Conservation Genet Resour DOI 10.1007/s12686-012-9650-5
TECHNICAL NOTE
Development and characterization of 15 polymorphic microsatellite loci in Sonneratia alba (Lythraceae) using next-generation sequencing Yoshimi Shinmura • Alison K. S. Wee • Koji Takayama • Takeshi Asakawa • Orlex Baylen Yllano • Severino G. Salmo III • Erwin Riyanto Ardli • Nguyen Xuan Tung • Norhaslinda Binti Malekal • Onrizal • Sankararamasubramanian Halasya Meenakshisundaram • Sarawood Sungkaew M. Nazre Saleh • Bayu Adjie • Khin Khin Soe • Emiko Oguri • Noriaki Murakami • Yasuyuki Watano • Shigeyuki Baba • Edward L. Webb • Tadashi Kajita
•
Received: 31 March 2012 / Accepted: 3 April 2012 Ó Springer Science+Business Media B.V. 2012
Abstract A set of 15 new microsatellite loci was developed and characterized for the widespread mangrove tree species Sonneratia alba (Lythraceae) by using next-generation sequencing. Forty-eight S. alba samples from seven populations in the Indo-West Pacific region were genotyped; all loci were polymorphic, with the number of alleles ranging from three to eight. The mean observed heterozygosity per locus was 0.21 for a population from Sabah, Malaysia. No null allele or significant linkage disequilibrium was detected, indicating the robustness of the markers. Only one locus (SA103) showed deviation from
Hardy–Weinberg equilibrium. As characterization of these microsatellite loci was done with samples covering most of the species’ distribution range, the markers can be applied to genetic diversity studies over the broad geographical range of the species.
Y. Shinmura T. Asakawa Y. Watano T. Kajita (&) Department of Biology, Graduate School of Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan e-mail:
[email protected]
N. X. Tung Mangrove Ecosystem Research Centre, Hanoi National University of Education, Hanoi, Vietnam
A. K. S. Wee E. L. Webb Department of Biological Sciences, National University of Singapore, Singapore, Singapore K. Takayama Department of Systematic and Evolutionary Botany, University of Vienna, Vienna, Austria O. B. Yllano Biology Department, College of Sciences and Technology, Adventist University of the Philippines, Cavite, Philippines S. G. Salmo III Department of Environmental Science, School of Science and Engineering, Ateneo de Manila University, Quezon City, Philippines E. R. Ardli Faculty of Biology, Jenderal Soedirman University, Purwokerto, Indonesia
Keywords Conservation Diversity Mangrove Pyrosequencing SSR
N. B. Malekal Institute for Tropical Biology and Conservation, Universiti Malaysia, Kota Kinabalu, Malaysia Onrizal Forestry Sciences Department, Universitas Sumatera Utara, Medan, Indonesia S. H. Meenakshisundaram Biotechnology Programme, M.S. Swaminathan Research Foundation, Chennnai, India S. Sungkaew Forest Biology Department, Faculty of Forestry, Kasetsart University, Bangkok, Thailand M. N. Saleh Department of Forest Production, Faculty of Forestry, Universiti Putra Malaysia, Serdang, Malaysia
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Conservation Genet Resour Table 1 Primer sequences Locus SA102
Primer sequence (50 –30 )
Repeat motif
Accession No.
F: GGTTTTCCCAGTCACGACGTCGACCGTATATGCCACCT
(AG)13
AB712237
(AG)13
AB712238
(AG)13
AB712239
(AG)12
AB712240
(ACC)12
AB712241
F: GGTTTTCCCAGTCACGACGAGCAGGAGCTGATGGAGAC R: GTTTGTGCACAGAGGCTATGAGG
(AAG)12
AB712242
F: GGTTTTCCCAGTCACGACGGATTCATACACAGATGGTGGA
(AT)11
AB712243
(AT)11
AB712244
(AG)11
AB712245
(AT)11
AB712246
(AG)11
AB712247
(AG)11
AB712248
(AGG)11
AB712249
F: GGTTTTCCCAGTCACGACGACCGTAGCAATGGTAGGTCG R: GTTTTCAACACGTGTCCTTGCTCT
(AT)11
AB712250
F: GGTTTTCCCAGTCACGACGCAAAGCAACACGCAATAGGA
(AG)10
AB712251
R: GTTTCGGGACGTAAGCCTAAATG SA103
F: GGTTTTCCCAGTCACGACGATCATTGGTCAGGGAGGCTT R: GTTTCAAAGTACCTGAGGGACCCA
SA105
F: GGTTTTCCCAGTCACGACGCATCAGAGGCACACGAAGAA R: GTTTGTCTTTCGAAGTCGCTGA
SA106
F: GGTTTTCCCAGTCACGACGATTCGGCTCCACTGCAATCT R: GTTTAGTCAATGTTCTTGGAGCGG
SA108
F: GGTTTTCCCAGTCACGACGCCTCCATGATGGTGATGAT R: GTTTGACGGTGAGGATGCTTATG
SA109 SA110
R: GTTTAGTCGGTCGTGCATGGATA SA111
F: GGTTTTCCCAGTCACGACGTGACAATGACGCTTGGATG R: GTTTAATGGGCTGACCCATAACTG
SA112
F: GGTTTTCCCAGTCACGACGATGATTGAGTCCGTTCCGTC R: GTTTAGCAGAGCAGTCCACTCGAT
SA113
F: GGTTTTCCCAGTCACGACGCTTTCCCACTATCATTGGC R: GTTTAGTCCCTCATGACGCATTTA
SA114
F: GGTTTTCCCAGTCACGACGGGAGTTCTGAAGCTGAACG R: GTTTGAAGCTATGGAGACACGGG
SA115
F: GGTTTTCCCAGTCACGACGTGAGAGGACCAGCAGAGTGA R: GTTTGAATTAGGGACCTCGGTG
SA117
F: GGTTTTCCCAGTCACGACGCCTCGAAGACGCAGTAAACC R: GTTTCGACGACAAGTGTGAAAGG
SA118 SA123
R: GTTTGAACTATCGCCCAACCCTA
Mangroves are intertidal, water-dispersed plants across the tropical and subtropical region and comprise a small number major component species (Spalding et al. 2010).
B. Adjie Bali Botanical Garden, Indonesian Institute of Science, Bali, Indonesia K. K. Soe Department of Botany, University of Yangon, Yangon, Myanmar E. Oguri N. Murakami Makino Herbarium, Graduate School of Science and Engineering, Tokyo Metropolitan University, Tokyo, Japan S. Baba Tropical Biosphere Research Center, University of the Ryukyus, Iriomote, Okinawa, Japan
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Although having exceptional ecological and economic value, mangrove systems are highly threatened globally (Duke et al. 2007), and particularly in Asia (Giri et al. 2008). Sonneratia alba Griff. (Lythraceae) is found in the Indo-West Pacific (IWP) region, with a distribution ranging from East Africa to the Pacific islands of Vanuatu (Spalding et al. 2010). Although it is not listed as threatened (Polidoro et al. 2010), there are important long-term genetic implications for all IWP mangroves under the current conditions of rapid deforestation and degradation (Giri et al. 2008). Baseline genetic diversity information is lacking for most IWP species, which is crucial because previous studies suggest strong genetic structure among mangrove populations from different subregions of the IWP (Triest 2008). Moreover, several molecular phylogenetic studies revealed the presence of cryptic species in wide-spread mangrove species [e.g., Kandelia (Sheue et al.
Conservation Genet Resour Table 2 Characteristics of 15 microsatellite loci in for 7 populations of Sonneratia alba Locus
Allele size range (bp)
All
Sabah Malaysia (n = 24)
NA
NA
HO
Mumbai India (n = 4)
Sumatra Indonesia (n = 4)
Java Indonesia (n = 4)
Panay Philippines (n = 4)
Ca Mau Vietnam (n = 4)
Trat Thailand (n = 4)
HE
NA
NA
NA
NA
NA
NA
SA102
161–175
5
2
0.04
0.04
3
2
1
3
1
1
SA103
198–204
4
3
0.58
0.54
3
2
2
2
1
1
SA105
194–202
4
1
0.00
0.00
3
1
2
2
1
1
SA108
144–150
4
1
0.00
0.00
2
1
2
3
1
1
SA109
191–212
7
1
0.00
0.00
3
2
2
4
1
1
SA110
144–152
7
2
0.50
0.48
4
2
3
4
2
3
SA111
255–263
6
4
0.33
0.44
3
3
4
3
2
3
SA112
103–115
5
3
0.38
0.57
3
1
2
2
1
1
SA113
190–206
8
2
0.67
0.50
4
3
3
1
4
2
SA114
295–299
3
1
0.00
0.00
2
2
2
3
2
2
SA115
194–200
4
1
0.00
0.00
2
2
3
3
1
1
SA117
179–187
5
2
0.04
0.04
1
1
2
2
1
2
SA118
208–222
6
1
0.00
0.00
2
2
1
6
1
2
SA119 SA123
286–292 203–207
4 3
2 2
0.04 0.50
0.12 0.50
2 3
1 2
1 2
3 1
1 3
1 1
Mean
5.0
1.9
0.21
0.21
2.7
1.8
2.1
2.8
1.5
1.5
NA number of alleles, HO observed heterozygosity, HE expected heterozygosity
2003) and Ceriops (Ballment et al. 1988)]. To fill this gap, we characterized a set of polymorphic microsatellite loci using samples across a broad area of the IWP region. Marker development and characterization was conducted using 48 samples of S. alba from seven populations in S-SE Asia: 24 individuals from Sabah (Malaysia) and four from each of six populations at Panay (Philippines), Trat (Thailand), Ca Mau (Vietnam), Sumatra (Indonesia), Java (Indonesia) and Mumbai (India). Samples were first dried in silica gel, followed by genomic DNA extraction using the CTAB method (Doyle and Doyle 1987) or the DNeasy Plant mini kit (QIAGEN) according to the manufacturer’s protocol. Total DNA of one sample from Sabah was subjected to shotgun sequencing using a Roche 454 Genome Sequencer Junior with the GS Junior Titanium Sequencing Kit at Tokyo Metropolitan University, following the manufacturer’s protocol. The sample was sequenced with one-third of the run by using Multiplex Identifier (MID) adaptors (see Margulies et al. 2005). The program QDD v.2.1 (Megle´cz et al. 2010) was used to detect and select microsatellite sequences and to design primer pairs. A total of 510 perfect microsatellite sequences with repeat motifs of two to six nucleotides were selected for further analysis from in total 18,358 sequence reads. The criterion for selection was a minimum of five repeats for all motifs. Detection of sequence similarity and establishment of contigs and consensus sequences was conducted according to the procedure of Takayama et al. (2011).
Primer pairs were designed with Primer3 (Rozen and Skaletsky 1999), implemented in QDD with the following criteria: (1) PCR product size of 80–300 bp; (2) flanking regions containing B5 repetitions of any di- to hexanucleotide motifs; and (3) primers with length 18–27 bp, annealing temperature 57–63 °C, and GC content 20–80 %. Twenty-four primer pairs having[9 repeats with various fragment sizes appropriate for multiplex PCR were selected. The 50 -tailed primer method (Schuelke 2000) was used to label PCR amplicons of the selected primers for visualization. The 50 -tail of forward primers was added with the 19-bp U19 sequence (GGTTTTCCCAGTCACGACG). The GTTT ‘‘pigtails’’ were added to the 50 end of the reverse primer to reduce stuttering by facilitating the addition of adenosine by Taq polymerase (Brownstein et al. 1996). PCR amplification, reproducibility and level of polymorphism of the 24 primer pairs were tested on all 48 samples. PCR was conducted in a final volume of 5 ll using the standard protocol of Qiagen Multiplex PCR Kit with simplex PCR. For the 50 -tailed primer method, three primers were used in the reaction—0.2 lM reverse primer, 0.2 lM fluorescent dye labeled primer (6-FAM, VIC, NED, or PET), and 0.04 lM forward primer with the following PCR protocol: initial denaturation at 95 °C for 15 min; 25 cycles of denaturation at 95 °C for 30 s, annealing at 63 °C for 90 s (decrement of 0.5 °C per cycle), extension at 72 °C for 60 s; and 20 cycles of denaturation at 95 °C for 30 s,
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Conservation Genet Resour
annealing at 53 °C for 90 s, extension at 72 °C for 60 s; final extension at 60 °C for 30 min. Genotyping of PCR amplicons was conducted with the ABI 3130xl automated DNA sequencer (Applied Biosystems) under standard conditions. Results were analyzed with GeneMapper v4.1 (Applied Biosystems). Fifteen loci showed polymorphism among the seven populations (Tables 1, 2). The number of alleles per locus (NA) varied from three to eight with a mean of 5.0. All populations were polymorphic with the mean NA ranging from 1.5 to 2.8. We performed validation tests [Hardy-Winberg equilibrium (HWE), null alleles and linkage disequilibrium (LD)] on the population from Sabah. In that population the NA varied from one to four (mean 1.9) and the observed heterozygosity ranged from 0 to 0.67. Only SA103 significantly departed from HWE. Testing for the presence of null alleles was accomplished using Micro-checker v2.2.3 (Van Oosterhout et al. 2004), and was not significant at all loci. The LD test was performed using GENEPOP 4.0 (Raymond and Rousset 1995) and FSTAT (Goudet 2001). No LD was observed for any loci pair, with the exception of SA111 and SA112. Because the set of 15 polymorphic microsatellites were developed from samples covering most of the species’ distribution range, these markers can be applied to phylogeographic and conservation genetic studies of S. alba. Acknowledgments The authors thank Drs. Sanjay Deshmukh (Mumbai), Romanus Prabowo (Java), Jurgenne Primavera (Panay), Monica Suleiman (Sabah), Mr. Seigo Murakami (KKWC) and Department of Environment and Natural Resources, Region VI (Panay) for field works to collect materials. We also thank Mr. Junya Ono, Mr. Tsuyoshi Takano and Mr. Futa Yamaji for their help in DNA extraction. This work was supported by the Singapore Ministry of Education (grant number R154-000-440-112), by the JSPS KAKENHI 22405005 to TK, and JSPS JENESYS Programme 2009 and 2011 to the Graduate School of Science, Chiba University.
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