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Norway spruce (Picea abies (L.) Karst.) is a conifer species of substanital economic and ecological importance. In common with most conifers, the P. abies genome is very large (∼20 Gbp) and contains a high fraction of repetitive DNA. The current P. abies genome assembly (v1.0) covers approximately 60% of the total genome size but is highly fragmented, consisting of >10 million scaffolds. The genome annotation contains 66,632 gene models that are at least partially validated (www.congenie.org), however, the fragmented nature of the assembly means that there is currently little information available on how these genes are physically distributed over the 12 P. abies chromosomes. By creating an ultra-dense genetic linkage map, we anchored and ordered scaffolds into linkage groups, which complements the fine-scale information available in assembly contigs. Our ultra-dense haploid consensus genetic map consists of 21,056 markers derived from 14,336 scaffolds that contain 17,079 gene models (25.6% of the validated gene models) that we have anchored to the 12 linkage groups. We used data from three independent component maps, as well as comparisons with previously published Picea maps to evaluate the accuracy and marker ordering of the linkage groups. We demonstrate that approximately 3.8% of the anchored scaffolds and 1.6% of the gene models covered by the consensus map have likely assembly errors as they contain genetic markers that map to different regions within or between linkage groups. We further evaluate the utility of the genetic map for the conifer research community by using an independent data set of unrelated individuals to assess genome-wide variation in genetic diversity using the genomic regions anchored to linkage groups. The results show that our map is sufficiently dense to enable detailed evolutionary analyses across the P. abies genome.

Despite the economic, ecological, and scientific importance of the genera Salix L. (willows) and Populus L. (poplars, cottonwoods, and aspens) Salicaceae, we know little about the sources of differences in species diversity between the genera and of the phylogenetic conflict that often confounds estimating phylogenetic trees. Salix subgenera and sections, in particular, have been difficult to classify, with one recent attempt termed a "spectacular failure" due to a speculated radiation of the subgenera Vetrix and Chamaetia. Here, we use targeted sequence capture to understand the evolutionary history of this portion of the Salicaceae plant family. Our phylogenetic hypothesis was based on 787 gene regions and identified extensive phylogenetic conflict among genes. Our analysis supported some previously described subgeneric relationships and confirmed the polyphyly of others. Using an fbranch analysis, we identified several cases of hybridization in deep branches of the phylogeny, which likely contributed to discordance among gene trees. In addition, we identified a rapid increase in diversification rate near the origination of the Vetrix-Chamaetia clade in Salix. This region of the tree coincided with several nodes that lacked strong statistical support, indicating a possible increase in incomplete lineage sorting due to rapid diversification. The extraordinary level of both recent and ancient hybridization in both Salix and Populus have played important roles in the diversification and diversity in these two genera., (© The Author(s) 2023. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The sustainability of many crops is hindered by the lack of genomic resources and a poor understanding of natural genetic diversity. Particularly, application of modern breeding requires high-density linkage maps that are integrated into a highly contiguous reference genome. Here, we present a rapid method for deriving haplotypes and developing linkage maps, and its application to Mentha suaveolens, one of the diploid progenitors of cultivated mints. Using sequence-capture via DNA hybridization to target single nucleotide polymorphisms (SNPs), we successfully genotyped ∼5000 SNPs within the genome of >400 individuals derived from a self cross. After stringent quality control, and identification of nonredundant SNPs, 1919 informative SNPs were retained for linkage map construction. The resulting linkage map defined a total genetic space of 942.17 cM divided among 12 linkage groups, ranging from 56.32 to 122.61 cM in length. The linkage map is in good agreement with pseudomolecules from our preliminary genome assembly, proving this resource effective for the correction and validation of the reference genome. We discuss the advantages of this method for the rapid creation of linkage maps., (© The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.)

Marker selection has emerged as an important component of phylogenomic study design due to rising concerns of the effects of gene tree estimation error, model misspecification, and data-type differences. Researchers must balance various trade-offs associated with locus length and evolutionary rate among other factors. The most commonly used reduced representation data sets for phylogenomics are ultraconserved elements (UCEs) and Anchored Hybrid Enrichment (AHE). Here, we introduce Rapidly Evolving Long Exon Capture (RELEC), a new set of loci that targets single exons that are both rapidly evolving (evolutionary rate faster than RAG1) and relatively long in length (>1,500 bp), while at the same time avoiding paralogy issues across amniotes. We compare the RELEC data set to UCEs and AHE in squamate reptiles by aligning and analyzing orthologous sequences from 17 squamate genomes, composed of 10 snakes and 7 lizards. The RELEC data set (179 loci) outperforms AHE and UCEs by maximizing per-locus genetic variation while maintaining presence and orthology across a range of evolutionary scales. RELEC markers show higher phylogenetic informativeness than UCE and AHE loci, and RELEC gene trees show greater similarity to the species tree than AHE or UCE gene trees. Furthermore, with fewer loci, RELEC remains computationally tractable for full Bayesian coalescent species tree analyses. We contrast RELEC to and discuss important aspects of comparable methods, and demonstrate how RELEC may be the most effective set of loci for resolving difficult nodes and rapid radiations. We provide several resources for capturing or extracting RELEC loci from other amniote groups., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Sequencing of target-enriched libraries is an efficient and cost-effective method for obtaining DNA sequence data from hundreds of nuclear loci for phylogeny reconstruction. Much of the cost of developing targeted sequencing approaches is associated with the generation of preliminary data needed for the identification of orthologous loci for probe design. In plants, identifying orthologous loci has proven difficult due to a large number of whole-genome duplication events, especially in the angiosperms (flowering plants). We used multiple sequence alignments from over 600 angiosperms for 353 putatively single-copy protein-coding genes identified by the One Thousand Plant Transcriptomes Initiative to design a set of targeted sequencing probes for phylogenetic studies of any angiosperm group. To maximize the phylogenetic potential of the probes, while minimizing the cost of production, we introduce a k-medoids clustering approach to identify the minimum number of sequences necessary to represent each coding sequence in the final probe set. Using this method, 5-15 representative sequences were selected per orthologous locus, representing the sequence diversity of angiosperms more efficiently than if probes were designed using available sequenced genomes alone. To test our approximately 80,000 probes, we hybridized libraries from 42 species spanning all higher-order groups of angiosperms, with a focus on taxa not present in the sequence alignments used to design the probes. Out of a possible 353 coding sequences, we recovered an average of 283 per species and at least 100 in all species. Differences among taxa in sequence recovery could not be explained by relatedness to the representative taxa selected for probe design, suggesting that there is no phylogenetic bias in the probe set. Our probe set, which targeted 260 kbp of coding sequence, achieved a median recovery of 137 kbp per taxon in coding regions, a maximum recovery of 250 kbp, and an additional median of 212 kbp per taxon in flanking non-coding regions across all species. These results suggest that the Angiosperms353 probe set described here is effective for any group of flowering plants and would be useful for phylogenetic studies from the species level to higher-order groups, including the entire angiosperm clade itself., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Society of Systematic Biologists.)

Norway spruce ( Picea abies (L.) Karst.) is a conifer species of substanital economic and ecological importance. In common with most conifers, the P. abies genome is very large (∼20 Gbp) and contains a high fraction of repetitive DNA. The current P. abies genome assembly (v1.0) covers approximately 60% of the total genome size but is highly fragmented, consisting of >10 million scaffolds. The genome annotation contains 66,632 gene models that are at least partially validated (www.congenie.org), however, the fragmented nature of the assembly means that there is currently little information available on how these genes are physically distributed over the 12 P. abies chromosomes. By creating an ultra-dense genetic linkage map, we anchored and ordered scaffolds into linkage groups, which complements the fine-scale information available in assembly contigs. Our ultra-dense haploid consensus genetic map consists of 21,056 markers derived from 14,336 scaffolds that contain 17,079 gene models (25.6% of the validated gene models) that we have anchored to the 12 linkage groups. We used data from three independent component maps, as well as comparisons with previously published Picea maps to evaluate the accuracy and marker ordering of the linkage groups. We demonstrate that approximately 3.8% of the anchored scaffolds and 1.6% of the gene models covered by the consensus map have likely assembly errors as they contain genetic markers that map to different regions within or between linkage groups. We further evaluate the utility of the genetic map for the conifer research community by using an independent data set of unrelated individuals to assess genome-wide variation in genetic diversity using the genomic regions anchored to linkage groups. The results show that our map is sufficiently dense to enable detailed evolutionary analyses across the P. abies genome., (Copyright © 2019 Bernhardsson et al.)

DNA methylation can contribute to the maintenance of genome integrity and regulation of gene expression. In most situations, DNA methylation patterns are inherited quite stably. However, changes in DNA methylation can occur at some loci as a result of tissue culture resulting in somaclonal variation. To investigate heritable epigenetic changes as a consequence of tissue culture, a sequence-capture bisulfite sequencing approach was implemented to monitor context-specific DNA methylation patterns in ∼15 Mb of the maize genome for a population of plants that had been regenerated from tissue culture. Plants that have been regenerated from tissue culture exhibit gains and losses of DNA methylation at a subset of genomic regions. There was evidence for a high rate of homozygous changes to DNA methylation levels that occur consistently in multiple independent tissue culture lines, suggesting that some loci are either targeted or hotspots for epigenetic variation. The consistent changes inherited following tissue culture include both gains and losses of DNA methylation and can affect CG, CHG, or both contexts within a region. Only a subset of the tissue culture changes observed in callus plants are observed in the primary regenerants, but the majority of DNA methylation changes present in primary regenerants are passed onto offspring. This study provides insights into the susceptibility of some loci and potential mechanisms that could contribute to altered DNA methylation and epigenetic state that occur during tissue culture in plant species., (Copyright © 2018 by the Genetics Society of America.)

The advent of next-generation sequencing technology has allowed for thecollection of large portions of the genome for phylogenetic analysis. Hybrid enrichment and transcriptomics are two techniques that leverage next-generation sequencing and have shown much promise. However, methods for processing hybrid enrichment data are still limited. We developed a pipeline for anchored hybrid enrichment (AHE) read assembly, orthology determination, contamination screening, and data processing for sequences flanking the target "probe" region. We apply this approach to study the phylogeny of butterflies and moths (Lepidoptera), a megadiverse group of more than 157,000 described species with poorly understood deep-level phylogenetic relationships. We introduce a new, 855 locus AHE kit for Lepidoptera phylogenetics and compare resulting trees to those from transcriptomes. The enrichment kit was designed from existing genomes, transcriptomes, and expressed sequence tags and was used to capture sequence data from 54 species from 23 lepidopteran families. Phylogenies estimated from AHE data were largely congruent with trees generated from transcriptomes, with strong support for relationships at all but the deepest taxonomic levels. We combine AHE and transcriptomic data to generate a new Lepidoptera phylogeny, representing 76 exemplar species in 42 families. The tree provides robust support for many relationships, including those among the seven butterfly families. The addition of AHE data to an existing transcriptomic dataset lowers node support along the Lepidoptera backbone, but firmly places taxa with AHE data on the phylogeny. Combining taxa sequenced for AHE with existing transcriptomes and genomes resulted in a tree with strong support for (Calliduloidea $+$ Gelechioidea $+$ Thyridoidea) $+$ (Papilionoidea $+$ Pyraloidea $+$ Macroheterocera). To examine the efficacy of AHE at a shallow taxonomic level, phylogenetic analyses were also conducted on a sister group representing a more recent divergence, the Saturniidae and Sphingidae. These analyses utilized sequences from the probe region and data flanking it, nearly doubled the size of the dataset; resulting trees supported new phylogenetics relationships, especially within the Saturniidae and Sphingidae (e.g., Hemarina derived in the latter). We hope that our data processing pipeline, hybrid enrichment gene set, and approach of combining AHE data with transcriptomes will be useful for the broader systematics community., (© The Author(s) 2017. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A growing number of genes responsible for reproductive incompatibilities between species (barrier loci) exhibit the signals of positive selection. However, the possibility that genes experiencing positive selection diverge early in speciation and commonly cause reproductive incompatibilities has not been systematically investigated on a genome-wide scale. Here, I outline a research program for studying the genetic basis of speciation in broadcast spawning marine invertebrates that uses a priori genome-wide information on a large, unbiased sample of genes tested for positive selection. A targeted sequence capture approach is proposed that scores single-nucleotide polymorphisms (SNPs) in widely separated species populations at an early stage of allopatric divergence. The targeted capture of both coding and non-coding sequences enables SNPs to be characterized at known locations across the genome and at genes with known selective or neutral histories. The neutral coding and non-coding SNPs provide robust background distributions for identifying F ST -outliers within genes that can, in principle, identify specific mutations experiencing diversifying selection. If natural hybridization occurs between species, the neutral coding and non-coding SNPs can provide a neutral admixture model for genomic clines analyses aimed at finding genes exhibiting strong blocks to introgression. Strongylocentrotid sea urchins are used as a model system to outline the approach but it can be used for any group that has a complete reference genome available.

The anomaly zone, defined by the presence of gene tree topologies that are more probable than the true species tree, presents a major challenge to the accurate resolution of many parts of the Tree of Life. This discrepancy can result from consecutive rapid speciation events in the species tree. Similar to the problem of long-branch attraction, including more data via loci concatenation will only reinforce the support for the incorrect species tree. Empirical phylogenetic studies often employ coalescent-based species tree methods to avoid the anomaly zone, but to this point these studies have not had a method for providing any direct evidence that the species tree is actually in the anomaly zone. In this study, we use 16 species of lizards in the family Scincidae to investigate whether nodes that are difficult to resolve place the species tree within the anomaly zone. We analyze new phylogenomic data (429 loci), using both concatenation and coalescent-based species tree estimation, to locate conflicting topological signal. We then use the unifying principle of the anomaly zone, together with estimates of ancestral population sizes and species persistence times, to determine whether the observed phylogenetic conflict is a result of the anomaly zone. We identify at least three regions of the Scincidae phylogeny that provide demographic signatures consistent with the anomaly zone, and this new information helps reconcile the phylogenetic conflict in previously published studies on these lizards. The anomaly zone presents a real problem in phylogenetics, and our new framework for identifying anomalous relationships will help empiricists leverage their resources appropriately for investigating and overcoming this challenge., (© The Author(s) 2016. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science., (Copyright © 2016 by the Genetics Society of America.)

Microbial enzyme diversity is a key to understand many ecosystem processes. Whole metagenome sequencing (WMG) obtains information on functional genes, but it is costly and inefficient due to large amount of sequencing that is required. In this study, we have applied a captured metagenomics technique for functional genes in soil microorganisms, as an alternative to WMG. Large-scale targeting of functional genes, coding for enzymes related to organic matter degradation, was applied to two agricultural soil communities through captured metagenomics. Captured metagenomics uses custom-designed, hybridization-based oligonucleotide probes that enrich functional genes of interest in metagenomic libraries where only probe-bound DNA fragments are sequenced. The captured metagenomes were highly enriched with targeted genes while maintaining their target diversity and their taxonomic distribution correlated well with the traditional ribosomal sequencing. The captured metagenomes were highly enriched with genes related to organic matter degradation; at least five times more than similar, publicly available soil WMG projects. This target enrichment technique also preserves the functional representation of the soils, thereby facilitating comparative metagenomics projects. Here, we present the first study that applies the captured metagenomics approach in large scale, and this novel method allows deep investigations of central ecosystem processes by studying functional gene abundances., (© The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Eukaryotic microbial communities play key functional roles in soil biology and potentially represent a rich source of natural products including biocatalysts. Culture-independent molecular methods are powerful tools to isolate functional genes from uncultured microorganisms. However, none of the methods used in environmental genomics allow for a rapid isolation of numerous functional genes from eukaryotic microbial communities. We developed an original adaptation of the solution hybrid selection (SHS) for an efficient recovery of functional complementary DNAs (cDNAs) synthesized from soil-extracted polyadenylated mRNAs. This protocol was tested on the Glycoside Hydrolase 11 gene family encoding endo-xylanases for which we designed 35 explorative 31-mers capture probes. SHS was implemented on four soil eukaryotic cDNA pools. After two successive rounds of capture, >90% of the resulting cDNAs were GH11 sequences, of which 70% (38 among 53 sequenced genes) were full length. Between 1.5 and 25% of the cloned captured sequences were expressed in Saccharomyces cerevisiae. Sequencing of polymerase chain reaction-amplified GH11 gene fragments from the captured sequences highlighted hundreds of phylogenetically diverse sequences that were not yet described, in public databases. This protocol offers the possibility of performing exhaustive exploration of eukaryotic gene families within microbial communities thriving in any type of environment., (© The Author 2014. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Targeted sequence capture is a promising technology in many areas in biology. These methods enable efficient and relatively inexpensive sequencing of hundreds to thousands of genes or genomic regions from many more individuals than is practical using whole-genome sequencing approaches. Here, we demonstrate the feasibility of target enrichment using sequence capture in polyploid cotton. To capture and sequence both members of each gene pair (homeologs) of wild and domesticated Gossypium hirsutum, we created custom hybridization probes to target 1000 genes (500 pairs of homeologs) using information from the cotton transcriptome. Two widely divergent samples of G. hirsutum were hybridized to four custom NimbleGen capture arrays containing probes for targeted genes. We show that the two coresident homeologs in the allopolyploid nucleus were efficiently captured with high coverage. The capture efficiency was similar between the two accessions and independent of whether the samples were multiplexed. A significant amount of flanking, nontargeted sequence (untranslated regions and introns) was also captured and sequenced along with the targeted exons. Intraindividual heterozygosity is low in both wild and cultivated Upland cotton, as expected from the high level of inbreeding in natural G. hirsutum and bottlenecks accompanying domestication. In addition, levels of heterozygosity appeared asymmetrical with respect to genome (A(T) or D(T)) in cultivated cotton. The approach used here is general, scalable, and may be adapted for many different research inquiries involving polyploid plant genomes.