Stacey L. Lance

Rapid microsatellite identification from Illumina paired-end genomic sequencing in two birds and a snake.

Identification of microsatellites, or simple sequence repeats (SSRs), can be a time-consuming and costly investment requiring enrichment, cloning, and sequencing of candidate loci. Recently, however, high throughput sequencing (with or without prior enrichment for specific SSR loci) has been utilized to identify SSR loci. The direct “Seq-to-SSR” approach has an advantage over enrichment-based strategies in that it does not require a priori selection of particular motifs, or prior knowledge of genomic SSR content. It has been more expensive per SSR locus recovered, however, particularly for genomes with few SSR loci, such as bird genomes. The longer but relatively more expensive 454 reads have been preferred over less expensive Illumina reads. Here, we use Illumina paired-end sequence data to identify potentially amplifiable SSR loci (PALs) from a snake (the Burmese python, Python molurus bivittatus), and directly compare these results to those from 454 data. We also compare the python results to results from Illumina sequencing of two bird genomes (Gunnison Sage-grouse, Centrocercus minimus, and Clarks Nutcracker, Nucifraga columbiana), which have considerably fewer SSRs than the python. We show that direct Illumina Seq-to-SSR can identify and characterize thousands of potentially amplifiable SSR loci for as little as

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

10 per sample – a fraction of the cost of 454 sequencing. Given that Illumina Seq-to-SSR is effective, inexpensive, and reliable even for species such as birds that have few SSR loci, it seems that there are now few situations for which prior hybridization is justifiable.

Isolation and characterization of 17 polymorphic microsatellite loci in the kangaroo mouse, genus Microdipodops (Rodentia: Heteromyidae)

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

Impacts of degraded DNA on restriction enzyme associated DNA sequencing (RADSeq)

We isolated and characterized 17 microsatellite loci from kangaroo mice, Microdipodops megacephalus and M. pallidus. Loci were screened in 24 individuals from 21 general localities across their distributional range in the Great Basin Desert. In total, the number of alleles per locus ranged from 4 to 16, observed heterozygosity ranged from 0.333 to 1, and the probability of identity values ranged from 0.013 to 1. These loci provide new tools for examining the biogeographic history and population dynamics of Microdipodops in the context of molecular ecology.

Multiyear multiple paternity and mate fidelity in the American alligator, Alligator mississippiensis.

Degraded DNA from suboptimal field sampling is common in molecular ecology. However, its impact on techniques that use restriction site associated next‐generation DNA sequencing (RADSeq, GBS) is unknown. We experimentally examined the effects of in situDNA degradation on data generation for a modified double‐digest RADSeq approach (3RAD). We generated libraries using genomic DNA serially extracted from the muscle tissue of 8 individual lake whitefish (Coregonus clupeaformis) following 0‐, 12‐, 48‐ and 96‐h incubation at room temperature posteuthanasia. This treatment of the tissue resulted in input DNA that ranged in quality from nearly intact to highly sheared. All samples were sequenced as a multiplexed pool on an Illumina MiSeq. Libraries created from low to moderately degraded DNA (12–48 h) performed well. In contrast, the number of RADtags per individual, number of variable sites, and percentage of identical RADtags retained were all dramatically reduced when libraries were made using highly degraded DNA (96‐h group). This reduction in performance was largely due to a significant and unexpected loss of raw reads as a result of poor quality scores. Our findings remained consistent after changes in restriction enzymes, modified fold coverage values (2‐ to 16‐fold), and additional read‐length trimming. We conclude that starting DNA quality is an important consideration for RADSeq; however, the approach remains robust until genomic DNA is extensively degraded.

32 species validation of a new Illumina paired-end approach for the development of microsatellites

We examined multiple paternity during eight breeding events within a 10‐year period (1995–2005) for a total of 114 wild American alligator nests in Rockefeller Wildlife Refuge in south‐west Louisiana. Our goals included examining (i) within population variation in multiple paternity among years, (ii) variation in multiple paternity in individual females and (iii) the potential for mate fidelity. To accomplish this, in the current study, eggs were sampled from 92 nests over 6 years and analysed along with 22 nests from a previous 2‐year study. Genotypes at five microsatellite loci were generated for 1802 alligator hatchlings. Multiple paternity was found in 51% of clutches and paternal contributions to these clutches were highly skewed. Rates of multiple paternity varied widely among years and were consistently higher in the current study than previously reported for the same population. Larger females have larger clutches, but are not more likely to have multiply sired nests. However, small females are unlikely to have clutches with more than two sires. For 10 females, nests from multiple years were examined. Seven (70%) of these females exhibited long‐term mate fidelity, with one female mating with the same male in 1997, 2002 and 2005. Five females exhibiting partial mate fidelity (71%) had at least one multiple paternity nest and thus mated with the same male, but not exclusively. These patterns of mate fidelity suggest a potential role for mate choice in alligators.

A genetic linkage map for the saltwater crocodile (Crocodylus porosus)

Development and optimization of novel species-specific microsatellites, or simple sequence repeats (SSRs) remains an important step for studies in ecology, evolution, and behavior. Numerous approaches exist for identifying new SSRs that vary widely in terms of both time and cost investments. A recent approach of using paired-end Illumina sequence data in conjunction with the bioinformatics pipeline, PAL_FINDER, has the potential to substantially reduce the cost and labor investment while also improving efficiency. However, it does not appear that the approach has been widely adopted, perhaps due to concerns over its broad applicability across taxa. Therefore, to validate the utility of the approach we developed SSRs for 32 species representing 30 families, 25 orders, 11 classes, and six phyla and optimized SSRs for 13 of the species. Overall the IPE method worked extremely well and we identified 1000s of SSRs for all species (mean = 128,485), with 17% of loci being potentially amplifiable loci, and 25% of these met our most stringent criteria designed to that avoid SSRs associated with repetitive elements. Approximately 61% of screened primers yielded strong amplification of a single locus.

Effects of chronic copper exposure on development and survival in the southern leopard frog (Lithobates [Rana] sphenocephalus)

BackgroundGenome elucidation is now in high gear for many organisms, and whilst genetic maps have been developed for a broad array of species, surprisingly, no such maps exist for a crocodilian, or indeed any other non-avian member of the Class Reptilia. Genetic linkage maps are essential tools for the mapping and dissection of complex quantitative trait loci (QTL), and in order to permit systematic genome scans for the identification of genes affecting economically important traits in farmed crocodilians, a comprehensive genetic linage map will be necessary.ResultsA first-generation genetic linkage map for the saltwater crocodile (Crocodylus porosus) was constructed using 203 microsatellite markers amplified across a two-generation pedigree comprising ten full-sib families from a commercial population at Darwin Crocodile Farm, Northern Territory, Australia. Linkage analyses identified fourteen linkage groups comprising a total of 180 loci, with 23 loci remaining unlinked. Markers were ordered within linkage groups employing a heuristic approach using CRIMAP v3.0 software. The estimated female and male recombination map lengths were 1824.1 and 319.0 centimorgans (cM) respectively, revealing an uncommonly large disparity in recombination map lengths between sexes (ratio of 5.7:1).ConclusionWe have generated the first genetic linkage map for a crocodilian, or indeed any other non-avian reptile. The uncommonly large disparity in recombination map lengths confirms previous preliminary evidence of major differences in sex-specific recombination rates in a species that exhibits temperature-dependent sex determination (TSD). However, at this point the reason for this disparity in saltwater crocodiles remains unclear.This map will be a valuable resource for crocodilian researchers, facilitating the systematic genome scans necessary for identifying genes affecting complex traits of economic importance in the crocodile industry. In addition, since many of the markers placed on this genetic map have been evaluated in up to 18 other extant species of crocodilian, this map will be of intrinsic value to comparative mapping efforts aimed at understanding genome content and organization among crocodilians, as well as the molecular evolution of reptilian and other amniote genomes. As researchers continue to work towards elucidation of the crocodilian genome, this first generation map lays the groundwork for more detailed mapping investigations, as well as providing a valuable scaffold for future genome sequence assembly.

Cross-species amplification of microsatellites in crocodilians: assessment and applications for the future

Exposure to environmental contaminants contributes to the global decline of amphibian populations. The impacts of organic contaminants on amphibians are well documented. However, substantially less is known concerning the potential effects of metals on amphibian populations. Copper (Cu) is an essential element, but it can be toxic at concentrations only slightly higher than the normal physiological range. The present study examines the effects of chronic Cu exposure on embryos and larvae of southern leopard frogs, Lithobates (Rana) sphenocephalus. Groups of eggs from multiple clutches were collected from two wetlands and exposed to a range of Cu concentrations (0-150 µg/L) until they reached the free-swimming stage, and then individual larvae were reared to metamorphosis. Higher Cu concentrations significantly reduced embryo survival to the free-swimming stage but did not further reduce survival to metamorphosis. Larval period was affected by Cu treatment, but the clutch from which larvae originated (i.e., parentage) explained a higher proportion of the variation. Embryo survival to hatching varied significantly among clutches, ranging from 42.9 to 79.2%. Measurable levels of Cu were found in larvae with body burdens up to 595 µg Cu/g dry mass in the 100 µg/L treatment, and larval Cu body burdens were higher than in metamorphs. The present study also demonstrated that higher initial egg density ameliorated embryo mortality at higher Cu levels and should be accounted for in future studies.

Development and characterization of twenty-two novel microsatellite markers for the mountain whitefish, Prosopium williamsoni and cross-amplification in the round whitefish, P. cylindraceum, using paired-end Illumina shotgun sequencing

Microsatellite DNA loci have emerged as the dominant genetic tool for addressing questions associated with genetic diversity in many wildlife species, including crocodilians. Despite their usefulness, their isolation and development can be costly, as well as labour intensive, limiting their wider use in many crocodilian species. In this study, we investigate the cross-species amplification success of 82 existing microsatellites previously isolated for the saltwater crocodile (Crocodylus porosus) in 18 non-target crocodilian species; Alligator sinensis, Caiman crocodylus, Caiman latirostris, Caiman yacare, Melanosuchus niger, Paleosuchus palpebrosus, Crocodylus acutus, Mecistops cataphractus, Crocodylus intermedius, Crocodylus johnstoni, Crocodylus mindorensis, Crocodylus moreletii, Crocodylus niloticus, Crocodylus novaeguineae, Crocodylus palustis, Crocodylus rhombifer, Crocodylus siamensis, and Osteolaemus tetraspis. Our results show a high level of microsatellites cross-amplification making available polymorphic markers for a range of crocodilian species previously lacking informative genetic markers.