Conservation genomics of the wild pumpkin Cucurbita radicans in Central Mexico: The influence of a changing environment on the genetic diversity and differentiation of a rare species.

The genetic diversity found in natural populations is the result of the evolutionary forces in response to historical and contemporary factors. The environmental characteristics and geological history of Mexico promoted the evolution and diversification of plant species, including wild relatives of crops such as the wild pumpkins (Cucurbita). Wild pumpkin species are found in a variety of habitats, evidencing their capability to adapt to different environments. Despite the potential value of wild Cucurbita as a genetic reservoir for crops, there is a lack of studies on their genetic diversity. Cucurbita radicans is an endangered species threatened by habitat destruction leading to low densities in small and isolated populations. Here, we analyze Genotype by Sequencing genomic data of the wild pumpkin C. radicans to evaluate the influence of factors like isolation, demographic history, and the environment shaping the amount and distribution of its genetic variation. We analyzed 91 individuals from 14 localities along its reported distribution. We obtained 5,107 SNPs and found medium-high levels of genetic diversity and genetic structure distributed in four main geographic areas with different environmental conditions. Moreover, we found signals of demographic growth related to historical climatic shifts. Outlier loci analysis showed significant association with the environment, principally with precipitation variables. Also, the outlier loci displayed differential changes in their frequencies in response to future global climate change scenarios. Using the results of genetic structure, outlier loci and multivariate analyses of the environmental conditions, we propose priority localities for conservation that encompass most of the genetic diversity of C. radicans.

Genome-Wide Association Study (GWAS) for Identifying SNPs and Genes Related to Phosphate-Induced Phenotypic Traits in Tomato (Solanum lycopersicum L.).

Phosphate (P) is a crucial macronutrient for normal plant growth and development. The P availability in soils is a limitation factor, and understanding genetic factors playing roles in plant adaptation for improving P uptake is of great biological importance. Genome-wide association studies (GWAS) have become indispensable tools in unraveling the genetic basis of complex traits in various plant species. In this study, a comprehensive GWAS was conducted on diverse tomato (Solanum lycopersicum L.) accessions grown under normal and low P conditions for two weeks. Plant traits such as shoot height, primary root length, plant biomass, shoot inorganic content (SiP), and root inorganic content (RiP) were measured. Among several models of GWAS tested, the Bayesian-information and linkage disequilibrium iteratively nested keyway (BLINK) models were used for the identification of single nucleotide polymorphisms (SNPs). Among all the traits analyzed, significantly associated SNPs were recorded for PB, i.e., 1 SNP (SSL4.0CH10_49261145) under control P, SiP, i.e., 1 SNP (SSL4.0CH08_58433186) under control P and 1 SNP (SSL4.0CH08_51271168) under low P and RiP i.e., 2 SNPs (SSL4.0CH04_37267952 and SSL4.0CH09_4609062) under control P and 1 SNP (SSL4.0CH09_3930922) under low P condition. The identified SNPs served as genetic markers pinpointing regions of the tomato genome linked to P-responsive traits. The novel candidate genes associated with the identified SNPs were further analyzed for their protein-protein interactions using STRING. The study provided novel candidate genes, viz. Solyc10g050370 for PB under control, Solyc08g062490, and Solyc08g062500 for SiP and Solyc09g010450, Solyc09g010460, Solyc09g010690, and Solyc09g010710 for RiP under low P condition. These findings offer a glimpse into the genetic diversity of tomato accessions' responses to P uptake, highlighting the potential for tailored breeding programs to develop P-efficient tomato varieties that could adapt to varying soil conditions, making them crucial for sustainable agriculture and addressing global challenges, such as soil depletion and food security.

Molecular characterization and SNP identification using genotyping-by-sequencing in high-yielding mutants of proso millet.

Proso millet (Panicummiliaceum L.) is a short-duration C4 crop that is drought tolerant and nutritionally rich and can grow well in marginal lands. Though the crop has many climate-resilient traits like tolerance to drought and heat, its yield is lower than that of common cereals like rice, wheat, and maize. Being an underutilized crop, the molecular resources in the crop are limited. The main aim of the present study was to develop and characterize contrasting mutants for yield and generate functional genomic information for the trait in proso millet. Gamma irradiation-induced mutant population was screened to identify high-yielding mutants, which were evaluated up to M4 generation. One mutant with a dense panicle and high yield (ATL_hy) and one with a lax panicle and low yield (ATL_ly) along with the wild type were sequenced using the genotyping-by-sequencing approach. The variants detected as single nucleotide polymorphisms (SNPs) and insertions-deletions (InDels) were annotated against the reference genome of proso millet. Bioinformatic analyses using the National Center for Biotechnology Information (NCBI) and UniProt databases were performed to elucidate genetic information related to the SNP variations. A total of 25,901, 30,335, and 31,488 SNPs, respectively, were detected in the wild type, ATL_hy mutants, and ATL_ly mutants. The total number of functional SNPs identified in high-yielding and low-yielding mutants was 84 and 171, respectively. Two functional SNPs in the high-yielding mutant (ATL_hy) and one in the low-yielding mutant (ATL_ly) corresponded to the gene coding for "E3 ubiquitin-protein ligase UPL7". Pathway mapping of the functional SNPs identified that two SNPs in ATL_ly were involved in the starch biosynthetic pathway coding for the starch synthase enzyme. This information can be further used in identifying genes responsible for various metabolic processes in proso millet and in designing useful genetic markers.

Speciation in Coastal Basins Driven by Staggered Headwater Captures: Dispersal of a Species Complex, Leporinus bahiensis, as Revealed by Genome-wide SNP Data.

Past sea level changes and geological instability along watershed boundaries have largely influenced fish distribution across coastal basins, either by dispersal via palaeodrainages now submerged or by headwater captures, respectively. Accordingly, the South American Atlantic coast encompasses several small and isolated drainages that share a similar species composition, representing a suitable model to infer historical processes. Leporinus bahiensis is a freshwater fish species widespread along adjacent coastal basins over narrow continental shelf with no evidence of palaeodrainage connections at low sea level periods. Therefore, this study aimed to reconstruct its evolutionary history to infer the role of headwater captures in the dispersal process. To accomplish this, we employed molecular-level phylogenetic and population structure analyses based on Sanger sequences (5 genes) and genome-wide SNP data. Phylogenetic trees based on Sanger data were inconclusive, but SNPs data did support the monophyletic status of L. bahiensis. Both COI and SNP data revealed structured populations according to each hydrographic basin. Species delimitation analyses revealed from 3 (COI) to 5 (multilocus approach) MOTUs, corresponding to the sampled basins. An intricate biogeographic scenario was inferred and supported by Approximate Bayesian Computation (ABC) analysis. Specifically, a staggered pattern was revealed and characterized by sequential headwater captures from basins adjacent to upland drainages into small coastal basins at different periods. These headwater captures resulted in dispersal throughout contiguous coastal basins, followed by deep genetic divergence among lineages. To decipher such recent divergences, as herein represented by L. bahiensis populations, we used genome-wide SNPs data. Indeed, the combined use of genome-wide SNPs data and ABC method allowed us to reconstruct the evolutionary history and speciation of L. bahiensis. This framework might be useful in disentangling the diversification process in other neotropical fishes subject to a reticulate geological history.

Genomic selection for genotype performance and environmental stability in Coffea canephora.

Coffee is one of the most important beverages and trade products in the world. Among the multiple research initiatives focused on coffee sustainability, plant breeding provides the best means to increase phenotypic performance and release cultivars that could meet market demands. Since coffee is well adapted to a diversity of tropical environments, an important question for those confronting the problem of evaluating phenotypic performance is the relevance of genotype-by-environment interaction. As a perennial crop with a long juvenile phase, coffee is subjected to significant temporal and spatial variations. Such facts not only hinder the selection of promising materials but also cause a majority of complaints among growers. In this study, we hypothesized that trait stability in coffee is genetically controlled and therefore is predictable using molecular information. To test it, we used genome-based methods to predict stability metrics computed with the primary goal of selecting coffee genotypes that combine high phenotypic performance and stability for target environments. Using 2 populations of Coffea canephora, evaluated across multiple years and locations, our contribution is 3-fold: (1) first, we demonstrated that the number of harvest evaluations may be reduced leading to accelerated implementation of molecular breeding; (2) we showed that stability metrics are predictable; and finally, (3) both stable and high-performance genotypes can be simultaneously predicted and selected. While this research was carried out on representative environments for coffee production with substantial crossover in genotypic ranking, we anticipate that genomic prediction can be an efficient tool to select coffee genotypes that combine high performance and stability across years and the target locations here evaluated.

QTL analysis for sodium removal ability in rice leaf sheaths under salinity using an IR-44595/318 F2 population.

Over-accumulation of salt in rice plants is an effect of salt stress which decreases growth and grain yield. Salt removal ability in leaf sheaths is a tolerance mechanism to decrease salt entry and accumulation in leaf blades and maintain photosynthesis under salinity. In this study, a QTL analysis of removal ability of sodium ions (Na+) in leaf sheaths and Na+ accumulation-related traits, was conducted using F2 population between two rice varieties, IR-44595 with superior Na+ removal ability, and 318 with contrasting Na+ removal ability in leaf sheaths under salinity. Suggestive QTLs for Na+ removal ability in leaf sheaths were found on chromosomes 4 and 11. The suggestive QTL on chromosome 11 overlapped with other significant QTLs for Na+ concentration in shoots, leaf blades and leaf sheaths, and Na+/K+ ratio in leaf blades. Correlation analysis indicated that Na+ removal ability in leaf sheaths is important in reducing Na+ accumulation in leaf blades. The varietal difference of Na+ removal ability in leaf sheaths at the whole plant level was greater at lower NaCl concentrations and became smaller as the treatment NaCl concentration increased. Although the Na+ removal ability in leaf sheath was comparable between IR-44595 and 318 under high salinity at the whole plant level, the younger leaves of IR-44595 still showed a higher Na+ sheath-blade ratio than 318, which implied the Na+ removal ability functions in the younger leaves in IR-44595 to reduce Na+ entry in young leaf blades even under high salinity.

Genetic variation and clonal diversity in floating aquatic plants: Comparative genomic analysis of water hyacinth species in their native range.

Many eukaryotic organisms reproduce by sexual and asexual reproduction. Genetic diversity in populations can be strongly dependent on the relative importance of these two reproductive modes. Here, we compare the amounts and patterns of genetic diversity in related water hyacinths that differ in their propensity for clonal propagation - highly clonal Eichhornia crassipes and moderately clonal E. azurea (Pontederiaceae). Our comparisons involved genotype-by-sequencing (GBS) of 137 E. crassipes ramets from 60 locations (193,495 nucleotide sites) and 118 E. azurea ramets from 53 locations (198,343 nucleotide sites) among six hydrological basins in central South America, the native range of both species. We predicted that because of more prolific clonal propagation, E. crassipes would exhibit lower clonal diversity than E. azurea. This prediction was supported by all measures of clonal diversity that we examined. Eichhornia crassipes also had a larger excess of heterozygotes at variant sites, another signature of clonality. However, genome-wide heterozygosity was not significantly different between the species. Eichhornia crassipes had weaker spatial genetic structure and lower levels of differentiation among hydrological basins than E. azurea, probably because of higher clonality and more extensive dispersal of its free-floating life form. Our findings for E. crassipes contrast with earlier studies from the invasive range which have reported very low levels of clonal diversity and extensive geographic areas of genetic uniformity.

Comparative phylogenomic patterns in the Baja California avifauna, their conservation implications, and the stages in lineage divergence.

Comparative phylogeography explores the historical congruence of co-distributed species to understand the factors that led to their current genetic and phenotypic structures. Even species that span the same biogeographic barrier can exhibit different phylogeographic structures owing to differences in effective population sizes, genetic marker bias, and dispersal abilities. The Baja California peninsula and adjacent desert regions include several biogeographic barriers, including the Vizcaíno Desert and Sierra de la Laguna (Cape District), that have left phylogeographic patterns in some but not all species. We used genome-wide SNP data to test the hypothesis that the diverse phylogeographic patterns inferred from prior studies were supported. We found that mitochondrial DNA, single nuclear gene, and genome-wide SNP data show that the cactus wren and LeConte's thrasher have a concordant historical division at or near the Vizcaíno Desert in north-central Baja California, the Gila woodpecker is at an intermediate stage of divergence, and the California gnatcatcher lacks phylogeographic structure. None of these four species are classified taxonomically in a way that captures their evolutionary history with the exception of the LeConte's thrasher. We also analyzed mtDNA data on samples of nine other species that span the Vizcaíno Desert, with four showing no apparent division, and six additional species from the Sierra de la Laguna, all but one of which are differentiated. Reasons for contrasting phylogeographic patterns among these species should be explored further with genomic data to test the extent of concordant phylogeographic patterns. The evolutionary division at the Vizcaíno desert is well known in other vertebrate species, and our study further corroborates the extent, profound effect, and importance of this biogeographic boundary. The areas north and south of the Vizcaíno Desert, which contains considerable diversity, should be recognized as historically significant areas for conservation.

Reduced genetic diversity associated with the northern expansion of an amphibian species with high habitat specialization, Ascaphus truei, resolved using two types of genetic markers.

Reconstruction of historical relationships between geographic regions within a species' range can indicate dispersal patterns and help predict future responses to shifts in climate. Ascaphus truei (coastal tailed frog) is an indicator species of the health of forests and perennial streams in the Coastal and Cascade Mountains of the Pacific Northwest of North America. We used two genetic techniques-microsatellite and genotype-by-sequencing (GBS)-to compare the within-region genetic diversity of populations near the northern extent of the species' range (British Columbia, Canada) to two geographic regions in British Columbia and two in Washington, USA, moving toward the core of the range. Allelic richness and heterozygosity declined substantially as latitude increased. The northernmost region had the lowest mean expected heterozygosities for both techniques (microsatellite, M = 0.20, SE = 0.080; GBS, M = 0.025, SE = 0.0010) and the southernmost region had the highest (microsatellite, M = 0.88, SE = 0.054; GBS, M = 0.20, SE = 0.0029). The northernmost regions (NC and MC) clustered together in population structure models for both genetic techniques. Our discovery of reduced diversity may have important conservation and management implications for population connectivity and the response of A. truei to climate change.

Rice height QTLs in KDML105 chromosome segment substitution lines.

Rice is an important crop that is consumed by approximately half of the world's population on a regular basis. Plant height is an important characteristic with shorter rice often having higher lodging resistance and better soil nutrient utilization allowing for lower fertilizer use. We used a Chromosome Segment Substitution Line (CSSL) population generated by introgressing segments of CT9993 and IR62266 into KDML 105. We identified height QTLs on chromosomes 1 and 4. We performed whole genome sequencing of the parental lines and found that IR62266 has the deletion in Gibberellin 20-oxidase 2 corresponding to the semi-dwarf 1 locus. However, short height on chromosome 1 came from CT9993 with no mutation in Gibberellin 20-oxidase 2, or any known height genes. The height QTL on chromosome 4 contains mutations in Peroxisome biogenesis protein 6, which has been linked to a reduced growth phenotype in A. thaliana, making this a good candidate height gene.

Oxidative stress response and programmed cell death guided by NAC013 modulate pithiness in radish taproots.

Radish, Raphanus sativus L., is an important root crop that is cultivated worldwide. Owing to its evolutionary proximity to Arabidopsis thaliana, radish can be used as a model root crop in research on the molecular basis of agronomic traits. Pithiness is a significant defect that reduces the production of radish with commercial value; however, traditional breeding to eliminate this trait has thus far been unsuccessful. Here, we performed transcriptomics and genotype-by-sequencing (GBS)-based quantitative trait locus (QTL) analyses of radish inbred lines to understand the molecular basis of pithiness in radish roots. The transcriptome data indicated that pithiness likely stems from the response to oxidative stress, leading to cell death of the xylem parenchyma during the root-thickening process. Subsequently, we narrowed down a list of candidates responsible for pithiness near a major QTL and found polymorphisms in a radish homologue of Arabidopsis ANAC013 (RsNAC013), an endoplasmic reticulum bound NAC transcription factor that is targeted to the nucleus to mediate the mitochondrial retrograde signal. We analysed the effects of polymorphisms in RsNAC013 using Arabidopsis transgenic lines overexpressing RsNAC013 alleles as well as in radish inbred lines bearing these alleles. This analysis indicated that non-synonymous variations within the coding sequence result in different levels of RsNAC013 activities, thereby providing a genetic condition for root pithiness. The elevated oxidative stress or hypoxia that activates RsNAC013 for mitochondrial signalling enhances this process. Collectively, this study serves as an exemplary case of translational research taking advantage of the extensive information available from a model organism.

Complex introgression among three diverged largemouth bass lineages.

Hybrid zones between diverged lineages offer a unique opportunity to study evolutionary processes related to speciation. Natural and anthropogenic hybridization in the black basses (Micropterus spp.) is well documented, including an extensive intergrade zone between the widespread northern Largemouth Bass (M. salmoides) and the Florida Bass (M. floridanus). Phenotypic surveys have identified an estuarine population of Largemouth Bass (M. salmoides) in the Mobile-Tensaw Delta, with larger relative weight and smaller adult size compared to inland populations, suggesting a potential third lineage of largemouth bass. To determine the evolutionary relationships among these Mobile Delta bass populations, M. salmoides and M. floridanus, putative pure and intergrade populations of all three groups were sampled across the eastern United States. Phylogenetic analyses of 8582 nuclear SNPs derived from genotype-by-sequencing and the ND2 mitochondrial gene determined that Delta bass populations stem from a recently diverged lineage of Largemouth Bass. Using a novel quantitative pipeline, a panel of 73 diagnostic SNPs was developed for the three lineages, evaluated for accuracy, and then used to screen 881 samples from 52 sites for genetic integrity and hybridization on the Agena MassARRAY platform. These results strongly support a redrawing of native ranges for both the intergrade zone and M. floridanus, which has significant implications for current fisheries management. Furthermore, Delta bass ancestry was shown to contribute significantly to the previously described intergrade zone between northern Largemouth Bass and Florida Bass, suggesting a more complex pattern of secondary contact and introgression among these diverged Micropterus lineages.

Comparative Analysis of Genotyping by Sequencing and Whole-Genome Sequencing Methods in Diversity Studies of Olea europaea L.

Olive, Olea europaea L., is a tree of great economic and cultural importance in the Mediterranean basin. Thousands of cultivars have been described, of which around 1200 are conserved in the different olive germplasm banks. The genetic characterisation of these cultivars can be performed in different ways. Whole-genome sequencing (WGS) provides more information than the reduced representation methods such as genotype by sequencing (GBS), but at a much higher cost. This may change as the cost of sequencing continues to drop, but, currently, genotyping hundreds of cultivars using WGS is not a realistic goal for most research groups. Our aim is to systematically compare both methodologies applied to olive genotyping and summarise any possible recommendations for the geneticists and molecular breeders of the olive scientific community. In this work, we used a selection of 24 cultivars from an olive core collection from the World Olive Germplasm Collection of the Andalusian Institute of Agricultural and Fisheries Research and Training (WOGBC), which represent the most of the cultivars present in cultivated fields over the world. Our results show that both methodologies deliver similar results in the context of phylogenetic analysis and popular population genetic analysis methods such as clustering. Furthermore, WGS and GBS datasets from different experiments can be merged in a single dataset to perform these analytical methodologies with proper filtering. We also tested the influence of the different olive reference genomes in this type of analysis, finding that they have almost no effect when estimating genetic relationships. This work represents the first comparative study between both sequencing techniques in olive. Our results demonstrate that the use of GBS is a perfectly viable option for replacing WGS and reducing research costs when the goal of the experiment is to characterise the genetic relationship between different accessions. Besides this, we show that it is possible to combine variants from GBS and WGS datasets, allowing the reuse of publicly available data.

Land use and life history constrain adaptive genetic variation and reduce the capacity for climate change adaptation in turtles.

BACKGROUND: Rapid anthropogenic climate change will require species to adapt to shifting environmental conditions, with successful adaptation dependent upon current patterns of genetic variation. While landscape genomic approaches allow for exploration of local adaptation in non-model systems, most landscape genomics studies of adaptive capacity are limited to exploratory identification of potentially important functional genes, often without a priori expectations as to the gene functions that may be most important for climate change responses. In this study, we integrated targeted sequencing of genes of known function and genotyping of single-nucleotide polymorphisms to examine spatial, environmental, and species-specific patterns of potential local adaptation in two co-occuring turtle species: the Blanding's turtle (Emydoidea blandingii) and the snapping turtle (Chelydra serpentina). RESULTS: We documented divergent patterns of spatial clustering between neutral and putatively adaptive genetic variation in both species. Environmental associations varied among gene regions and between species, with stronger environmental associations detected for genes involved in stress response and for the more specialized Blanding's turtle. Land cover appeared to be more important than climate in shaping spatial variation in functional genes, indicating that human landscape alterations may affect adaptive capacity important for climate change responses. CONCLUSIONS: Our study provides evidence that responses to climate change will be contingent on species-specific adaptive capacity and past history of exposure to human land cover change.

Translation of continuous artificial selection on phenotype into genotype during rice breeding programs.

Understanding genetic diversity among local populations is a primary goal of modern crop breeding programs. Here, we demonstrated the genetic relationships of rice varieties in Hokkaido, Japan, one of the northern limits of rice cultivation around the world. Furthermore, artificial selection during rice breeding programs has been characterized using genome sequences. We utilized 8,565 single nucleotide polymorphisms and insertion/deletion markers distributed across the genome in genotype-by-sequencing for genetic diversity analyses. Phylogenetics, genetic population structure, and principal component analysis showed that a total of 110 varieties were classified into four distinct clusters according to different populations geographically and historically. Furthermore, the genome sequences of 19 rice varieties along with historic representations in Hokkaido, nucleotide diversity and FST values in each cluster revealed that artificial selection of elite phenotypes focused on chromosomal regions. These results clearly demonstrated the history of the selections on agronomic traits as genome sequences among current rice varieties from Hokkaido.

Understanding the Effectiveness of Genomic Prediction in Tetraploid Potato.

Use of genomic prediction (GP) in tetraploid is becoming more common. Therefore, we think it is the right time for a comparison of GP models for tetraploid potato. GP models were compared that contrasted shrinkage with variable selection, parametric vs. non-parametric models and different ways of accounting for non-additive genetic effects. As a complement to GP, association studies were carried out in an attempt to understand the differences in prediction accuracy. We compared our GP models on a data set consisting of 147 cultivars, representing worldwide diversity, with over 39 k GBS markers and measurements on four tuber traits collected in six trials at three locations during 2 years. GP accuracies ranged from 0.32 for tuber count to 0.77 for dry matter content. For all traits, differences between GP models that utilised shrinkage penalties and those that performed variable selection were negligible. This was surprising for dry matter, as only a few additive markers explained over 50% of phenotypic variation. Accuracy for tuber count increased from 0.35 to 0.41, when dominance was included in the model. This result is supported by Genome Wide Association Study (GWAS) that found additive and dominance effects accounted for 37% of phenotypic variation, while significant additive effects alone accounted for 14%. For tuber weight, the Reproducing Kernel Hilbert Space (RKHS) model gave a larger improvement in prediction accuracy than explicitly modelling epistatic effects. This is an indication that capturing the between locus epistatic effects of tuber weight can be done more effectively using the semi-parametric RKHS model. Our results show good opportunities for GP in 4x potato.

Digital PCR for Genotype Quantification: A Case Study in a Pasta Production Chain.

Digital polymerase chain reaction (dPCR) is a breakthrough technology based on the partitioning of the analytical sample and detection of individual end-point amplifications into the separate compartments. Among the numerous applications of this technology, its suitability in mutation detection is relevant and characterized by unprecedented levels of precision. The actual applicability of this analytical technique to quantify the presence of a specific plant genotype, in both raw materials and transformed products, by exploiting a point polymorphism has been evaluated. As proof of concept, an Italian premium pasta production chain was considered and a dPCR assay based on a durum wheat target variety private point mutation was designed and evaluated in supply-chain samples. From the results obtained, the assay can be applied to confirm the presence of a target variety and to quantify it in raw materials and transformed products, such as commercial grain lots and pasta. The performance, costs, and applicability of the assay has been compared to analytical alternatives, namely simple sequence repeats (SSRs) and genotype-by-sequencing based on Diversity Arrays Technology sequencing (DArTseqTM).

Standing genetic variation in laboratory populations of insecticide-susceptible Phlebotomus papatasi and Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) for the evolution of resistance.

Insecticides can exert strong selection on insect pest species, including those that vector diseases, and have led to rapid evolution of resistance. Despite such rapid evolution, relatively little is known about standing genetic variation for resistance in insecticide-susceptible populations of many species. To help fill this knowledge gap, we generated genotyping-by-sequencing data from insecticide-susceptible Phlebotomus papatasi and Lutzomyia longipalpis sand flies that survived or died from a sub-diagnostic exposure to either permethrin or malathion using a modified version of the Centers for Disease Control and Prevention bottle bioassay. Multi-locus genome-wide association mapping methods were used to quantify standing genetic variation for insecticide resistance in these populations and to identify specific alleles associated with insecticide survival. For each insecticide treatment, we estimated the proportion of the variation in survival explained by the genetic data (i.e., "chip" heritability) and the number and contribution of individual loci with measurable effects. For all treatments, survival to an insecticide exposure was heritable with a polygenic architecture. Both P. papatasi and L. longipalpis had alleles for survival that resided within many genes throughout their genomes. The implications for resistance conferred by many alleles, as well as inferences made about the utility of laboratory insecticide resistance association studies compared to field observations, are discussed.

Base-substitution mutation rate across the nuclear genome of Alpheus snapping shrimp and the timing of isolation by the Isthmus of Panama.

BACKGROUND: The formation of the Isthmus of Panama and final closure of the Central American Seaway (CAS) provides an independent calibration point for examining the rate of DNA substitutions. This vicariant event has been widely used to estimate the substitution rate across mitochondrial genomes and to date evolutionary events in other taxonomic groups. Nuclear sequence data is increasingly being used to complement mitochondrial datasets for phylogenetic and evolutionary investigations; these studies would benefit from information regarding the rate and pattern of DNA substitutions derived from the nuclear genome. RESULTS: To estimate the genome-wide neutral mutation rate (µ), genotype-by-sequencing (GBS) datasets were generated for three transisthmian species pairs in Alpheus snapping shrimp. A range of bioinformatic filtering parameters were evaluated in order to minimize potential bias in mutation rate estimates that may result from SNP filtering. Using a Bayesian coalescent approach (G-PhoCS) applied to 44,960 GBS loci, we estimated µ to be 2.64E-9 substitutions/site/year, when calibrated with the closure of the CAS at 3 Ma. Post-divergence gene flow was detected in one species pair. Failure to account for this post-split migration inflates our substitution rate estimates, emphasizing the importance of demographic methods that can accommodate gene flow. CONCLUSIONS: Results from our study, both parameter estimates and bioinformatic explorations, have broad-ranging implications for phylogeographic studies in other non-model taxa using reduced representation datasets. Our best estimate of µ that accounts for coalescent and demographic processes is remarkably similar to experimentally derived mutation rates in model arthropod systems. These results contradicted recent suggestions that the closure of the Isthmus was completed much earlier (around 10 Ma), as mutation rates based on an early calibration resulted in uncharacteristically low genomic mutation rates. Also, stricter filtering parameters resulted in biased datasets that generated lower mutation rate estimates and influenced demographic parameters, serving as a cautionary tale for the adherence to conservative bioinformatic strategies when generating reduced-representation datasets at the species level. To our knowledge this is the first use of transisthmian species pairs to calibrate the rate of molecular evolution from GBS data.