Restriction Site-associated DNA Research Articles

A Phylogenetic and Morphological Evolution Study of Ribes L. in China Using RAD-Seq

Ribes L. belongs to the Grossulariaceae family and has important edible, medicinal, ornamental, and landscaping values. Taxonomic classification within this genus is difficult due to its large variety of species, wide distribution, large morphological variations, and presence of two complex taxonomic groups with bisexual or unisexual flowers. Our study aims to clarify the phylogenetic relationships of Ribes L. taxa in China, and further, to provide a reference for a revised global classification of it. The phylogenetic analysis of 52 Ribes L. samples from 30 species was constructed based on restriction site-associated DNA sequencing and single nucleotide polymorphisms. Afterward, two important taxonomic characters were selected for ancestral state reconstruction over the molecular phylogeny. The results showed that the 52 samples could be divided into six branches, i.e., six subgenera, which caused some controversy regarding the morphological classification of Ribes L. in China. The molecular phylogeny supported the separation of subg. Coreosma from subg. Ribesia and subg. Hemibotrya from subg. Berisia and validated the rationale for recognizing subg. Grossularia as an independent subgenus, the rationality of which was further verified by the reconstruction of ancestor traits. Gene flow among Ribes L. was identified and further confirmed our results.

Genome-wide analysis facilitates estimation of the amount of male contribution in meiotic gynogenetic three-spined stickleback (Gasterosteus aculeatus).

Gynogenetic embryos - those inheriting only maternal DNA - can be experimentally created by fertilizing eggs with radiation-treated sperm containing inactivated paternal chromosomes. Diploidy in the zygotes can be maintained through prevention of the second meiosis or restored by preventing the first mitosis after the maternal chromosome complement has been replicated. These gynogenetic organisms are useful in many fields including aquaculture, evolutionary biology and genomics. Although gynogenetic organisms have been created in numerous species, the completeness of uni-parental inheritance has often been assumed rather than thoroughly quantified across the genome. Instead, when tests of uni-parental inheritance occur, they typically rely on well-studied genetically determined phenotypes that represent a very small sub-set of the genome. Only assessing small genomic regions for paternal inheritance leaves the question of whether some paternal contributions to offspring might still have occurred. In this study, the authors quantify the efficacy of creating gynogenetic diploid three-spined stickleback fish (Gasterosteus aculeatus). To this end, the authors mirrored previous assessments of paternal contribution using well-studied genetically determined phenotypes including sex and genetically dominant morphological traits but expanded on previous studies using dense restriction site-associated DNA sequencing (RAD-seq) markers in parents and offspring to assess paternal inheritance genome-wide. In the gynogenetic diploids, the authors found no male genotypes underlying their phenotypes of interest - sex and dominant phenotypic traits. Using genome-wide assessments of paternal contribution, nevertheless, the authors found evidence of a small, yet potentially important, amount of paternally "leaked" genetic material. The application of this genome-wide approach identifies the need for more widespread assessment of paternal contributions to gynogenetic animals and promises benefits for many aspects of aquaculture, evolutionary biology and genomics.

High-throughput sequencing confirms the boundary between traditionally considered species pairs in a group of lichenized fungi (Peltigeraceae, Pseudocyphellaria)

Abstract Accurate species delimitations are fundamental to our understanding of the genetic diversity on Earth and a vital part in evolutionary and conservation biology research. In lichenized fungi, species pairs have the same morphology and chemistry. They only differ in how they reproduce with one species using sexual reproductive structures and the other using asexual propagules. To classify these as one species or two has been a point of contention, and conclusions based on Sanger sequencing, where sequence data are limited and species boundaries are usually not observed, have been refuted after analysis with genome-scale data such as restriction site-associated DNA sequencing that tends to find fixed genetic differences between the two morphs. Pseudocyphellaria glabra and P. homoeophylla have long been considered a species pair that differ in geographical ranges but co-occur in New Zealand. We used restriction site-associated DNA sequencing data and generated thousands of genetic loci across 53 individuals. The RADseq data provided high-resolution phylogenetic and population genomic information. A maximum-likelihood phylogenetic reconstruction recovered both species as separate lineages, whereas population genetics indicated some evidence for admixture among P. glabra and P. homoeophylla from New Zealand. It is not clear whether the latter is due to ancient polymorphism or recent gene flow. Our study represents another example of the usefulness of RADseq to test species boundaries that segregate closely related species in lichenized fungi.

Characterization of a sex-determining locus and development of early molecular assays in Telfairia occidentalisHook. F., a dioecious cucurbit

Although there exist over 7000 crop species, only a few are commercially valuable and grown on a large scale in monocultures worldwide. However, underutilised crops (also called orphan crops) have significant potential for food security and Telfairia occidentalis Hook. F. (Cucurbitaceae) is one such orphan crop grown in West Africa for its nutritious leaves, oil and protein-rich seeds. In this dioecious crop, farmers like to eliminate male plants and keep mostly females to increase their yield. However, they face the challenge of determining sex due to limited morphological differences between females and males before flowering. This study used double digested restriction site-associated DNA sequencing data (ddRADseq) to examine the genetic diversity within and among landraces of T. occidentalis, identify common sex-determining loci, and establish reliable assays to characterize the sex of immature plants in the vegetative state. To differentiate males from females of T. occidentalis, two molecular assays were thereupon developed based on polymerase chain reaction (PCR) to genotype sex-specific sequence variation either through restriction by Mfe1 or the direct use of sex-specific primers. Both assays require standard laboratory conditions to reach a certainty of 94.3% for females and 95.7% for males from the studied samples. With the inclusion of additional landraces, medium to largescale farms growing T. occidentalis as a crop can readily benefit from an early determination of the sex of plants.

Multispectral Phenotyping and Genetic Analyses of Spring Appearance in Greening Plant, Phedimus spp.

The change in appearance during the seasonal transitions in ornamental greening plants is an important characteristic. In particular, the early onset of green leaf color is a desirable trait for a cultivar. In this study, we established a method for phenotyping leaf color change by multispectral imaging and performed genetic analysis based on the phenotypes to clarify the potential of the approach in breeding greening plants. We performed multispectral phenotyping and quantitative trait locus (QTL) analysis of an F1 population derived from 2 parental lines of Phedimus takesimensis, known to be a drought and heat-tolerant rooftop plant species. The imaging was conducted in April of 2019 and 2020 when dormancy breakage occurs and growth extension begins. Principal component analysis of 9 different wavelength values showed a high contribution from the first principal component (PC1), which captured variation in the visible light range. The high interannual correlation in PC1 and in the intensity of visible light indicated that the multispectral phenotyping captured genetic variation in the color of leaves. We also performed restriction site-associated DNA sequencing and obtained the first genetic linkage map of Phedimus spp. QTL analysis revealed 2 QTLs related to early dormancy breakage. Based on the genotypes of the markers underlying these 2 QTLs, the F1 phenotypes with early (late) dormancy break, green (red or brown) leaves, and a high (low) degree of vegetative growth were classified. The results suggest the potential of multispectral phenotyping in the genetic dissection of seasonal leaf color changes in greening plants.

Genetic diversity and population structure of the upriver orange mangrove Bruguiera sexangula along the coastlines of Thailand

Upriver orange mangrove Bruguiera sexangula is a member of the Rhizophoraceae family and is usually distributed in the intertidal zones of the tropical and subtropical coastal areas. The analyses of genetic diversity and population structure of B. sexangula are essential for their conservation and management. In the present study, the genetic diversity and structure of 101 B. sexangula individuals from mangrove forests in Thailand were evaluated using single nucleotide polymorphisms (SNPs) through restriction site-associated DNA sequencing (RAD-seq). A total of 3482 high-quality SNPs were obtained after filtration for genetic study. All 3 genetic structure analyses (Bayesian analysis, principal components analysis, and maximum likelihood tree) revealed that these individuals can be clustered into 2 groups corresponding to geographical regions, namely the Andaman Sea and Gulf of Thailand. The level of genetic differentiation between the clusters was moderate (FST = 0.122, p < 0.001), and the analysis of molecular variance (AMOVA) indicated that the individual differences within groups were greater than the differences between the 2 groups. A moderate level of genetic diversity (Shannon's information index, I = 0.458; observed heterozygosity, Ho = 0.416; expected heterozygosity, He = 0.295) of B. sexangula was observed. These results give us a better understanding of the genetic variations and will provide a framework for the conservation of B. sexangula.

SNPs Detection and Genetic Analysis of Chionanthus retusus via Genotyping-by-Sequencing

Abstract Chionanthus retusus is one of the well-known ornamental trees in East Asia and America. Not only its value in the market but also had the potential as a source for producing antioxidant. However, due to uncontrolled exploitation, the number of wild C. retusus in China is decreasing rapidly. The genetic study of C. retusus is limited. In order to investigate the genetic diversity and the distribution of C. retusus in China, 47 samples from 8 different provinces have been sequenced via restriction site-associated DNA sequencing (RAD-seq). Totally, 31, 402 high-quality single nucleotide polymorphisms (SNP) were obtained. According to the phylogenetic tree and the principal component analysis, the samples were divided into four populations, including 3 major populations and 1 hybrid population. Population1 were the samples from Jiangsu and Yunnan province and the Population2 were mainly from northern and northeast of China including Liaoning and Hubei province, while the Population4 were from Shandong and Henan province, which were in central China. As the admixture showed, the population3 were the offspring of the other 3 populations by hybridization. The mean heterozygosity of Chinese Fringe tree from different province is 0.42 %, with the highest heterozygosity, which is as high as 0.63 %, from Jiangsu province and the lowest heterozygosity, which is only 0.19, from Henan province. This is the first report about the genetic diversity and relationship of Chionanthus retusus, which will provide value information for further genetic study, genomic study, conservation and breeding.

Genomic differentiation with isolation by distance along a latitudinal gradient in the spotted-leg treefrog Polypedates megacephalus.

The patterns of isolation by distance (IBD) entailing increased genetic differentiation among populations have aroused extensive concerns for evolutionary biologists. Although the IBD may act on spatial processes contributing to the genetic differentiation among populations in anuran species, the factors shaping the IBD of frogs among populations in natural systems are largely unknown. Here, we studied the genetic differentiation among six populations with 24 individuals of the spotted-leg treefrog along a latitudinal gradient (1860.31 km) based on 1020 single nucleotide polymorphisms from restriction site-associated DNA sequencing. The results showed that the genetic diversity differed significantly among populations and that the insular populations had higher genetic diversity than the mainland populations. Furthermore, we also found a significant genetic differentiation among populations (FST = 0.277) and no sign of inbreeding (FUNI = -0.145). The IBD was detected for all populations, and a higher degree of the IBD was indicated when controlling for the effects of the isolation between Hainan and mainland populations caused by the Qiongzhou Strait. Our findings suggest that the form of the Qiongzhou Strait plays a key role in shaping the genetic diversity and population differentiation in treefrogs.

Morphology, nuclear SNPs and mate selection reveal that COI barcoding overestimates species diversity in a Mediterranean freshwater amphipod by an order of magnitude.

DNA sequence information has revealed many morphologically cryptic species worldwide. For animals, DNA-based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, a growing amount of evidence indicate that mitochondrial markers alone can lead to misleading species diversity estimates due to mito-nuclear discordance. Therefore, reports of putative species based solely on mitochondrial DNA should be verified by other methods, especially in cases where COI sequences are identical for different morphospecies or where divergence within the same morphospecies is high. Freshwater amphipods are particularly interesting in this context because numerous putative cryptic species have been reported. Here, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within Echinogammarus sicilianus. We used an integrative approach combining DNA barcoding with mate selection observations, detailed morphometrics and genome-wide double digest restriction site-associated DNA sequencing (ddRAD-seq). Within a relatively small sampling area, we detected twelve COI MOTUs (divergence=1.8-20.3%), co-occurring in syntopy at two-thirds of the investigated sites. We found that pair formation was random and there was extensive nuclear gene flow among the ten MOTUs co-occurring within the same river stretch. The four most common MOTUs were also indistinguishable with respect to functional morphology. Therefore, the evidence best fits the hypothesis of a single, yet genetically diverse, species within the main river system. The only two MOTUs sampled outside the focal area were genetically distinct at the nuclear level and may represent distinct species. Our study reveals that COI-based species delimitation can significantly overestimate species diversity, highlighting the importance of integrative taxonomy for species validation, especially in hyperdiverse complexes with syntopically occurring mitochondrial MOTUs.

Cryptic persistence and loss of local endemism in Lake Constance charr subject to anthropogenic disturbance.

In the welcome circumstance that species believed extinct are rediscovered, it is often the case that biological knowledge acquired before the presumed extinction is limited. Efforts to address these knowledge gaps, in particular to assess the taxonomic integrity and conservation status of such species, can be hampered by a lack of genetic data and scarcity of samples in museum collections. Here, we present a proof-of-concept case study based on a multidisciplinary data evaluation approach to tackle such problems. The approach was developed after the rediscovery, 40 years after its presumed extinction, of the enigmatic Lake Constance deep-water charr Salvelinus profundus. Targeted surveys led to the capture of further species and additional sympatric normal charr, Salvelinus cf. umbla. Since the lake had been subject to massive stocking in the past, an evaluation of the genetic integrity of both extant forms was called for in order to assess possible introgression. A two-step genomic approach was developed based on restriction site associated DNA (RAD). Diagnostic population genomic (single nucleotide polymorphism [SNP]) data were harvested from contemporary samples and used for RNA bait design to perform target capture in DNA libraries of archival scale material, enabling a comparison between extant and historic samples. Furthermore, life history traits and morphological data for both extant forms were gathered and compared with historical data from the past 60-120 years. While extant deep-water charr matched historical deep-water specimens in body shape, gill raker count, and growth rates, significant differences were discovered between historical and extant normal charr. These resulted were supported by genomic analyses of contemporary samples, revealing the two extant forms to be highly divergent. The results of population assignment tests suggest that the endemic deep-water charr persisted in Lake Constance during the eutrophic phase, but not one of the historical genomic samples could be assigned to the extant normal charr taxon. Stocking with non-endemic charr seems to be the most likely reason for these changes. This proof-of-concept study presents a multidisciplinary data evaluation approach that simultaneously tests population genomic integrity and addresses some of the conservation issues arising from rediscovery of a species characterized by limited data availability.

Haploid gynogens facilitate disomic marker development in paleotetraploid sturgeons.

Acipenseriformes (sturgeons and paddlefishes) are of substantial conservation concern, and development of genomic resources for these species is difficult due to past whole genome duplication. Development of disomic markers for polyploid organisms can be challenging due to difficulty in resolving alleles at a single locus from those among duplicated loci. In this study, we detail the development of disomic markers for the endangered pallid sturgeon (Scaphirhynchus albus) found in North America. One of the strategies for pallid sturgeon conservation is to stock U.S. rivers with offspring of pure pallid sturgeon, but introgression with the sympatric shovelnose sturgeon (S.platorynchus) threatens pallid sturgeon genetic integrity. Currently, 19 microsatellite loci are used to differentiate between both species and their hybrids, but the markers are insufficient to robustly identify backcrosses. We performed double digest restriction site-associated DNA sequencing (ddRADseq) on shovelnose sturgeon haploid gynogens to produce a reduced-representation genomic reference. Contiguous sequences that were heterozygous within a haploid individual were flagged as potentially encompassing multiple loci. Approximately 60 individuals of each species from two management units were sequenced, and reads were mapped to the haploid reference to identify single nucleotide polymorphisms (SNPs) at individual loci. The final data set contained 11,082 microhaplotyped loci which offer at least an order of magnitude greater resolution for species discrimination than the current panel of 19 microsatellites. These markers will be used to examine a larger sample of Scaphirhynchus individuals throughout their ranges to determine the extent and trajectory of hybridization.

Unequal Reproduction Early in a Social Transition: Insights from Invasive Wasps.

AbstractIn eusocial insects, nestmate queens can differ in their reproductive output, causing asymmetries in the distribution of mutual benefits. However, little is known about how reproductive success is partitioned in incipiently polygynous species, which would provide clues about the evolutionary forces shaping the emergence of polygyny. Here, we leverage a recent transition from predominantly single-queen (monogyne) to multiple-queen (polygyne) colonies in an invasive yellowjacket species to investigate whether queens in incipiently polygyne colonies invest equally in reproductive effort or vary in their relative investment in each caste. We excavated nine polygyne Vespula pensylvanica colonies in Hawaii and used restriction site-associated DNA sequencing to infer the parentage of worker, male, and gyne (daughter queen) pupae from each nest comb. In four colonies with at least eight gyne pupae, a single queen produced most or all gynes. These queens had no male offspring and few worker offspring, suggesting that a subset of nestmate queens might exploit the collective benefits of newly polygyne societies. In contrast to most queens, gyne producers had offspring distributed nonrandomly across nest combs. Nestmate queens generally exhibited low relatedness levels. Our results suggest that rapid, ecologically driven transitions to polygyny among unrelated queens may, at their onset, be vulnerable to reproductive asymmetries that are likely evolutionarily unstable. More broadly, this study contributes to the understanding of social evolution by uncovering asymmetric partitioning of reproduction in a population with newly evolved polygyny and raises questions about the future trajectories of introduced populations.

Molecular ecology of the sleeper shark subgenus Somniosus (Somniosus) reveals genetic homogeneity within species and lack of support for S. antarcticus.

Inferences made from molecular data support regional stock assessment goals by providing insights into the genetic population dynamics of enigmatic species. Population genomics metrics, such as genetic diversity and population connectivity, serve as useful proxies for species health and stability. Sleeper sharks (genus Somniosus) are ecologically important deep-sea predators, estimated to reach ages of 250 to 300 yr and taking decades to reach sexual maturity. The subgenus Somniosus (Somniosus) is comprised of 3 species: S. pacificus, S. microcephalus, and S. antarcticus. Given the life history strategy of somniosids, they are vulnerable to overfishing and population declines. Further, data to assess the stocks of these species are limited. To address this deficiency, we used the reduced representation library method Restriction-site Associated DNA sequencing (RADseq) to conduct phylogenomic and population genomics analyses, providing novel information for use in stock assessments. Our results strongly support the species status of S. microcephalus (N = 79), but recover S. antarcticus (N = 2) intermixed within the S. pacificus (N = 170) clade. Population genomics analyses reveal genetic homogeneity within S. pacificus and S. microcephalus, and estimates of effective population size were in the hundreds for both species. Kinship analysis identified 2 first-degree relative pairs within our dataset (1 within each species). Our results contribute new information for stock assessments of these uniquely long-lived species by providing the strongest molecular evidence to date for the synonymization of S. antarcticus and S. pacificus, as well as estimating population genomic metrics for each supported species within the Somniosus (Somniosus) subgenus.

The anthropogenic effect of land use on population genetics of Malcus inconspicuus.

Since the beginning of the Holocene era, human activities have seriously impacted animal habitats and vegetative environments. Species that are dependent on natural habitats or with narrow niches might be more severely affected by habitat changes. Malcus inconspicuus is distributed in subtropical China and highly dependent on the mountain environment. Our study investigated the role of the mountainous landscape in the historical evolution of M. inconspicuus and the impact of Holocene human activities on it. A phylogeographical approach was implemented with integrative datasets including double-digest restriction site-associated DNA (ddRAD), mitochondrial data, and distribution data. Three obvious clades and an east-west phylogeographical pattern were found in subtropical China. Mountainous landscape has "multifaceted" effects on the evolutionary history of M. inconspicuus, it has contributed to population differentiation, provided glacial refuges, and provided population expansion corridors during the postglacial period. The effective population size (Ne) of M. inconspicuus showed a sharp decline during the Holocene era, which revealed a significantly negative correlation with the development of cropland in a hilly area at the same time and space. It supported that the species which are highly dependent on natural habitats might undergo greater impact when the habitat was damaged by agricultural activities and we should pay more attention to them, especially in the land development of their distribution areas.

Detecting a novel locus associated with pellicle peelability in Japanese chestnut populations preselected for a major peelability gene

• The distribution of phenotypic values of peelability in Japanese chestnut populations preselected for a major peelability gene was biased toward maximum values. • Use of rank-based inverse normal transformation and categorization of peelability phenotypes was applied for GWAS to further identify QTLs associated with peelability. • A single QTL for peelability was identified at CCR1.0F_56177061 on chromosome F. Peelability is one of the most important traits in Japanese chestnut ( Castanea crenata Sieb. et Zucc.) breeding. Previous studies revealed that the peelability of Japanese chestnut is controlled by a major recessive peeling gene (MRPG). This locus has been used effectively for marker-assisted selection in chestnut breeding programs, but there are also likely to be minor loci that would further improve the efficiency of breeding for peelability. We conducted a genome-wide association study (GWAS) based on a variational approximation version of BayesB using 27 cultivars and MRPG-preselected populations consisting of 520 F 1 seedlings from 22 families. Using double-digest restriction site-associated DNA sequencing (ddRAD-Seq), we obtained 9164 tag single-nucleotide polymorphisms. Because the phenotypic values of peelability were distributed very asymmetrically, with a peak at the high end due to the preselection for MRPG, we converted the original data set to both inverse normal transformed values and categorical scores, and we analyzed both sets of converted data by GWAS. In both analyses, a quantitative trait locus (QTL) for peelability at CCR1.0F_56177061 was identified with posterior probability values of 0.68 and 1.00, respectively. The average values of genotypes at CCR1.0F_56177061 for cultivars and seedlings in this study were 60.4% (peelability rate) for CC, 63.7% for CT, and 86.5% for TT. This analysis reveals a significant association between CCR1.0F_56177061 genotype and peelability, indicating that this newly identified QTL will be useful for improving peelability in chestnut breeding programs.

A low-density single nucleotide polymorphism panel for brown trout (Salmo trutta L.) suitable for exploring genetic diversity at a range of spatial scales.

The rivers of southern England and northern France which drain into the English Channel contain several genetically unique groups of trout (Salmo trutta L.) that have suffered dramatic declines in numbers over the past 40 years. Knowledge of levels and patterns of genetic diversity is essential for effective management of these vulnerable populations. Using restriction site-associated DNA sequencing (RADseq) data, we describe the development and characterisation of a panel of 95 single nucleotide polymorphism (SNP) loci for trout from this region and investigate their applicability and variability in both target (i.e., southern English) and non-target trout populations from northern Britain and Ireland. In addition, we present three case studies which demonstrate the utility and resolution of these genetic markers at three levels of spatial separation:(a) between closely related populations in nearby rivers, (b) within a catchment and (c) when determining parentage and familial relationships between fish sampled from a single site, using both empirical and simulated data. The SNP loci will be useful for population genetic and assignment studies on brown trout within the UK and beyond.

Species delimitation using genomic data to resolve taxonomic uncertainties in a speciation continuum of pelagic seabirds

Speciation is a continuous and complex process shaped by the interaction of numerous evolutionary forces. Despite the continuous nature of the speciation process, the implementation of conservation policies relies on the delimitation of species and evolutionary significant units (ESUs). Puffinus shearwaters are globally distributed and threatened pelagic seabirds. Due to remarkable morphological status the group has been under intense taxonomic debate for the past three decades. Here, we use double digest Restriction-Site Associated DNA sequencing (ddRAD-Seq) to genotype species and subspecies of North Atlantic and Mediterranean Puffinus shearwaters across their entire geographical range. We assess the phylogenetic relationships and population structure among and within the group, evaluate species boundaries, and characterise the genomic landscape of divergence. We find that current taxonomies are not supported by genomic data and propose a more accurate taxonomy by integrating genomic information with other sources of evidence. Our results show that several taxon pairs are at different stages of a speciation continuum. Our study emphasises the potential of genomic data to resolve taxonomic uncertainties, which can help to focus management actions on relevant taxa, even if they do not necessarily coincide with the taxonomic rank of species.

Detecting clonemate pairs in multicellular diploid clonal species based on a shared heterozygosity index.

Clonal reproduction, the formation of nearly identical individuals via mitosis in the absence of genetic recombination, is a very common reproductive mode across plants, fungi and animals. To detect clonal genetic structure, genetic similarity indices based on shared alleles are widely used, such as the Jaccard index, or identity by state. Here we propose a new pairwise genetic similarity index, the SH index, based on segregating genetic marker loci (typically single nucleotide polymorphisms) that are identically heterozygous for pairs of samples (NSH ). To test our method, we analyse two old seagrass clones (Posidonia australis, estimated to be around 8500 years old; Zostera marina, >750 years old) along with two young Z. marina clones of known age (17 years old). We show that focusing on shared heterozygosity amplifies the power to distinguish sample pairs belonging to different clones compared to methods focusing on all shared alleles. Our proposed workflow can successfully detect clonemates at a location dominated by a single clone. When the collected samples involve two or more clones, the SH index shows a clear gap between clonemate pairs and interclone sample pairs. Ideally NSH should be on the order of approximately ≥3000, a number easily achievable via restriction-site associated DNA (RAD) sequencing or whole-genome resequencing. Another potential application of the SH index is to detect possible parent-descendant pairs under selfing. Our proposed workflow takes advantage of the availability of the larger number of genetic markers in the genomic era, and improves the ability to distinguish clonemates from nonclonemates in multicellular diploid clonal species.

Evidence of backcross inviability and mitochondrial DNA paternal leakage in sea turtle hybrids.

Hybridization is known to be part of many species' evolutionary history. Sea turtles have a fascinating hybridization system in which species separated by as much as 43million years are still capable of hybridizing. Indeed, the largest nesting populations in Brazil of loggerheads (Caretta caretta) and hawksbills (Eretmochelys imbricata) have a high incidence of hybrids between these two species. A third species, olive ridleys (Lepidochelys olivacea), is also known to hybridize although at a smaller scale. Here, we used restriction site-associated DNA sequencing (RAD-Seq) markers, mitogenomes, and satellite-telemetry to investigate the patterns of hybridization and introgression in the Brazilian sea turtle population and their relationship with the migratory behaviours between feeding and nesting aggregations. We also explicitly test if the mixing of two divergent genomes in sea turtle hybrids causes mitochondrial paternal leakage. We developed a new species-specific PCR-assay capable of detecting mitochondrial DNA (mtDNA) inheritance from both parental species and performed ultra-deep sequencing to estimate the abundance of each mtDNA type. Our results show that all adult hybrids are first generation (F1) and most display a loggerhead migratory behaviour. We detected paternal leakage in F1 hybrids and different proportions of mitochondria from maternal and paternal species. Although previous studies showed no significant fitness decrease in hatchlings, our results support genetically-related hybrid breakdown possibly caused by cytonuclear incompatibility. Further research on hybrids from other populations in addition to Brazil and between different species will show if backcross inviability and mitochondrial paternal leakage is observed across sea turtle species.

Molecular typing and genome sequencing allow the identification of persistent Listeria monocytogenes strains and the tracking of the contamination source in food environments

The presence of Listeria monocytogenes (Lm) in the food processing environment (facilities and products) is a challenging problem in food safety management. Lm is one of the main causes of mortality in foodborne infections, and the trend is continuously increasing. In this study, a collection of 323 Lm strain isolates recovered from food matrices and food industry environments (surfaces and equipment) over four years from 80 food processing facilities was screened using a restriction site-associated tag sequencing (2b-RAD) typing approach developed for Lm. Thirty-six different restriction site-associated DNA (RAD) types (RTs) were identified, most of which correspond to lineage II. RT1, the most represented genotype in our collection and already reported as one of the most prevalent genotypes in the food environment, was significantly associated with meat processing facilities. The sequencing of the genomes of strains belonging to the same RT and isolated in the same facility in different years revealed several clusters of persistence. The definition of the persistent strains (PSs) allowed the identification of the potential source of contamination in the incoming raw meat that is introduced in the facility to be processed. The slaughterhouses, which, according to the European Union (EU) regulation, are not inspected for the presence of Lm could be hotspots for the persistence of Lm PSs.