Restriction-site Associated DNA Markers Research Articles

Exploring Genomics and Microbial Ecology: Analysis of Bidens pilosa L. Genetic Structure and Soil Microbiome Diversity by RAD-Seq and Metabarcoding.

Bidens pilosa L., native to South America and commonly used for medicinal purposes, has been understudied at molecular and genomic levels and in its relationship with soil microorganisms. In this study, restriction site-associated DNA markers (RADseq) techniques were implemented to analyze genetic diversity and population structure, and metabarcoding to examine microbial composition in soils from Palmira, Sibundoy, and Bogotá, Colombia. A total of 2,984,123 loci and 3485 single nucleotide polymorphisms (SNPs) were identified, revealing a genetic variation of 12% between populations and 88% within individuals, and distributing the population into three main genetic groups, FST = 0.115 (p < 0.001) and FIT = 0.013 (p > 0.05). In the soil analysis, significant correlations were found between effective cation exchange capacity (ECEC) and apparent density, soil texture, and levels of Mg and Fe, as well as negative correlations between ECEC and Mg, and Mg, Fe, and Ca. Proteobacteria and Ascomycota emerged as the predominant bacterial and fungal phyla, respectively. Analyses of alpha, beta, and multifactorial diversity highlight the influence of ecological and environmental factors on these microbial communities, revealing specific patterns of clustering and association between bacteria and fungi in the studied locations.

Population and conservation genetics using RAD sequencing in four endemic conifers from South America

The emblematic conifer flora of Chile has experienced wide-scale habitat loss and fragmentation. Coupled with the complex topography of the region, this leads to a prediction of high levels of genetic drift and isolation resulting in strong population differentiation, and the potential for negative genetic consequences. To address the degree to which these predictions are realised, we conducted a comparative population genetic survey in four endemic conifers from South America, each of conservation concern and with a restricted distribution area. Between seven and ten populations per species were sampled, covering their entire natural distribution in Chile. We used restriction site-associated DNA markers (RAD-seq), with de novo assembly and optimisation, to accommodate the large and complex genome of conifers. The main finding was low levels of genetic structure in all four conifer species (FST = 0.017–0.062). This pattern was strikingly consistent across all four species. These results are likely due to the extreme longevity of individuals restricting divergence due to genetic drift. Thus, despite the currently highly fragmented ranges of these conifer species, we did not detect evidence for high levels of population differentiation and genetic isolation. This suggests some resilience to negative genetic impacts of habitat fragmentation, and the longevity of the individual trees may provide considerable buffering capacity. We recommend follow-up studies focusing on genotyping the seedling generation, to assess whether there is any evidence for the early warning of genetic isolation and/or elevated inbreeding in the currently established cohorts.

Insights from a comprehensive study of Trypanosoma cruzi: A new mitochondrial clade restricted to North and Central America and genetic structure of TcI in the region.

More than 100 years since the first description of Chagas Disease and with over 29,000 new cases annually due to vector transmission (in 2010), American Trypanosomiasis remains a Neglected Tropical Disease (NTD). This study presents the most comprehensive Trypanosoma cruzi sampling in terms of geographic locations and triatomine species analyzed to date and includes both nuclear and mitochondrial genomes. This addresses the gap of information from North and Central America. We incorporate new and previously published DNA sequence data from two mitochondrial genes, Cytochrome oxidase II (COII) and NADH dehydrogenase subunit 1 (ND1). These T. cruzi samples were collected over a broad geographic range including 111 parasite DNA samples extracted from triatomines newly collected across North and Central America, all of which were infected with T. cruzi in their natural environment. In addition, we present parasite reduced representation (Restriction site Associated DNA markers, RAD-tag) genomic nuclear data combined with the mitochondrial gene sequences for a subset of the triatomines (27 specimens) collected from Guatemala and El Salvador. Our mitochondrial phylogenetic reconstruction revealed two of the major mitochondrial lineages circulating across North and Central America, as well as the first ever mitochondrial data for TcBat from a triatomine collected in Central America. Our data also show that within mtTcIII, North and Central America represent an independent, distinct clade from South America, named here as mtTcIIINA-CA, geographically restricted to North and Central America. Lastly, the most frequent lineage detected across North and Central America, mtTcI, was also an independent, distinct clade from South America, noted as mtTcINA-CA. Furthermore, nuclear genome data based on Single Nucleotide Polymorphism (SNP) showed genetic structure of lineage TcI from specimens collected in Guatemala and El Salvador supporting the hypothesis that genetic diversity at a local scale has a geographical component. Our multiscale analysis contributes to the understanding of the independent and distinct evolution of T. cruzi lineages in North and Central America regions.

Evolution, systematics and historical biogeography of sand flies of the subgenus Paraphlebotomus (Diptera, Psychodidae, Phlebotomus) inferred using restriction-site associated DNA markers.

Phlebotomine sand flies are the main natural vectors of Leishmania, which cause visceral and tegumentary tropical diseases worldwide. However, their taxonomy and evolutionary history remain poorly studied. Indeed, as for many human disease vectors, their small size is a challenge for morphological and molecular works. Here, we successfully amplified unbiased copies of whole genome to sequence thousands of restriction-site associated DNA (RAD) markers from single specimens of phlebotomines. RAD markers were used to infer a fully resolved phylogeny of the subgenus Paraphlebotomus (11 species + 5 outgroups, 32 specimens). The subgenus was not recovered as monophyletic and we describe a new subgenus Artemievus subg. nov. Depaquit for Phlebotomus alexandri. We also confirm the validity of Ph. riouxi which is reinstated as valid species. Our analyses suggest that Paraphlebotomus sensu nov. originated ca 12.9-8.5 Ma and was possibly largely distributed from peri-Mediterranean to Irano-Turanian regions. Its biogeographical history can be summarized into three phases: i) a first split between Ph. riouxi + Ph. chabaudi and other species that may have resulted from the rise of the Saharan belt ca 8.5 Ma; ii) a Messinian vicariant event (7.3-5.3 Ma) during which the prolonged drought could have resulted in the divergence of main lineages; iii) a recent radiation event (3-2 Ma) that correspond to cycles of wet and dry periods in the Middle East and the East African subregions during the Pleistocene. Interestingly these cycles are also hypothetical drivers of the diversification of rodents, in the burrows of which Paraphlebotomus larvae develop. By meeting the challenge of sequencing pangenomics markers from single, minute phlebotomines, this work opens new avenues for improving our understanding of the epidemiology of leishmaniases and possibly other human diseases transmitted by arthropod vectors.

Quantitative Trait Locus Analysis in Squash (Cucurbita moschata) Based on Simple Sequence Repeat Markers and Restriction Site-Associated DNA Sequencing Analysis

Squash (Cucurbita moschata) displays wide morphological and genetic variations; however, limited information is available regarding the genetic loci of squash that control its agronomic traits. To obtain basic genetic information for C. moschata, an F2 population was prepared derived from a cross between the Vietnamese cultivar ‘Bí Hồ Lô TN 6 (TN 6)’ and the Japanese cultivar ‘Shishigatani’, and flowering and fruit traits were examined. Overall, the traits showed a continuous distribution in the F2 population, suggesting that they were quantitative traits. A linkage map was constructed based on simple sequence repeat and restriction site-associated DNA (RAD) markers to detect quantitative trait loci (QTLs). Twelve QTLs for flowering and fruit traits, as well as one phenotypic trait locus, were successfully localized on the map. The present QTLs explained the phenotypic variations at a moderate to relatively high level (16.0%–47.3%). RAD markers linked to the QTLs were converted to codominant cleaved amplified polymorphic sequence (CAPS) and derived CAPS markers for the easy detection of alleles. The information reported here provides useful information for understanding the genetics of Cucurbita and other cucurbit species, and for the selection of individuals with ideal traits during the breeding of Cucurbita vegetables.

Genomic differentiation in an endemic Philippine genus (Aves: Sarcophanops) owing to geographical isolation on recently disassociated islands.

Phylogeographical studies of Philippine vertebrates have demonstrated that genetic variation is broadly partitioned by Pleistocene island aggregation. Contemporary island discontinuity is expected to influence genetic differentiation but remains relatively undocumented, perhaps because the current episode of island isolation started in relatively recent times. We investigated inter- and intra-island population structure in a Philippine endemic bird genus (Sarcophanops) to determine whether genetic differentiation has evolved during the recent period of isolation. We sequenced thousands of genome-wide restriction site associated DNA (RAD) markers from throughout the Mindanao group to assess fine-scale genetic structure across islands. Specifically, we investigated patterns of gene flow and connectivity within and between taxonomic and geographical bounds. A previous assessment of mitochondrial DNA detected deep structure between Sarcophanops samarensis and a sister species, Sarcophanops steerii, but was insufficient to detect differentiation within either species. Analysis of RAD markers, however, revealed structure within S. samarensis between the islands of Samar/Leyte and Bohol. This genetic differentiation probably demonstrates an effect of recent geographical isolation (after the Last Glacial Maximum) on the genetic structure of Philippine avifauna. We suggest that the general lack of evidence for differentiation between recently isolated populations is a failure to detect subtle population structure owing to past genetic sampling constraints, rather than the absence of such structure.

Quantitative trait loci involved in the reproductive success of a parasitoid wasp.

Dissecting the genetic basis of intraspecific variations in life history traits is essential to understand their evolution, notably for potential biocontrol agents. Such variations are observed in the endoparasitoid Cotesia typhae (Hymenoptera: Braconidae), specialized on the pest Sesamia nonagrioides (Lepidoptera: Noctuidae). Previously, we identified two strains of C. typhae that differed significantly for life history traits on an allopatric host population. To investigate the genetic basis underlying these phenotypic differences, we used a quantitative trait locus (QTL) approach based on restriction site-associated DNA markers. The characteristic of C. typhae reproduction allowed us generating sisters sharing almost the same genetic content, named clonal sibship. Crosses between individuals from the two strains were performed to generate F2 and F8 recombinant CSS. The genotypes of 181 clonal sibships were determined as well as the phenotypes of the corresponding 4,000 females. Informative markers were then used to build a high-quality genetic map. These 465 markers spanned a total length of 1,300cM and were organized in 10 linkage groups which corresponded to the number of C. typhae chromosomes. Three QTLs were detected for parasitism success and two for offspring number, while none were identified for sex ratio. The QTLs explained, respectively, 27.7% and 24.5% of the phenotypic variation observed. The gene content of the genomic intervals was investigated based on the genome of C. congregata and revealed 67 interesting candidates, as potentially involved in the studied traits, including components of the venom and of the symbiotic virus (bracovirus) shown to be necessary for parasitism success in related wasps.

Introgression Leads to Genomic Divergence and Responsible for Important Traits in Upland Cotton.

Understanding the genetic diversity and population structure of germplasms is essential when selecting parents for crop breeding. The genomic changes that occurred during the domestication and improvement of Upland cotton (Gossypium hirsutum) remains poorly understood. Besides, the available genetic resources from cotton cultivars are limited. By applying restriction site-associated DNA marker sequencing (RAD-seq) technology to 582 tetraploid cotton accessions, we confirmed distinct genomic regions on chromosomes A06 and A08 in Upland cotton cultivar subgroups. Based on the pedigree, reported QTLs, introgression analyses, and genome-wide association study (GWAS), we suggest that these divergent regions might have resulted from the introgression of exotic lineages of G. hirsutum landraces and their wild relatives. These regions were the typical genomic signatures that might be responsible for maturity and fiber quality on chromosome A06 and chromosome A08, respectively. Moreover, these genomic regions are located in the putative pericentromeric regions, implying that their application will be challenging. In the study, based on high-density SNP markers, we reported two genomic signatures on chromosomes A06 and A08, which might originate from the introgression events in the Upland cotton population. Our study provides new insights for understanding the impact of historic introgressions on population divergence and important agronomic traits of modern Upland cotton cultivars.

New genomic resources for three exploited Mediterranean fishes

Extensive fishing has led to fish stock declines throughout the last decades. While clear stock identification is required for designing management schemes, stock delineation is problematic due to generally low levels of genetic structure in marine species. The development of genomic resources can help to solve this issue. Here, we present the first mitochondrial and nuclear draft genome assemblies of three economically important Mediterranean fishes, the white seabream, the striped red mullet, and the comber. The assemblies are between 613 and 785 Mbp long and contain between 27,222 and 32,375 predicted genes. They were used as references to map Restriction-site Associated DNA markers, which were developed with a single-digest approach. This approach provided between 15,710 and 21,101 Single Nucleotide Polymorphism markers per species. These genomic resources will allow uncovering subtle genetic structure, identifying stocks, assigning catches to populations and assessing connectivity. Furthermore, the annotated genomes will help to characterize adaptive divergence.

Genotyping by RAD Sequencing Analysis Assessed the Genetic Distinctiveness of Experimental Lines and Narrowed Down the Genomic Region Responsible for Leaf Shape in Endive (Cichorium endivia L.).

The characterization of genetic diversity in elite breeding stocks is crucial for the registration and protection of new varieties. Moreover, experimental population structure analysis and information about the genetic distinctiveness of commercial materials are essential for crop breeding programs. The purpose of our research was to assess the genetic relationships of 32 endive (Cichorium endivia L.) breeding lines, 18 from var. latifolium (escarole) and 14 from var. crispum (curly), using heterologous Cichorium intybus-derived simple sequence repeats (SSR) markers and single-nucleotide polymorphisms (SNP) markers. We found that 14 out of 29 SSR markers were successfully amplified, but only 8 of them were related to polymorphic loci. To overcome the limitation of the low number of informative SSR marker loci, an alternative SNP-based approach was employed. The 4621 SNPs produced by a restriction site-associated DNA marker sequencing approach were able to fully discriminate the 32 endive accessions; most importantly, as many as 50 marker loci were found to distinguish the curly group from the escarole group. Interestingly, 24 of the marker loci mapped within a peripheral segment of chromosome 8 of lettuce (Lactuca sativa L.), spanning a chromosomal region of 49.6 Mb. Following Sanger sequencing-based validation, three genes were determined to carry nonsynonymous SNPs, and one of them matched a putative ortholog of AtELP1, subunit 1 of the Elongator complex. Considering that several previously characterized Elongator complex subunit mutants exhibited elongated and/or curly leaf phenotypes, this gene should be taken into consideration for a better understanding of the underlying mechanism controlling leaf shape in endive.

Comparative phylogeographic inference with genome‐wide data from aggregated population pairs

Comparing divergences across multiple sister population pairs has been a focus in phylogeography since its inception. Initial approaches used organelle genetic data and involved qualitative comparisons of phylogenetic patterns to evaluate hypotheses of shared and variable evolutionary responses. This endeavor has progressed with coalescent model-based statistical techniques and advances in next-generation sequencing, yet there remains a need for methods that can exploit aggregated genomic-scale data within a unified analytical framework. To this end, we introduce the aggregate joint site frequency spectrum (ajSFS) by validating its use within a hierarchical Bayesian framework through several in silico experiments. Subsequently, we applied our method against two published restriction site-associated DNA marker datasets consisting of eight local replicates of a lamprey species pair and six co-distributed passerine taxon pairs, respectively, with the aim of inferring variability in co-divergence and co-migration histories. We found that the lamprey population pairs exhibited temporal synchrony in both co-divergence and collective secondary contact times, yet an idiosyncratic pattern in secondary migration intensities. In contrast, the bird population pairs displayed thoroughly asynchronous co-divergence histories. Our results demonstrate that the ajSFS can be exploited for complex and flexible co-demographic inference, opening up new possibilities for comparative phylogeography and population genomic studies.

Identifying Diagnostic Genetic Markers for a Cryptic Invasive Agricultural Pest: A Test Case Using the Apple Maggot Fly (Diptera: Tephritidae)

AbstractInsect pests destroy ~15% of all U.S. crops, resulting in losses of $15 billion annually. Thus, developing cheap, quick, and reliable methods for detecting harmful species is critical to curtail insect damage and lessen economic impact. The apple maggot fly, Rhagoletis pomonella, is a major invasive pest threatening the multibillion-dollar apple industry in the Pacific Northwest United States. The fly is also sympatric with a benign but morphologically similar and genetically closely related species, R. zephyria, which attacks noncommercial snowberry. Unambiguous species identification is essential due to a zero-infestation policy of apple maggot for fruit export. Mistaking R. zephyria for R. pomonella triggers unnecessary and costly quarantines, diverting valuable control resources. Here we develop and apply a relatively simple and cost-effective diagnostic approach using Illumina sequencing of double-digest restriction site-associated DNA markers. We identified five informative single-nucleotide polymorphisms (SNPs) and designed a diagnostic test based on agarose gel electrophoresis of restriction enzyme-digested polymerase chain reaction amplification products (RFLPs) to distinguish fly species. We demonstrated the utility of this approach for immediate, 1-d species identification by scoring apple- and snowberry-infesting flies from known hosts, reared from fruit collected at 11 sites throughout Washington. However, if immediate diagnosis is not required, or hundreds to thousands of specimens must be assessed, then a direct Illumina-based sequencing strategy, similar to that used here for diagnostic SNP identification, can be powerful and cost-effective. The genomic strategy we present is effective for R. pomonella and also transferable to many cryptic pests.

Museum specimens provide reliable SNP data for population genomic analysis of a widely distributed but threatened cockatoo species.

Natural history museums harbour a plethora of biological specimens which are of potential use in population and conservation genetic studies. Although technical advancements in museum genomics have enabled genome-wide markers to be generated from aged museum specimens, the suitability of these data for robust biological inference is not well characterized. The aim of this study was to test the utility of museum specimens in population and conservation genomics by assessing the biological and technical validity of single nucleotide polymorphism (SNP) data derived from such samples. To achieve this, we generated thousands of SNPs from 47 red-tailed black cockatoo (Calyptorhychus banksii) traditional museum samples (i.e. samples that were not collected with the primary intent of DNA analysis) and 113 fresh tissue samples (cryopreserved liver/muscle) using a restriction site-associated DNA marker approach (DArTseq™ ). Thousands of SNPs were successfully generated from most of the traditional museum samples (with a mean age of 44years, ranging from 5 to 123years), although 38% did not provide useful data. These SNPs exhibited higher error rates and contained significantly more missing data compared with SNPs from fresh tissue samples, likely due to considerable DNA fragmentation. However, based on simulation results, the level of genotyping error had a negligible effect on inference of population structure in this species. We did identify a bias towards low diversity SNPs in older samples that appears to compromise temporal inferences of genetic diversity. This study demonstrates the utility of a RADseq-based method to produce reliable genome-wide SNP data from traditional museum specimens.

Restriction-site associated DNA markers provide new insights into the evolutionary history of the bark beetle genus Dendroctonus

The bark beetle genus Dendroctonus contains some of the most economically important pests of conifers worldwide. Despite many attempts, there is no agreement today on the phylogenetic relationships within the genus, which limits our understanding of its evolutionary history. Here, using restriction-site associated DNA (RAD) markers from 70 specimens representing 17 species (85% of the known diversity) we inferred the phylogeny of the genus, its time of origin and biogeographic history, as well as the evolution of key ecological traits (host plants, larval behavior and adults’ attack strategies). For all combinations of tested parameters (from 6444 to 23,570 RAD tags analyzed), the same, fully resolved topology was inferred. Our analyses suggest that the most recent common ancestor (mrca) of all extant Dendroctonus species was widely distributed across eastern Palearctic and western Nearctic during the early Miocene, from where species dispersed to other Holarctic regions. A first main inter-continental vicariance event occurred during early Miocene isolating the ancestors of D. armandi in the Palearctic, which was followed by the radiation of the main Dendroctonus lineages in North America. During the Late Miocene, the ancestor of the ‘rufipennis’ species group colonized north-east Palearctic regions from western North America, which was followed by a second main inter-continental vicariance event isolating Pleistocene populations in Asia (D. micans) and western North America (D. murrayanae and D. punctatus). The present study supports previous hypotheses explaining intercontinental range disjunctions across the Northern Hemisphere by the fragmentation of a continuous distribution due to climatic cooling, host range fragmentation and geological changes during the late Cenozoic. The reconstruction of ancestral ecological traits indicates that the mrca bored individual galleries and mass attacked the boles of pines. The gregarious feeding behavior of the larvae as well as the individual attack of the base of trees have apparently independently evolved twice in North America (in the ‘rufipennis’ and the ‘valens’ species groups), which suggests a higher adaptive potential than previously thought and may be of interest for plant protection and biodiversity conservation in a rapidly changing world.

Influence of a climatic gradient on genetic exchange between two oak species.

In plant groups with limited intrinsic barriers to gene flow, it is thought that environmental conditions can modulate interspecific genetic exchange. Oaks are known for limited barriers to gene flow among closely related species. Here, we use Quercus as a living laboratory in which to pursue a fundamental question in plant evolution: Do environmental gradients restrict or promote genetic exchange between species? We focused on two North American oaks, the rare Quercus dumosa and the widespread Q. berberidifolia. We sampled intensively along a contact zone in California, USA. We sequenced restriction site-associated DNA markers and measured vegetative phenotype. We tested for genetic exchange, the association with climate, and the effect on phenotype. There is evidence for genetic exchange between the species. Admixed plants are found in areas of intermediate climate, while less admixed plants are found at the extremes of the climatic gradient. Genetic and phenotypic patterns are out of phase in the contact zone; some plants display the phenotype of one species but are genetically associated with another. Our results support the hypothesis that a strong climatic gradient can promote genetic exchange between species. The overall weak correlation between genotype and phenotype in the contact zone between the species suggests that genetic exchange can lead to the breakdown of trait combinations used to define species. This incongruency predicts ongoing problems for conservation of Q. dumosa, with implications for conservation of other oaks.

Conservation Genetics of the Endangered Del Mar Manzanita (Arctostaphylos glandulosasubsp.Crassifolia) Based On Rad Sequencing Data

Del Mar manzanita (Arctostaphylos glandulosa Eastw. subsp. crassifolia (Jeps.) P.V. Wells) is a federally listed endangered shrub found in San Diego County, California and Baja California, Mexico. This manzanita forms part of the imperiled southern maritime chaparral of southwestern California and adjacent Baja California, Mexico. Del Mar manzanita is problematic to identify because of morphological intergradation with other subspecies of A. glandulosa. Such intergradation could result from biological phenomena, such as gene flow among subspecies. Alternatively, it could be that the current circumscription of the Del Mar manzanita is not correct, and that the morphological characters used to diagnose this subspecies are inaccurate indicators of underlying genetics. This situation leads to problems for conservation planning, where accurate identification of individual plants is essential. Here, we used high-throughput sequencing of restriction-site associated DNA markers (RADseq) to develop single nucleotide polymorphism (SNP) data for a large sample of putative Del Mar manzanita, and a small sample of closely related subspecies of A. glandulosa. We analyzed genetic relationships using a total of 65,964 SNPs, with the aim of testing whether morphological traits used to identify Del Mar manzanita are an accurate reflection of underlying genetic patterns. We conclude that vegetative morphology is a poor predictor of genetic patterns, and that the current morphology-based circumscription of Del Mar manzanita is probably in need of some change. However, due to the limited sampling of A. glandulosa subspecies in this study, it is not possible to determine the taxonomic limits of Del Mar manzanita using our SNP data.

Restriction site-associated DNA sequencing for SNP discovery and high-density genetic map construction in southern catfish (Silurus meridionalis).

Single-nucleotide polymorphism (SNP) markers and high-density genetic maps are important resources for marker-assisted selection, mapping of quantitative trait loci (QTLs) and genome structure analysis. Although linkage maps in certain catfish species have been obtained, high-density maps remain unavailable in the economically important southern catfish (Silurus meridionalis). Recently developed restriction site-associated DNA (RAD) markers have proved to be a promising tool for SNP detection and genetic map construction. The objective of the present study was to construct a high-density linkage map using SNPs generated by next-generation RAD sequencing in S. meridionalis for future genetic and genomic studies. An F1 population of 100 individuals was obtained by intraspecific crossing of two wild heterozygous individuals. In total, 77 634 putative high-quality bi-allelic SNPs between the parents were discovered by mapping the parents' paired-end RAD reads onto the reference contigs from both parents, of which 54.7% were transitions and 45.3% were transversions (transition/transversion ratio of 1.2). Finally, 26 714 high-quality RAD markers were grouped into 29 linkage groups by using de novo clustering methods (Stacks). Among these markers, 4514 were linked to the female genetic map, 23 718 to the male map and 6715 effective loci were linked to the integrated map spanning 5918.31 centimorgans (cM), with an average marker interval of 0.89 cM. High-resolution genetic maps are a useful tool for both marker-assisted breeding and various genome investigations in catfish, such as sequence assembly, gene localization, QTL detection and genome structure comparison. Hence, such a high-density linkage map will serve as a valuable resource for comparative genomics and fine-scale QTL mapping in catfish species.

MsgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package (https://bioconductor.org/packages/release/bioc/html/msgbsR.html).

Construction of an SSR and RAD Marker-Based Genetic Linkage Map for Carnation (Dianthus caryophyllus L.)

A high-density genetic map, an essential tool for comparative genomic studies and quantitative trait locus fine mapping, can also facilitate genome sequence assembly. The sequence-based marker technology known as restriction site-associated DNA (RAD) enables synchronous, single nucleotide polymorphism marker discovery, and genotyping using massively parallel sequencing. We constructed a high-density linkage map for carnation (Dianthus caryophyllus L.) based on simple sequence repeat (SSR) markers in combination with RAD markers developed by double-digest RAD sequencing (ddRAD-seq). A total of 2404 (285 SSR and 2119 RAD) markers could be assigned to 15 linkage groups spanning 971.5 cM, with an average marker interval of 0.4 cM. The total length of scaffolds with identified map positions was 95.6 Mb, which is equivalent to 15.4 % of the estimated genome size. The generated map is the first SSR and RAD marker-based high-density linkage map reported for carnation. The ddRAD-seq pipeline developed in this study should also help accelerate genetic and genomics analyses and molecular breeding of carnation and other non-model crops.

A RAD-based linkage map of kiwifruit (Actinidia chinensis Pl.) as a tool to improve the genome assembly and to scan the genomic region of the gender determinant for the marker-assisted breeding

Kiwifruit breeding still largely relies on phenotypic observation of cross progeny grown in the field to fruiting maturity, without any selection prior to the juvenility being overcome. Developing markers for the selection of traits of interest would greatly help breeders to rapidly screen breeding populations. With the aim of mapping several traits of interest in kiwifruit, a F1 population of diploid (2n = 58) Actinidia chinensis was produced by combining parents with contrasting phenotypic traits. Ninety-four individuals were preliminarily analyzed to obtain a saturated genetic map based on 167 SSRs from the literature and 12,586 segregating restriction-site-associated DNA (RAD) loci obtained through an approach known as genotyping-by-sequencing (GBS) based on haplotype calling of SNP markers identified by a modified double digest restriction-associated DNA sequencing (ddRADseq) protocol as proposed by Peterson et al. (2012). To improve the accuracy of genotype calling, restriction-site-associated reads were aligned to the scaffolds of the recently published kiwifruit genome (Huang et al. 2013). This strategy provided genetic anchoring to 557 Mbp (90 %) of the assembly, helping also to anchor some 120 unmapped Mbp and to identify some mis-joined scaffolds. The analysis of the region controlling the dioecy in kiwifruit, spanning 16 scaffolds in the pseudomolecule 25 of the genome assembly (approximately 4.9 Mbp), with RAD markers that co-segregated with the gender determinant, allowed to sort out markers suitable for marker-assisted selection for the gender in the mapping population with successful extension to further controlled crosses having parents at different ploidy level and belonging to the A. chinensis/Actinidia deliciosa complex.