Abstract
We characterized 37 new microsatellite markers in the Pacific lion-paw scallop (Nodipecten subnodosus) and tested for cross-amplification in four other species. Genetic diversity was estimated using 24 individuals from the Lagoon Ojo de Liebre, B.C.S., Mexico. Allelic richness varied from 5 to 27 alleles per locus and the average expected heterozygosity was 0.76. Ten loci exhibited significant departure from Hardy–Weinberg equilibrium likely due to the presence of null alleles. Sixteen of these markers cross-amplified in closely related N. nodosus, while little or no amplification was observed in three Argopecten species.
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Prior work identified 35 polymorphic microsatellite loci in the Pacific lion-paw scallop (Nodipecten subnodosus) (Ibarra et al. 2006) for use in conservation and population genetics of this large pectinid. Aggregations of this species are found around the coast of the Baja California Peninsula, Mexico, where they are both fished and cultured for human consumption. Like many marine species, populations have recently declined due to harvest pressures. In addition to characterizing the genetic diversity within and between populations, the development of microsatellite markers will allow for genetic mapping of the genome and eventually the discovery of the genetic basis of traits such as fitness and growth. With 19 chromosomes (N), and only 35 markers identified prior to this work, these additional markers are necessary to enable genetic mapping of this species and will also be valuable for continued population level investigations in the wild and in aquaculture.
Clones (n = 480) enriched for the repeats (TAGA)n, (TGAC)n, (TACA)n, and (ATC)n from the mixed scallop-crayfish library described in Ibarra et al. (2006) were sequenced. These sequences were aligned with previously identified N. subnodosus microsatellites using Sequencher v4.7 (Gene Codes) to avoid duplication. Novel sequences were input into mReps (Kolpakov et al. 2003) to determine repeat regions and primers flanking the repeats were designed with Primer 3 (Rozen and Skaletsky 2000).
Because the enriched library was created from two species, the primers were first screened for amplification using genomic DNA of four N. subnodosus and four Shasta crayfish (Pacifastacus fortis) individuals. PCR reactions for the initial screening were identical to those in Ibarra et al. (2006). Thermalcycler conditions consisted of 3 min at 94°C followed by 31 cycles of 94°C for 30 s, 50°C for 30 s, 72°C for 30 s, and a final extension of 10 min at 72°C. PCR products were separated and visualized as described in (Ibarra et al. 2006).
Of the 106 designed primer pairs, 37 (34.9%) amplified and were polymorphic in N. subnodosus. For population screening and cross-amplification tests, the forward primer of each pair was either labeled with a 5′ fluorophore, or one of four 5′ modifications were added (universal primers: T7T, T7P, M13, SP6) using the method of (Schuelke 2000). In this method, the “tail” consisting of the universal primer sequence is incorporated into the template during early rounds of PCR. An additional third labeled oligo then targets these tails, incorporating the fluorophore and allowing for visualization of the fragment. The four tails were each labeled with a different fluorophore (T7T = PET, T7P = NED, SP6 = VIC, M13 = 6-FAM) allowing for multiplexing of PCR reactions.
Twenty-four N. subnodosus collected from the Lagoon Ojo de Liebre on the Pacific coast of the Baja California Peninsula, Mexico, were genotyped at all 37 markers. Amplification was attempted for three individuals each of Argopecten irradians (USA, west Atlantic), Argopecten purpuratus (Chile, east Pacific), Argopecten ventricosus (Mexico, east Pacific), and Nodipecten nodosus (Venezuela, west Atlantic) to assess cross-amplification success.
For primers directly labeled with 5′- 6-FAM, VIC, NED, or PET, 10 μl PCR reactions included 5–10 ng of genomic DNA, 1X buffer (Roche), 2 mM MgCl2, 0.2 mM of each dNTP (Promega), 10 pmol each primer, and 0.4U FastStart Taq DNA polymerase (Roche). The thermal cycler profile was identical to the initial screen, except annealing temperatures varied depending upon the marker (Table 1).
PCR reactions for tailed primers differed by using 7.5 pmol tailed forward primer, 22.5 pmol reverse primer, and 1.5 pmol of fluorescently labeled oligo. The thermal cycler profile for tailed primers consisted of 5 min at 94°C followed by a marker dependent number of cycles of 94°C for 45 s, Ta for 90 s, and 72°C for 1 min, additional cycles of 94°C for 45 s, Ta for 90 s, and 72°C for 1 min, and a 10 min final extension at 72°C (see Table 1 for Ta and cycle number).
PCR products were diluted with 70 μl nanopure water and 1.5 μl was added to 8.8 μl of highly deionized formamide (Gel Company) and 0.2 μl of LIZ600 size standard (ABI). The samples were denatured for 3 min at 95°C before electrophoresis on an ABI 3130xl Genetic Analyzer. Fragments were scored using GeneMapper 4.0 (ABI). GDA (Lewis and Zaykin 2001) was used to calculate observed and expected heterozygosities, allelic richness, and to check for deviations from Hardy–Weinberg Equilibrium (HWE) using Fisher’s exact test with 10,000 permutations.
The 37 loci showed high genetic diversity in N. subnodosus with 5–27 alleles per locus (avg = 15.3) and average expected heterozygosity of 0.76 (Table 1). Ten loci deviated significantly from HWE, all in the direction of heterozygote excess. Though other explanations are possible this is likely due to null alleles, prevalent in marine mollusks (Hedgecock et al. 2004; McGoldrick et al. 2000; Reece et al. 2004), and observed in prior parentage analysis of this species (Petersen et al. 2008). Cross amplification was not observed in A. ventricosus but was successful at 16 loci in N. nodosus and five and two loci, respectively, in A. irradians and A. purpuratus (Table 2).
References
Hedgecock D, Li G, Hubert S, Bucklin K, Ribes V (2004) Widespread null alleles and poor cross-species amplification of microsatellite DNA loci cloned from the Pacific oyster, Crassostrea gigas. J Shellfish Res 23:379–385
Ibarra AM, Petersen JL, Famula TR, May B (2006) Characterization of 35 microsatellite loci in the Pacific lion-paw scallop (Nodipecten subnodosus) and their cross-species amplification in four other scallops of the pectinidae family. Mol Ecol Notes 6:153–156
Kolpakov R, Bana G, Kucherov G (2003) mreps: efficient and flexible detection of tandem repeats in DNA. Nucleic Acid Res 31:3672–3678
Lewis PO, Zaykin D (2001) Genetic data analysis: computer program for the analysis of allelic data. http://lewis.eeb.uconn.edu/lewishome/software.html
McGoldrick DJ, Hedgecock D, English LJ, Baoprasertkul P, Ward RD (2000) The transmission of microsatellite alleles in Australian and North American stocks of the Pacific oyster (Crassostrea gigas): selection and null alleles. J Shellfish Res 19:779–788
Petersen JL, Ibarra AM, Ramirez JL, May B (2008) An induced mass spawn of the hermaphroditic lion-paw scallop, Nodipecten subnodosus: genetic assignment of maternal and paternal parentage. J Hered 99:337–348
Reece KS, Ribeiro WL, Gaffney PM, Carnegie RB, Allen SK Jr (2004) Microsatellite marker development and analysis in the Eastern oyster (Crassostrea virginica): confirmation of null alleles and non-Mendelian segregation ratios. J Hered 95:346–352
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Methods in Molecular Biology. Humana Press, Totowa, p 365
Schuelke M (2000) An economic method for the fluorescent labeling of PCR fragments. Nat Biotechnol 18:233–234
Acknowledgements
Funding: 2007 UCMEXUS Dissertation Grant to JLP; CONACYT 2005, grant 48443 and SEMARNAT S0010-2006-1, grant 23397 to AMI. Samples from other species were obtained from Amy Wilbur, Iker Uriarte, and Cesar Lodeiros. John Pedroia assisted with data collection and Melinda Baerwald provided helpful comments on the manuscript.
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Open Access This is an open access article distributed under the terms of the Creative Commons Attribution Noncommercial License (https://creativecommons.org/licenses/by-nc/2.0), which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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Petersen, J.L., Ibarra, A.M. & May, B. Thirty-seven additional microsatellite loci in the Pacific lion-paw scallop (Nodipecten subnodosus) and cross-amplification in other pectinids. Conservation Genet Resour 1, 101 (2009). https://doi.org/10.1007/s12686-009-9025-8
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DOI: https://doi.org/10.1007/s12686-009-9025-8