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1
Doublet, Mathilde, Fabien Degalez, Sandrine Lagarrigue, et al. "Variant calling and genotyping accuracy of ddRAD-seq: Comparison with 20X WGS in layers." PLOS ONE 19, no. 7 (2024): e0298565. http://dx.doi.org/10.1371/journal.pone.0298565.
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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.
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Puchta-Jasińska, Marta, Paulina Bolc, Urszula Piechota, and Maja Boczkowska. "Optimized In Vitro Restriction Digestion Protocol for Preparing Maize and Barley ddRAD-Seq Libraries." Agronomy 13, no. 12 (2023): 2956. http://dx.doi.org/10.3390/agronomy13122956.
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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.
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Ruperao, Pradeep, Prasad Bajaj, Rajkumar Subramani, et al. "A pilot-scale comparison between single and double-digest RAD markers generated using GBS strategy in sesame (Sesamum indicum L.)." PLOS ONE 18, no. 6 (2023): e0286599. http://dx.doi.org/10.1371/journal.pone.0286599.
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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.
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Okuyama, Yudai, Nana Goto, Atsushi J. Nagano, et al. "Radiation history of Asian Asarum (sect. Heterotropa, Aristolochiaceae) resolved using a phylogenomic approach based on double-digested RAD-seq data." Annals of Botany 126, no. 2 (2020): 245–60. http://dx.doi.org/10.1093/aob/mcaa072.
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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.
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Sooriyabandara, M. G. C., J. M. S. M. Jayasundara, M. S. L. R. P. Marasinghe, et al. "Genetic features of Sri Lankan elephant, Elephas maximus maximus Linnaeus revealed by high throughput sequencing of mitogenome and ddRAD-seq." PLOS ONE 18, no. 6 (2023): e0285572. http://dx.doi.org/10.1371/journal.pone.0285572.
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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.
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Darschnik, Sonja, Florian Leese, Martina Weiss, and Hannah Weigand. "When barcoding fails: development of diagnostic nuclear markers for the sibling caddisfly species Sericostoma personatum (Spence in Kirby & Spence, 1826) and Sericostoma flavicorne Schneider, 1845." ZooKeys 872 (August 20, 2019): 57–68. http://dx.doi.org/10.3897/zookeys.872.34278.
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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.
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Huang, Shih-Jie, Jheng-Yang Ou, Yao-Cheng Lin, et al. "KASP Markers for Identifying Roselle (Hibiscus sabdariffa L.) Key Varieties Based on Genetic Polymorphisms Revealed by ddRAD-Seq." Horticulturae 10, no. 12 (2024): 1325. https://doi.org/10.3390/horticulturae10121325.
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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.
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Darschnik, Sonja, Florian Leese, Martina Weiss, and Hannah Weigand. "When barcoding fails: development of diagnostic nuclear markers for the sibling caddisfly species Sericostoma personatum (Spence in Kirby & Spence, 1826) and Sericostoma flavicorne Schneider, 1845." ZooKeys 872 (August 20, 2019): 57–68. https://doi.org/10.3897/zookeys.872.34278.
Full textAbstract:
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.
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Janjua, Safia, Jeffrey L. Peters, Byron Weckworth, et al. "Improving our conservation genetic toolkit: ddRAD-seq for SNPs in snow leopards." Conservation Genetics Resources 12, no. 2 (2019): 257–61. http://dx.doi.org/10.1007/s12686-019-01082-2.
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Mehravi, Shaghayegh, Gholam Ali Ranjbar, Ghader Mirzaghaderi, et al. "De Novo SNP Discovery and Genotyping of Iranian Pimpinella Species Using ddRAD Sequencing." Agronomy 11, no. 7 (2021): 1342. http://dx.doi.org/10.3390/agronomy11071342.
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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.
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Peres, Pedro A., Heather Bracken-Grissom, Laura E. Timm, and Fernando L. Mantelatto. "Genomic Analyses Implicate the Amazon–Orinoco Plume as the Driver of Cryptic Speciation in a Swimming Crab." Genes 13, no. 12 (2022): 2263. http://dx.doi.org/10.3390/genes13122263.
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The Amazon–Orinoco plume (AOP) is the world’s largest freshwater and sediment discharge into the ocean. Previous studies limited to mtDNA suggest that the swimming crab Callinectes ornatus Ordway, 1863 exists as two distinct genetic clusters separated by the AOP. However, questions concerning migration, diversification time, and species delimitation are unresolved. Densely sampling markers across the genome (SNPs) could elucidate the evolutionary processes within this species. Here, we combined mtDNA data and ddRAD-seq to explore the diversification patterns and processes within the swimming crab C. ornatus. We show great genetic differentiation between groups on the north and south sides of the plume but also signs of hybridization. Demographic modeling indicates the divergence between groups starting around 8 Mya following the AOP’s formation. After a period of isolation, we detect two incidences of secondary contact with stronger migration in concordance with the North Brazil Current flow. Our results suggest speciation with gene flow explained by the interplay among the AOP, oceanographic currents, and long larval dispersal. This work represents the first investigation employing ddRAD-seq in a marine invertebrate species with distribution encompassing the north and south Atlantic and sheds light on the role of the AOP in the diversification of a marine species.
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de Medeiros, Bruno A. S., and Brian D. Farrell. "Whole-genome amplification in double-digest RADseq results in adequate libraries but fewer sequenced loci." PeerJ 6 (July 17, 2018): e5089. http://dx.doi.org/10.7717/peerj.5089.
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Whole-genome amplification by multiple displacement amplification (MDA) is a promising technique to enable the use of samples with only limited amount of DNA for the construction of RAD-seq libraries. Previous work has shown that, when the amount of DNA used in the MDA reaction is large, double-digest RAD-seq (ddRAD) libraries prepared with amplified genomic DNA result in data that are indistinguishable from libraries prepared directly from genomic DNA. Based on this observation, here we evaluate the quality of ddRAD libraries prepared from MDA-amplified genomic DNA when the amount of input genomic DNA and the coverage obtained for samples is variable. By simultaneously preparing libraries for five species of weevils (Coleoptera, Curculionidae), we also evaluate the likelihood that potential contaminants will be encountered in the assembled dataset. Overall, our results indicate that MDA may not be able to rescue all samples with small amounts of DNA, but it does produce ddRAD libraries adequate for studies of phylogeography and population genetics even when conditions are not optimal. We find that MDA makes it harder to predict the number of loci that will be obtained for a given sequencing effort, with some samples behaving like traditional libraries and others yielding fewer loci than expected. This seems to be caused both by stochastic and deterministic effects during amplification. Further, the reduction in loci is stronger in libraries with lower amounts of template DNA for the MDA reaction. Even though a few samples exhibit substantial levels of contamination in raw reads, the effect is very small in the final dataset, suggesting that filters imposed during dataset assembly are important in removing contamination. Importantly, samples with strong signs of contamination and biases in heterozygosity were also those with fewer loci shared in the final dataset, suggesting that stringent filtering of samples with significant amounts of missing data is important when assembling data derived from MDA-amplified genomic DNA. Overall, we find that the combination of MDA and ddRAD results in high-quality datasets for population genetics as long as the sequence data is properly filtered during assembly.
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Kobayashi, Honoka, Yuka Haino, Takaya Iwasaki, Ayumi Tezuka, Atsushi J. Nagano, and Satoshi Shimada. "ddRAD-seq based phylogeographic study of Sargassum thunbergii (Phaeophyceae, Heterokonta) around Japanese coast." Marine Environmental Research 140 (September 2018): 104–13. http://dx.doi.org/10.1016/j.marenvres.2018.05.021.
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Ksouri, N., M. M. Benítez, M. M. Aballay, G. Sanchez, B. Contreras-Moreira, and Y. Gogorcena. "ddRAD-seq variant calling in peach and the effect of removing PCR duplicates." Acta Horticulturae, no. 1352 (December 2022): 405–12. http://dx.doi.org/10.17660/actahortic.2022.1352.56.
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Liu, Nian, Jianbin Guo, Xiaojing Zhou, et al. "High-resolution mapping of a major and consensus quantitative trait locus for oil content to a ~ 0.8-Mb region on chromosome A08 in peanut (Arachis hypogaea L.)." Theoretical and Applied Genetics 133, no. 1 (2019): 37–49. http://dx.doi.org/10.1007/s00122-019-03438-6.
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Key message ddRAD-seq-based high-density genetic map comprising 2595 loci identified a major and consensus QTL with a linked marker in a 0.8-Mb physical interval for oil content in peanut. Abstract Enhancing oil content is an important breeding objective in peanut. High-resolution mapping of quantitative trait loci (QTLs) with linked markers could facilitate marker-assisted selection in breeding for target traits. In the present study, a recombined inbred line population (Xuhua 13 × Zhonghua 6) was used to construct a genetic map based on double-digest restriction-site-associated DNA sequencing (ddRAD-seq). The resulting high-density genetic map contained 2595 loci, and spanned a length of 2465.62 cM, with an average distance of 0.95 cM/locus. Seven QTLs for oil content were identified on five linkage groups, including the major and stable QTL qOCA08.1 on chromosome A08 with 10.14–27.19% phenotypic variation explained. The physical interval of qOCA08.1 was further delimited to a ~ 0.8-Mb genomic region where two genes affecting oil synthesis had been annotated. The marker SNPOCA08 was developed targeting the SNP loci associated with oil content and validated in peanut cultivars with diverse oil contents. The major and stable QTL identified in the present study could be further dissected for gene discovery. Furthermore, the tightly linked marker for oil content would be useful in marker-assisted breeding in peanut.
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Wang, Wencai, Guoqian Yang, Xin Deng, et al. "Molecular Sex Identification in the Hardy Rubber Tree (Eucommia ulmoides Oliver) via ddRAD Markers." International Journal of Genomics 2020 (May 16, 2020): 1–10. http://dx.doi.org/10.1155/2020/2420976.
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Eucommia ulmoides, also known as the industrially and medicinally important hardy rubber tree, is the sole species of Eucommiaceae. Nevertheless, its dioecious property hinders sex recognition by traditional morphological observation at very early developmental stages, thus inhibiting breeding and economic cropping. In this study, double-digest restriction site-associated DNA sequencing (ddRAD-seq) was applied to screen sex-linked molecular markers for sex identification and investigation of the sex determination system in 20 male and female E. ulmoides individual plants, respectively. In consequence, five candidate male-specific loci but no female-specific loci were predicated among the 183,752 male and 147,122 female catalogue loci by bioinformatics analysis. Subsequent PCR (polymerase chain reaction) amplification and Sanger sequencing examinations were performed on another 24 individuals, 12 for each sex, from a separate population. One ideal sex-linked locus, MSL4, was identified among the five putative male-specific loci that were found using ddRAD data. MSL4 is 479 bp in length and highly conserved in all the male individuals, suggesting its feature of being stable and repeatable. Our results also indicated that the sex of E. ulmoides is likely determined genetically. In short, this study provides a consistent and reproducible ddRAD marker (MSL4) that is able to discriminate male from female seedlings in E. ulmoides, which will be valuable for rapid breeding practice and better commercial production of this economically important tree.
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Morinha, Francisco, James Ernest, Ana Archer-Taveira, Ana M. Rocha, and Robert T. Iwamasa. "Molecular and morphological data support the synonymy of Muricanthus radix Gmelin, 1791 and Muricanthus ambiguus Reeve, 1845 (Gastropoda, Muricidae)." ZooKeys 1239 (May 28, 2025): 281–303. https://doi.org/10.3897/zookeys.1239.143837.
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The Muricanthus radix/ambiguus/nigritus complex includes species with a great diversity of shell shapes and shared habitats in various regions, which has raised questions and doubts about the current taxonomic classification of these species. Muricanthus nigritus, M. radix, and M. ambiguus are three similar-looking black and white murex found commonly on the west coast of North and South America. The wide variety of morphological patterns within and between these species makes the classification of specimens difficult by visual observation. To this day, controversy persists over whether M. radix and M. ambiguus are one or two distinct species. Molecular genetic data have helped clarify the taxonomic classification of many mollusk species in recent decades, contributing to a more accurate understanding of biodiversity and ecosystems. In this study, DNA barcoding and double digest restriction-site associated DNA sequencing (ddRAD-seq) methodologies were applied to complement morphological data, establishing for the first time the phylogenetic relationships between M. nigritus, M. ambiguus and M. radix. The classic mitochondrial and nuclear barcodes obtained from 80 specimens collected from three different geographic locations differentiated only two phylogenetic clades (M. nigritus and M. radix/ambiguus from Mexico differentiated from M. radix/ambiguus from Mexico and Panama). High levels of mitochondrial DNA introgression have been observed between M. nigritus and M. radix/ambiguus. The deep-level approach performed using 3692 loci obtained from ddRAD-seq also differentiated only two genetic clusters (M. nigritus and M. radix/ambiguus). Our results clearly support the proposal that M. ambiguus should be synonymized with M. radix.
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Wang, Qijian, Xiaoni Zhang, Shengnan Lin, et al. "Mapping a double flower phenotype-associated gene DcAP2L in Dianthus chinensis." Journal of Experimental Botany 71, no. 6 (2020): 1915–27. http://dx.doi.org/10.1093/jxb/erz558.
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Abstract The double flower is a highly important breeding trait that affects the ornamental value in many flowering plants. To get a better understanding of the genetic mechanism of double flower formation in Dianthus chinensis, we have constructed a high-density genetic map using 140 F2 progenies derived from a cross between a single flower genotype and a double flower genotype. The linkage map was constructed using double-digest restriction site-associated DNA sequencing (ddRAD-seq) with 2353 single nucleotide polymorphisms (SNPs). Quantitative trait locus (QTL) mapping analysis was conducted for 12 horticultural traits, and major QTLs were identified for nine of the 12 traits. Among them, two major QTLs accounted for 20.7% and 78.1% of the total petal number variation, respectively. Bulked segregant RNA-seq (BSR-seq) was performed to search accurately for candidate genes associated with the double flower trait. Integrative analysis of QTL mapping and BSR-seq analysis using the reference genome of Dianthus caryophyllus suggested that an SNP mutation in the miR172 cleavage site of the A-class flower organ identity gene APETALA2 (DcAP2L) is responsible for double flower formation in Dianthus through regulating the expression of DcAG genes.
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Dong, Xue, Wenbo Yi, Chenguang Zheng, et al. "Species delimitation of rice seed bugs complex: Insights from mitochondrial genomes and ddRAD‐seq data." Zoologica Scripta 51, no. 2 (2021): 185–98. http://dx.doi.org/10.1111/zsc.12523.
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Qin, Hantao, Guoqian Yang, Jim Provan, Jie Liu, and Lianming Gao. "Using Mi ddRAD-seq data to develop polymorphic microsatellite markers for an endangered yew species." Plant Diversity 39, no. 5 (2017): 294–99. http://dx.doi.org/10.1016/j.pld.2017.05.008.
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Tan, Shao-Lin, Peter M. Hollingsworth, Han-Tao Qin, Lin-Jiang Ye, Jia-Yun Zou, and Lian-Ming Gao. "Development of polymorphic microsatellite markers for tree peony Paeonia delavayi (Paeoniaceae) using ddRAD-seq data." Molecular Biology Reports 46, no. 4 (2019): 4605–10. http://dx.doi.org/10.1007/s11033-019-04831-6.
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Morinha, Francisco, James Ernest, Ana Archer-Taveira, Ana M. Rocha, and Robert T. Iwamasa. "Molecular and morphological data support the synonymy of Muricanthus radix Gmelin, 1791 and Muricanthus ambiguus Reeve, 1845 (Gastropoda, Muricidae)." ZooKeys 1239 (May 28, 2025): 281–303. https://doi.org/10.3897/zookeys.1239.143837.
Full textAbstract:
The <i>Muricanthus radix</i>/<i>ambiguus</i>/<i>nigritus</i> complex includes species with a great diversity of shell shapes and shared habitats in various regions, which has raised questions and doubts about the current taxonomic classification of these species. <i>Muricanthus nigritus</i>, <i>M. radix</i>, and <i>M. ambiguus</i> are three similar-looking black and white murex found commonly on the west coast of North and South America. The wide variety of morphological patterns within and between these species makes the classification of specimens difficult by visual observation. To this day, controversy persists over whether <i>M. radix</i> and <i>M. ambiguus</i> are one or two distinct species. Molecular genetic data have helped clarify the taxonomic classification of many mollusk species in recent decades, contributing to a more accurate understanding of biodiversity and ecosystems. In this study, DNA barcoding and double digest restriction-site associated DNA sequencing (ddRAD-seq) methodologies were applied to complement morphological data, establishing for the first time the phylogenetic relationships between <i>M. nigritus</i>, <i>M. ambiguus</i> and <i>M. radix</i>. The classic mitochondrial and nuclear barcodes obtained from 80 specimens collected from three different geographic locations differentiated only two phylogenetic clades (<i>M. nigritus</i> and <i>M. radix/ambiguus</i> from Mexico differentiated from <i>M. radix</i>/<i>ambiguus</i> from Mexico and Panama). High levels of mitochondrial DNA introgression have been observed between <i>M. nigritus</i> and <i>M. radix/ambiguus</i>. The deep-level approach performed using 3692 loci obtained from ddRAD-seq also differentiated only two genetic clusters (<i>M. nigritus</i> and <i>M. radix/ambiguus</i>). Our results clearly support the proposal that <i>M. ambiguus</i> should be synonymized with <i>M. radix</i>.
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Mladineo, Ivona, Jerko Hrabar, Željka Trumbić, et al. "Community Parameters and Genome-Wide RAD-Seq Loci of Ceratothoa oestroides Imply Its Transfer between Farmed European Sea Bass and Wild Farm-Aggregating Fish." Pathogens 10, no. 2 (2021): 100. http://dx.doi.org/10.3390/pathogens10020100.
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Wild fish assemblages that aggregate within commercial marine aquaculture sites for feeding and shelter have been considered as a primary source of pathogenic parasites vectored to farmed fish maintained in net pens at an elevated density. In order to evaluate whether Ceratothoa oestroides (Isopoda, Cymothoidae), a generalist and pestilent isopod that is frequently found in Adriatic and Greek stocks of farmed European sea bass (Dicentrarchus labrax), transfers between wild and farmed fish, a RAD-Seq (restriction-site-associated DNA sequencing)-mediated genetic screening approach was employed. The double-digest RAD-Seq of 310 C. oestroides specimens collected from farmed European sea bass (138) and different wild farm-aggregating fish (172) identified 313 robust SNPs that evidenced a close genetic relatedness between the “wild” and “farmed” genotypes. ddRAD-Seq proved to be an effective method for detecting the discrete genetic structuring of C. oestroides and genotype intermixing between two populations. The parasite prevalence in the farmed sea bass was 1.02%, with a mean intensity of 2.0 and mean abundance of 0.02, while in the wild fish, the prevalence was 8.1%; the mean intensity, 1.81; and the mean abundance, 0.15. Such differences are likely a consequence of human interventions during the farmed fish’s rearing cycle that, nevertheless, did not affect the transfer of C. oestroides.
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Lin, Lidong, Fei Wang, Mingjiang Wu, and Shengqin Wang. "ddRAD Sequencing-Based Scanning of Genetic Variants in Sargassum fusiforme." Journal of Marine Science and Engineering 10, no. 7 (2022): 958. http://dx.doi.org/10.3390/jmse10070958.
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Sargassum fusiforme is a commercially important brown seaweed that has experienced significant population reduction both from heavy exploitation and degradation of the environment. Cultivated breed strains are also in a state of population mixing. These population stressors make it necessary to investigate the population genetics to discover best practices to conserve and breed this seaweed. In this study, the genetic diversity and population structure of S. fusiforme were investigated by the genome-wide SNP data acquired from double digest restriction site-associated DNA sequencing (ddRAD-seq). We found a low genetic diversity and a slight population differentiation within and between wild and cultivated populations, and the effective population size of S. fusiforme had experienced a continuous decline. Tajima’s D analysis showed the population contraction in wild populations may be related to copper pollution which showed a consistent trend with the increase of the sea surface temperature. The potential selection signatures may change the timing or level of gene expression, and further experiments are needed to investigate the effect of the mutation on relevant pathways. These results suggest an urgent need to manage and conserve S. fusiforme resources and biodiversity considering the accelerating change of the environment.
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DaCosta, Jeffrey M., and Michael D. Sorenson. "ddRAD-seq phylogenetics based on nucleotide, indel, and presence–absence polymorphisms: Analyses of two avian genera with contrasting histories." Molecular Phylogenetics and Evolution 94 (January 2016): 122–35. http://dx.doi.org/10.1016/j.ympev.2015.07.026.
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Shepherd, Lara D., Mariana Bulgarella, Oliver Haddrath, and Colin M. Miskelly. "Genetic analyses reveal an unexpected refugial population of subantarctic snipe (Coenocorypha aucklandica)." Notornis 67, no. 1 (2020): 403. https://doi.org/10.63172/035228znelna.
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Auckland Island snipe (Coenocorypha aucklandica aucklandica) are presumed to have occurred throughout the Auckland Island archipelago but became restricted to a subset of the islands following mammal introductions. Snipe were known to have survived on Adams Island, Ewing Island, and Disappointment Island. However, it is uncertain whether snipe were continually present on Enderby Island and/or adjacent Rose Island. These islands lie near Ewing Island, and both hosted a suite of introduced mammals until the last species were eradicated in 1993. Using SNPs generated by ddRAD-Seq we identified four genetically distinct groups of snipe that correspond to the expected three refugia, plus a fourth comprised of Enderby Island and Rose Island. Each genetic group also exhibited private microsatellite alleles. We suggest that snipe survived in situ on Rose and/or Enderby Island in the presence of mammals, and discuss the conservation implications of our findings.
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Liang, Heng, Jiabin Deng, Gang Gao, Chunbang Ding, Li Zhang, and Ruiwu Yang. "Incompatibility Phylogenetic Signals between Double-Digest Restriction Site-Associated DNA Sequencing and Plastid Genomes in Chinese Curcuma (Zingiberaceae)—A Recent Qinghai–Tibetan Plateau Diversification Genera." Forests 13, no. 2 (2022): 280. http://dx.doi.org/10.3390/f13020280.
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Curcuma is of high economic value, credited to its medicinal, edible, and ornamental properties, which possess all signatures of adaptability, and rapid radiation, especially species of Curcuma (Chinese Curcuma, a recent Qinghai–Tibetan Plateau diversification genera) scattered in China. However, little is known about the incongruent phylogenetic signals within this genera from different inheritance patterns that will militate against the further development of this genera. In this research, we applied complete chloroplast genome data together with double-digest restriction site-associated DNA sequencing data (ddRAD-seq) strategy to investigate phylogenetic signals of Chinese Curcuma species, clustering using two RAD analysis pipelines (STACKS and pyRAD). Phylogenetic trees were obtained from each locus based on the maximum likelihood (ML) and multispecies coalescent (BEAST) methods. For visual comparison, multi-method and different datasets were used to infer the phylogeny. We discovered inconsistent relationships for the Chinese Curcuma with varying degrees of support using different methods and datasets.
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Geletu, Temesgen Tola, Shoujie Tang, and Jinliang Zhao. "Genetic diversity and differentiation of cultured Nile tilapia populations from Ethiopia revealed by ddRAD-seq: implications for better hatchery management." Aquatic Living Resources 38 (2025): 2. https://doi.org/10.1051/alr/2024018.
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Sub-Saharan Africa, including Ethiopia, is a center of native Nile tilapia populations, which are important for conservation and aquaculture development. Nile tilapia aquaculture in Ethiopia is dominated by small-scale fish farming in ponds, with seeds from poorly managed hatcheries and wild sources. Hence, the development of aquaculture in Ethiopia faces a major hurdle owing to the absence of good-quality seeds, largely because of the lack of genetic management practices within hatchery centers. This study aimed to assess the genetic diversity and differentiation among farmed Nile tilapia populations to inform genetic management strategies and support the development of robust strains for aquaculture advancement. Using ddRAD-seq technology for SNP discovery, we assessed genetic diversity metrics across three farmed populations, Sebeta, Batu, and Aweday, comprising 20, 21, and 15 individuals, respectively. Expected heterozyosity (He), observed heterozygosity (Ho) and nucleotide diversity (π) estimates indicated moderate within-population genetic diversity (mean: He = 0.24, Ho = 0.25, π = 0.25). Pairwise FST values revealed the highest genetic distance (FST = 0.067) between Batu and Aweday populations, while the lowest genetic distance (FST = 0.027) was observed between Sebeta and Aweday populations. STRUCTURE analysis identified two genetic clusters, with the first cluster including Batu individuals and some from Sebeta and Aweday. Overall, our results show moderate within-population genetic variation and weak genetic differentiation among the populations. This study underscores the importance of documentation of broodstock backgrounds and formulation of reasonable hatchery practices to assist in aquaculture development and conservation of native genetic resources in Ethiopia.
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Yamashita, Hiroto, Hideyuki Katai, Lina Kawaguchi, et al. "Analyses of single nucleotide polymorphisms identified by ddRAD-seq reveal genetic structure of tea germplasm and Japanese landraces for tea breeding." PLOS ONE 14, no. 8 (2019): e0220981. http://dx.doi.org/10.1371/journal.pone.0220981.
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KOZLOV, MIKHAIL V., MARKO MUTANEN, KYUNG MIN LEE, and PETER HUEMER. "Cryptic diversity in the long-horn mothNemophora degeerella(Lepidoptera: Adelidae) revealed by morphology, DNA barcodes and genome-wide ddRAD-seq data." Systematic Entomology 42, no. 2 (2016): 329–46. http://dx.doi.org/10.1111/syen.12216.
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Casavecchia, Simona, Francesco Giannelli, Massimo Giovannotti, et al. "Morphological and Genomic Differences in the Italian Populations of Onopordum tauricum Willd.—A New Source of Vegetable Rennet." Plants 13, no. 5 (2024): 654. http://dx.doi.org/10.3390/plants13050654.
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Onopordum tauricum Willd., a species distributed in Eastern Europe, has been the subject of various research endeavors aimed at assessing its suitability for extracting vegetable rennet for use in the production of local cheeses as a substitute for animal-derived rennet. In Italy, the species has an extremely fragmented and localized distribution in six locations scattered across the central-northern Apennines and some areas of southern Italy. In this study, both the morphology and genetic diversity of the six known Italian populations were investigated to detect putative ecotypes. To this end, 33 morphological traits were considered for morphometric measurements, while genetic analysis was conducted on the entire genome using the ddRAD-Seq method. Both analyses revealed significant differences among the Apennine populations (SOL, COL, and VIS) and those from southern Italy (ROT, PES, and LEC). Specifically, the southern Italian populations appear to deviate significantly in some characteristics from the typical form of the species. Therefore, its attribution to O. tauricum is currently uncertain, and further genetic and morphological analyses are underway to ascertain its systematic placement within the genus Onopordum.
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Shimomura, Koichiro, Mitsuhiro Sugiyama, Yoichi Kawazu, and Yosuke Yoshioka. "Identification of quantitative trait loci for powdery mildew resistance in highly resistant cucumber (Cucumis sativus L.) using ddRAD-seq analysis." Breeding Science 71, no. 3 (2021): 326–33. http://dx.doi.org/10.1270/jsbbs.20141.
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Li, Li-Li, Song Huang, Juan Hou, et al. "Construction of a high-density genetic map for melon using ddRAD-Seq technology from a population derived from flexuosus and reticulatus botanical groups." Scientia Horticulturae 272 (October 2020): 109531. http://dx.doi.org/10.1016/j.scienta.2020.109531.
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Lavretsky, Philip, Jeffrey M. DaCosta, Michael D. Sorenson, Kevin G. McCracken, and Jeffrey L. Peters. "ddRAD‐seq data reveal significant genome‐wide population structure and divergent genomic regions that distinguish the mallard and close relatives in North America." Molecular Ecology 28, no. 10 (2019): 2594–609. http://dx.doi.org/10.1111/mec.15091.
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Huang, Yongrong, Yu Li, Xiaojie Hong, et al. "Genetic Variation for Wild Populations of the Rare and Endangered Plant Glyptostrobus pensilis Based on Double-Digest Restriction Site-Associated DNA Sequencing." Current Issues in Molecular Biology 47, no. 1 (2024): 12. https://doi.org/10.3390/cimb47010012.
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Glyptostrobus pensilis is an endangered tree species, and detecting its genetic diversity can reveal the mechanisms of endangerment, providing references for the conservation of genetic resources. Samples of 137 trees across seven populations within Fujian Province were collected and sequenced using double-digest restriction site-associated DNA (ddRAD-seq). A total of 3,687,189 single-nucleotide polymorphisms (SNPs) were identified, and 15,158 high-quality SNPs were obtained after filtering. The genetic diversity in the populations was found to be low (Ho = 0.08630, He = 0.03475, π = 0.07239), with a high genetic differentiation coefficient (Fst). When K = 4, the coefficient of variation (CV) error value was minimized, suggesting that the 137 individuals could be divided into four groups, with frequent gene flow between them. Principal component analysis (PCA) divided the seven populations into two major categories based on their north–south geographic location. The clustering was consistent with those obtained from the PCA. The main reasons for the endangerment of G. pensilis are likely to be poor natural regeneration, human disturbances, and climatic factors. It is recommended that methods such as in situ conservation, ex situ conservation, and the establishment of germplasm banks be implemented to maintain the genetic diversity of G. pensilis populations.
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Li, Fengjuan, Linyuan Fan, Jingli Zhang, et al. "Genetic Diversity and Structure of a Critically Endangered Ornamental Species, Rhododendron farinosum, with Extremely Small Populations." Horticulturae 11, no. 1 (2025): 51. https://doi.org/10.3390/horticulturae11010051.
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A comprehensive study of the genetic characteristics of endangered species is a prerequisite for their effective conservation and management. Rhododendron farinosum is an endangered ornamental species with extremely small populations located in northeastern Yunnan Province. To unravel the reasons behind the endangerment of this species and provide guidance for the rational conservation of this species, this study obtained a large number of SNP loci by using double-digest restriction-site-associated DNA sequencing (ddRAD-seq) to evaluate the genetic diversity and genetic structure of R. farinosum, as well as to infer the population history of this species. Our findings reveal that, at the population level, R. farinosum exhibited a high genetic diversity (π = 0.1948 ± 0.0020, HE = 0.1880 ± 0.0020). The FST values (0.1383–0.2231) indicated high genetic differentiation among the three populations. The AMOVA revealed that 62.83% of the genetic variation originated within populations and 37.17% between populations. The PCA, Structure, and UPGMA consistently depicted that the three populations of R. farinosum are clearly distinguished into three clusters. Furthermore, the effective population size of R. farinosum was inferred to date back to 95,000 years ago using the stairway plot, with a continuous decline from 3292 years. Based on these findings, we propose conservation strategies and management measures for R. farinosum.
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Jackson, George D., Timothy Standish, Ortaç Çetintaş, Oleksandr Zinenko, Asilatu H. Shechonge, and Alexey Yanchukov. "Population Genetics and Gene Flow in Cyphotilapia frontosa and Cyphotilapia gibberosa Along the East Coast of Lake Tanganyika." Fishes 9, no. 12 (2024): 481. http://dx.doi.org/10.3390/fishes9120481.
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The radiation of cichlid species in the East African Great Lakes is remarkable and rapid. The population genetics of two deep-water Cyphotilapia species along the east coast of Lake Tanganyika from Burundi to southern Tanzania was determined using ddRAD-seq. A combination of ADMIXTURE, PCA, genome polarization, and 2D site frequency spectrum analyses confirmed the presence of two species, C. frontosa in the north and C. gibberosa in the south, as documented in other studies. We also found evidence of a potential hybrid zone connecting the two species at a sharp genetic cline centered in the middle of the lake and apparent introgression in both directions, but predominantly from ‘gibberosa’ into ‘frontosa’. The highest proportion of introgressed ‘gibberosa’ ancestry was present in the southernmost populations of C. frontosa collected near Karilani Island and Cape Kabogo. At the intra-specific level, there was support for between 1 and 3 populations of C. frontosa, whereas the results indicated only a single homogeneous population of C. gibberosa. The presence of different morphs in the lake despite the low levels of heterozygosity suggests that a small number of loci may be involved in the morphological variation and/or that there is a more complex interplay between genetics and the environment in different locations.
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Shi, Hongyu, Linling Liu, Peter Foged Larsen, et al. "Genomic Regions Associated with Growth and Reproduction Traits in Pink-Eyed White Mink." Genes 15, no. 9 (2024): 1142. http://dx.doi.org/10.3390/genes15091142.
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In mink breeding, balanced selection for growth and reproductive features is essential because these traits are contradictory. The variables of total number born (TNB), number born alive (NBA), and body weight (BW) are highly valuable in terms of their importance in mink production. A comprehensive understanding of the molecular mechanisms that drive these features could offer vital insights into their genetic compositions. In the present study, the single-nucleotide polymorphism (SNP) genotypes of 219 minks were obtained via double digest restriction-site associated DNA sequencing (ddRAD-seq). Following several rounds of screening, about 2,415,121 high-quality SNPs were selected for a genome-wide association study (GWAS). The GWAS was used to determine BW and reproductive traits in pink-eyed white mink. It was suggested that SLC26A36, STXBP5L, and RPS 29 serve as potential genes for the total number of kits born (TNB), while FSCB, PDPN, NKX 2-1, NFKB 1, NFKBIA, and GABBR1 are key genes for the number born alive (NBA). Moreover, RTTN, PRPF31, MACROD1, and KYAT1 are possible BW genes based on association results and available functional data from gene and mammalian phenotype databases. These results offer essential information about the variety of mink and theoretical principles for applying mink breeds.
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Méndez-Cea, Belén, Isabel García-García, Raúl Sánchez-Salguero, Víctor Lechuga, Francisco Javier Gallego, and Juan C. Linares. "Tree-Level Growth Patterns and Genetic Associations Depict Drought Legacies in the Relict Forests of Abies marocana." Plants 12, no. 4 (2023): 873. http://dx.doi.org/10.3390/plants12040873.
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The frequency and intensity of drought events are increasing worldwide, challenging the adaptive capacity of several tree species. Here, we evaluate tree growth patterns and climate sensitivity to precipitation, temperature, and drought in the relict Moroccan fir Abies marocana. We selected two study sites, formerly stated as harboring contrasting A. marocana taxa (A. marocana and A. tazaotana, respectively). For each tree, dendrochronological methods were applied to quantify growth patterns and climate–growth sensitivity. Further, ddRAD-seq was performed on the same trees and close saplings to obtain single nucleotide polymorphisms (SNPs) and related genotype–phenotype associations. Genetic differentiation between the two studied remnant populations of A. marocana was weak. Growth patterns and climate–growth relationships were almost similar at the two sites studied, supporting a negative effect of warming. Growth trends and tree size showed associations with SNPs, although there were no relationships with phenotypes related to climatic sensitivity. We found significant differences in the SNPs subjected to selection in the saplings compared to the old trees, suggesting that relict tree populations might be subjected to genetic differentiation and local adaptation to climate dryness. Our results illustrate the potential of tree rings and genome-wide analysis to improve our understanding of the adaptive capacity of drought-sensitive forests to cope with ongoing climate change.
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Reyes-Velasco, Jacobo, Sandra Goutte, Xenia Freilich, and Stéphane Boissinot. "Mitogenomics of historical type specimens clarifies the taxonomy of Ethiopian Ptychadena Boulenger, 1917 (Anura, Ptychadenidae)." ZooKeys 1070 (November 12, 2021): 135–49. https://doi.org/10.3897/zookeys.1070.66598.
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The taxonomy of the Ptychadena neumanni species complex, a radiation of grass frogs inhabiting the Ethiopian highlands, has puzzled scientists for decades because of the morphological resemblance among its members. Whilst molecular phylogenetic methods allowed the discovery of several species in recent years, assigning pre-existing and new names to clades was challenged by the unavailability of molecular data for century-old type specimens. We used Illumina short reads to sequence the mitochondrial DNA of type specimens in this group, as well as ddRAD-seq analyses to resolve taxonomic uncertainties surrounding the P. neumanni species complex. The phylogenetic reconstruction revealed recurrent confusion between Ptychadena erlangeri (Ahl, 1924) and P. neumanni (Ahl, 1924) in the literature. The phylogeny also established that P. largeni Perret, 1994 represents a junior synonym of P. erlangeri (Ahl, 1924) and distinguished between two small species, P. nana Perret, 1994, restricted to the Arussi Plateau, and P. robeensis Goutte, Reyes-Velasco, Freilich, Kassie & Boissinot, 2021, which inhabits the Bale Mountains. The phylogenetic analyses of mitochondrial DNA from type specimens also corroborate the validity of seven recently described species within the group. Our study shows how modern molecular tools applied to historical type specimens can help resolve long-standing taxonomic issues in cryptic species complexes.
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Teng, Changcai, Hongyan Zhang, Wanwei Hou, Ping Li, Xianli Zhou, and Yujiao Liu. "High-Density Genetic Map Construction and QTL Detection for Cotyledon Color in Faba Bean Based on Double Digest Restriction-Site Associated DNA Sequencing (ddRAD-Seq)." Agronomy 15, no. 1 (2025): 193. https://doi.org/10.3390/agronomy15010193.
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Cotyledon color is one of the important indices for identifying faba bean variety purity and measuring processing quality. Therefore, an in-depth study of the genetic mechanism of cotyledon color is vital for promoting faba bean industry development. We used the yellow cotyledon variety Qingcan 16 and the green cotyledon variety Qingcan 17 as parent plants to construct hybrid combinations. F1-, F2-, BC1F1-, and BC2F1-generation single-plant cotyledon colors were counted to clarify cotyledon color inheritance. F2-generation individuals were genotyped using ddRAD-Seq to construct a genetic linkage map and identify QTLs for cotyledon color. Green cotyledons were controlled by one pair of recessive nuclear genes. Using the screened 1991 SNP markers, a high-density linkage map was constructed, with a coverage length of 1476.95 cM and an average map distance of 0.96 cM. The green cotyledon trait was located using WinQTL Cart, and a vfGC candidate interval explaining 34.30 to 49.40% of the phenotypic variation was identified at LG02 (101.952 cM to 115.493 cM) and at LOD = 16.0, corresponding to chr1L 1,077,051,302 bp to 1,636,400,339 bp (559.35 Mb). The above interval contained 2021 genes, 20 of which were involved in photosynthesis, but no SGR or genes with similar functions were identified. However, the published faba bean vfSGR was located within the vfGC candidate interval, confirming that our localization interval was reliable. The above findings provided further clues for the fine localization of genes regulating green cotyledons and the development of molecular linked markers in faba bean.
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Rick, Kate, Kym Ottewell, Cheryl Lohr, Rujiporn Thavornkanlapachai, Margaret Byrne, and W. Jason Kennington. "Population Genomics of Bettongia lesueur: Admixing Increases Genetic Diversity with no Evidence of Outbreeding Depression." Genes 10, no. 11 (2019): 851. http://dx.doi.org/10.3390/genes10110851.
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Small and isolated populations are subject to the loss of genetic variation as a consequence of inbreeding and genetic drift, which in turn, can affect the fitness and long-term viability of populations. Translocations can be used as an effective conservation tool to combat this loss of genetic diversity through establishing new populations of threatened species, and to increase total population size. Releasing animals from multiple genetically diverged sources is one method to optimize genetic diversity in translocated populations. However, admixture as a conservation tool is rarely utilized due to the risks of outbreeding depression. Using high-resolution genomic markers through double-digest restriction site-associated sequencing (ddRAD-seq) and life history data collected over nine years of monitoring, this study investigates the genetic and fitness consequences of admixing two genetically-distinct subspecies of Bettongia lesueur in a conservation translocation. Using single nucleotide polymorphisms (SNPs) identified from 215 individuals from multiple generations, we found an almost 2-fold increase in genetic diversity in the admixed translocation population compared to the founder populations, and this was maintained over time. Furthermore, hybrid class did not significantly impact on survivorship or the recruitment rate and therefore we found no indication of outbreeding depression. This study demonstrates the beneficial application of mixing multiple source populations in the conservation of threatened species for minimizing inbreeding and enhancing adaptive potential and overall fitness.
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Guzinski, Jaromir, Paolo Ruggeri, Marion Ballenghien, et al. "Seascape Genomics of the Sugar Kelp Saccharina latissima along the North Eastern Atlantic Latitudinal Gradient." Genes 11, no. 12 (2020): 1503. http://dx.doi.org/10.3390/genes11121503.
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Temperature is one of the most important range-limiting factors for many seaweeds. Driven by the recent climatic changes, rapid northward shifts of species’ distribution ranges can potentially modify the phylogeographic signature of Last Glacial Maximum. We explored this question in detail in the cold-tolerant kelp species Saccharina latissima, using microsatellites and double digest restriction site-associated DNA sequencing ( ddRAD-seq) derived single nucleotide polymorphisms (SNPs) to analyze the genetic diversity and structure in 11 sites spanning the entire European Atlantic latitudinal range of this species. In addition, we checked for statistical correlation between genetic marker allele frequencies and three environmental proxies (sea surface temperature, salinity, and water turbidity). Our findings revealed that genetic diversity was significantly higher for the northernmost locality (Spitsbergen) compared to the southern ones (Northern Iberia), which we discuss in light of the current state of knowledge on phylogeography of S. latissima and the potential influence of the recent climatic changes on the population structure of this species. Seven SNPs and 12 microsatellite alleles were found to be significantly associated with at least one of the three environmental variables. We speculate on the putative adaptive functions of the genes associated with the outlier markers and the importance of these markers for successful conservation and aquaculture strategies for S. latissima in this age of rapid global change.
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Zhao, Renfen, Nian Huang, Zhiyan Zhang, et al. "Genetic Diversity Analysis and Prediction of Potential Suitable Areas for the Rare and Endangered Wild Plant Henckelia longisepala." Plants 13, no. 15 (2024): 2093. http://dx.doi.org/10.3390/plants13152093.
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Henckelia longisepala (H. W. Li) D. J. Middleton & Mich. Möller is a rare and endangered plant species found only in Southeastern Yunnan, China, and Northern Vietnam. It is listed as a threatened species in China and recognized as a plant species with extremely small populations (PSESP), while also having high ornamental value and utilization potential. This study used ddRAD-seq technology to quantify genetic diversity and structure for 32 samples from three extant populations of H. longisepala. The H. longisepala populations were found to have low levels of genetic diversity (Ho = 0.1216, He = 0.1302, Pi = 0.1731, FIS = 0.1456), with greater genetic differentiation observed among populations (FST = 0.3225). As indicated by genetic structure and phylogenetic analyses, samples clustered into three distinct genetic groups that corresponded to geographically separate populations. MaxEnt modeling was used to identify suitable areas for H. longisepala across three time periods and two climate scenarios (SSP1-2.6, SSP5-8.5). High-suitability areas were identified in Southeastern Yunnan Province, Northern Vietnam, and Eastern Laos. Future H. longisepala distribution was predicted to remain centered in these areas, but with a decrease in the total amount of suitable habitat. The present study provides key data on H. longisepala genetic diversity, as well as a theoretical basis for the conservation, development, and utilization of its germplasm resources.
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Muharromah, Atikah Fitria, Jerica Isabel L. Reyes, Ngure Kagia, and Kozo Watanabe. "Genome-wide detection of Wolbachia in natural Aedes aegypti populations using ddRAD-Seq." Frontiers in Cellular and Infection Microbiology 13 (December 14, 2023). http://dx.doi.org/10.3389/fcimb.2023.1252656.
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BackgroundWolbachia, an endosymbiotic bacterium, is globally used to control arboviruses because of its ability to block arboviral replication and manipulate the reproduction of Wolbachia host, Aedes aegypti. Polymerase chain reaction (PCR)-based Wolbachia detection has been recently reported from natural Ae. aegypti populations. However, due to the technical limitations of PCR, such as primer incompatibility, PCR-based assays are not sufficiently reliable or accurate. In this study, we examined double digestion restriction site-associated DNA sequencing (ddRAD-Seq) efficiency and limitations in Wolbachia detection and quantification in field-collected Ae. aegypti natural populations in Metro Manila, the Philippines, compared with PCR-based assays.MethodsA total of 217 individuals Ae. aegypti were collected from Metropolitan Manila, Philippines. We separated it into 14 populations consisting of 7 female and male populations. We constructed a library for pool ddRAD-Seq per population and also screened for Wolbachia by PCR assays using wsp and 16S rRNA. Wolbachia density per population were measured using RPS17 as the housekeeping gene.ResultsFrom 146,239,637 sequence reads obtained, 26,299 and 43,778 reads were mapped across the entire Wolbachia genome (with the wAlbA and wAlbB strains, respectively), suggesting that ddRAD-Seq complements PCR assays and supports more reliable Wolbachia detection from a genome-wide perspective. The number of reads mapped to the Wolbachia genome per population positively correlated with the number of Wolbachia-infected individuals per population based on PCR assays and the relative density of Wolbachia in the Ae. aegypti populations based on qPCR, suggesting ddRAD-Seq-based semi-quantification of Wolbachia by ddRAD-Seq. Male Ae. aegypti exhibited more reads mapped to the Wolbachia genome than females, suggesting higher Wolbachia prevalence rates in their case. We detected 150 single nucleotide polymorphism loci across the Wolbachia genome, allowing for more accurate the detection of four strains: wPip, wRi, TRS of Brugia malayi, and wMel.ConclusionsTaken together, our results demonstrate the feasibility of ddRAD-Seq-based Wolbachia detection from field-collected Ae. aegypti mosquitoes.
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Nishimura, Kazusa, Maho Okuma, Junko Kaneyoshi, et al. "Workflow for development of CAPS markers with one type of restriction enzyme to identify citrus cultivars." Tree Genetics & Genomes 20, no. 5 (2024). http://dx.doi.org/10.1007/s11295-024-01661-x.
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AbstractGiven the ease of propagating fruit tree species through cloning, the economic viability of their breeding programs hinges on protecting breeders' rights. This necessitates the development of highly accurate DNA markers for cultivar identification. Here, we present a methodology for the rapid design of cleaved amplified polymorphic sequence (CAPS) markers to discriminate newly bred Japanese citrus cultivars from genetically related cultivars. We first compared the performance of ddRAD-seq and MIG-seq in citrus germplasm. The ddRAD-seq libraries generated using EcoRI and HindIII restriction enzymes yielded the highest number of polymorphisms. Subsequently, ddRAD-seq with EcoRI and HindIII was employed to analyze 29 citrus cultivars and thus identify 331,801 genome-wide polymorphisms. A semi-automated bioinformatics pipeline was then utilized to identify candidate CAPS markers, resulting in the discovery of 14,072 potential markers. Of these candidates, 52 were chosen for validation based on their recognition by the PstI restriction enzyme. This evaluation resulted in the development of 11 highly discriminative CAPS markers. Remarkably, a combination of only six such markers was sufficient to differentiate newly bred cultivars from their genetically related parents. The single restriction enzyme employed for these markers facilitates straightforward multiplexing. Finally, a combination of one multiplex marker testing two loci and four singleplex markers was successfully selected that completely discriminated the cultivars other than the bud sports used in this study. The pipeline established here extends beyond citrus and has the potential to simplify marker development and cultivar protection in various plant species.
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Sumitomo, Katsuhiko, Kenta Shirasawa, Sachiko Isobe, et al. "A genome-wide association and fine-mapping study of white rust resistance in hexaploid chrysanthemum cultivars with a wild diploid reference genome." Horticulture Research, August 3, 2022. http://dx.doi.org/10.1093/hr/uhac170.
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Abstract White rust caused by Puccinia horiana is one of the most serious diseases of chrysanthemum (Chrysanthemum × morifolium). In this study, we report the DNA markers associated with resistance against P. horiana via a simple approach using the genome of a wild diploid relative, Chrysanthemum seticuspe. First, we identified the important region of the genome in the resistant cultivar “Ariesu” via a genome-wide association study. Simplex single nucleotide polymorphism (SNP) markers mined from ddRAD-Seq were used in a biparental population originating from crosses between resistant “Ariesu” and susceptible “Yellow Queen”. The C. seticuspe genome was used as a reference. For the fine mapping of P. horiana resistance locus 2 (Phr2), a comparative whole genome sequencing study was conducted. Although the genome sequences of chrysanthemum cultivars assembled via the short-read approach were fragmented, reliable genome alignments were reconstructed by mapping onto the chromosome level of the C. seticuspe pseudomolecule. Base variants were then identified by comparing the assembled genome sequences of resistant “Ariesu” and susceptible “Yellow Queen”. Consequently, SNP markers that were closer to Phr2 compared with ddRAD-Seq markers were obtained. These SNP markers co-segregated with resistance in F1 progenies originating from resistant “Ariesu” and showed robust transferability for detecting Phr2-conferring resistance among chrysanthemum genetic resources. The wild C. seticuspe pseudomolecule, a de facto monoploid genome used for ddRAD-Seq analysis and assembled genome sequence comparison, demonstrated this method’s utility as a model for developing DNA markers in hexaploid chrysanthemum cultivars.
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Muharromah, Atikah Fitria, Thaddeus M. Carvajal, Maria Angenica F. Regilme, and Kozo Watanabe. "Fine-scale adaptive divergence and population genetic structure of Aedes aegypti in Metropolitan Manila, Philippines." Parasites & Vectors 17, no. 1 (2024). http://dx.doi.org/10.1186/s13071-024-06300-x.
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Abstract Background The adaptive divergence of Aedes aegypti populations to heterogeneous environments can be a driving force behind the recent expansion of their habitat distribution and outbreaks of dengue disease in urbanized areas. In this study, we investigated the population genomics of Ae. aegypti at a regional scale in Metropolitan Manila, Philippines. Methods We used the Pool-Seq double digestion restriction-site association DNA sequencing (ddRAD-Seq) approach to generate a high number of single nucleotide polymorphisms (SNPs), with the aim to determine local adaptation and compare the population structure with 11 microsatellite markers. A total of 217 Ae. aegypti individuals from seven female and seven male populations collected from Metropolitan Manila were used in the assays. Results We detected 65,473 SNPs across the populations, of which 76 were non-neutral SNPs. Of these non-neutral SNPs, the multivariate regression test associated 50 with eight landscape variables (e.g. open space, forest, etc.) and 29 with five climate variables (e.g. air temperature, humidity, etc.) (P-value range 0.005–0.045) in female and male populations separately. Male and female populations exhibited contrasting spatial divergence, with males exhibiting greater divergence than females, most likely reflecting the different dispersal abilities of male and female mosquitoes. In the comparative analysis of the same Ae. aegypti individuals, the pairwise FST values of 11 microsatellite markers were lower than those of the neutral SNPs, indicating that the neutral SNPs generated via pool ddRAD-Seq were more sensitive in terms of detecting genetic differences between populations at fine-spatial scales. Conclusions Overall, our study demonstrates the utility of pool ddRAD-Seq for examining genetic differences in Ae. aegypti populations in areas at fine-spatial scales that could inform vector control programs such as Wolbachia-infected mosquito mass-release programs. This in turn would provide information on mosquito population dispersal patterns and the potential barriers to mosquito movement within and around the release area. In addition, the potential of environmental adaptability observed in Ae. aegypti could help population control efforts. Graphical Abstract
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Aballay, Maximiliano Martín, Natalia Cristina Aguirre, Carla Valeria Filippi, Gabriel Hugo Valentini, and Gerardo Sánchez. "Fine-tuning the performance of ddRAD-seq in the peach genome." Scientific Reports 11, no. 1 (2021). http://dx.doi.org/10.1038/s41598-021-85815-0.
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AbstractThe advance of Next Generation Sequencing (NGS) technologies allows high-throughput genotyping at a reasonable cost, although, in the case of peach, this technology has been scarcely developed. To date, only a standard Genotyping by Sequencing approach (GBS), based on a single restriction with ApeKI to reduce genome complexity, has been applied in peach. In this work, we assessed the performance of the double-digest RADseq approach (ddRADseq), by testing 6 double restrictions with the restriction profile generated with ApeKI. The enzyme pair PstI/MboI retained the highest number of loci in concordance with the in silico analysis. Under this condition, the analysis of a diverse germplasm collection (191 peach genotypes) yielded 200,759,000 paired-end (2 × 250 bp) reads that allowed the identification of 113,411 SNP, 13,661 InDel and 2133 SSR. We take advantage of a wide sample set to describe technical scope of the platform. The novel platform presented here represents a useful tool for genomic-based breeding for peach.
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Zhu, Hong, Juan Liu, Meirong Gao, Chunlei Yue, and Hepeng Li. "Population genetic assessment of Viburnum japonicum in China using ddRAD-seq." Frontiers in Genetics 14 (June 1, 2023). http://dx.doi.org/10.3389/fgene.2023.1150437.
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Viburnum japonicum is a rare plant species and endemic to the coastal region of Eastern Asia with extremely small populations. Within mainland China, this species can be only found in narrow habitats of the northeast coastal islands of Zhejiang Province. However, there are scarce conservation genetic studies on V. japonicum, which has limited the effective conservation and management of this rare species. Here, 51 individuals in four natural populations covering the Chinese geographic range of the species were sampled to assess the genetic diversity and population structure. A total of 445,060 high-quality single nucleotide polymorphisms (SNPs) were identified using double digest restriction-site associated sequencing (ddRAD-seq). The overall average values of observed heterozygosity (Ho), expected heterozygosity (He), and average nucleotide diversity (π), were 0.2207, 0.2595, and 0.2741, respectively. The DFS-2 population exhibited the highest level of genetic diversity among all the populations. Genetic differentiation between populations was moderate (FST = 0.1425), and there was selfing between populations (FIS = 0.1390, S = 24.52%). Of the total genetic variation, 52.9% was found among populations through AMOVA analysis. The Mantel test (r = 0.982, p = 0.030) combined with analyses of the Maximum Likelihood (ML) phylogenetic tree, ADMIXTURE, and principal component analysis (PCA), revealed that populations of V. japonicum were genetically segregated and significantly correlated with their geographical distribution. Our study demonstrated that V. japonicum maintained a medium level of genetic diversity and differentiation with a strong population structure, and the results were mainly affected by its island distribution pattern and self-crossing characteristics. These results provide insights into the genetic diversity and population history of V. japonicum, critical information for conserving and sustainably developing its genetic resources.