Restriction Site-associated DNA Sequencing Research Articles

Intergeneric hybrid origin of the invasive tetraploid Cirsium vulgare.

The invasive tetraploid Cirsium vulgare hybridizes with both Cirsium and Lophiolepis. Its conflicted position in molecular phylogenies, and its peculiar combination of morphological, anatomical, and genomic features that are alternatively shared with representatives of Cirsium or Lophiolepis, strongly suggest its intergeneric hybrid origin. Genetic relationships of C. vulgare (8 samples) with genus Lophiolepis (11 species) and other representatives of genus Cirsium (12 species) were evaluated using restriction site-associated DNA sequencing (RADseq) and examined using analytical and imaging approaches, such as NeighborNet, Heatmap, and STRUCTURE, to identify nuclear genomes admixture. Estimation of the intensity of spontaneous hybridization within and between Cirsium and Lophiolepis was based on herbarium revisions and published data for all reported hybrids pertinent to taxa currently included in Cirsium or Lophiolepis. The genome of any examined Cirsium species is more similar to C. vulgare than to any Lophiolepis species, and vice versa. The nuclear genome of the tetraploid C. vulgare is composed of two equivalent parts, each attributable either to Lophiolepis or to Cirsium; the organellar RADseq data clustered C. vulgare with the genus Cirsium. Spontaneous hybridization between Cirsium and Lophiolepis is significantly less intensive than within these genera. Our analyses provide compelling evidence that the invasive species C. vulgare has an allotetraploid intergeneric origin, with the maternal parent from Cirsium and the paternal from Lophiolepis. For the purpose of delimiting monophyletic genera, we propose keeping Lophiolepis separate from Cirsium and segregating C. vulgare into the hybridogenous genus Ascalea.

Different reference genomes determine different results: Comparing SNP calling in RAD-seq of Engelhardia roxburghiana using different reference genomes

Advances in next-generation sequencing (NGS) have significantly reduced the cost and improved the efficiency of obtaining single nucleotide polymorphism (SNP) markers, particularly through restriction site-associated DNA sequencing (RAD-seq). Meanwhile, the progression in whole genome sequencing has led to the utilization of an increasing number of reference genomes in SNP calling processes. This study utilized RAD-seq data from 242 individuals of Engelhardia roxburghiana, a tropical tree of the walnut family (Juglandaceae), with SNP calling conducted using the STACKS pipeline. We aimed to compare both reference-based approaches, namely, employing a closely related species as the reference genome versus the species itself as the reference genome, to evaluate their respective merits and limitations. Our findings indicate a substantial discrepancy in the number of obtained SNPs between using a closely related species as opposed to the species itself as reference genomes, the former yielded approximately an order of magnitude fewer SNPs compared to the latter. While the missing rate of individuals and sites of the final SNPs obtained in the two scenarios showed no significant difference. The results showed that using the reference genome of the species itself tends to be prioritized in RAD-seq studies. However, if this is unavailable, considering closely related genomes is feasible due to their wide applicability and low missing rate as alternatives. This study contributes to enrich the understanding of the impact of SNP acquisition when utilizing different reference genomes.

Niche conservatism and spread explain introgression between native and invasive fish.

Hybridisation can be an important driver of evolutionary change, but hybridisation with invasive species can have adverse effects on native biodiversity. While hybridisation has been documented across taxa, there is limited understanding of ecological factors promoting patterns of hybridisation and the spatial distribution of hybrid individuals. We combined the results of ecological niche modelling (ENM) and restriction site-associated DNA sequencing to test theories of niche conservatism and biotic resistance on the success of invasion, admixture, and extent of introgression between native and non-native fishes. We related Maxent predictions of habitat suitability based on the native ranges of invasive Eastern Banded Killifish (Fundulus diaphanus diaphanus Lesueur 1817) and native Western Banded Killifish (Fundulus diaphanus menona Jordan and Copeland 1877) to admixture indices of individual Banded Killifish. We found that Eastern Banded Killifish predominated at sites predicted as suitable from their ENM, consistent with niche conservatism. Admixed individuals were more common as Eastern Banded Killifish habitat suitability declined. We also found that Eastern Banded Killifish were most common at sites closest to the presumed source of this invasion, whereas the proportion of admixed individuals increased with distance from the source of invasion. Lastly, we found little evidence that habitat suitability for Western Banded Killifish provides biotic resistance from either displacement by, or admixture with, invasive Eastern Banded Killifish. Our study demonstrates that ENMs can inform conservation-relevant outcomes between native and invasive taxa while emphasising the importance of protecting isolated Western Banded Killifish populations from invasive conspecifics.

Comprehensive genome assembly reveals genetic diversity and carcass consumption insights in critically endangered Asian king vultures

The Asian king vulture (AKV), a vital forest scavenger, is facing globally critical endangerment. This study aimed to construct a reference genome to unveil the mechanisms underlying its scavenger abilities and to assess the genetic relatedness of the captive population in Thailand. A reference genome of a female AKV was assembled from sequencing reads obtained from both PacBio long-read and MGI short-read sequencing platforms. Comparative genomics with New World vultures (NWVs) and other birds in the Family Accipitridae revealed unique gene families in AKV associated with retroviral genome integration and feather keratin, contrasting with NWVs’ genes related to olfactory reception. Expanded gene families in AKV were linked to inflammatory response, iron regulation and spermatogenesis. Positively selected genes included those associated with anti-apoptosis, immune response and muscle cell development, shedding light on adaptations for carcass consumption and high-altitude soaring. Using restriction site-associated DNA sequencing (RADseq)-based genome-wide single nucleotide polymorphisms (SNPs), genetic relatedness and inbreeding status of five captive AKVs were determined, revealing high genomic inbreeding in two females. In conclusion, the AKV reference genome was established, providing insights into its unique characteristics. Additionally, the potential of RADseq-based genome-wide SNPs for selecting AKV breeders was demonstrated.

Development and utilization of genome-wide InDel markers in Sorghum [Sorghum bicolor (L.) Moench]

Sorghum (Sorghum bicolor L. Moench) has become an increasingly valuable crop for food, feed, and especially bioenergy feedstock production, which makes the crop extremely attractive for studying genomics and genetic diversity. Molecular markers and genomics play essential roles in sorghum breeding. The rapid development of next-generation sequencing technology has facilitated the identification of genome-wide insertion-deletion (InDel) polymorphisms, enabling the efficient construction of InDel markers that are suitable for user-friendly PCR. This study was conducted with the objective of discovering and developing InDel markers using double digest restriction site-associated DNA sequencing (ddRAD-Seq) data. A total of 19,226 InDels distributed across 10 chromosomes in the sorghum genome was identified. Of those, deletions constituted 65.7% while the remain was insertions. A comprehensive analysis of all the chromosomes revealed a total of 80 InDel sites with a minimum length of 10 bp. For a good conversion of the InDel regions to beneficial molecular markers, specific primers were designed for the amplification of 47 InDel regions that were selected for further investigation. A diverse panel of sorghum consisting of 16 accessions served a source for the developed InDel markers validation. Of the 47 InDel markers, 14 were tested across 16 sorghum accessions and were demonstrated their helpfulness for marker-assisted selection in sorghum. The polymorphic information content (PIC) values of the 16 markers varied between 0.11 and 0.38, with an average of 0.28. The findings of this study indicated that the identification of InDels and the development of molecular markers for sorghum were accomplished using the ddRAD-Seq data.

Genetic linkage mapping and quantitative trait locus (QTL) analysis of sweet basil (Ocimum basilicum L.) to identify genomic regions associated with cold tolerance and major volatiles.

Chilling sensitivity is one of the greatest challenges affecting the marketability and profitability of sweet basil (Ocimum basilicum L.) in the US and worldwide. Currently, there are no sweet basils commercially available with significant chilling tolerance and traditional aroma profiles. This study was conducted to identify quantitative trait loci (QTLs) responsible for chilling tolerance and aroma compounds in a biparental mapping population, including the Rutgers advanced breeding line that served as a chilling tolerant parent, 'CB15', the chilling sensitive parent, 'Rutgers Obsession DMR' and 200 F2 individuals. Chilling tolerance was assessed by percent necrosis using machine learning and aroma profiling was evaluated using gas chromatography (GC) mass spectrometry (MS). Single nucleotide polymorphism (SNP) markers were generated from genomic sequences derived from double digestion restriction-site associated DNA sequencing (ddRADseq) and converted to genotype data using a reference genome alignment. A genetic linkage map was constructed and five statistically significant QTLs were identified in response to chilling temperatures with possible interactions between QTLs. The QTL on LG24 (qCH24) demonstrated the largest effect for chilling response and was significant in all three replicates. No QTLs were identified for linalool, as the population did not segregate sufficiently to detect this trait. Two significant QTLs were identified for estragole (also known as methyl chavicol) with only qEST1 on LG1 being significant in the multiple-QTL model (MQM). QEUC26 was identified as a significant QTL for eucalyptol (also known as 1,8-cineole) on LG26. These QTLs may represent key mechanisms for chilling tolerance and aroma in basil, providing critical knowledge for future investigation of these phenotypic traits and molecular breeding.

A comprehensive framework for the delimitation of species within the Bemisia tabaci cryptic complex, a global pest-species group.

Identifying cryptic species poses a substantial challenge to both biologists and naturalists due to morphological similarities. Bemisia tabaci is a cryptic species complex containing more than 44 putative species; several of which are currently among the world's most destructive crop pests. Interpreting and delimiting the evolution of this species complex has proved problematic. To develop a comprehensive framework for species delimitation and identification, we evaluated the performance of distinct data sources both individually and in combination among numerous samples of the B. tabaci species complex acquired worldwide. Distinct datasets include full mitogenomes, single-copy nuclear genes, restriction site-associated DNA sequencing, geographic range, host speciation, and reproductive compatibility datasets. Phylogenetically, our well-supported topologies generated from threedense molecular markers highlighted the evolutionary divergence of species of the B. tabaci complex and suggested that the nuclear markers serve as a more accurate representation of B. tabaci species diversity. Reproductive compatibility datasets facilitated the identification of at least 17 different cryptic species within our samples. Native geographic range information provides a complementary assessment of species recognition, while the host range datasets provide low rate of delimiting resolution. We further summarized different data performances in species classification when compared with reproductive compatibility, indicating that combination of mtCOI divergence, nuclear markers, geographic range provide a complementary assessment of species recognition. Finally, we represent a model for understanding and untangling the cryptic species complexes based on the evidence from this study and previously published articles.

Plant kleptomaniacs: geographic genetic patterns in the amphi-apomictic Rubus ser. Glandulosi (Rosaceae) reveal complex reticulate evolution of Eurasian brambles.

Rubus ser. Glandulosi represents a unique model of geographic parthenogenesis on a homoploid (2n = 4x) level. We aim to characterize evolutionary and phylogeographic patterns in this taxon and shed light on the geographic differentiation of apomicts and sexuals. Ultimately, we aim to evaluate the importance of phylogeography in the formation of geographic parthenogenesis. R. ser. Glandulosi was sampled across its Eurasian range together with other co-occurring Rubus taxa (587 individuals in total). Double digest restriction-site associated DNA sequencing (ddRADseq) and modelling of suitable climate were employed for evolutionary inferences. Six ancestral species were identified that contributed to the contemporary gene pool of R. ser. Glandulosi. While sexuals were introgressed from R. dolichocarpus and R. moschus in West Asia and from R. ulmifolius agg., R. canescens and R. incanescens in Europe, apomicts were characterized by alleles of R. subsect. Rubus. Gene flow between sexuals and apomicts was also detected, as well as occasional hybridization with other taxa. We hypothesize that sexuals survived the last glacial period in several large southern refugia, whereas apomicts were mostly restricted to southern France from whence they quickly recolonized Central and Western Europe. The secondary contact of sexuals and apomicts was probably the principal factor that established geographic parthenogenesis in R. ser. Glandulosi. Sexual populations are not impoverished in genetic diversity along their borderline with apomicts and maladaptive population genetic processes likely did not shape the geographic patterns.

Hybridization, polyploidization, and morphological convergence make dozens of taxa into one chaotic genetic pool: a phylogenomic case of the Ficus erecta species complex (Moraceae).

The Ficus erecta complex, characterized by its morphological diversity and frequent interspecific overlap, shares pollinating fig wasps among several species. This attribute, coupled with its intricate phylogenetic relationships, establishes it as an exemplary model for studying speciation and evolutionary patterns. Extensive researches involving RADseq (Restriction-site associated DNA sequencing), complete chloroplast genome data, and flow cytometry methods were conducted, focusing on phylogenomic analysis, genetic structure, and ploidy detection within the complex. Significantly, the findings exposed a pronounced nuclear-cytoplasmic conflict. This evidence, together with genetic structure analysis, confirmed that hybridization within the complex is a frequent occurrence. The ploidy detection revealed widespread polyploidy, with certain species exhibiting multiple ploidy levels, including 2×, 3×, and 4×. Of particular note, only five species (F. abelii, F. erecta, F. formosana, F. tannoensis and F. vaccinioides) in the complex were proved to be monophyletic. Species such as F. gasparriniana, F. pandurata, and F. stenophylla were found to encompass multiple phylogenetically distinct lineages. This discovery, along with morphological comparisons, suggests a significant underestimation of species diversity within the complex. This study also identified F. tannoensis as an allopolyploid species originating from F. vaccinioide and F. erecta. Considering the integration of morphological, molecular systematics, and cytological evidences, it is proposed that the scope of the F. erecta complex should be expanded to the entire subsect. Frutescentiae. This would redefine the complex as a continuously evolving group comprising at least 33 taxa, characterized by blurred species boundaries, frequent hybridization and polyploidization, and ambiguous genetic differentiation.

Genomic and common garden data reveal significant genetic differentiation in the endangered San Fernando Valley spineflower Chorizanthe parryi var. fernandina

AbstractSan Fernando Valley spineflower (Chorizanthe parryi var. fernandina [S. Watson] Jeps.) (Polygonaceae) is an herbaceous annual plant, endemic to California, and until rediscovered in 1999 had been thought to be extinct for almost seven decades. Historically documented at 10 locations, it currently persists at 2, separated by approximately 27 km. State listed as endangered, a description of its genetic diversity and structure is of conservation interest. After determining a lack of variation in ploidy, we examined genetic variation from samples within both populations: a common garden study for potentially adaptive genetic variation in selected growth and phenological traits and analysis of single nucleotide polymorphisms identified through restriction-site associated DNA sequencing. Both measures indicated that this highly restricted taxon nevertheless harbors substantial levels of genetic diversity and has significant between- and within-population genetic structure. Combining approaches from population genomics and common garden studies provided more insight into the patterns and basis of genetic diversity than is typical for studies of non-model species. Although local adaptation was not specifically studied (i.e., via reciprocal transplant studies), the differences determined from these two independent lines of evidence indicate that mixing gene pools between populations is not recommended at this time. Further, with significant differences revealed among subpopulations, we caution against mixing genotypes across subpopulations for the most part, and without much more evidence that this would not pose a risk of outbreeding depression. The importance of supporting pollinator health and diversity is highlighted. With genetic diversity—particularly with an annual species—being dynamic, fluctuating with the usual processes and with contributions from the soil seedbank, we recommend periodic resampling to monitor genetic diversity and structure. Climate change is anticipated to contribute to this variability.

Comparison of ddRADseq and EUChip60K SNP genotyping systems for population genetics and genomic selection in Eucalyptus dunnii (Maiden).

Eucalyptus dunnii is one of the most important Eucalyptus species for short-fiber pulp production in regions where other species of the genus are affected by poor soil and climatic conditions. In this context, E. dunnii holds promise as a resource to address and adapt to the challenges of climate change. Despite its rapid growth and favorable wood properties for solid wood products, the advancement of its improvement remains in its early stages. In this work, we evaluated the performance of two single nucleotide polymorphism, (SNP), genotyping methods for population genetics analysis and Genomic Selection in E. dunnii. Double digest restriction-site associated DNA sequencing (ddRADseq) was compared with the EUChip60K array in 308 individuals from a provenance-progeny trial. The compared SNP set included 8,011 and 19,008 informative SNPs distributed along the 11 chromosomes, respectively. Although the two datasets differed in the percentage of missing data, genome coverage, minor allele frequency and estimated genetic diversity parameters, they revealed a similar genetic structure, showing two subpopulations with little differentiation between them, and low linkage disequilibrium. GS analyses were performed for eleven traits using Genomic Best Linear Unbiased Prediction (GBLUP) and a conventional pedigree-based model (ABLUP). Regardless of the SNP dataset, the predictive ability (PA) of GBLUP was better than that of ABLUP for six traits (Cellulose content, Total and Ethanolic extractives, Total and Klason lignin content and Syringyl and Guaiacyl lignin monomer ratio). When contrasting the SNP datasets used to estimate PAs, the GBLUP-EUChip60K model gave higher and significant PA values for six traits, meanwhile, the values estimated using ddRADseq gave higher values for three other traits. The PAs correlated positively with narrow sense heritabilities, with the highest correlations shown by the ABLUP and GBLUP-EUChip60K. The two genotyping methods, ddRADseq and EUChip60K, are generally comparable for population genetics and genomic prediction, demonstrating the utility of the former when subjected to rigorous SNP filtering. The results of this study provide a basis for future whole-genome studies using ddRADseq in non-model forest species for which SNP arrays have not yet been developed.

A complex biosystematic approach to reveal evolutionary and diversity patterns in West Asian brambles (Rubus subgen. Rubus, Rosaceae)

West Asia, with its high mountain ranges and glacial refugia, stands out as a biodiversity hotspot for various plant taxa, including the taxonomically complex Rubus subgen. Rubus. Despite this significance, our understanding of the evolutionary processes shaping this group in the region has been limited. We employed an integrative approach combining flow cytometry, Sanger sequencing of two plastid regions, microsatellite genotyping, and double digest restriction-site associated DNA sequencing to characterize evolutionary and diversity patterns in West Asian brambles. We identified four diploid and two sexual tetraploid species, with the remaining diversity comprising apomictic tetra- and triploids, mostly belonging to R. ser. Discolores, and sexual pentaploid hybrids/hybridogens derived from R. caesius. Rubus dolichocarpus, newly reported here as diploid, emerged as a significant ancestor for many polyploids. Most apomictic genotypes are locally distributed and only three genotypes exhibit extremely large ranges from Armenia to Kyrgyzstan and from Georgia to Iran. These genotypes are probably of an ancient (Pleistocene) European origin and likely laid the foundations for the evolution of apomicts in West Asia, whose diversification was subsequently boosted by the genetic contribution of sexual taxa. DNA markers confirmed the West Asian origin of Rubus armeniacus, a globally significant invasive species, yet rare in its native range. We report on the first indigenous occurrence of this species based on microsatellite genotyping. Our findings contribute to filling existing gaps in understanding the evolution and diversity of Rubus subgen. Rubus in West Asia.

Sequencing vs. amplification for the estimation of allele dosages in sugarcane (Saccharum spp.)

AbstractPremiseDetecting single‐nucleotide polymorphisms (SNPs) in a cost‐effective way is fundamental in any plant breeding pipeline. Here, we compare three genotyping techniques for their ability to reproduce the allele dosage of SNPs of interest in sugarcane (Saccharum spp.).MethodsTo identify a reproducible technique to estimate allele dosage for the validation of SNP markers, the correlation between Flex‐Seq, kompetitive allele‐specific PCR (KASP), and genotyping‐by‐sequencing and restriction site–associated DNA sequencing (GBS+RADseq) was determined for a set of 76 SNPs. To find alternative methodologies for allele dosage estimation, the KASP and Flex‐Seq techniques were compared for the same set of SNPs. For the three techniques, a population of 53 genotypes from the diverse sugarcane panel of the Centro de Investigación de la Caña de Azúcar (Cenicaña), Colombia, was selected.ResultsThe average Pearson correlation coefficients between GBS+RADseq and Flex‐Seq, GBS+RADseq and KASP, and Flex‐Seq and KASP were 0.62 ± 0.27, 0.38 ± 0.27, and 0.38 ± 0.30, respectively.DiscussionFlex‐Seq reproduced the allele dosages determined using GBS+RADseq with good levels of precision because of its depth of sequencing and ability to target specific positions in the genome. Additionally, Flex‐Seq outperformed KASP by allowing the conversion of a higher number of SNPs and a more accurate estimation of the allele dosage. Flex‐Seq has therefore become the genotyping methodology of choice for marker validation at Cenicaña.

Shared phylogeographic patterns and environmental responses of co-distributed soybean pests: Insights from comparative phylogeographic studies of Riptortus pedestris and Riptortus linearis in the subtropics of East Asia

Comparative phylogeographic studies of closely related species sharing co-distribution areas can elucidate the role of shared historical factors and environmental changes in shaping their phylogeographic pattern. The bean bugs, Riptortus pedestris and Riptortus linearis, which both inhabit subtropical regions in East Asia, are recognized as highly destructive soybean pests. Many previous studies have investigated the biological characteristics, pheromones, chemicals and control mechanisms of these two pests, but few studies have explored their phylogeographic patterns and underlying factors. In this study, we generated a double-digest restriction site-associated DNA sequencing (ddRAD-seq) dataset to investigate phylogeographic patterns and construct ecological niche models (ENM) for both Riptortus species. Our findings revealed similar niche occupancies and population genetic structures between the two species, with each comprising two phylogeographic lineages (i.e., the mainland China and the Indochina Peninsula clades) that diverged approximately 0.1 and 0.3 million years ago, respectively. This divergence likely resulted from the combined effects of temperatures variation and geographical barriers in the mountainous regions of Southwest China. Further demographic history and ENM analyses suggested that both pests underwent rapid expansion prior to the Last Glacial Maximum (LGM). Furthermore, ENM predicts a northward shift of both pests into new soybean-producing regions due to global warming. Our study indicated that co-distribution soybean pests with overlapping ecological niches and similar life histories in subtropical regions of East Asia exhibit congruent phylogeographic and demographic patterns in response to shared historical biogeographic drivers.

Strong philopatry in an estuarine-dependent fish.

Understanding fish movement is critical in determining the spatial scales in which to appropriately manage wild populations. Genetic markers provide a natural tagging approach to assess the degree of gene flow and population connectivity across a species distribution. We investigated the genetic structure of black bream Acanthopagrus butcheri across its entire distribution range in Australia, as well as regional scale gene flow across south-eastern Australia by undertaking a comprehensive analysis of the populations in estuaries across the region. We applied genome-wide sampling of single-nucleotide polymorphism (SNP) markers generated from restriction site-associated DNA sequencing. Genetic structure and potential gene flow was assessed using principal component analyses and admixture analyses (STRUCTURE). Using 33,493 SNPs, we detected broad scale genetic structuring, with limited gene flow among regional clusters (i.e. Western Australia, South Australia and western Victoria; and eastern Victoria, Tasmania and New South Wales). This is likely the result of unsuitable habitats, strong ocean currents (e.g. the Leeuwin Current and the East Australian Current), large water bodies (e.g. Bass Strait) and known biogeographical provinces across the continent. Local-scale genetic structuring was also identified across the south-eastern Australian estuaries sampled, reflecting that the coexistence of both migratory and resident individuals within populations (i.e. partial migration), and the movement of fish into coastal waters, still results in strong philopatry across the region. Instances of movement among estuaries at this spatial scale were primarily found between adjacent estuaries and were likely attributed to lone migrants utilising inshore coastal currents for movement beyond nearby habitats. Targeting SNP markers in A. butcheri at this continental scale highlighted how neither spatial proximity of estuaries nor black bream's ability to move into coastal waters reflects increased gene flow. Overall, our findings highlight the importance of location-specific management.

Weighted single step GWAS reveals genomic regions associated with economic traits in Murrah buffaloes

The objective of the present study was to identify genomic regions influencing economic traits in Murrah buffaloes using weighted single step Genome Wide Association Analysis (WssGWAS). Data on 2000 animals, out of which 120 were genotyped using a double digest Restriction site Associated DNA (ddRAD) sequencing approach. The phenotypic data were collected from NDRI, India, on growth traits, viz., body weight at 6M (month), 12M, 18M and 24M, production traits like 305D (day) milk yield, lactation length (LL) and dry period (DP) and reproduction traits like age at first calving (AFC), calving interval (CI) and first service period (FSP). The biallelic genotypic data consisted of 49353 markers post-quality check. The heritability estimates were moderate to high, low to moderate, low for growth, production, reproduction traits, respectively. Important genomic regions explaining more than 0.5% of the total additive genetic variance explained by 30 adjacent SNPs were selected for further analysis of candidate genes. In this study, 105 genomic regions were associated with growth, 35 genomic regions with production and 42 window regions with reproduction traits. Different candidate genes were identified in these genomic regions, of which important are OSBPL8, NAP1L1 for growth, CNTNAP2 for production and ILDR2, TADA1 and POGK for reproduction traits.

A comparison of the prevalence of respiratory pathogens and opportunistic respiratory pathogenic profile of ‘clean’ and ‘unclean’ removable dental prostheses

ObjectivesTo determine and compare the opportunistic respiratory pathogenic index (ORPI) and prevalence of respiratory pathogens between clean and unclean removable prostheses. MethodsA cross-sectional study was conducted among 97 removable prosthesis wearers at a teaching dental hospital. Participants’ prosthesis hygiene was grouped into clean and unclean. After prosthesis plaque samples were sequenced using the Type IIB Restriction-site Associated DNA Sequencing for Microbiome method, the prevalence was assessed for the presence of respiratory pathogens on each sample. The ORPIs for clean and unclean prostheses were quantified based on the sum of the relative abundance of respiratory pathogenic bacteria in a microbiome using a reference database that contains opportunistic respiratory pathogens and disease-associated information. ResultsA total of 30 opportunistic respiratory pathogens were identified on the removable prostheses. Eighty-one (83.5%) removable prostheses harboured respiratory pathogenic bacteria. Stenotrophomonas maltophilia (34.0%), Pseudomonas aeruginosa (27.8%), and Streptococcus agalactiae (27.8%) were the top three prevalent respiratory pathogens detected in plaque samples. There was a significantly higher prevalence of respiratory pathogens residing on unclean than clean prostheses (P = 0.046). However, the ORPIs in both groups showed no statistically significant difference (P = 0.516). ConclusionsThe ORPIs for both clean and unclean prostheses demonstrated a similar abundance of respiratory pathogens. However, the high prevalence of respiratory pathogens residing on unclean prostheses should not be underestimated. Therefore, maintaining good prosthesis hygiene is still important for overall oral and systemic health, even though the direct link between prosthesis cleanliness and reduced abundance of respiratory pathogens has not been established. Clinical significanceThe association between the prevalence of respiratory pathogens and unclean removable prostheses has been demonstrated and might increase the theoretical risk of respiratory disease development.

Rare, long-distance dispersal underpins genetic connectivity in the pink sea fan, Eunicella verrucosa.

Characterizing patterns of genetic connectivity in marine species is of critical importance given the anthropogenic pressures placed on the marine environment. For sessile species, population connectivity can be shaped by many processes, such as pelagic larval duration, oceanographic boundaries and currents. This study combines restriction-site associated DNA sequencing (RADseq) and passive particle dispersal modelling to delineate patterns of population connectivity in the pink sea fan, Eunicella verrucosa, a temperate octocoral. Individuals were sampled from 20 sites covering most of the species' northeast Atlantic range, and a site in the northwest Mediterranean Sea to inform on connectivity across the Atlantic-Mediterranean transition. Using 7510 neutral SNPs, a geographic cline of genetic clusters was detected, partitioning into Ireland, Britain, France, Spain (Atlantic), and Portugal and Spain (Mediterranean). Evidence of significant inbreeding was detected at all sites, a finding not detected in a previous study of this species based on microsatellite loci. Genetic connectivity was characterized by an isolation by distance pattern (IBD) (r 2 = 0.78, p < 0.001), which persisted across the Mediterranean-Atlantic boundary. In contrast, exploration of ancestral population assignment using the program ADMIXTURE indicated genetic partitioning across the Bay of Biscay, which we suggest represents a natural break in the species' range, possibly linked to a lack of suitable habitat. As the pelagic larval duration (PLD) is unknown, passive particle dispersal simulations were run for 14 and 21 days. For both modelled PLDs, inter-annual variations in particle trajectories suggested that in a long-lived, sessile species, range-wide IBD is driven by rare, longer dispersal events that act to maintain gene flow. These results suggest that oceanographic patterns may facilitate range-wide stepping-stone genetic connectivity in E. verrucosa and highlight that both oceanography and natural breaks in a species' range should be considered in the designation of ecologically coherent MPA networks.

New linkage maps for the Cupressaceae species Chamaecyparis obtusa and Thujopsis dolabrata var. hondae and comparison with the linkage map of Cryptomeria japonica

ABSTRACT Comparative mapping studies, which can determine whether the genome structure differs among genera, have yet to be conducted in Cupressaceae. This study analyzed three important forestry species in Japan: Cryptomeria japonica, Chamaecyparis obtusa, and Thujopsis dolabrata var. hondae. To date, linkage maps have only been constructed for C. japonica. We constructed the first linkage maps for C. obtusa and T. dolabrata var. hondae using double digest restriction-site associated DNA sequencing (ddRAD-Seq) data, expressed sequence tag-derived simple sequence repeat (EST-SSR) markers, and single-nucleotide polymorphism (SNP) markers designed from homologous unigenes obtained from RNA-seq data. The lengths of the linkage maps were 1,835 cM (1,981 markers) for C. obtusa and 1,833 cM (1,295 markers) for T. dolabrata var. hondae. A total of 11 distinct linkage groups were identified in these maps, corresponding to the haploid number of Cupressaceae. Using four published linkage maps for C. japonica, we then constructed a composite linkage map with 7,784 markers. Finally, the three linkage maps were compared using 207 SNP markers designed from homologous unigenes. Although most markers were mapped to the same LG despite representing different species, indicating strong synteny. The high level of interspecific and intergeneric synteny and macro-collinearity detected in Cupressaceae were similar to those of other trees. We also detected three major chromosomal rearrangements between but not within subfamilies, which may have been caused by the taxonomic distance. This is the first example of comparative mapping for Cupressaceae species.

Characterization of Eighty-Eight Single-Nucleotide Polymorphism Markers in the Manila Clam Ruditapes philippinarum Based on High-Resolution Melting (HRM) Analysis.

Single-nucleotide polymorphisms (SNPs) are the most commonly used DNA markers in population genetic studies. We used the Illumina HiSeq4000 platform to develop single-nucleotide polymorphism (SNP) markers for Manila clam Ruditapes philippinarum using restriction site-associated DNA sequencing (RAD-seq) genotyping. Eighty-eight SNP markers were successfully developed by using high-resolution melting (HRM) analysis, with a success rate of 44%. SNP markers were analyzed for genetic diversity in two clam populations. The observed heterozygosity per locus ranged from 0 to 0.9515, while the expected heterozygosity per locus ranged from 0.0629 to 0.4997. The value of FIS was estimated to be from -0.9643 to 1.0000. The global Fst value was 0.1248 (p < 0.001). After Bonferroni correction, 15 loci deviated significantly from the Hardy-Weinberg equilibrium (p < 0.0006). These SNP markers provide a valuable resource for population and conservation genetics studies in this commercially important species.