Restriction Site-associated DNA Research Articles

A Diverging Species within the Stewartia gemmata (Theaceae) Complex Revealed by RAD-Seq Data

Informed species delimitation is crucial in diverse biological fields; however, it can be problematic for species complexes. Showing a peripatric distribution pattern, Stewartia gemmata and S. acutisepala (the S. gemmata complex) provide us with an opportunity to study species boundaries among taxa undergoing nascent speciation. Here, we generated genomic data from representative individuals across the natural distribution ranges of the S. gemmata complex using restriction site-associated DNA sequencing (RAD-seq). Based on the DNA sequence of assembled loci containing 41,436 single-nucleotide polymorphisms (SNPs) and invariant sites, the phylogenetic analysis suggested strong monophyly of both the S. gemmata complex and S. acutisepala, and the latter was nested within the former. Among S. gemmata individuals, the one sampled from Mt. Tianmu (Zhejiang) showed the closest evolutionary affinity with S. acutisepala (which is endemic to southern Zhejiang). Estimated from 2996 high-quality SNPs, the genetic divergence between S. gemmata and S. acutisepala was relatively low (an Fst of 0.073 on a per-site basis). Nevertheless, we observed a proportion of genomic regions showing relatively high genetic differentiation on a windowed basis. Up to 1037 genomic bins showed an Fst value greater than 0.25, accounting for 8.31% of the total. After SNPs subject to linkage disequilibrium were pruned, the principal component analysis (PCA) showed that S. acutisepala diverged from S. gemmata along the first and the second PCs to some extent. By applying phylogenomic analysis, the present study determines that S. acutisepala is a variety of S. gemmata and is diverging from S. gemmata, providing empirical insights into the nascent speciation within a species complex.

Genetic divergence and adaptation of an isolated European lobster population in the Netherlands

Abstract Identifying isolated populations is a key step towards enacting effective conservation management. European lobsters (Homarus gammarus) from Oosterschelde in the Netherlands are subject to fishery pressure and have previously been reported as genetically differentiated. They are also putatively of transplanted origin and have subsequently endured recent bottlenecking and environmental change. We assessed Oosterschelde lobsters to evaluate their demographic independence and appraise potential founder effects and evolutionary responses to isolation. Using restriction-site associated DNA sequencing, we genotyped 6185 single nucleotide polymorphisms across 188 individuals from 27 sites across the Atlantic range of H. gammarus to investigate population genetic diversity, structure, and potential adaptation. Our results show that Oosterschelde lobsters are genetically divergent from other stocks. We evidence extensive differentiation via both neutral and outlier loci, indicative of strong biophysical and demographic isolation, and detect signatures of reduced genetic diversity that may reflect weak founder effects or subsequent population contractions. Among outlier loci, we identify candidates for range-wide local adaptation via variants in genes of important biological functionality and link a private allele of Oosterschelde to a locus potentially conveying adaptive tolerance to environmental hypoxia. Given our findings, we advise proactive monitoring of Oosterschelde lobsters to explore whether existing management measures effectively conserve this discrete, self-recruiting population.

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.

Genome-wide association study reveals candidate genes critical for skin pigmentation in common carp (Cyprinus carpio) strains including koi

The differentiation and variation of body color in fish is an important component of biological evolution and phylogenetic research, and is influenced by several factors such as heredity, nutrition, physiological hormones and rearing conditions, with heredity being the most critical factor. An improved comprehension of the genetic regulation in different carp populations will facilitate future applications of precision breeding and pigment cell development-related genes editing. In this study, we used restriction site-associated DNA sequencing (2d-RAD) to determine the distribution characteristics of single nucleotide polymorphisms (SNPs) in eight common carp strains including six koi strains, i.e., Kohaku (Hb), Taisho (Dz), Showa (Zh), Golden (Hj), Matsukawabake (Sch) and Five-color (Ws), and two genetically improved breeding strains, i.e., FFRC No. 2 strain (Fr) and red FFRC strain (Hfr), and then SNP markers to resolve genetic information on the population structure and skin color formation of these strains. We found that most individuals of eight strains were clearly divided into eight clades. Genetic diversity within Fr, Sch and Ws is relatively high, while it is low within Dz, Hb and Hfr. A comparison between the populations revealed that Hfr and Ws have the highest degree of genetic differences, while Hb and Dz have the lowest. The GWAS analysis for two strategies identified several SNPs that are significantly related to skin color variation and affect melanin synthesis (ephrinb2, slc22a23, herc2, ephb2), carotenoid metabolism (oca2, bcmo1, bco2) and circadian rhythm(cipc). To summarize, our study provides some information on the genetic structure and diversity of Japanese koi and Chinese FFRC carp. Furthermore, based on the data obtained on relevant genes related to skin color differentiation and pigmentation, it can provide a theoretical basis for the subsequent integration of dominant traits and precision breeding in carps.

Genetic diversity and population structure of four Anatolian sheep revealed by genome-wide ddRADseq data

This is the first wide-scale study to reveal next-generation sequencing-based and comprehensive study to reveal genetic diversity, population structure, and phylogenetic relationships of some of Türkiye's indigenous fat-tailed sheep breeds. A total of 80 animals belonging to Akkaraman (AKR), Güney Karaman (GKR), Morkaraman (MKR), and Karakaş (KRK) sheep breeds were genotyped with genome-wide of 296.097 single nucleotide polymorphisms (SNPs) data recovered from double digest restriction site-associated DNA sequencing (ddRADseq) libraries processed by Illumina HiSeq X Ten instrument. Minor allele frequency (MAF) ranged from 0.311 (MKR) to 0.316 (AKR and GKR). Observed heterozygosity (HO) values were higher than expected values (HE) across all populations with a mean of 0.307. Negative inbreeding coefficient (FIS) values were observed in four sheep populations varying between −0.034 (AKR) to −0.060 (MKR). Cross-entropy criterion-based ADMIXTURE analysis demonstrated that four Anatolian sheep breeds were represented by two ancestral populations in which GKR and AKR were distinct, while KRK and MKR were mixed populations. Genetic distance-based tree, discriminant analysis of principal components (DAPC), and ADMIXTURE analyses confirmed that GKR, which is believed to be derived from the AKR breed, has become genetically different from other sheep populations. Moreover, no migration from and into the GKR breed was detected, while a migration edge was drawn from the KRK and MKR clade into the AKR breed by the TreeMix algorithm. This study confirmed that high-density SNP data obtained from advanced next-generation sequencing (NGS) platforms are efficient in genetically distinguishing animal breeds, even varieties. It is recommended that further NGS-based studies could be carried out to better characterize different indigenous livestock populations raised in Türkiye.

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.

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.

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.

Gallbladder microbial species and host bile acids biosynthesis linked to cholesterol gallstone comparing to pigment individuals.

Gallstones are crystalline deposits in the gallbladder that are traditionally classified as cholesterol, pigment, or mixed stones based on their composition. Microbiota and host metabolism variances among the different types of gallstones remain largely unclear. Here, the bile and gallstone microbial species spectra of 29 subjects with gallstone disease (GSD, 24 cholesterol and 5 pigment) were revealed by type IIB restriction site-associated DNA microbiome sequencing (2bRAD-M). Among them (21 subjects: 18 cholesterol and 3 pigment), plasma samples were subjected to liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics. The microbiome yielded 896 species comprising 882 bacteria, 13 fungi, and 1 archaeon. Microbial profiling revealed significant enrichment of Cutibacterium acnes and Microbacterium sp005774735 in gallstone and Agrobacterium pusense and Enterovirga sp013044135 in the bile of cholesterol GSD subjects. The metabolome revealed 2296 metabolites, in which malvidin 3-(6''-malonylglucoside), 2-Methylpropyl glucosinolate, and ergothioneine were markedly enriched in cholesterol GSD subjects. Metabolite set enrichment analysis (MSEA) demonstrated enriched bile acids biosynthesis in individuals with cholesterol GSD. Overall, the multi-omics analysis revealed that microbiota and host metabolism interaction perturbations differ depending on the disease type. Perturbed gallstone type-related microbiota may contribute to unbalanced bile acids metabolism in the gallbladder and host, representing a potential early diagnostic marker and therapeutic target for GSD.

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.

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.

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.

Characterization of pathogenic microbiome on removable prostheses with different levels of cleanliness using 2bRAD-M metagenomic sequencing

ABSTRACT Background The microbiomes on the surface of unclean removable prostheses are complex and yet largely underexplored using metagenomic sequencing technology. Objectives To characterize the microbiome of removable prostheses with different levels of cleanliness using Type IIB Restriction-site Associated DNA for Microbiome (2bRAD-M) sequencing and compare the Microbial Index of Pathogenic Bacteria (MIP) between clean and unclean prostheses. Materials and Methods Ninety-seven removable prostheses were classified into ‘clean’ and ‘unclean’ groups. All prosthesis plaque samples underwent 2bRAD metagenomic sequencing to characterize the species-resolved microbial composition. MIPs for clean and unclean prostheses were calculated based on the sum of the relative abundance of pathogenic bacteria in a microbiome using a reference database that contains opportunistic pathogenic bacteria and disease-associated information. Results Beta diversity analyses based on Jaccard qualitative and Bray-Curtis quantitative distance matrices identified significant differences between the two groups (p < 0.05). There was a significant enrichment of many pathogenic bacteria in the unclean prosthesis group. The MIP for unclean prostheses (0.47 ± 0.25) was significantly higher than for clean prostheses (0.37 ± 0.29), p = 0.029. Conclusions The microbial community of plaque samples from ‘unclean’ prostheses demonstrated compositional differences compared with ‘clean’ prostheses. In addition, the pathogenic microbiome in the ‘unclean’ versus ‘clean’ group differed.

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.

New perspectives on the genetic structure of dotted gizzard shad (Konosirus punctatus) based on RAD-seq

To maintain, develop and rationally utilize marine organisms, understanding their genetic structure and habitat adaptation pattern is necessary. Konosirus punctatus, which is a commercial fish species inhabiting the Indo-west Pacific Ocean, has shown an obvious annual global capture and aquaculture production decline due to climate changes and human activities. In the present study, restriction-site associated DNA sequencing (RAD-seq) was used to describe its genome-wide single nucleotide polymorphisms panel (SNPs). Among 146 individuals collected at nine locations scattered in China, Korea and Japan, a set of 632,090 SNPs were identified. Population genetic analysis showed that K. punctatus individuals were divided into two significant genetic clusters. Meanwhile, potential genetic differentiation between northern and southern population of K. punctatus was found. Treemix results indicated that gene flow existed among sampling locations of K. punctatus, especially from southern Japan to others. Moreover, candidate genes associated with habitat adaptations of K. punctatus were identified, which are involved in diverse physiological processes of K. punctatus including growth and development (e.g., KIDINS220, PAN3), substance metabolism (e.g., PGM5) and immune response (e.g., VAV3, CCT7, HSPA12B). Our findings may aid in understanding the possible mechanisms for the population genetic structure and local adaptation of K. punctatus, which is beneficial to establish the management and conservation units of K. punctatus, guiding the rational use of resources, with reference significance for a profound understanding of the adaptative mechanisms of other marine organisms to the environment.

RADseq data reveal widespread historical introgression in four familiar North American songbirds.

Population genetic structure is influenced by a combination of contemporary and historical events; however, this structure can be complicated by ongoing gene flow. While it is well known that contemporary hybridization occurs frequently among many closely related species, it often remains uncertain as to which populations are involved in introgression events, and this can be even more difficult to infer when introgression is historical. Here we use restriction-site associated DNA sequencing (RADseq) to look at the level of introgression among four species of songbirds in North America: the black-capped, mountain, boreal, and chestnut-backed chickadee. Samples from both sympatric and allopatric sites across the species' ranges supported limited ongoing mixing among the four species with Bayesian clustering and PCA. In contrast, f4-statistics and admixture graphs revealed extensive historical introgression among geographically structured populations. Almost all historical admixture events were among populations west of the Rocky Mountains, and almost all populations west of the Rocky Mountains, excluding island and coastal populations, showed evidence of historical admixture. The inclusion of all four chickadee species proved crucial in differentiating which species were involved in hybridization events to avoid erroneous conclusions. Taken together, the results suggest a complex pattern of divergence with gene flow.