Neotropical nutmeg (Virola sebifera Aubl.; Myristicaceae) is a wide-ranging canopy tree found in mature tropical forests from Central America to the Amazon Basin and Guiana Shield. Like other species in its genus, V. sebifera is dioecious, pollinated by small insects, and dispersed by vertebrates (primarily large birds) that consume the nutrient-rich red aril covering its seeds (Howe, 1981). Given the high mobility and considerable seed loads of large avian dispersers, seed-mediated gene flow in V. sebifera may play an important role in maintaining genetic variation within and among populations. However, as increasing anthropogenic activities (e.g., hunting and landscape change) adversely impact the abundance and/or habitat of frugivores (Wright, 2003; Vetter et al., 2011), it is important to investigate how changing vertebrate densities may impact gene flow and population structure in V. sebifera and other tropical forest tree species.
To address these and other questions, we developed a set of polymorphic microsatellite DNA markers for V. sebifera, based on genomic DNA libraries obtained from French Guiana samples by shotgun 454 pyrosequencing (Gardner et al., 2011a, b).
METHODS AND RESULTS
Previously developed genomic libraries of V. sebifera (Gardner et al., 2011a, b) were obtained using the combined genomic DNA of six French Guiana individuals, sampled from tagged trees in trails or permanent forest inventory plots in three localities: Sentier la Mirande (4°51′N, 52°20′W; tag no. S35, S31), Sentier Rorota (4°52′N, 52°15′W; S104, S110), and Iracoubo (5°25′N, 53°5′W; S230, S235). Genomic DNA was isolated from each individual using Nucleo-Spin Plant II (Macherey-Nagel, Düren, Germany), then pooled with equal concentrations (∼0.8 µg/individual) for subsequent 454 pyrosequencing. Standard GS-FLX Titanium library preparation was adopted. After DNA nebulization, small fragments of length <350 bp were removed. Fragmented DNA was then ligated with MID-tagged (MID5, ACGAGTAGACT) adapters. This barcoded V. sebifera DNA library was multiplexed with seven other species in a single ran of GS-FLX Titanium, which rendered V. sebifera 12.5% of the picotiter plate.
We used the program QDD version 2 (Meglécz et al., 2010), set at default parameters, to search for simple sequence repeat (SSR) loci with ≥5 uninterrupted motif repeats from 90,164 read sequences (mean read length = 367 bp) (Gardner et al., 2011a, b). The SSR marker output was further restricted to A and B primer designs in QDD version 2, so as to exclude loci with complex flanking regions (i.e., containing repeat units). We obtained a total of 526 SSR loci, of which 315 contained dinucleotide motifs, followed by 182 tri-, 21 tetra-, six penta-, and two hexanucleotide motifs. Following the suggestions of Gardner et al. (2011a), we first focused on loci containing at least 10 pure repeat units of di-, tetra-, and pentanucleotide SSR motifs, which were expected to be more polymorphic than other motifs. However, because of an unexpected low rate of amplification success and polymorphism, we also included compound motifs, and tri- and hexanucleotide microsatellite loci of ≥9 repeats. The final testing array contained 61 candidate SSR markers (57% in di-, 36% in tri-, 3% in tetra-, 2% in penta-, and 2% in hexanucleotide motifs).
Characteristics of 10 polymorphic SSR markers developed in Virola sebifera.
We checked the amplification rate and polymorphism of the 61 SSR primer pairs in 42 V. sebifera adult trees (diameter at breast height [dbh] ≥20 cm; voucher no. Pérez 1806 and Pérez 1930, STRI herbarium, Panama), which were randomly collected from the 50-ha Forest Dynamics Plot in the plateau of Barro Colorado Island (9°10′N, 79°51′W), Panama. Genomic DNA was isolated from silica-dried leaves using the DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA), quantified using NanoDrop 2000 (Thermo Scientific, Wilmington, Delaware, USA), and diluted to 1.5 ng/µL for subsequent PCR. The 6-µL PCR cocktail contained 1.5 ng of DNA template, 0.05 µM M13-tagged (5′-TGTAAAACGACGGCCAGT-3′) forward primer, 0.4 µM reverse primer, 0.017 µM 6FAM-labeled M13 primer (5′-TGTAAAACGACGGCCAGT-3′), 4 mM MgCl2, 3 µL GoTaq Colorless Master Mix (Promega Corporation, Madison, Wisconsin, USA) with buffer (pH 8.5), 200 µM of each dNTP, and 1 U Taq DNA polymerase. PCRs were carried out in a Mastercycler ep thermocycler (Eppendorf, Hamburg, Germany) following an initial denaturation at 94°C for 4 min; 28 cycles of 94°C for 30 s, 55°C for 40 s, and 72°C for 60 s; 10 cycles of 94°C for 30 s, 52°C for 40 s, and 72°C for 60 s; and a final extension at 72°C for 10 min. PCR product of 1.5 µL was added to 12 µL Hi-Di formamide (Applied Biosystems, Carlsbad, California, USA) and 0.05 µL GeneScan 500 Rox Standard (Applied Biosystems) for subsequent fragment sizing in an ABI 3730 DNA Analyzer (Applied Biosystems) by the DNA Sequencing Core Laboratory at the University of Michigan. Alleles were visualized and scored using GeneMarker version 3.7 (SoftGenetics, State College, Pennsylvania, USA). Marker polymorphism, including the number of alleles per locus, observed and expected heterozygosity, exclusion probability with one parent known, and Hardy– Weinberg equilibrium (HWE), was estimated in GenAlEx version 6.4 (Peakall and Smouse, 2006). Significance levels for multiple tests of HWE (α-level = 0.05) were adjusted by sequential Bonferroni procedure (Rice, 1989). In addition, polymorphism information content of each locus was measured using PowerMarker version 3.0 (Liu and Muse, 2005). We tested for the presence of null alleles, allelic dropout, and scoring errors (due to stuttering) using MICRO-CHECKER version 2.2.3 (Van Oosterhout et al., 2004).
Our results showed that 17 (49%) di-, 13 (59%) tri-, 1 (50%) tetra-, 0 penta-, and 1 (100%) hexanucleotide markers were amplifiable; but 3 (9%) di-, 6 (27%) tri-, 0 tetra-, 0 penta-, and 1 (100%) hexanucleotide SSRs were considered as polymorphic (≥6 alleles per locus) in the current study. These 10 polymorphic markers (Table 1) had mean allelic richness of 10.3 alleles per locus (Table 2). Observed heterozygosity ranged from 0.465 to 0.905, and expected heterozygosity was between 0.477 and 0.876. PIC per locus averaged 0.694 (Table 2). No allelic dropout or scoring errors were detected, but one locus (VSE02) appeared to contain null alleles. Two (VSE02 and VSE36) of the 10 loci showed deviation from Hardy–Weinberg proportions after sequential Bonferroni correction (P < 0.006). The overall exclusion probability with one parent known was 0.992.
We found that trinucleotide SSR loci exhibited better marker properties, such as higher probability of polymorphism and less stuttering, than the other motifs, particularly dinucleotide SSRs. Although the 454 genomic libraries were obtained from French Guiana samples, the markers were developed for Panamanian individuals, despite the probable high levels of genomic divergence between populations located east and west of the Andean Cordilleras. Genomic divergence may partly explain the unexpected low rate of amplification (52%) and polymorphism (16%) of the markers. Although one marker (VSE02) showed evidence of null alleles, and one other marker showed deviation from HWE, these markers may perform well in the South American populations. The 10 polymorphic loci characterized here will be useful for studies of gene flow and population structure in this widespread, vertebrate-dispersed, dioecious tree species.
Summary statistics of SSR marker polymorphism screened in 42 Virola sebifera individuals located in the 50-ha Forest Dynamics Plot on Barro Colorado Island, Panama.