Academic literature on the topic 'DdRAD-seq'

Whole Genome Sequencing (WGS) remains a costly or unsuitable method for routine genotyping of laying hens. Until now, breeding companies have been using or developing SNP chips. Nevertheless, alternatives methods based on sequencing have been developed. Among these, reduced representation sequencing approaches can offer sequencing quality and cost-effectiveness by reducing the genomic regions covered by sequencing. The aim of this study was to evaluate the ability of double digested Restriction site Associated DNA sequencing (ddRAD-seq) to identify and genotype SNPs in laying hens, by comparison with a presumed reliable WGS approach. Firstly, the sensitivity and precision of variant calling and the genotyping reliability of ddRADseq were determined. Next, the SNP Call Rate (CRSNP) and mean depth of sequencing per SNP (DPSNP) were compared between both methods. Finally, the effect of multiple combinations of thresholds for these parameters on genotyping reliability and amount of remaining SNPs in ddRAD-seq was studied. In raw form, the ddRAD-seq identified 349,497 SNPs evenly distributed on the genome with a CRSNP of 0.55, a DPSNP of 11X and a mean genotyping reliability rate per SNP of 80%. Considering genomic regions covered by expected enzymatic fragments (EFs), the sensitivity of the ddRAD-seq was estimated at 32.4% and its precision at 96.4%. The low CRSNP and DPSNP values were explained by the detection of SNPs outside the EFs theoretically generated by the ddRAD-seq protocol. Indeed, SNPs outside the EFs had significantly lower CRSNP (0.25) and DPSNP (1X) values than SNPs within the EFs (0.7 and 17X, resp.). The study demonstrated the relationship between CRSNP, DPSNP, genotyping reliability and the number of SNPs retained, to provide a decision-support tool for defining filtration thresholds. Severe quality control over ddRAD-seq data allowed to retain a minimum of 40% of the SNPs with a CcR of 98%. Then, ddRAD-seq was defined as a suitable method for variant calling and genotyping in layers.

In recent years, high-throughput sequencing methods have become increasingly popular in molecular biology laboratories, mainly due to the relatively low cost of small, benchtop platforms, the simplicity of library preparation, and the low price per unit of information. Sequencing huge and complex genomes, such as cereal genomes, remains challenging and may not always be necessary. Therefore, several techniques have been developed to sequence a reduced representation of the genome. The most flexible and widely used of these is ddRAD-Seq, which uses a pair of restriction enzymes to generate a pool of DNA fragments. The aim of this study was to validate in vitro the efficacy of different combinations of restriction enzymes for ddRAD-Seq library construction in barley and maize. Eleven pairs of restriction enzymes were selected and tested to determine the concentrations of fragments with the expected length range and to select suitable pairs for sampling the genomes of these two cereals using ddRAD-Seq. For the selected pairs, i.e., PstI—MspI and HindIII—FspBI for barley and maize, respectively, libraries were prepared for NGS sequencing on Illumina MiSeq. Sequencing confirmed the suitability of the selected enzymes to perform ddRAD-Seq in different genotypes. The results presented can be used for extensive research on these important cereal species.

To reduce the genome sequence representation, restriction site-associated DNA sequencing (RAD-seq) protocols is being widely used either with single-digest or double-digest methods. In this study, we genotyped the sesame population (48 sample size) in a pilot scale to compare single and double-digest RAD-seq (sd and ddRAD-seq) methods. We analysed the resulting short-read data generated from both protocols and assessed their performance impacting the downstream analysis using various parameters. The distinct k-mer count and gene presence absence variation (PAV) showed a significant difference between the sesame samples studied. Additionally, the variant calling from both datasets (sdRAD-seq and ddRAD-seq) exhibits a significant difference between them. The combined variants from both datasets helped in identifying the most diverse samples and possible sub-groups in the sesame population. The most diverse samples identified from each analysis (k-mer, gene PAV, SNP count, Heterozygosity, NJ and PCA) can possibly be representative samples holding major diversity of the small sesame population used in this study. The best possible strategies with suggested inputs for modifications to utilize the RAD-seq strategy efficiently on a large dataset containing thousands of samples to be subjected to molecular analysis like diversity, population structure and core development studies were discussed.

Abstract Background and Aims The genus Asarum sect. Heterotropa (Aristolochiaceae) probably experienced rapid diversification into 62 species centred on the Japanese Archipelago and Taiwan, providing an ideal model for studying island adaptive radiation. However, resolving the phylogeny of this plant group using Sanger sequencing-based approaches has been challenging. To uncover the radiation history of Heterotropa, we employed a phylogenomic approach using double-digested RAD-seq (ddRAD-seq) to yield a sufficient number of phylogenetic signals and compared its utility with that of the Sanger sequencing-based approach. Methods We first compared the performance of phylogenetic analysis based on the plastid matK and trnL–F regions and nuclear ribosomal internal transcribed spacer (nrITS), and phylogenomic analysis based on ddRAD-seq using a reduced set of the plant materials (83 plant accessions consisting of 50 species, one subspecies and six varieties). We also conducted more thorough phylogenomic analyses including the reconstruction of biogeographic history using comprehensive samples of 135 plant accessions consisting of 54 species, one subspecies, nine varieties of Heterotropa and six outgroup species. Key Results Phylogenomic analyses of Heterotropa based on ddRAD-seq were superior to Sanger sequencing-based approaches and resulted in a fully resolved phylogenetic tree with strong support for 72.0–84.8 % (depending on the tree reconstruction methods) of the branches. We clarified the history of Heterotropa radiation and found that A. forbesii, the only deciduous Heterotropa species native to mainland China, is sister to the evergreen species (core Heterotropa) mostly distributed across the Japanese Archipelago and Taiwan. Conclusions The core Heterotropa group was divided into nine subclades, each of which had a narrow geographic distribution. Moreover, most estimated dispersal events (22 out of 24) were between adjacent areas, indicating that the range expansion has been geographically restricted throughout the radiation history. The findings enhance our understanding of the remarkable diversification of plant lineages in the Japanese Archipelago and Taiwan.

Elephas maximus maximus Linnaeus, the Sri Lankan subspecies is the largest and the darkest among Asian elephants. Patches of depigmented areas with no skin color on the ears, face, trunk, and belly morphologically differentiate it from the others. The elephant population in Sri Lanka is now limited to smaller areas and protected under Sri Lankan law. Despite its ecological and evolutionary importance, the relationship between Sri Lankan elephants and their phylogenetic position among Asian elephants remains controversial. While identifying genetic diversity is the key to any conservation and management strategies, limited data is currently available. To address such issues, we analyzed 24 elephants with known parental lineages with high throughput ddRAD-seq. The mitogenome suggested the coalescence time of the Sri Lankan elephant at ~0.2 million years, and sister to Myanmar elephants supporting the hypothesis of the movement of elephants in Eurasia. The ddRAD-seq approach identified 50,490 genome-wide SNPs among Sri Lankan elephants. The genetic diversity within Sri Lankan elephants assessed with identified SNPs suggests a geographical differentiation resulting in three main clusters; north-eastern, mid-latitude, and southern regions. Interestingly, though it was believed that elephants from the Sinharaja rainforest are of an isolated population, the ddRAD-based genetic analysis clustered it with the north-eastern elephants. The effect of habitat fragmentation on genetic diversity could be further assessed with more samples with specific SNPs identified in the current study.

The larval stages of the central European sibling caddisfly species Sericostoma personatum (Spence in Kirby and Spence, 1826) and S. flavicorne Schneider, 1845 are morphologically similar and can only be distinguished by differences in coloration in late larval instars. Identification using the mitochondrial barcoding gene, i.e., the Cytochrome c Oxidase 1, is impossible, as both species share the same highly differentiated haplotypes due to introgression. Nuclear gene markers obtained through double digest restriction site associate sequencing (ddRAD seq), however, can reliably distinguish both species, yet the method is expensive as well as time-consuming and therefore not practicable for species determination. To facilitate accurate species identification without sequencing genome-wide markers, we developed nine diagnostic nuclear RFLP markers based on ddRAD seq data. The markers were successfully tested on geographically distinct populations of the two Sericostoma species in western Germany, on known hybrids, and on another sericostomatid caddisfly species, Oecismus monedula (Hagen, 1859) that sometimes shares the habitat and can be morphologically confounded with Sericostoma. We describe a simple and fast protocol for reliable species identification of S. personatum and S. flavicorne independent of the life cycle stage of the specimens.

Variety characterization is crucial in the seed trade, particularly for protecting variety rights. However, the identification of roselle (Hibiscus sabdariffa L.) varieties, known for their beneficial effects on human health and high processing potential, has traditionally relied on morphological traits due to limited genetic information. To investigate genetic polymorphisms of roselle germplasms and to develop breeder-accessible genotyping tools, this study first phenotyped a roselle collection from diverse geographical origins for the selection of core varieties, and then utilized double-digest restriction-associated DNA sequencing (ddRAD-seq) to identify 53,746 single nucleotide polymorphisms (SNPs) across 17 core varieties. Cluster analysis of the SNP data effectively grouped varieties with similar genetic backgrounds. From this genetic information, we selected nine SNPs as a toolkit to simplify core variety discrimination. These SNPs were then converted into breeder-friendly kompetitive allele-specific PCR (KASP) markers, facilitating the classification of an additional 54 roselle accessions. In conclusion, this research contributes novel insights into the genetic relationships among roselle varieties, and establishes a robust framework utilizing ddRAD-seq and KASP markers for improved genetic resource identification and application in breeding programs.

The larval stages of the central European sibling caddisfly species Sericostoma personatum (Spence in Kirby and Spence, 1826) and S. flavicorne Schneider, 1845 are morphologically similar and can only be distinguished by differences in coloration in late larval instars. Identification using the mitochondrial barcoding gene, i.e., the Cytochrome c Oxidase 1, is impossible, as both species share the same highly differentiated haplotypes due to introgression. Nuclear gene markers obtained through double digest restriction site associate sequencing (ddRAD seq), however, can reliably distinguish both species, yet the method is expensive as well as time-consuming and therefore not practicable for species determination. To facilitate accurate species identification without sequencing genome-wide markers, we developed nine diagnostic nuclear RFLP markers based on ddRAD seq data. The markers were successfully tested on geographically distinct populations of the two Sericostoma species in western Germany, on known hybrids, and on another sericostomatid caddisfly species, Oecismus monedula (Hagen, 1859) that sometimes shares the habitat and can be morphologically confounded with Sericostoma. We describe a simple and fast protocol for reliable species identification of S. personatum and S. flavicorne independent of the life cycle stage of the specimens.

The species of Pimpinella, one of the largest genera of the family Apiaceae, are traditionally cultivated for medicinal purposes. In this study, high-throughput double digest restriction-site associated DNA sequencing technology (ddRAD-seq) was used to identify single nucleotide polymorphisms (SNPs) in eight Pimpinella species from Iran. After double-digestion with the enzymes HpyCH4IV and HinfI, a total of 334,702,966 paired-end reads were de novo assembled into 1,270,791 loci with an average of 28.8 reads per locus. After stringent filtering, 2440 high-quality SNPs were identified for downstream analysis. Analysis of genetic relationships and population structure, based on these retained SNPs, indicated the presence of three major groups. Gene ontology and pathway analysis were determined by using comparison SNP-associated flanking sequences with a public non-redundant database. Due to the lack of genomic resources in this genus, our present study is the first report to provide high-quality SNPs in Pimpinella based on a de novo analysis pipeline using ddRAD-seq. This data will enhance the molecular knowledge of the genus Pimpinella and will provide an important source of information for breeders and the research community to enhance breeding programs and support the management of Pimpinella genomic resources.

The aim of the current thesis was the analysis of the genetics of sex determination of farmed fish with sexual dimorphism, using Next Generation Sequencing. Three different species of farmed fish with sex-determining systems of varying complexity were studied. Both full-sibs and more distantly related specimens of Atlantic halibut (Hippoglossus hippoglossus), Nile tilapia (Oreochromis niloticus) and European sea bass (Dicentrarchus labrax) were used for this study. Application of Restriction-site Associated DNA sequencing (RAD-seq) and double digest Restriction-site Associated DNA sequencing (ddRAD-seq), two related techniques based on next generation sequencing, allowed the identification of thousands of Single Nucleotide Polymorphisms (SNPs; > 3,000) for each of the above species. The first SNP-based genetic maps for the above species were constructed during the current study. The first evidence concerning the location of the sex-determining region of Atlantic halibut is provided in this study. In the case of Nile tilapia both novel sex-determining regions and fine mapping of the major sex-determining region are presented. In the study of European sea bass evidence concerning the absence of a major sex-determining gene was provided. Indications of putative sex-determining regions in this species are also provided. The results of the current thesis help to broaden current knowledge concerning sex determination in three important farmed fish. In addition the results of the current thesis have practical applications as well, towards the production of mono-sex stocks of those species for the aquaculture industry.

Seagrass ecosystems are highly productive and fulfil vital roles in the coastal environment as ecosystem engineers and keystone species. Unfortunately, anthropogenic pressures have resulted in a significant loss of seagrass habitat and associated ecosystem services. The complex and unpredictable nature of these pressures, and their increasing extent and intensity globally, makes resilience-based ecosystem management a valuable approach. Resilience-based management requires knowledge on processes enhancing ecosystem resilience and on actions supporting these processes. Genetic diversity, which is the allelic variation among individuals of a population, has been recognised as one of the pillars of resilient ecosystems. Genetic diversity within a population can contribute to its capacity to resist and recover from disturbances and to adapt to changing conditions. Thus far, however, our understanding of the spatial and temporal variation in genetic diversity in seagrasses has been very limited, hindering the use of genetic data in seagrass management. This knowledge gap was addressed within this thesis, through three separate approaches: a global review on population genetic diversity of seagrass populations and two field studies which focused on variation in diversity in two seagrass species across different spatial and temporal scales. The review aimed to assess global variation in the genetic diversity of seagrasses across key biological attributes and geographical distributions. The first field study aimed to investigate temporal variation in clonal and genetic diversity and clone composition of the widely distributed, colonising tropical seagrass Halodule uninervis. The second field study aimed to characterise the spatial patterns in genetic diversity and population genetic structure of Amphibolis antarctica across its distribution along a strong salinity gradient in Shark Bay, Western Australia. The review explored how commonly reported clonal and genetic diversity metrics varied across family, life history strategy, reproductive mode, bioregion, and latitude. This was achieved through a global systematic literature review focused on publications with data on genetic diversity of natural seagrass populations. A total of 154 articles were found with genetic diversity data from 1622 populations, of which the majority (1483 populations) used microsatellite markers. There was large variation in diversity across populations and weak spatial patterns, indicating that local conditions appear to strongly influence genetic diversity as they overwhelm global patterns. There were also significant effects of life history strategy, reproductive mode, and family on clonal and genetic diversity metrics. Lower clonal richness and higher inbreeding coefficient were found in colonising compared to opportunistic and persistent taxa and higher clonal richness was observed in monoecious compared to dioecious and hermaphroditic taxa. The first field study assessed temporal variation in clonal and genetic diversity, and clone composition of the colonising seagrass H. uninervis in the short (6 months) and long-term (4-6 years) along ~1000 km of West Australian coastline at 15 sites and across a range of disturbance conditions. A total of 44 Single Nucleotide Polymorphisms (SNPs) were used to estimate population genetic diversity. Diversity varied among sites, but varied more for clonal than genetic diversity metrics, e.g. clonal richness ranged from 0.00-0.83 and observed heterozygosity ranged from 0.27-0.34. Clonal and genetic diversity showed unexpected temporal stability over both timescales for all populations, despite temporal changes in clone composition. Furthermore, 19% of clones persisted over 4-6 years, and a maximum clonal age up to 3905 years was estimated. The second field study characterised genetic diversity and structure, and tested for evidence of local adaptation, in the temperate seagrass A. antarctica across an extreme environmental salinity gradient (> 20) in the Shark Bay World Heritage Site, Australia. In addition, this salinity gradient was characterised and hydrodynamic connectivity among sites was explored through modelling. Amphibolis antarctica was genotyped at 10 sites across this gradient using Double digest Restriction site Associated DNA sequencing (ddRADseq), resulting in 6523 loci. Intermediate levels of genotypic diversity (global R = 0.50 ± 0.06 SE) and observed heterozygosity (global HO = 0.30 ± 0.00 SE), and high genetic connectivity among most sites indicates potential for genetic resilience at a population level with pathways for recovery. Modelled salinity patterns showed extreme differences in mean monthly salinity among (> 20) and within (> 15) sites. Only 11 of the 6523 loci identified as potentially adaptive, but were not associated with salinity, providing no evidence of local adaption, and suggesting phenotypic plasticity to salinity variation instead. These studies provide important insights into the genetic diversity, population dynamics and longevity of seagrasses and the potential to contribute to population resilience, with implications for seagrass management. First, the finding of large variation in genetic diversity measures and population dynamics, like genet persistence, among populations within species indicate that it is important to collect site-specific genetic data and to understand local conditions when incorporating genetic diversity into seagrass management. Second, the unexpected longevity in a colonising taxa rivalled that found in persistent seagrasses. Since longevity contributes to the stability and persistence of populations over time, these findings indicate that a re-evaluation of the persistence of genets and populations in seagrasses across life history strategies is needed. Third, the temporal stability in diversity observed in H. uninervis indicates it is feasible to include clonal and genetic diversity in seagrass monitoring since genetic assessments can provide an estimate of genetic diversity that is valid for multiple years. Fourth, the persistence of individual genets observed in a colonising taxa means there are opportunities for the assisted recovery of colonising seagrass meadows through the introduction of new clones. Finally, this research showed that the estimated natural recovery period for seagrass can be tens to thousands of years, which indicates that assisted recovery could be a valuable tool within seagrass management.