We developed an in-solution gluten exome capture system called GlutEnSeq (Gluten gene Enrichment and Sequencing), covering the sequence variation of thousands of prolamin genes from various Triticeae species and cultivars. We assessed the efficacy of this capture system in hexaploid wheat (Triticum aestivum L.) using Illumina sequencing. On-target regions were determined based on the Chinese Spring (CS) reference genome sequence. Gluten gene sequences were generally enriched around 10,000-fold. The loss of gluten genes in CS deletion line 1BS-19/6DS-4 was detected as absence of gluten gene coverage on chromosomes 1B and Un (containing the Unmapped α-gliadin genes of chromosome 6D). Two γ-irradiated lines of cultivar Paragon, shown to be affected in their gliadin protein profile, were found to contain homozygous deletions for the α-gliadins on 6A and the γ-gliadins on 1B. Four Fielder CRISPR/Cas9 gliadin gene-edited lines revealed homozygous deletions of the γ-gliadins on 1B and heterozygous deletions for the α-gliadins on 6A. We also detected a decrease of gluten gene coverage within some gluten genes. The bioinformatics pipeline developed here will be further optimised to enable characterisation of small indels within individual gluten genes, in order to fully analyse CRISPR/Cas9 mutant lines for their decrease in immunogenicity for Coeliac patients.

In the age of next-generation sequencing, the number of loci available for phylogenetic analyses has increased by orders of magnitude. But despite this dramatic increase in the amount of data, some phylogenomic studies have revealed rampant gene-tree discordance that can be caused by many historical processes, such as rapid diversification, gene duplication, or reticulate evolution. We used a target enrichment approach to sample 400 single-copy nuclear genes and estimate the phylogenetic relationships of 13 genera in the lichen-forming family Lobariaceae to address the effect of data type (nucleotides and amino acids) and phylogenetic reconstruction method (concatenation and species tree approaches). Furthermore, we examined datasets for evidence of historical processes, such as rapid diversification and reticulate evolution. We found incongruence associated with sequence data types (nucleotide vs. amino acid sequences) and with different methods of phylogenetic reconstruction (species tree vs. concatenation). The resulting phylogenetic trees provided evidence for rapid and reticulate evolution based on extremely short branches in the backbone of the phylogenies. The observed rapid and reticulate diversifications may explain conflicts among gene trees and the challenges to resolving evolutionary relationships. Based on divergence times, the diversification at the backbone occurred near the Cretaceous-Paleogene (K-Pg) boundary (65 Mya) which is consistent with other rapid diversifications in the tree of life. Although some phylogenetic relationships within the Lobariaceae family remain with low support, even with our powerful phylogenomic dataset of up to 376 genes, our use of target-capturing data allowed for the novel exploration of the mechanisms underlying phylogenetic and systematic incongruence.

Targeted genome sequencing approaches allow characterization of evolutionary relationships using a considerable number of nuclear genes and informative characters. However, most phylogenomic analyses only utilize single nucleotide polymorphisms (SNPs). Studies at the species level, especially in groups that have recently radiated, often recover low amounts of phylogenetically informative variation in coding regions, and require non-coding sequences, which are richer in indels, to resolve gene trees. Here, NGS-Indel Coder, a pipeline to detect and omit false positive indels inferred from assemblies of short read sequence data was developed to resolve the relationships among and within major clades of the American milkweeds (Asclepias), which are the result of a rapid and recent evolutionary radiation, and whose phylogeny has been difficult to resolve. This pipeline was applied to a Hyb-Seq data set of 768 loci including targeted exons and flanking intron regions from 33 milkweed species. Robust species tree inference was improved by excluding small alignment partitions (<100 bp) that increased gene tree ambiguity and incongruence. To further investigate the robustness of indel coding, data sets that included small and large indels were explored, and species trees derived from concatenated loci versus coalescent methods based on gene trees were compared. The phylogeny of Asclepias obtained using nuclear data was well resolved, and phylogenetic information from indels improved resolution of specific nodes. The Temperate North American, Mexican Highland, and Incarnatae clades were well supported as monophyletic. Asclepias coulteri, which has been considered part of the Sonoran Desert clade based on plastome analyses, was placed as sister to all the other milkweed species studied here, rather than as a member of that clade. Two groups within the Temperate North American and Mexican clades were not resolved, and the inferred relationships strongly conflicted when comparing results based on data sets that did or did not include indel characters. This new pipeline represents a step forward in making maximal use of the information content in phylogenomic data sets.

Madagascar is known as a biodiversity hotspot, providing an ideal natural laboratory for investigating the processes of avian diversification. Yet, the phylogeography of Madagascar’s avifauna is still largely unexamined. In this study, we evaluated phylogeographic patterns and species limits within the Rufous Vanga, Schetba rufa, a monotypic genus of forest-dwelling birds endemic to the island. Using an integrative taxonomic approach, we synthesized data from over 4,000 ultra-conserved element (UCE) loci, mitochondrial DNA, multivariate morphometrics, and ecological niche modeling to uncover two reciprocally monophyletic, geographically circumscribed, and morphologically distinct clades of Schetba. The two lineages are restricted to eastern and western Madagascar, respectively, with distributions broadly consistent with previously described subspecies. Based on their genetic and morphological distinctiveness, the two subspecies merit recognition as separate species. The bioclimatic transition between the humid east and dry west of Madagascar likely promoted population subdivision and drove speciation in Schetba during the Pleistocene. Our study is the first evidence that an East-West bioclimatic transition zone played a role in the speciation of birds within Madagascar.

Summary Living sloths represent two distinct lineages of small-sized mammals that independently evolved arboreality from terrestrial ancestors. The six extant species are the survivors of an evolutionary radiation marked by the extinction of large terrestrial forms at the end of the Quaternary. Until now, sloth evolutionary history has mainly been reconstructed from phylogenetic analyses of morphological characters. Here, we used ancient DNA methods to successfully sequence 10 extinct sloth mitogenomes encompassing all major lineages. This includes the iconic continental ground sloths Megatherium, Megalonyx, Mylodon, and Nothrotheriops and the smaller endemic Caribbean sloths Parocnus and Acratocnus. Phylogenetic analyses identify eight distinct lineages grouped in three well-supported clades, whose interrelationships are markedly incongruent with the currently accepted morphological topology. We show that recently extinct Caribbean sloths have a single origin but comprise two highly divergent lineages that are not directly related to living two-fingered sloths, which instead group with Mylodon. Moreover, living three-fingered sloths do not represent the sister group to all other sloths but are nested within a clade of extinct ground sloths including Megatherium, Megalonyx, and Nothrotheriops. Molecular dating also reveals that the eight newly recognized sloth families all originated between 36 and 28 million years ago (mya). The early divergence of recently extinct Caribbean sloths around 35 mya is consistent with the debated GAARlandia hypothesis postulating the existence at that time of a biogeographic connection between northern South America and the Greater Antilles. This new molecular phylogeny has major implications for reinterpreting sloth morphological evolution, biogeography, and diversification history.

Comparative Analysis of Whole-Genome Sequence of African Swine Fever Virus Belgium 2018/1

Thousands of varieties of wine grapes have been recorded and described in historical accounts, some going back as far as the Middle Ages, but genetic relationships between ancient and modern varieties were unknown. Genomic sequencing of 28 seeds, dating back as far as the Iron Age, finds close relationships to today’s cultivars, including a genetic match to Savagnin Blanc from 1100 ce.

Unraveling genetic population structure is challenging in species potentially characterized by large population size and high dispersal rates, often resulting in weak genetic differentiation. Genotyping a large number of samples can improve the detection of subtle genetic structure, but this may substantially increase sequencing cost and downstream bioinformatics computational time. To overcome this challenge, alternative, cost‐effective sequencing approaches, namely Pool‐seq and Rapture, have been developed. We empirically measured the power of resolution and congruence of these two methods in documenting weak population structure in nonmodel species with high gene flow comparatively to a conventional genotyping‐by‐sequencing (GBS) approach. For this, we used the American lobster (Homarus americanus) as a case study. First, we found that GBS, Rapture, and Pool‐seq approaches gave similar allele frequency estimates (i.e., correlation coefficient over 0.90) and all three revealed the same weak pattern of population structure. Yet, Pool‐seq data showed FST estimates three to five times higher than GBS and Rapture, while the latter two methods returned similar FST estimates, indicating that individual‐based approaches provided more congruent results than Pool‐seq. We conclude that despite higher costs, GBS and Rapture are more convenient approaches to use in the case of species exhibiting very weak differentiation. While both GBS and Rapture approaches provided similar results with regard to estimates of population genetic parameters, GBS remains more cost‐effective in project involving a relatively small numbers of genotyped individuals (e.g., <1,000). Overall, this study illustrates the complexity of estimating genetic differentiation and other summary statistics in complex biological systems characterized by large population size and migration rates.