Development of microsatellite markers from four Hawaiian corals: Acropora cytherea, Fungia scutaria, Montipora capitata and Porites lobata
Comprehensive population genetic studies of coral communities are comparatively rare, because of the scarcity of population genetic markers. The Hawaiian archipelago offers a unique perspective into understanding the population genetic structuring of this ecologically important group of organisms. Here we report the development of microsatellite marker libraries from holobiont extracts of four corals: Acropora cytherea (n = 50), Fungia scutaria (n = 118), Montipora capitata (n = 140) and Porites lobata (n = 149). Blast searches indicate that these libraries contain microsatellites from both the coral host and Symbiodinium endosymbionts from each coral. In addition, we also present redesigned primers for the nuclear coding region (atpsβ) for use in M. capitata. We report testing and optimization for seven of these microsatellites from A. cytherea, and eight microsatellites and the atpsβ locus from M. capitata. Using 25 individuals per species collected from each French Frigate Shoals (FF) and Johnston Atoll (JO), the number of alleles per locus ranged from 2 to 9. Expected heterozygosities ranged from 0.38 to 0.85 and 0.08 to 0.87 for A. cytherea and M. capitata, respectively. We expect that these libraries will be a valuable resource and provide additional useful microsatellite markers for both the coral host and zooxanthellae.
KeywordsScleractinian Zooxanthellae Symbiodinium Connectivity Population genetics Phylogeography
Spanning 2,600 km, and ranging in age from a few hundred thousand to more than 60 million years, the isolated volcanic archipelago of Hawai‘i offers a unique opportunity to understand population connectivity in the marine environment. For sessile benthic marine organisms, the larval phase of the life history is the only part of the life cycle that allows for gene flow between populations, but it is difficult to track miniscule larvae across oceanic scales (Levin 2006). In order to gain insight into the historical patterns of coral colonization of the archipelago, microsatellite markers are incredibly useful tool for understanding population genetic structuring and inferring patterns of connectivity (Selkoe and Toonen 2006).
Locus name, repeat motif, primer sequences, annealing temperature (Ta) and approximate size of expected product for one nuclear coding region and eight microsatellites for M.capitata and seven microsatellites from A. cytherea
Primer sequence (5′-3′)
F: TGA TTG TGT CTG GTG TAA TCA GC
R: CGG GCA CGG GCG CCG GGG GGT TCG TTC AT
F: T3—TGA AAT AAG CAG GAT CCA TGT G
R: AGG TAA ATG CCA GAA TTG GAA A
F: T1—AAG AAC ACC AAA CAA CCG AAC T
R: GGT TAG CGC TCT TGT GCT AAA T
F: T4—TTA TTT CTC GTG TAT CGC CCT T
R: AGA CAG AGC GGT TGG TGT AAA T
F: T2—CAG TGC GAG AAC GAC TAG AGA C
R: AAA ATG ACA AGC ATG TTG GTG T
F: T3—CTT TCA AGG TGT TGA TGC CAT A
R: GGC ACA TCA TGA GAA CAT CAG T
F: T4—TGG CCA GCT TAG TGT TAG TTG A
R: GTT CTT GTA TTT GAC TTG CCC C
F: T1—CAT CTA GAA TTA GCG GGA TGC T
R: CAG AAG TTC CGA CTT TCG ACT T
F: T2—TAG GGG TAA GGA AGG TTG AAC A
R: AAG GGA AAC GGT AAG ACA TGA A
F: T2—GCG AAA GAG ATT CCG TTA GAG A
R: AAT GGG CTC AAT TTC CCT TAA T
F: T3—TTT TAG CTG GAG ATG ACG ATG A
R: TAA CAG GAA AAG GGA AAC AAG G
F: T1—ACA AAA TAA CCC CTT CTA CCT
R: CTT CAT CTC TAC AGC CGA TT
F: T2—CTT GAC CTA AAA AAC TGT CGT ACA A
R: GTT ATT ACT AAA AAG GAC GAG AAT AAC TTT
F: T4—TAA TGA GCA AAC TCA TTC ATG G
R: CTT TTC CAA GAG AAG TCA AGA A
F: T4—CTG TGG CCT TGT TAG ATA GC
R: AGA TTT GTG TTG TCC TGC TT
F: T3—GCA AGT GTT ACT GCA TCA AA
R: TCA TGA TGC TTT ACA GGT GA
Due to time constraints and cost, primers were ordered and tested for only the first 50 putative loci from M. capitata and A. cytherea. Primers were screened with a low annealing temperature (48°C) PCR against DNA extracts from pure zooxanthellae cultures provided by RA Kinzie believed to represent clades A, B and C isolated from Cassiopea sp. (KB8), Aiptasia pulchella (HIAp) and Montipora verrucosa (Mv), respectively. They were subsequently screened against host genomic DNA with an annealing temperature of 55°C. We used the tailed three primer method described by Gaither et al. (2009). Tails were added to the 5′ end of all forward primers that successfully amplified host genomic DNA, but not symbiont DNA (Table 1). PCRs were as follows: each 10 μl reaction contained: 1 μl 10×NH4 reaction buffer, 0.6 μl 50 mM MgCl2, 0.4 μl 10 mM total dNTPs (2.5 mM each), 0.35 pmol tailed forward primer, 1.5 pmol reverse primer, 1.5 pmol oligonucleotide dye label, 2–25 ng of template DNA, 0.1 μl of Biolase polymerase (Bioline Inc.), and deionized water to volume. PCR amplification was performed on a BioRad MyCycler™ as follows: 95°C for 10 min (1 cycle), 94°C for 30 s, 55°C for 30 s, 72°C for 30 s (35 cycles), followed by a final extension of 72°C for 30 min (1 cycle).
From each sampling location for each of eight microsatellites and one nuclear coding region isolated for Montipora capitata and two microsatellites isolated from Acropora cytherea plus five additional microsatellites isolated from congener Acropora millepora, we report sample size (N), number of alleles (Na), observed heterozygosity (Ho); expected heterozygosity (Ne); inbreeding coefficient (Fis), P-values for tests of departure from HWE (Phwe), and Brookfield-1 estimator of null alleles (Nullest). Significant values for Phwe after correction with a false discovery rate α = 0.05 shown in bold. Corrected α for M. capitata (P < 0.006) and A. cytherea (P < 0.014)
Additionally, forward and reverse primers for ATP synthethase β (atpsβ) (Jarman et al. 2002) were redesigned for specificity in the genus Montipora with PCR conditions following Concepcion et al. (2008). This locus was also sequenced directly for each individual from each population (n = 50). Computational methods for determining phase of diploid sequence data is more cost effective and can be as accurate as cloning (Harrigan et al. 2008). Therefore, we used Phase (Stephens et al. 2001; Stephens and Donnelly 2003) as implemented in DnaSP (Librado and Rozas 2009) for determining gametic phases (Table 2).
The use of microsatellites to study coral population genetics is still in its infancy. We hope this library of both characterized, and untested microsatellite loci will provide a wealth of population genetic tools to aid studies both inside and outside of Hawai‘i, in species of corals other than the four for which they were isolated, as well as providing possible microsatellite markers for the symbionts (Symbiodinium spp.)
We’d like to thank R. Brainard, M. Crepeau, E. Franklin, S. Godwin, M. Iacchei, J. Maragos, J. Salerno, D. Skillings, M. Stat, M. Timers, the staff of the Papahānaumokuākea Marine National Monument, and the crew of the R/V Hi’ialakai for sample collection, field and laboratory assistance. We also thank members of the Toonen-Bowen laboratory for their discussion, advice and support, and the NSF-EPSCoR Evolutionary Genetics facility at HIMB. This research was funded in part by the National Science Foundation (Grant OCE-0550294 to IBB, OCE- 0623678 to RJT, an NSF pre-doctoral fellowship to NRP, and an NSF EPSCoR pre-doctoral fellowship to GTC) and the National Oceanic and Atmospheric Administration (NMSP MOA#2005-008/66882). This is contribution 1366 from the Hawai‘i Institute of Marine Biology and SOEST 7818.
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