Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2010-30 September 2010.

This article documents the addition of 229 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acacia auriculiformis × Acacia mangium hybrid, Alabama argillacea, Anoplopoma fimbria, Aplochiton zebra, Brevicoryne brassicae, Bruguiera gymnorhiza, Bucorvus leadbeateri, Delphacodes detecta, Tumidagena minuta, Dictyostelium giganteum, Echinogammarus berilloni, Epimedium sagittatum, Fraxinus excelsior, Labeo chrysophekadion, Oncorhynchus clarki lewisi, Paratrechina longicornis, Phaeocystis antarctica, Pinus roxburghii and Potamilus capax. These loci were cross-tested on the following species: Acacia peregrinalis, Acacia crassicarpa, Bruguiera cylindrica, Delphacodes detecta, Tumidagena minuta, Dictyostelium macrocephalum, Dictyostelium discoideum, Dictyostelium purpureum, Dictyostelium mucoroides, Dictyostelium rosarium, Polysphondylium pallidum, Epimedium brevicornum, Epimedium koreanum, Epimedium pubescens, Epimedium wushanese and Fraxinus angustifolia.

Post-mating reproductive barriers in two unidirectionally hybridizing sunfish (Centrarchidae: Lepomis).

The evolutionary sequence of events in the evolution of reproductive barriers between species is at the core of speciation biology. Where premating barriers fail, post-mating barriers, such as conspecific sperm precedence (CSP), gamete incompatibility (GI) and hybrid inviability (HI) may evolve to prevent the production of (often) costly hybrid offspring with reduced fitness. We tested the role of post-mating mechanisms for the reproductive isolation between two sunfish species [bluegill (BG) Lepomis macrochirus and pumpkinseed (PS) Lepomis gibbosus] and their first-generation hybrids. Performing in vitro sperm competition experiments, we observed asymmetric CSP as main post-mating isolation mechanism when BG and PS sperm were competing for PS eggs, whereas when sperm from both species were competing for BG eggs it was HI. Furthermore, hybrid sperm--although fertile in the absence of competition--were outcompeted by sperm of either parental species. This result may at least partly explain previous observations that natural hybridization in the study system is unidirectional.

A finite locus effect diffusion model for the evolution of a quantitative trait.

A diffusion model is constructed for the joint distribution of absolute locus effect sizes and allele frequencies for loci contributing to an additive quantitative trait under selection in a haploid, panmictic population. The model is designed to approximate a discrete model exactly in the limit as both population size and the number of loci affecting the trait tend to infinity. For the case when all loci have the same absolute effect size, formal multiple-timescale asymptotics are used to predict the long-time response of the population trait mean to selection. For the case where loci can take on either of two distinct effect sizes, not necessarily with equal probability, numerical solutions of the system indicate that response to selection of a quantitative trait is insensitive to the variability of the distribution of effect sizes when mutation is negligible.

Reconciling nuclear microsatellite and mitochondrial marker estimates of population structure: breeding population structure of Chesapeake Bay striped bass (Morone saxatilis).

Comparative analyses of nuclear and organelle genetic markers may help delineate evolutionarily significant units or management units, although population differentiation estimates from multiple genomes can also conflict. Striped bass (Morone saxatilis) are long-lived, highly migratory anadromous fish recently recovered from a severe decline in population size. Previous studies with protein, nuclear DNA and mitochondrial DNA (mtDNA) markers produced discordant results, and it remains uncertain if the multiple tributaries within Chesapeake Bay constitute distinct management units. Here, 196 young-of-the-year (YOY) striped bass were sampled from Maryland's Choptank, Potomac and Nanticoke Rivers and the north end of Chesapeake Bay in 1999 and from Virginia's Mataponi and Rappahannock Rivers in 2001. A total of 10 microsatellite loci exhibited between two and 27 alleles per locus with observed heterozygosities between 0.255 and 0.893. The 10-locus estimate of R(ST) among the six tributaries was -0.0065 (95% confidence interval -0.0198 to 0.0018). All R(ST) and all but one theta estimates for pairs of populations were not significantly different from zero. Reanalysis of Chesapeake Bay striped bass mtDNA data from two previous studies estimated population differentiation between theta=-0.002 and 0.160, values generally similar to mtDNA population differentiation predicted from microsatellite R(ST) after adjusting for reduced effective population size and uniparental inheritance in organelle genomes. Based on mtDNA differentiation, breeding sex ratios or gene flow may have been slightly male biased in some years. The results reconcile conflicting past studies based on different types of genetic markers, supporting a single Chesapeake Bay management unit encompassing a panmictic striped bass breeding population.

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation.

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.

Cloned microsatellite repeats differ between 4-base restriction endonucleases.

Simple sequence repeat (SSR) loci are an important marker type for population genetic studies despite the limitation that development of novel loci requires construction and screening of genomic DNA libraries. The common practice of size fractioning genomic DNA before cloning could lead to differential representation of SSR loci within genomic libraries. In addition, linkage mapping studies have shown that small numbers of SSR markers are not randomly distributed within the genomes from which they are isolated. From attempts to clone five SSR repeat sequences in two wild plant species we show that the numbers and repeat type of potential SSR markers depend on the restriction endonuclease used to sample the genome when constructing DNA libraries. This observation is consistent with unequal sampling of the genome by different restriction enzymes. However, as a group the five SSR repeat sequences are not associated with a given restriction enzyme, suggesting they are not clumped within the genome. Use of multiple restriction enzymes to construct DNA libraries may help ensure that cloned SSR loci are drawn from diverse locations in the genome, helping to meet the assumption of randomly located marker loci required for population genetic inferences.

Universal linker and ligation procedures for construction of genomic DNA libraries enriched for microsatellites.

Microsatellite loci are highly informative genetic markers useful for population genetic studies, linkage mapping and parentage determination. Methods to identify novel microsatellite loci commonly use subtractive hybridization to enrich small-insert genomic libraries for repeat sequences. A critical step in enrichment is attachment of an oligonucleotide linker to genomic DNA fragments so that repeat-containing sequences can be recovered by PCR for cloning. Current linkers and ligation methods rely on single restriction enzymes to size-fraction genomic DNA and generate complementary ends. These restriction enzyme/linker combinations are often species-specific, give poor recovery of repeat-enriched DNA and yield library inserts that are not a broad sample of the genome. We have developed a blunt-end linker, named SNX for its restriction sites, that allows the use of combinations of restriction enzymes to digest the majority of genomic DNA into the 200-1000-bp range. SNX is attached to genomic DNA with a simultaneous ligation/restriction reaction that is highly efficient and improves recovery of sequences after subtractive hybridization. SNX can be used for microsatellite enrichment in any species, since ligation is independent of the restriction enzymes used to size-fraction genomic DNA. These methods improve current repeat-enrichment strategies, resulting in representative small-insert libraries with a very high proportion of positive clones.

Relatedness measured by oligonucleotide probe DNA fingerprints and an estimate of the mating system of Sea Lavender (Limonium carolinianum).

Using DNA fingerprint markers within species and populations of wild plants requires information on the relationship between fingerprint similarity and relatedness. We identified a hypervariable marker based on oliog(GATA)4-hybridization of DpnII-cut genomic DNA from Sea Lavender (Limonium carolinianum). Banding patterns were somatically stable and highly variable among unrelated individuals. Band molecular-weight sizing errors (as a percent of band molecular weight) were estimated at 0.44%±0.003 within gels and 0.76%±0.964 between gels. Band sizing errors defined a 99% confidence bin of ±0.95% (1.90% total) of molecular weight. Band-sharing estimates were based on this bin size and on variance estimates that compensate for non-independent comparisons. Band-sharing among nine unrelated individuals (θ) was 0.198±0.O11. Experimental pollinations designed to produce selfed, fulland half-sib progeny groups led to five selfed progeny groups and no outcrossed progeny (mean band-sharing, ovS=0.468±0.074). A linear regression between band-sharing (S) and relatedness (r) assuming 17% inbreeding was r=0.006+0.914*S (R(2)=0.973) and established the maximum amount of inbreeding. ovS(0.392±0.022) estimated from wild pollinated seeds from four maternal families was intermediate to unrelated individuals and experimental selfed progeny, giving evidence for mixed mating in wild plants. More extensive plant pedigrees with known levels of inbreeding will be needed to measure variation in the relationship between S and r among populations and families.