|Microsatellite multiplex assay for the coral-eating crown-of-thorns starfish, Acanthaster cf. planci
Hugo B. Harrison1,*, Pablo Saenz-Agudelo2,3, Manalle Al-Salamah2, Vanessa Messmer1, Morgan S. Pratchett1, Michael L. Berumen2
1 Centre of Excellence for Coral Reef Studies, James Cook University, Townsville, Queensland 4811, Australia
2 Red Sea Research Center, King Abdullah University of Science and Technology, 23955-6900 Thuwal, Kingdom of Saudi Arabia
3 Instituto de Ciencias Ambientales y Evolutivas, Universidad Austral de Chile, Valdivia, Chile
* Corresponding author: firstname.lastname@example.org
Keywords Acanthasteridae, COTS, population outbreak, Great Barrier Reef
Population outbreaks of crown-of-thorns starfish (Acanthaster spp.) represent one of the most significant biological disturbances on Indo-Pacific coral reefs. Here, we combine 15 published and 11 newly isolated polymorphic microsatellite markers from the coral-eating starfish, A. cf. planci and describe their integration into four multiplex PCRs. All markers were polymorphic with a mean of 11.7 1.9 SE alleles per locus and an average observed heterozygosity of 0.619 0.049 SE across 195 genotyped individuals from the Great Barrier Reef. This multiplex assay provides an effective means of investigating the population dynamics of crown-of-thorns starfish and the initiation and spread of population outbreaks.
Population outbreaks of coral-eating crown-of-thorns starfish (COTS), Acanthaster cf. planci, are responsible for sustained declines in the cover and abundance of reef-building corals throughout the Indo-Pacific region (Pratchett et al. 2014). A lack of understanding of the potential causes and spread of outbreaks remains a major challenge for management interventions attempting to control active outbreaks. Here we describe the isolation and characterization of 26 polymorphic microsatellite markers in four multiplex PCRs. This novel set of markers is aimed at exploring the spatial and temporal dynamics of the initiation and spread of outbreaks of COTS within the northern section of the Great Barrier Reef, Australia.
Tissue samples for 195 COTS were collected from reefs in the northern section of the Great Barrier Reef between November 2013 and September 2014 under the Great Barrier Reef Marine Parks permit G13/36401.1. Individual COTS were collected on SCUBA and tube feet were removed and preserved in 95-100% ethanol. DNA extractions were performed from single tube feet following procedures described in the Nucleospin-96 Tissue kit (Macherey-Nagel, Germany).
All novel microsatellite markers were isolated from a shot-gun genomic library obtained from Ion PGM technology (Ion Plus 400bp fragment library Kit, 318 chip v2). The library was prepared from genomic DNA from an individual collected from the Red Sea (Thuwal). Primers from previously published libraries (Yasuda et al. 2006, 2007; Wainwright et al. 2012) were sometimes redesigned to satisfy the requirements of multiplex PCRs (Table 1). For each marker, the forward primer was labelled with one of the fluorescent dies 6-FAM, VIC, NED or PET.
Selected primer pairs were combined in a primer premix for in-reaction concentrations ranging from 0.02 to 0.06 μM (Table 1), adjusted for even amplification. All four multiplex reactions were performed using the QIAGEN Microsatellite Type-it kit (QIAGEN, Germany) in a total volume of 10 l containing 5 l of QIAGEN Multiplex Master Mix (2x), 1 l QIAGEN Q-solution, 1 l of distilled water, 2 l of primer premix, and 1 l template DNA. Multiplex PCRs were performed on Eppendorf thermal cyclers with the following sequence: 15min initial denaturation at 95˚C, 7 cycles of 30 seconds at 95˚C, 90 seconds at 62˚C, and 60 seconds at 72˚C, then 7 cycles of 30 seconds at 95˚C, 90 seconds at 60˚C, and 60 seconds at 72˚C, then 28 cycles of 30 seconds at 95˚C, 90 seconds at 58˚C, and 60 seconds at 72˚C, followed by 30 minutes at 60˚C. PCR products were screened on an ABI 3370xl DNA Analyzer (Applied Biosystems) with the GeneScan 500 LIZ (Applied Biosystems) internal size standard following a 1:15 dilution. Individual genotypes were scored in genemapper v4.0 and unique alleles were distinguished using marker specific binsets in msatallele (Alberto 2009).
Among 195 individuals genotyped with all 26 loci, the mean number of alleles per locus was 11.7 1.9 SE with an average observed heterozygosity of 0.619 0.049 SE across all samples (Table 1). Five loci showed significant departure from Hardy-Weinberg expectations due to heterozygote deficiency as determined in genepop on the web. Significance levels of 0.05 were adjusted for a given false discovery rate of 10% to account for multiple testing. No loci showed significant pairwise linkage disequilibrium as determined in genepop on the web. When combined, the four multiplex kits provide a cost effective means to investigate a range of population and evolutionary processes in populations of A. cf. planci to inform management actions for coral reef conservation.
We are grateful to volunteers who assisted with sample collections. This project was supported by funding from the Commonwealth Department of Environment (Reef Plan) and ARC Centre of Excellence for Coral Reef Studies to MSP, and KAUST Competitive Research Grant (URF/1/1389-01-01) and baseline research funds to MLB.
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Table 1 Description of 26 microsatellite loci in four multiplex PCRs for the crown-of-thorns starfish, Acanthaster cf. planci.