RADseq Markers Research Articles

Polyploidy and hybridization in the Mediterranean: unravelling the evolutionary history of Centaurium (Gentianaceae).

Polyploidy is considered one of the main mechanisms of plant evolution and speciation. In the Mediterranean Basin, polyploidy has contributed to making this region a biodiversity hotspot, along with its geological and climatic history and other ecological and biogeographical factors. The Mediterranean genus Centaurium (Gentianaceae) comprises ~25 species, of which 60 % are polyploids, including tetraploids and hexaploids. To date, the evolutionary history of centauries has been studied using Sanger sequencing phylogenies, which have been insufficient to fully understand the phylogenetic relationships in this lineage. The goal of this study is to gain a better understanding of the evolutionary history of Centaurium by exploring the mechanisms that have driven its diversification, specifically hybridization and polyploidy. We aim to identify the parentage of hybrid species, at the species or clade level, as well as assessing whether morphological traits are associated with particular ploidy levels. We sequenced RADseq markers from 42 samples of 28 Centaurium taxa, and performed phylogenomic analyses using maximum likelihood, summary coalescent SVDquartets and Neighbor-Net approaches. To identify hybrid taxa, we used PhyloNetworks and the fastSTRUCTURE algorithm. To infer the putative parental species of the allopolyploids, we employed genomic analyses (SNIPloid). The association between different traits and particular ploidy levels was explored with non-metric multidimensional scaling. Our phylogenetic analyses confirmed the long-suspected occurrence of recurrent hybridization. The allopolyploid origin of the tetraploid C. serpentinicola and the hexaploids C. mairei, C. malzacianum and C. centaurioides was also confirmed, unlike that of C. discolor. We inferred additional signatures of hybridization events within the genus and identified morphological traits differentially distributed in different ploidy levels. This study highlights the important role that hybridization has played in the evolution of a Mediterranean genus such as Centaurium, leading to a polyploid complex, which facilitated its diversification and may exemplify that of other Mediterranean groups.

Cytotaxonomic characterization and estimation of migration patterns of onchocerciasis vectors (Simulium damnosum sensu lato) in northwestern Ethiopia based on RADSeq data.

While much progress has been made in the control and elimination of onchocerciasis across Africa, the extent to which vector migration might confound progress towards elimination or result in re-establishment of endemism in areas where transmission has been eliminated remains unclear. In Northern Ethiopia, Metema and Metekel-two foci located near the Sudan border-exhibit continuing transmission. While progress towards elimination has been faster in Metema, there remains a problematic hotspot of transmission. Whether migration from Metekel contributes to this is currently unknown. To assess the role of vector migration from Metekel into Metema, we present a population genomics study of 151 adult female vectors using 47,638 RADseq markers and mtDNA CoI sequencing. From additional cytotaxonomy data we identified a new cytoform in Metema, closely related to S. damnosum s.str, here called the Gondar form. RADseq data strongly indicate the existence of two distinctly differentiated clusters within S. damnosum s.l.: one genotypic cluster found only in Metema, and the second found predominantly in Metekel. Because blackflies from both clusters were found in sympatry (in all four collection sites in Metema), but hybrid genotypes were not detected, there may be reproductive barriers preventing interbreeding. The dominant genotype in Metema was not found in Metekel while the dominant genotype in Metekel was found in Metema, indicating that (at the time of sampling) migration is primarily unidirectional, with flies moving from Metekel to Metema. There was strong differentiation between clusters but little genetic differentiation within clusters, suggesting migration and gene flow of flies within the same genetic cluster are sufficient to prevent genetic divergence between sites. Our results confirm that Metekel and Metema represent different transmission foci, but also indicate a northward movement of vectors between foci that may have epidemiological importance, although its significance requires further study.

An evaluation of DNA sample source and molecular markers to determine gender in the short-beaked echidna (Tachyglossus aculeatus).

The short-beaked echidna is sexually monomorphic such that gender identification without veterinary intervention is challenging. The aim of this study was to evaluate and compare the most optimal noninvasive genetic source by extracting echidna genomic DNA(gDNA) from fecal scats, plucked hair, and quills to perform genetic sex testing using a range of molecular markers. Sex determination of 14 captive short-beaked echidnas was determined by amplifying isolated DNA from noninvasive samples, targeting two Y chromosome (male-specific) genes (mediator complex subunit 26 Y-gametologue [CRSPY] and anti-Müllerian hormone Y-gametologue [AMHY]), in addition to four confirmed sex-specific RADseq markers. Results of noninvasive samples were compared with blood samples and clinical records. Receiver operating characteristiccurves were used to assess accuracy of sex determination of markers for each sample type. The gender of the echidnas was successfully identified on 75% of occasions using fecal samples, 90.6% occasions using hair, and 84.6% occasions with quills. Overall, the male-specific RADseq markers accurately identified the sex of echidnas with all sample types for 90% of animals; compared with 81.5% using CRSPY, and 82.0% using AMHY to identify sex. Collection of hair, quills, and feces provides a useful alternative to invasively collected samples, however, the accuracy of results depends on sample type and genetic marker selected. We found gender determination in the short-beaked echidna was most accurate using four male-specific RADseq markers on gDNA isolated from blood and hair. The noninvasive genetic sexing techniques documented here will inform and facilitate husbandry and genetic management of captive echidna populations.

Two transects reveal remarkable variation in gene flow on opposite ends of a European toad hybrid zone.

Speciation entails a reduction in gene flow between lineages. The rates at which genomic regions become isolated varies across space and time. Barrier markers are linked to putative genes involved in (processes of) reproductive isolation, and, when observed over two transects, indicate species-wide processes. In contrast, transect-specific putative barrier markers suggest local processes. We studied two widely separated transects along the 900 km hybrid zone between Bufo bufo and B. spinosus, in northern and southern France, for ~1200 RADseq markers. We used genomic and geographic cline analyses to identify barrier markers based on their restricted introgression, and found that some markers are transect-specific, while others are shared between transects. Twenty-six barrier markers were shared across both transects, of which some are clustered in the same chromosomal region, suggesting that their associated genes are involved in reduced gene flow across the entire hybrid zone. Transect-specific barrier markers were twice as numerous in the southern than in the northern transect, suggesting that the overall barrier effect is weaker in northern France. We hypothesize that this is consistent with a longer period of secondary contact in southern France. The smaller number of introgressed genes in the northern transect shows considerably more gene flow towards the southern (B. spinosus) than the northern species (B. bufo). We hypothesize that hybrid zone movement in northern France and hybrid zone stability in southern France explain this pattern. The Bufo hybrid zone provides an excellent opportunity to separate a general barrier effect from localized gene flow-reducing conditions.

Identification of Iguania Ancestral Syntenic Blocks and Putative Sex Chromosomes in the Veiled Chameleon (Chamaeleo calyptratus, Chamaeleonidae, Iguania)

The veiled chameleon (Chamaeleo calyptratus) is a typical member of the family Chamaeleonidae and a promising object for comparative cytogenetics and genomics. The karyotype of C. calyptratus differs from the putative ancestral chameleon karyotype (2n = 36) due to a smaller chromosome number (2n = 24) resulting from multiple chromosome fusions. The homomorphic sex chromosomes of an XX/XY system were described recently using male-specific RADseq markers. However, the chromosomal pair carrying these markers was not identified. Here we obtained chromosome-specific DNA libraries of C. calyptratus by chromosome flow sorting that were assigned by FISH and sequenced. Sequence comparison with three squamate reptiles reference genomes revealed the ancestral syntenic regions in the C. calyptratus chromosomes. We demonstrated that reducing the chromosome number in the C. calyptratus karyotype occurred through two fusions between microchromosomes and four fusions between micro-and macrochromosomes. PCR-assisted mapping of a previously described Y-specific marker indicates that chromosome 5 may be the sex chromosome pair. One of the chromosome 5 conserved synteny blocks shares homology with the ancestral pleurodont X chromosome, assuming parallelism in the evolution of sex chromosomes from two basal Iguania clades (pleurodonts and acrodonts). The comparative chromosome map produced here can serve as the foundation for future genome assembly of chameleons and vertebrate-wide comparative genomic studies.

Genome-wide markers reveal differentiation between and within the cryptic sister species, sunset and vermilion rockfish

The vermilion rockfish complex, which consists of the cryptic sister species vermilion and sunset rockfish, is one of the most valuable recreational fisheries on the U.S. West Coast. These species are currently managed as a single complex, and because of uncertainty surrounding the relative contribution of each species within existing data sources, the stock status of each species is not fully known. A reliable and cost-effective method is needed to disentangle these species that will allow for the development of abundance indices, life history profiles, and catch histories that may potentially support species-specific stock assessments. Using restriction-site associated DNA sequence (RADseq) markers we generated 10,003 polymorphic loci to characterize the vermilion rockfish complex. PCA and Bayesian clustering approaches based on these loci clearly distinguished between sunset and vermilion rockfishes and identified hybrid individuals. These loci included 203 highly differentiated (FST ≥ 0.99) single nucleotide polymorphisms, which we consider candidates in the planned development of a diagnostic assay capable of distinguishing between these cryptic species. In addition to clearly delineating to species, subsets of the interspecific markers allowed for insight into intraspecific differentiation in both species. Population genetic analyses for sunset rockfish identified two weakly divergent genetic groups with similar levels of genetic diversity. Vermilion rockfish, however, were characterized by three distinct genetic groups with much stronger signals of differentiation and significantly different genetic diversities. Collectively, these data will contribute to well-informed, species-specific management strategies to protect this valuable species complex.

Two decades of DNA barcoding in the genus Usnea (Parmeliaceae): how useful and reliable is the ITS?

We present an exhaustive analysis of the ITS barcoding marker in the genus Usnea s.lat., separated into Dolichousnea, Eumitria, and Usnea including the subgenus Neuropogon, analyzing 1,751 accessions. We found only a few low-quality accessions, whereas information on voucher specimens and accuracy and precision of identifications was of subpar quality for many accessions. We provide an updated voucher table, alignment and phylogenetic tree to facilitate DNA barcoding of Usnea, either locally or through curated databases such as UNITE. Taxonomic and geographic coverage was moderate: while Dolichousnea and subgenus Neuropogon were well-represented among ITS data, sampling for Eumitria and Usnea s.str. was sparse and biased towards certain lineages and geographic regions, such as Antarctica, Europe, and South America. North America, Africa, Asia and Oceania were undersampled. A peculiar situation arose with New Zealand, represented by a large amount of ITS accessions from across both major islands, but most of them left unidentified. The species pair Usnea antarctica vs. U. aurantiacoatra was the most sampled clade, including numerous ITS accessions from taxonomic and ecological studies. However, published analyses of highly resolved microsatellite and RADseq markers showed that ITS was not able to properly resolve the two species present in this complex. While lack of resolution appears to be an issue with ITS in recently evolving species complexes, we did not find evidence for gene duplication (paralogs) or hybridization for this marker. Comparison with other markers demonstrated that particularly IGS and RPB1 are useful to complement ITS-based phylogenies. Both IGS and RPB1 provided better backbone resolution and support than ITS; while IGS also showed better resolution and support at species level, RPB1 was less resolved and delineated for larger species complexes. The nuLSU was of limited use, providing neither resolution nor backbone support. The other three commonly employed protein-coding markers, TUB2, RPB2, and MCM7, showed variable evidence of possible gene duplication and paralog formation, particularly in the MCM7, and these markers should be used with care, especially in multimarker coalescence approaches. A substantial challenge was provided by difficult morphospecies that did not form coherent clades with ITS or other markers, suggesting various levels of cryptic speciation, the most notorious example being the U. cornuta complex. In these cases, the available data suggest that multimarker approaches using ITS, IGS and RPB1 help to assess distinct lineages. Overall, ITS was found to be a good first approximation to assess species delimitation and recognition in Usnea s.lat., as long as the data are carefully analyzed, and reference sequences are critically assessed and not taken at face value. In difficult groups, we recommend IGS as a secondary barcode marker, with the option to employ more resource-intensive approaches, such as RADseq, in species complexes involving so-called species pairs or other cases of disparate morphology not reflected in the ITS or IGS. Attempts should be made to close taxonomic and geographic gaps especially for the latter two markers, in particular in Eumitria and Usnea s.str. and in the highly diverse areas of North America and Central America, Africa, Asia, and Oceania.

Rapid generation of ecologically relevant behavioral novelty in experimental cichlid hybrids.

The East African cichlid radiations are characterized by repeated and rapid diversification into many distinct species with different ecological specializations and by a history of hybridization events between nonsister species. Such hybridization might provide important fuel for adaptive radiation. Interspecific hybrids can have extreme trait values or novel trait combinations and such transgressive phenotypes may allow some hybrids to explore ecological niches neither of the parental species could tap into. Here, we investigate the potential of second‐generation (F2) hybrids between two generalist cichlid species from Lake Malawi to exploit a resource neither parental species is specialized on: feeding by sifting sand. Some of the F2 hybrids phenotypically resembled fish of species that are specialized on sand sifting. We combined experimental behavioral and morphometric approaches to test whether the F2 hybrids are transgressive in both morphology and behavior related to sand sifting. We then performed a quantitative trait loci (QTL) analysis using RADseq markers to investigate the genetic architecture of morphological and behavioral traits. We show that transgression is present in several morphological traits, that novel trait combinations occur, and we observe transgressive trait values in sand sifting behavior in some of the F2 hybrids. Moreover, we find QTLs for morphology and for sand sifting behavior, suggesting the existence of some loci with moderate to large effects. We demonstrate that hybridization has the potential to rapidly generate novel and ecologically relevant phenotypes that may be suited to a niche neither of the parental species occupies. Interspecific hybridization may thereby contribute to the rapid generation of ecological diversity in cichlid radiations.

Detection of quantitative trait loci for salt tolerance in zombi pea [Vigna vexillata (L.) A. Rich

Zombi pea [Vigna vexillata (L.) A. Rich] is an underutilized legume crop, with highly diverse wild species that carry genes conferring resistance to several biotic and abiotic stresses. The objective of this study was to construct a zombi pea genetic linkage map and locate quantitative trait loci related to salt resistance. A zombi pea F2 population (159 individuals) developed from a cross between the salt-resistant wild zombi pea accession JP235908 (var. ovata; female parent) and the salt-susceptible cultivated zombi pea accession TVNu240 (var. macrosperma; male parent) was genotyped with simple sequence repeat (SSR) and restriction site-associated DNA sequencing (RAD-seq) markers. A genetic linkage map with 10 linkage groups (LGs) was constructed based on 379 markers (136 SSR and 243 RAD-seq markers). The map spanned 793.25 cM, with a mean distance between adjacent markers of 2.14 cM. A comparative genome analysis revealed high macro-synteny between zombi pea and mung bean/adzuki bean, although several chromosomal translocations likely occurred in zombi pea. The salt tolerance of the F2:3 population was evaluated at the seedling stage under hydroponic conditions. Inclusive composite interval mapping identified three quantitative trait loci (QTLs) (qSaltol1.1 on LG1, qSaltol2.1 on LG2, and qSaltol6.1 on LG6) related to the salt resistance of the F2 population. The qSaltol1.1, qSaltol2.1, and qSaltol6.1 QTLs explained 13.3%, 7.6%, and 8.1% of the salt-resistance phenotypic variance, respectively. A comparative genome analysis revealed that qSaltol1.1 may correspond to the Saltol1.1 QTL conferring salt tolerance in beach cowpea [Vigna marina (Burm.) Merr.], which is a halophytic species. Moreover, two genes encoding a plasma membrane H+-ATPase and a gene encoding a cation/proton exchanger may contribute to the salt resistance of zombi pea and beach cowpea. These findings may be relevant for the breeding of salt-resistant zombi pea plants.

Limited gene exchange between two sister species of leaf beetles within a hybrid zone in the Alps.

Analysing genomic variation within and between sister species is a first step towards understanding species boundaries. We focused on two sister species of cold-resistant leaf beetles, Gonioctena quinquepunctata and G.intermedia, whose ranges overlap in the Alps. A previous study of DNA sequence variation had revealed multiple instances of mitochondrial genome introgression in this region, suggesting recent hybridization between the two species. To evaluate the extent of gene exchange resulting from these hybridization events, we sampled individuals of both species inside and outside the hybrid zone and analysed genomic variation among them using RAD-seq markers. Individual levels of introgression in the nuclear genome were estimated first by defining species-specific SNPs (displaying a fixed difference between species) a priori and second by using model-based methods. Both types of analyses indicated little gene exchange, if any, between species at the level of the nuclear genome. Whereas the first method suggested slightly more gene flow, we argue that it has likely overestimated introgression in the phylogeographic context of this study. We conclude that strong intrinsic barriers prevent genetic exchange at the level of the nuclear genome between the two species. The apparent discrepancy observed between introgression occurring in the nuclear and mitochondrial genomes could be explained by selection acting in favour of the latter. Also, these results have consequences for the phylogeographic study of each species, since we can assume that genetic diversity in the overlapping portion of their ranges is not the product of introgression.

Developing a High-Quality Linkage Map for the Atlantic Killifish Fundulus heteroclitus.

Killifish (Fundulus heteroclitus) are widely distributed among different aquatic environments where they demonstrate an impressive range of highly-plastic and locally adaptive phenotypes. High-throughput sequencing has begun to unravel the mechanisms and evolutionary history of these interesting features by establishing relationships in the genotype-phenotype map. However, some genotype-phenotype analyses require a higher order of contiguity than what initial scaffolded (fragmented genome assembly where contigs have been assemble into scaffolds) genome assemblies can provide. Here, we used 5,685 high-quality RAD-Seq markers from a single mapping family to order 84% of the scaffolded genome assembly to 24 chromosomes. This serves to: 1) expand the killifish genomic toolkit, 2) estimate genome-wide recombination rates, and 3) compare genome synteny to humans and other fishes. After initially building our map, we found that the selection of thresholds for sequence data filtration highly impacted scaffold placement in the map. We outline each step of the approach that dramatically improved our map to help guide others toward more effective linkage mapping for genome assembly. Our final map supports strong conservation of genomic synteny among closely related fish species and reveals previously described chromosomal rearrangements between more distantly related clades. However, we also commonly found minor scaffold misorientations in F. heteroclitus and in other assemblies, suggesting that further mapping (such as optical mapping) is necessary for finer scale resolution of genome structure. Lastly, we discuss the problems that would be expected from misoriented/unplaced scaffolds and stress the importance of a quality mapped genome as a key feature for further investigating population and comparative genomic questions with F. heteroclitus and other taxa.

Dense SNP panels resolve closely related Chinook salmon populations

Chinook salmon (Oncorhynchus tshawytscha) are migratory fish that are highly valued for subsistence, sport, and commercial fisheries throughout their native range. Populations of Chinook salmon in western Alaska have exhibited long-term declines, leading to restrictions on harvests. Management priorities require greater resolution for genetic stock identification (GSI) than is available with current methods. We leveraged RADseq, TaqMan, and GT-seq data originating from multiple sources, collected through time, to develop a set of GT-seq panels containing 1092 single nucleotide polymorphisms (SNPs) that improved GSI resolution in western Alaska for at-sea and in-river sampling. We generated a dense linkage map to ensure that markers selected for panels spanned the entire genome. In addition, we identified multiple RADseq markers that were associated with sex; these aligned to a 5-centimorgan (cM) region on the sex chromosome. Finally, we developed a bioinformatic pipeline to streamline analysis of GT-seq data that is capable of genotyping microhaplotypes and paralogs, both of which can improve GSI resolution over traditional single-SNP data. Our panels and pipeline provide tools for management agencies to rapidly and easily analyze large-scale genotyping projects.

Construction of a high-density genetic map of tree peony (Paeonia suffruticosa Andr. Moutan) using restriction site associated DNA sequencing (RADseq) approach

High-density genetic maps play a vital role in dissecting genetic components of biologically or agronomically important traits and molecular marker-assisted selection breeding, especially for a species with limited genomic information. Tree Peony (Paeonia suffruticosa Andr. Moutan DC.) is a traditional flowering plant in China, with important ornamental value, which have been spread around the world. However, less genetic and genomic information is available for the tree peony molecular studies. Here, we performed restriction site-associated DNA sequencing (RADseq) of an F1 population, derived from a traditional variety “Qing Long Wo Mo Chi (QL)” and a variety “Mo Zi Lian (MZL)”. High-density genetic maps were developed using RADseq markers for female (1471 markers) and male (793 markers) parents. They covered 965.69 and 870.21 cM, with the average marker intervals of 0.66 and 1.10 cM along the seven and five linkage groups (LGs), respectively. Furthermore, the identified markers were assigned to 1671 bins, representing the unique genetic positions in LGs, and inter-marker distances smaller than 5 cM covered 97.93% of genetic maps. This study suggests that rapid de novo construction of genetic maps in peony could be conducted through RADseq approach, which provides an important tool for further linkage mapping and genomic structure analysis, and enable development of molecular markers for molecular breeding in tree peony.

Genomic variation of introduced Salvinia minima in southeastern United States

Common salvinia, Salvinia minima Baker (Salviniaceae), is a small, floating aquatic fern native to Central and South America that has invaded fresh water bodies in southeastern United States since the 1930s. We examined genetic variation across much of the introduced range of this species in the United States using codominant RAD-seq markers. Data from over 600 variable loci showed a reduction in heterozygosity from east to west in addition to a corresponding trend in assignment of samples to one of two genetic groups. Our data are consistent with previous published work and with the hypothesis that common salvinia had a single introduction on the east end of its current range in the United States. From there it migrated westward, losing genetic diversity during this spread. The data are also consistent with sexual reproduction, although we are unable to estimate the extent of this relative to asexual spreading. Future genetic work should include sampling from the native range to help determine the original sources of North American common salvinia.

Investigation of Chinese Wolfberry (Lycium spp.) Germplasm by Restriction Site-Associated DNA Sequencing (RAD-seq)

Chinese wolfberry (Lycium spp.) is an important edible and medicinal plant, with a long cultivation history. The genetic relationships among wild Lycium species and landraces have been unclear for a number of reasons, which has hindered the breeding of modern Chinese wolfberry cultivars. In this study, we collected 19 accessions of Chinese wolfberry germplasm, and constructed the genetic relationship based on RAD-seq markers. We obtained 30.32 Gb of clean data, with the average value of each sample being 1.596 Gb. The average mapping rate was 85.7%, and the average coverage depth was 6.76 X. The phylogeny results distinguished all accessions clearly. All the studied landraces shared their most recent common ancestor with L. barbarum, which indicated that L. barbarum may be involved in cultivation of these landraces. The relationship of some landraces, namely the ‘Ningqi’ series, ‘Qingqi-1’ and ‘Mengqi-1,’ has been supported by the phylogeny results, while the triploid wolfberry was shown to be based on a hybrid between ‘Ningqi-1’ and a tetraploid wolfberry. This study uncovered the genetic background of Chinese wolfberry, and developed the foundation for species classification, accession identification and protection, and the production of hybrid cultivars of wolfberry.

XX/XY Sex Chromosomes in the South American Dwarf Gecko (Gonatodes humeralis).

Sex-specific genetic markers identified using restriction site-associated DNA sequencing, or RADseq, permits the recognition of a species' sex chromosome system in cases where standard cytogenetic methods fail. Thus, species with male-specific RAD markers have an XX/XY sex chromosome system (male heterogamety) while species with female-specific RAD markers have a ZZ/ZW sex chromosome (female heterogamety). Here, we use RADseq data from 5 male and 5 female South American dwarf geckos (Gonatodes humeralis) to identify an XX/XY sex chromosome system. This is the first confidently known sex chromosome system in a Gonatodes species. We used a low-coverage de novo G. humeralis genome assembly to design PCR primers to validate the male-specificity of a subset of the sex-specific RADseq markers and describe how even modest genome assemblies can facilitate the design of sex-specific PCR primers in species with diverse sex chromosome systems.

Deciphering the demographic history of allochronic differentiation in the pine processionary moth Thaumetopoea pityocampa.

Understanding the processes of adaptive divergence, which may ultimately lead to speciation, is a major question in evolutionary biology. Allochronic differentiation refers to a particular situation where gene flow is primarily impeded by temporal isolation between early and late reproducers. This process has been suggested to occur in a large array of organisms, even though it is still overlooked in the literature. We here focused on a well-documented case of incipient allochronic speciation in the winter pine processionary moth Thaumetopoea pityocampa. This species typically reproduces in summer and larval development occurs throughout autumn and winter. A unique, phenologically shifted population (SP) was discovered in 1997 in Portugal. It was proved to be strongly differentiated from the sympatric "winter population" (WP), but its evolutionary history could only now be explored. We took advantage of the recent assembly of a draft genome and of the development of pan-genomic RAD-seq markers to decipher the demographic history of the differentiating populations and develop genome scans of adaptive differentiation. We showed that the SP diverged relatively recently, that is, few hundred years ago, and went through two successive bottlenecks followed by population size expansions, while the sympatric WP is currently experiencing a population decline. We identified outlier SNPs that were mapped onto the genome, but none were associated with the phenological shift or with subsequent adaptations. The strong genetic drift that occurred along the SP lineage certainly challenged our capacity to reveal functionally important loci.

Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini.

BackgroundRust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field.ResultsTo facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein.ConclusionsThe M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3011-9) contains supplementary material, which is available to authorized users.

Tempo and mode of speciation in Holacanthus angelfishes based on RADseq markers

In this study we estimated the timing of speciation events in a group of angelfishes using 1186 RADseq markers corresponding to 94,880 base pairs. The genus Holacanthus comprises seven species, including two clades of Panama trans-Isthmian geminates, which diverged approximately 3–3.5Mya. These clades diversified within the Tropical Eastern Pacific (TEP, three species) and Tropical Western Atlantic (TWA, two species) which our data suggest to have occurred within the past 1.5My in both ocean basins, but may have proceeded via different mechanisms. In the TEP, speciation is likely to have followed a peripatric pathway, while in the TWA, sister species are currently partially sympatric, thus raising the possibility of sympatric speciation. This study highlights the use of RADseq markers for estimating both divergence times and modes of speciation at a 1–3My timescale.

High-density linkage maps fail to detect any genetic component to sex determination in a Rana temporaria family.

Sex chromosome differentiation in Rana temporaria varies strikingly among populations or families: whereas some males display well-differentiated Y haplotypes at microsatellite markers on linkage group 2 (LG2), others are genetically undistinguishable from females. We analysed with RADseq markers one family from a Swiss lowland population with no differentiated sex chromosomes, and where sibship analyses had failed to detect any association between the phenotypic sex of progeny and parental haplotypes. Offspring were reared in a common tank in outdoor conditions and sexed at the froglet stage. We could map a total of 2177 SNPs (1123 in the mother, 1054 in the father), recovering in both adults 13 linkage groups (= chromosome pairs) that were strongly syntenic to Xenopus tropicalis despite > 200 My divergence. Sexes differed strikingly in the localization of crossovers, which were uniformly distributed in the female but limited to chromosome ends in the male. None of the 2177 markers showed significant association with offspring sex. Considering the very high power of our analysis, we conclude that sex determination was not genetic in this family; which factors determined sex remain to be investigated.