot008
(CT)12
NGS
124–128
Cymrot009
(TC)5
C
125–129
F:U19-TTCTTTGGCCGTTGTTTGT R:GTACAGAACGGAAGTGCGTGT F:ACACACACACACACTCTCTC R:CTTGCATGGCTCCAACATGG
Cymrot015
(TC)12
NGS
144–158
F:U19-AGTTTATGGTGTCCCTGAGCAT R:GTGTTTGTTGATGCGGTGTACT
Cymrot017
(AT)13
NGS
308–312
Cymrot019
(CA)13
NGS
320–324
F:U19-CTTTTGCCCCTCCTATGAGTTA R:CACCTTCAAGTCCAAGTCCTTC F:U19-TCCTTGCCTTTTTAATTAGTTTTT R:AAGTTCACAAAGCTGATCCCAT
Cymrot027 Cymrot032
(AG)12 (TA)13
NGS NGS
311–315
F:GCTGTCCCCAGATAGACAAATC
3
0.313
0.385
0.189
AB845685
154–158
R:U19-TTTCTTATTCTGTGCAGGCTGA F:U19-AATTCAGTTTTTCGAAAGGTTTT
3
0.526
0.65
0.190
AB845686
6
0.313
0.713
0.562
AB845687
5
0.516
0.623
0.171
AB845688
2
0.313
0.451
0.307
AB845689
4
0.000
0.502
1.000
AB845690
3
0.156
0.5
0.688
AB845691
3
0.094
0.426
0.780
AB845692
R:AATGTAGGTATAGGGGAGCGGT Cymrot039
(AG)16
NGS
310–326
F:M13R-TAAAAATAATTGGTGCGGGAG R:TCTGATTTTTCACCCAATACCC
Cymrot040
(AT)13
NGS
186–196
Cymrot044
(TG)14
NGS
245–249
F:U19-GCAAATTAAGTGCATTTTTCGAT R:TCTATCTATCTCACGCGCTCTT F:AACGATTAGGGCGACAAATCTA R:U19-TAAGGATCAGAAGGGTCAACG
Cymrot144
(AG)23
NGS
332–346
F:U19-TTCATGAATAATCTTCTCGATGC R:AAAAAGTCAAATTTCAAGCCCA
Cymrot145
(CA)15
NGS
280–284
Cymrot149
(AG)19
NGS
188–192
F:U19-AATGAGGAGGTGGATTGAGAAA R:TATGATCCTGTGCTTGTGATCC F:U19-TGGATATGCTAGACTGAAGGCA R:AAAACTGCCAAAGAAGAACTGC
Cymrot150 Cymrot151
(AG)18 (TA)21
NGS NGS
412–418
F:U19-ATCAGAGGCTTTTGGACACATT
4
0.031
0.427
0.927
AB845693
242–244
R:TCGTTTGTCGTGCATCTTAATC F:TCAACAAAGCTGTGGGAAAGTA
2
0.040
0.113
0.645
AB845694
4
0.250
0.51
0.509
AB845695
3
0.222
0.623
0.643
AB845696
4
0.219
0.657
0.667
AB845697
2
0.000
0.117
1.000
AB845698
R:U19-AGATCTGTGAGACAAACGCAGA Cymrot152
(AT)15
NGS
186–192
F:ATGTATGCGCGTTTGTTTGTAG R:U19-GAAGCCCTTAGTTTGCCTATT
Cymrot153
(AT)19
NGS
116–122
Cymrot157
(AG)17
NGS
352–358
F:U19-CACTTCATCATCACTTGGAGGA R:AAGACACTGTAATGCCATGCTG F:U19-ATGTGAGAGTTGCATTTTCCAA R:AGATAGTCGGTTCGGTGTTGAT
Cymrot158
(GA)17
NGS
247–249
F:U19-ACCACAACTGCCTTCTCTCTT R:GGAGAACTGTAACCGACAGACC
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Author's personal copy Conservation Genet Resour Table 1 continued Locus name
Repeat motif
Protocol
Size range (bp)
Cymrot160
(AT)15
NGS
242–254
Primer sequences (50 –30 )
F:U19-AGCTCAAATTACGAAGCGGATA
Na
Ho
He
FIS
Accession no.
4
0.125
0.315
0.603
AB845699
14
0.469
0.732
0.360
AB845700
3
0.156
0.201
0.221
AB845701
3
0.276
0.482
0.428
AB845702
4
0.258
0.283
0.087
AB845703
R:TTAAATATGCTGGACCGTCAAA Cymrot162
(AT)16
NGS
293–327
F:U19-TGAGGCCGACCTTAATACTCAT R:CCGTCCAATGTCTGCAATACTA
Cymrot163
(TA)15
NGS
346–362
Cymrot164
(AT)18
NGS
193–197
F:ATGATGCCAAAAGTATGCGTAA R:U19-GGCAGATATGACATAATGGGT F:U19-AGTTAGCTTTCACCGCAGGTAG R:TATTGAAAGAAACGTGCATTGG
Cymrot166
(AT)16
NGS
360–366
F:U19-TGTCATTGCCTTAATGTTTTGC R:TGCCATAATGACAAAATTCACA
Number of samples genotyped is 32 individuals collected from different sites; Philippines (20 individuals), Japan (11 individuals) and China (1 individual) C compound method, NGS next generation sequencing, Na number of alleles, Ho observed heterozygosity, He expected heterozygosity, FIS deviation index from Hardy-Weinberg Equilibrium (HWE). FIS value in bold indicates significant deviation from Hardy–Weinberg equilibrium (P \ 0.05)
del Norte in the Philippines (8°350 000 N, 123°320 000 E), using a modified cetyltrimethylammonium bromide (CTAB) method (Lian et al. 2003). Two methods were employed to isolate SSR markers, a technique for isolating codominant compound SSR markers, and next-generation sequencing (NGS). DNA libraries were constructed to isolate codominant compound SSR markers, using two blunt-end restriction enzymes, EcoRV and HaeIII described by Lian et al. (2006). The adapter-ligated fragments were amplified from the EcoRV or HaeIII DNA library using compound SSR primers (AC)6(AG)5, (AG)6(AC)5, (AC)6(TC)5 or (TC)6(AC)5 and the adaptor primer AP2 (50 -CTATAGGGCACGCGTGGT30 ). The amplified fragments were subcloned and sequenced following the method described by Lian et al. (2006). A specific primer (IP1) was designed from the region flanking the compound SSR sequence. The IP1 and corresponding compound SSR primers were used as SSR markers. For next-generation sequencing, C. rotundata was sequenced using the GS-FLX ? system (454 Life Sciences by Roche Diagnosis, Basel, Switzerland) following the protocol for a one-fourth run. Sequence fragments ranging from ca. 300 to 800 bp were obtained for 5.7 9 105 reads. Sequences containing di-, tri-, tetra- and pentanucleotide repeats were screened and PCR primers were designed to amplify sequence regions containing microsatellites using Auto-primer (Nakamura et al. 2013). To examine polymorphisms of the isolated SSR markers, we performed a PCR in a 10-lL reaction mixture containing approximately 10 ng of template DNA, 1 9 PCR buffer II, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.125 U of Ampli Taq Gold (Applied Biosystems) and each of the designed primers. For the loci isolated by the
compound SSR method, the corresponding compound SSR markers were labeled with 6-FAM, VIC, NED, and PET fluorochromes (applied biosystems). For the loci developed by the next-generation sequencing method, a U19 (50 GGTTTTCCCAGTCACGACG-30 ) or M13 (50 -CAGGAAACAGCTATGAC-30 ) was tailed to the 50 end of one of the primer pairs, and the U19 or M13R primer labeled with 6-FAM, VIC, NED, or PET, was added to the PCR reaction mix. The PCR reaction was performed using the following cycling conditions; denaturation at 95 °C for 9 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C (all loci) for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 5 min. The PCR amplicons were electrophoresed and quantified using an ABI 3130 xl genetic analyzer and GENEMAPPERTM analysis software (Applied Biosystems), respectively. Of the SSR loci isolated, twenty-nine loci were successfully amplified and yielded one- or two-bands products. These 29 loci were polymorphic in the tested populations (Table 1). The observed and expected heterozygosity values ranged from 0 to 0.688 and 0.113 to 0.770, respectively. Deviation from the Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium between the loci were tested after Bonferroni correction using FSTAT, version 2.9.3 (Goudet 1995). The fixation index FIS was significantly different from zero with relatively high positive values. Fifteen loci significantly deviated from HWE (P \ 0.05) (Table 1), suggesting either selection or inbreeding in the natural environment. Three pairs of loci were found to be significantly linked (Cymrot019 and Cymrot044, Cymrot004 and Cymrot166, and Cymrot152 and Cymrot166) (P \ 0.01). The microsatellite markers
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Author's personal copy Conservation Genet Resour
described here will facilitate population genetic studies of C. rotundata for conservation and management. Acknowledgments This study was supported by the Japanese Science and Technology Agency/Japanese International Cooperation Agency-Science and Technology Research Partnership for Sustainable Development (JST/JICA-SATREPS) for the integrated Coastal Ecosystem Conservation and Adaptive Management project under Local and Global Environmental Impacts in the Philippines (CECAM).
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