skip to main content


Title: Comparative performance of double‐digest RAD sequencing across divergent arachnid lineages
Abstract

Next‐generation sequencing technologies now allow researchers of non‐model systems to perform genome‐based studies without the requirement of a (often unavailable) closely related genomic reference. We evaluated the role of restriction endonuclease (RE) selection in double‐digest restriction‐site‐associatedDNAsequencing (ddRADseq) by generating reduced representation genome‐wide data using four differentREcombinations. Our expectation was thatREselections targeting longer, more complex restriction sites would recover fewer loci thanREwith shorter, less complex sites. We sequenced a diverse sample of non‐model arachnids, including five congeneric pairs of harvestmen (Opiliones) and four pairs of spiders (Araneae). Sample pairs consisted of either conspecifics or closely related congeneric taxa, and in total 26 sample pair analyses were tested. Sequence demultiplexing, read clustering and variant calling were performed in thepyRADprogram. The 6‐base pair cutterEcoRIcombined with methylated site‐specific 4‐base pair cutterMspIproduced, on average, the greatest numbers of intra‐individual loci and shared loci per sample pair. As expected, the number of shared loci recovered for a sample pair covaried with the degree of genetic divergence, estimated with cytochrome oxidase I sequences, although this relationship was non‐linear. Our comparative results will prove useful in guiding protocol selection for ddRADseq experiments on many arachnid taxa where reference genomes, even from closely related species, are unavailable.

 
more » « less
Award ID(s):
1354558
NSF-PAR ID:
10034878
Author(s) / Creator(s):
 ;  ;  ;  ;  ;  
Publisher / Repository:
Wiley-Blackwell
Date Published:
Journal Name:
Molecular Ecology Resources
Volume:
17
Issue:
3
ISSN:
1755-098X
Page Range / eLocation ID:
p. 418-430
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Geographic variation in insect coloration is among the most intriguing examples of rapid phenotypic evolution and provides opportunities to study mechanisms of phenotypic change and diversification in closely related lineages. The bumble beeBombus bifariuscomprises two geographically disparate color groups characterized by red‐banded and black‐banded abdominal pigmentation, but with a range of spatially and phenotypically intermediate populations across western North America. Microsatellite analyses have revealed thatB. bifariusin the USA are structured into two major groups concordant with geography and color pattern, but also suggest ongoing gene flow among regional populations. In this study, we better resolve the relationships among major color groups to better understand evolutionary mechanisms promoting and maintaining such polymorphism. We analyze >90,000 and >25,000 single‐nucleotide polymorphisms derived from transcriptome (RNAseq) and double digest restriction site associatedDNAsequencing (ddRAD), respectively, in representative samples from spatial and color pattern extremes inB. bifariusas well as phenotypic and geographic intermediates. Both ddRADandRNAseq data illustrate substantial genome‐wide differentiation of the red‐banded (eastern) color form from both black‐banded (western) and intermediate (central) phenotypes and negligible differentiation among the latter populations, with no obvious admixture among bees from the two major lineages. Results thus indicate much stronger background differentiation amongB. bifariuslineages than expected, highlighting potential challenges for revealing loci underlying color polymorphism from population genetic data alone. These findings will have significance for resolving taxonomic confusion in this species and in future efforts to investigate color‐pattern evolution inB. bifariusand other polymorphic bumble bee species.

     
    more » « less
  2. Abstract

    The development of high‐throughput sequencing technologies is dramatically increasing the use of single nucleotide polymorphisms (SNPs) across the field of genetics, but most parentage studies of wild populations still rely on microsatellites. We developed a bioinformatic pipeline for identifyingSNPpanels that are informative for parentage analysis from restriction site‐associatedDNAsequencing (RADseq) data. This pipeline includes options for analysis with or without a reference genome, and provides methods to maximize genotyping accuracy and select sets of unlinked loci that have high statistical power. We test this pipeline on small populations of Mexican gray wolf and bighorn sheep, for which parentage analyses are expected to be challenging due to low genetic diversity and the presence of many closely related individuals. We compare the results of parentage analysis acrossSNPpanels generated with or without the use of a reference genome, and betweenSNPs and microsatellites. For Mexican gray wolf, we conducted parentage analyses for 30 pups from a single cohort where samples were available from 64% of possible mothers and 53% of possible fathers, and the accuracy of parentage assignments could be estimated because true identities of parents were known a priori based on field data. For bighorn sheep, we conducted maternity analyses for 39 lambs from five cohorts where 77% of possible mothers were sampled, but true identities of parents were unknown. Analyses with and without a reference genome producedSNPpanels with ≥95% parentage assignment accuracy for Mexican gray wolf, outperforming microsatellites at 78% accuracy. Maternity assignments were completely consistent across allSNPpanels for the bighorn sheep, and were 74.4% consistent with assignments from microsatellites. Accuracy and consistency of parentage analysis were not reduced when using as few as 284SNPs for Mexican gray wolf and 142SNPs for bighorn sheep, indicating our pipeline can be used to developSNPgenotyping assays for parentage analysis with relatively small numbers of loci.

     
    more » « less
  3. Premise

    The ability to sequence genome‐scale data from herbarium specimens would allow for the economical development of data sets with broad taxonomic and geographic sampling that would otherwise not be possible. Here, we evaluate the utility of a basic double‐digest restriction site–associatedDNAsequencing (ddRADseq) protocol usingDNAs from four genera extracted from both silica‐dried and herbarium tissue.

    Methods

    DNAs fromDraba,Boechera,Solidago, andIlexwere processed with a ddRADseq protocol. The effects ofDNAdegradation, taxon, and specimen age were assessed.

    Results

    Although taxon, preservation method, and specimen age affected data recovery, large phylogenetically informative data sets were obtained from the majority of samples.

    Discussion

    These results suggest that herbarium samples can be incorporated into ddRADseq project designs, and that specimen age can be used as a rapid on‐site guide for sample choice. The detailed protocol we provide will allow users to pursue herbarium‐based ddRADseq projects that minimize the expenses associated with fieldwork and sample evaluation.

     
    more » « less
  4. Abstract

    Molecular ecologists seek to genotype hundreds to thousands of loci from hundreds to thousands of individuals at minimal cost per sample. Current methods, such as restriction‐site‐associatedDNAsequencing (RADseq) and sequence capture, are constrained by costs associated with inefficient use of sequencing data and sample preparation. Here, we introduceRADcap, an approach that combines the major benefits ofRADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches.RADcap uses a new version of dual‐digestRADseq (3RAD) to identify candidateSNPloci for capture bait design and subsequently uses custom sequence capture baits to consistently enrich candidateSNPloci across many individuals. We combined this approach with a new library preparation method for identifying and removingPCRduplicates from 3RADlibraries, which allows researchers to processRADseq data using traditional pipelines, and we tested theRADcap method by genotyping sets of 96–384Wisteriaplants. Our results demonstrate that ourRADcap method: (i) methodologically reduces (to <5%) and allows computational removal ofPCRduplicate reads from data, (ii) achieves 80–90% reads on target in 11 of 12 enrichments, (iii) returns consistent coverage (≥4×) across >90% of individuals at up to 99.8% of the targeted loci, (iv) produces consistently high occupancy matrices of genotypes across hundreds of individuals and (v) costs significantly less than current approaches.

     
    more » « less
  5. Abstract

    Identifying genomic signatures of natural selection can be challenging against a background of demographic changes such as bottlenecks and population expansions. Here, we disentangle the effects of demography from selection in the House Finch (Haemorhous mexicanus) using samples collected before and after a pathogen‐induced selection event. Using ddRADseq, we genotyped over 18,000SNPs across the genome in native pre‐epizootic westernUSbirds, introduced birds from Hawaii and the eastern United States, post‐epizootic eastern birds, and western birds sampled across a similar time span. We found 14% and 7% reductions in nucleotide diversity, respectively, in Hawaiian and pre‐epizootic eastern birds relative to pre‐epizootic western birds, as well as elevated levels of linkage disequilibrium and other signatures of founder events. Despite finding numerous significant frequency shifts (outlier loci) between pre‐epizootic native and introduced populations, we found no signal of reduced genetic diversity, elevated linkage disequilibrium, or outlier loci as a result of the epizootic. Simulations demonstrate that the proportion of outliers associated with founder events could be explained by genetic drift. This rare view of genetic evolution across time in an invasive species provides direct evidence that demographic shifts like founder events have genetic consequences more widespread across the genome than natural selection.

     
    more » « less