AmpliRAD: A new method combining amplicon and RAD sequencing

At present, low-pass whole-genome sequencing (WGS) is frequently used in clinical research and in the screening of copy number variations (CNVs). However, there are still some challenges in the detection of triploids. Restriction site-associated DNA sequencing (RAD-Seq) technology is a reduced-representation genome sequencing technology developed based on next-generation sequencing. Here, we verified whether RAD-Seq could be employed to detect CNVs and triploids. In this study, genomic DNA of 11 samples was extracted employing a routine method and used to build libraries. Five cell lines of known karyotypes and 6 triploid abortion tissue samples were included for RAD-Seq testing. The triploid samples were confirmed by STR analysis and also tested by low-pass WGS. The accuracy and efficiency of detecting CNVs and triploids by RAD-Seq were then assessed, compared with low-pass WGS. In our results, RAD-Seq detected 11 out of 11 (100%) chromosomal abnormalities, including 4 deletions and 1 aneuploidy in the purchased cell lines and all triploid samples. By contrast, these triploids were missed by low-pass WGS. Furthermore, RAD-Seq showed a higher resolution and more accurate allele frequency in the detection of triploids than low-pass WGS. Our study shows that, compared with low-pass WGS, RAD-Seq has relatively higher accuracy in CNV detection at a similar cost and is capable of identifying triploids. Therefore, the application of this technique in medical genetics has a significant potential value.

Amorphophallus krausei is a perennial plant which is native to Southeast Asia. Wild populations of A. krausei are declining in China due to anthropogenic threats. Genetic monitoring is an important tool for the conservation of any threatened population, but genetic diversity studies of A. krausei are currently hindered by a lack of molecular markers. To help with conservation genetics studies of this species, we developed 41 single nucleotide polymorphism markers using restriction site-associated DNA sequencing (RAD-seq). The Shannon's information index of each locus ranged from 0.199 to 0.693, the observed heterozygosity ranged from 0.025 to 0.237, and the expected heterozygosity ranged from 0.095 to 0.500. Twenty-two loci showed signs of departure from Hardy–Weinberg equilibrium after Bonferroni corrections (P < 0.05). The developed loci will contribute to future conservation genetics studies of A. krausei.

The salmon louse (Lepeophtheirus salmonis (Krøyer, 1837)) is a parasitic copepod that can, if untreated, cause considerable damage to Atlantic salmon (Salmo salar Linnaeus, 1758) and incurs significant costs to the Atlantic salmon mariculture industry. Salmon lice are gonochoristic and normally show sex ratios close to 1:1. While this observation suggests that sex determination in salmon lice is genetic, with only minor environmental influences, the mechanism of sex determination in the salmon louse is unknown. This paper describes the identification of a sex-linked Single Nucleotide Polymorphism (SNP) marker, providing the first evidence for a genetic mechanism of sex determination in the salmon louse. Restriction site-associated DNA sequencing (RAD-seq) was used to isolate SNP markers in a laboratory-maintained salmon louse strain. A total of 85 million raw Illumina 100 base paired-end reads produced 281,838 unique RAD-tags across 24 unrelated individuals. RAD marker Lsa101901 showed complete association with phenotypic sex for all individuals analysed, being heterozygous in females and homozygous in males. Using an allele-specific PCR assay for genotyping, this SNP association pattern was further confirmed for three unrelated salmon louse strains, displaying complete association with phenotypic sex in a total of 96 genotyped individuals. The marker Lsa101901 was located in the coding region of the prohibitin-2 gene, which showed a sex-dependent differential expression, with mRNA levels determined by RT-qPCR about 1.8-fold higher in adult female than adult male salmon lice. This study’s observations of a novel sex-linked SNP marker are consistent with sex determination in the salmon louse being genetic and following a female heterozygous system. Marker Lsa101901 provides a tool to determine the genetic sex of salmon lice, and could be useful in the development of control strategies.

Intracellular bacteria are ubiquitous in the insect world, with perhaps the best-studied example being the alphaproteobacterium, Wolbachia. Like most endosymbionts, Wolbachia cannot be cultivated outside of its host cells, hindering traditional microbial characterization techniques. Furthermore, multiple Wolbachia strains can be present within a single host, and certain strains can be present in densities below the detection limit of current methods. To date, Wolbachia has most commonly been studied using polymerase chain reaction (PCR) amplification and Sanger DNA sequencing by targeting specific genes in the bacterium's genome. PCR amplification and Sanger sequencing of multiple Wolbachia strains requires analysis of individually cloned sequences, which is resource and labor intensive. To help mitigate these difficulties, we present a modified double digest restriction site associated DNA sequencing (ddRADseq) approach to target and sequence in parallel multiple genes by adding restriction enzyme recognition sites to gene-specific PCR primers. Adopting this strategy allows us to uniquely tag and sequence amplicons from multiple hosts simultaneously on an Illumina MiSeq platform. Our approach represents an efficient and cost-effective method to characterize multiple target genes in population surveys.

Understanding the diversification of biological lineages is central to evolutionary studies. To properly study the process of speciation, it is necessary to link micro-evolutionary studies with macro-evolutionary mechanisms. Micro-evolutionary studies require proper sampling across a taxon’s range to adequately infer genetic diversity. Here we use the grass frogs of the genus Ptychadena from the Ethiopian highlands as a model to study the process of lineage diversification in this unique biodiversity hotspot. We used thousands of genome-wide SNPs obtained from double digest restriction site associated DNA sequencing (ddRAD-seq) in populations of the Ptychadena neumanni species complex from the Ethiopian highlands in order to infer their phylogenetic relationships and genetic structure, as well as to study their demographic history. Our genome-wide phylogenetic study supports the existence of approximately 13 lineages clustered into 3 species groups. Our phylogenetic and phylogeographic reconstructions suggest that those endemic lineages diversified in allopatry, and subsequently specialized to different habitats and elevations. Demographic analyses point to a continuous decrease in the population size across the majority of lineages and populations during the Pleistocene, which is consistent with a continuous period of aridification that East Africa experienced since the Pliocene. We discuss the taxonomic implications of our analyses and, in particular, we warn against the recent practice to solely use Bayesian species delimitation methods when proposing taxonomic changes.

Transposable elements (TEs) - selfish DNA sequences that can move within the genome - comprise a large proportion of the genomes of many organisms. Although low-coverage whole-genome sequencing can be used to survey TE composition, it is noneconomical for species with large quantities of DNA. Here, we utilize restriction-site associated DNA sequencing (RADSeq) as an alternative method to survey TE composition. First, we demonstrate in silico that double digest restriction-site associated DNA sequencing (ddRADseq) markers contain the same TE compositions as whole genome assemblies across arthropods. Next, we show empirically using eight Synalpheus snapping shrimp species with large genomes that TE compositions from ddRADseq and low-coverage whole-genome sequencing are comparable within and across species. Finally, we develop a new bioinformatic pipeline, TERAD, to extract TE compositions from RADseq data. Our study expands the utility of RADseq to study the repeatome, making comparative studies of genome structure for species with large genomes more tractable and affordable.

Recent advances in high throughput sequencing have transformed the study of wild organisms by facilitating the generation of high quality genome assemblies and dense genetic marker datasets. These resources have the potential to significantly advance our understanding of diverse phenomena at the level of species, populations and individuals, ranging from patterns of synteny through rates of linkage disequilibrium (LD) decay and population structure to individual inbreeding. Consequently, we used PacBio sequencing to refine an existing Antarctic fur seal (Arctocephalus gazella) genome assembly and genotyped 83 individuals from six populations using restriction site associated DNA (RAD) sequencing. The resulting hybrid genome comprised 6,169 scaffolds with an N50 of 6.21 Mb and provided clear evidence for the conservation of large chromosomal segments between the fur seal and dog (Canis lupus familiaris). Focusing on the most extensively sampled population of South Georgia, we found that LD decayed rapidly, reaching the background level by around 400 kb, consistent with other vertebrates but at odds with the notion that fur seals experienced a strong historical bottleneck. We also found evidence for population structuring, with four main Antarctic island groups being resolved. Finally, appreciable variance in individual inbreeding could be detected, reflecting the strong polygyny and site fidelity of the species. Overall, our study contributes important resources for future genomic studies of fur seals and other pinnipeds while also providing a clear example of how high throughput sequencing can generate diverse biological insights at multiple levels of organization.

Exploring factors shaping genetic structure of marine fish is challenging due to fewer barriers to gene flow in the ocean. However, genome-wide sequence data can greatly enhance our ability to delineate previously unidentified population structure as well as potential adaptive divergence. The small yellow croaker (Larimichthys polyactis) is a commercially important fish species with high gene flow and its overwintering populations experience heterogeneous environment, suggesting possible population differentiation and adaptive divergence. To delineate patterns of population structure as well as test for signatures of local adaptation, a total of 68,666 quality filtered SNP markers were identified for 80 individuals from four overwintering populations by using restriction site-associated DNA sequencing (RAD-seq). Significant genetic differentiation among overwintering populations from the Central Yellow Sea, the South Yellow Sea and the North East China Sea were detected (Pair-wise FST: 0.00036–0.00390), which were consistent with population division of overwintering groups inferred from traditional ecological approaches. In addition, a total of 126 unique SNPs were detected to be significantly associated with environmental parameters (temperature, salinity and turbidity). These candidate SNPs were involved in multiple pathways such as energy metabolism and phagocytosis, suggesting they may play key roles in growth and innate immunity. Our results suggested the existence of hitherto unrecognized cryptic population structure and local adaptation in this high gene flow marine fish and thus gain new insights into the design of management strategies.

Japanese eel (Anguilla japonica) is an important food source in East Asia whose population has dramatically declined since the 1970s. Despite past analysis with DNA sequencing, microsatellite and isozyme methods, management decisions remain hampered by contradictory findings. For example, it remains unresolved whether Japanese eels are a single panmictic population or whether they harbor significant substructure. Accurate assessment of population genetic substructure, both spatial and temporal, is essential for determining the relevant number of distinct management units appropriate for this species. In the present study, we assayed genetic variation genome-wide using Restriction Site Associated DNA Sequencing (RAD-seq) technology to analyze the population genetic structure of Japanese eels. For analysis of temporal isolation, five “cohort” samples were collected yearly from 2005 to 2009 in the Yangtze River Estuary. For analysis of spatial structure, five “arrival wave” samples were collected in China in 2009, and two arrival wave samples were collected in Japan in 2001. In each cohort of each arrival wave, five individuals were collected for a total of 55 eels sampled. In total, 214,210 loci were identified from these individuals, 106,652 of which satisfied quality checks and were retained for further analysis. There was relatively little population differentiation between arrival waves and cohorts collected either at different locations during the same year (Fst = 0.077) or at the same location collected over subsequent years (Fst = 0.082), and locations displayed no consistent isolation-by-distance.

BackgroundThe eggplant (Solanum melongena L.) genome is relatively unexplored, especially compared to those of the other major Solanaceae crops tomato and potato. In particular, no SNP markers are publicly available; on the other hand, over 1,000 SSR markers were developed and publicly available. We have combined the recently developed Restriction-site Associated DNA (RAD) approach with Illumina DNA sequencing for rapid and mass discovery of both SNP and SSR markers for eggplant.ResultsRAD tags were generated from the genomic DNA of a pair of eggplant mapping parents, and sequenced to produce ~17.5 Mb of sequences arrangeable into ~78,000 contigs. The resulting non-redundant genomic sequence dataset consisted of ~45,000 sequences, of which ~29% were putative coding sequences and ~70% were in common between the mapping parents. The shared sequences allowed the discovery of ~10,000 SNPs and nearly 1,000 indels, equivalent to a SNP frequency of 0.8 per Kb and an indel frequency of 0.07 per Kb. Over 2,000 of the SNPs are likely to be mappable via the Illumina GoldenGate assay. A subset of 384 SNPs was used to successfully fingerprint a panel of eggplant germplasm, producing a set of informative diversity data. The RAD sequences also included nearly 2,000 putative SSRs, and primer pairs were designed to amplify 1,155 loci.ConclusionThe high throughput sequencing of the RAD tags allowed the discovery of a large number of DNA markers, which will prove useful for extending our current knowledge of the genome organization of eggplant, for assisting in marker-aided selection and for carrying out comparative genomic analyses within the Solanaceae family.

Craniosynostosis (CS) is a disorder that involves the premature ossification of one or more cranial sutures. Our research team has described a naturally occurring rabbit model of CS with a variable phenotype and unknown etiology. Restriction-site associated DNA (RAD) sequencing is a genomic sampling method for identifying genetic variants in species with little or no existing sequence data. RAD sequencing data was analyzed using a mixed linear model to identify single nucleotide polymorphisms (SNPs) associated with disease occurrence and onset in the rabbit model of CS. SNPs achieving a genome-wide significance of p ≤ 5 x 10−8 were identified on chromosome 2 in association with disease occurrence and on chromosomes 14 and 19 in association with disease onset. Genotyping identified a coding variant in fibroblast growth factor binding protein 1 (FGFBP-1) on chromosome 2 and a non-coding variant upstream of integrin alpha 3 (ITGA3) on chromosome 19 that associated with disease occurrence and onset, respectively. Retrospective analysis of patient data revealed a significant inverse correlation between FGFBP-1 and ITGA3 transcript levels in patients with coronal CS. FGFBP-1 and ITGA3 are genes with roles in early development that warrant functional study to further understand suture biology.

አህፅሮት ጤፍ በኢትዮጵያ የተገኘ እና በኢትዮጵያዊያን አርሶአደሮች ለብዙ አመታት ሲመረት የኖረ ዘርፈ ብዙ ጥቅሞች ያሉት የሰብል አይነት ነዉ፡፡ ጤፍ የሚመረተውና ጥቅም ላይ የሚውለው በአብዛኛው በኢትዮጵያ በመሆኑ በአለም አቀፍ ደረጃ ጤፍ ላይ የሚደረጉ ምርምሮች ውሱን ናቸው፡፡ በተለይ የጤፍን ምርምር ለማሳለጥ የተዘጋጁ የሞለኪውላር ሳይንስ ግብአቶች ውሱን በመሆናቸው በሌሎች ሰብሎች ላይ ጥቅም ላይ የዋሉ የዲ ኤን ኤ ሲኩዌንሲንግ ቴክኖሎጂን መሰረት ያደረጉ የምርምር አቅጣጫዎችን መከተል ወሳኝ ነው፡፡ እዚህ በቀረበው የምርምር ስራ የዲ ኤን ኤ ሲኩዌንሲንግ ቴክኖሎጂን መሰረት ያደረገን የምርምር ስልት በመከተል ከአገሪቱ የተለያዩ አካባቢዎች የተሰበሰቡ አርባ ሁለት የጤፍ ዝርያዎች፡ አንድ ሚውታንት ላይን እና ሁለት ለጤፍ ቅርብ የሆኑ ዋይልድ ሬላቲቭስ ላይ ጥናት ተደርጎ የጤፍን ምርምር በዘመናዊ መልኩ የሚያግዙ ግብአቶች ተገኝተዋል፡፡ Abstract Genome-wide knowledge about the nature and extent of genetic diversity present in tef (Eragrostis tef), the most consumed food grain in Ethiopia is limited. Adopting next generation sequencing (NGS) protocols to enhance its genomics and breeding is essential. Here, we applied the Restriction Site Associated DNA (RAD) sequencing protocol and surveyed the genomes of 43 tef landraces, one mutant line and two wild Eragrostis species. After mapping sequencing reads to the de novo assembled unitag and the tef reference genome, a total of 9,024 and 58,735 high quality single nucleotide polymorphisms (SNPs) were identified, respectively. We identified greater number of SNPs and greater nucleotide diversity in the two wild Eragrostis species than in the tef landraces. The tef landrace populations in this study were poorly differentiated with F ST values of 0.015. In the phylogenetic analysis, grouping of the landraces was not consistent with the area of collection, but few localized grouping of the landraces was evident, probably showing the communality of tef seed use across geographical boundaries. The improved tef varieties show reduced genetic diversity compared to the landraces and were all grouped into one cluster reflecting the nature of tef breeding which largely targets common genomic regions. We suggest that future work needs to aim beyond common genomic regions. The work presented here is a valuable addition to the growing molecular resources developed for tef genetic improvement.

Garcinia subelliptica Merr. (called “fukugi” in Japanese) is a dioecious tree species, which has traditionally been planted as windbreak forests in the coast of Ryukyu islands. Although effective afforestation and maintenance of G. subelliptica would greatly be assisted by early sex diagnosis and selection at the seedling stage, the genome and the sex-determination system of G. subelliptica are still poorly understood. In this study, we estimated the genome size of G. subelliptica to be approximately ~ 12 Gb for female and male, based on k-mer analysis. Furthermore, we developed a male-specific DNA marker for G. subelliptica, using the restriction site-associated DNA sequencing (RAD-seq) method. The 660 bp marker fragment, named FSDM (“fukugi” sex determination marker), was amplified in all males but not in the female G. subelliptica genome analyzed so far. In addition, the segregation ratio of the marker was 1:1 among the tested population. The DNA sequence of FSDM showed a significant similarity to the antifungal protein ginkbilobin-2 gene. The development of the sex-specific genetic marker of G. subelliptica may allow more effective conservation of traditional landscape and future design of villages around the northern part of the main island of Okinawa, a promising candidate site for World Natural Heritage.

Microsatellites have been a workhorse of evolutionary genetic studies for decades and are still commonly in use for estimating signatures of genetic diversity at the population and species level across a multitude of taxa. Yet, the very high mutation rate of these loci is a double-edged sword, conferring great sensitivity at shallow levels of analysis (e.g. paternity analysis) but yielding considerable uncertainty for deeper evolutionary comparisons. For the present study, we used reduced representation genome-wide data (restriction site-associated DNA sequencing (RADseq)) to test for patterns of interspecific hybridization previously characterized using microsatellite data in a contact zone between two closely related mouse lemur species in Madagascar (Microcebus murinus and Microcebus griseorufus). We revisit this system by examining populations in, near, and far from the contact zone, including many of the same individuals that had previously been identified as hybrids with microsatellite data. Surprisingly, we find no evidence for admixed nuclear ancestry. Instead, re-analyses of microsatellite data and simulations suggest that previously inferred hybrids were false positives and that the program NewHybrids can be particularly sensitive to erroneously inferring hybrid ancestry. Combined with results from coalescent-based analyses and evidence for local syntopic co-occurrence, we conclude that the two mouse lemur species are in fact completely reproductively isolated, thus providing a new understanding of the evolutionary rate whereby reproductive isolation can be achieved in a primate.

Burmese long-tailed macaque (Macaca fascicularis aurea) is the only Old World monkey that habitually uses stones as tools to access encased food. The natural range of M. f. aurea closely contacts to the common long-tailed macaque (M. f. fascicularis) in southern Thailand at 8°10'-12°24'N, where the intraspecific hybridization between the two subspecies was reported. While M. f. aurea x M. f. fascicularis hybrids were expressed a stone-tool use behavior, the behavior has never been seen in the wild or captive M. f. fascicularis. It is interesting to understand if the genetic factor plays a role on the emergence of this behavior. This study surveyed the populations of M. f. aurea, M. f. fascicularis and hybrids at the intraspecific hybrid zone and vicinity, identified morphological subspecies, observed/tested the stone-tool use behaviors, collected blood and fecal samples for genetic analyses, and genetic subspecies identification using mtDNA, Y-chromsome genes (SRY and TSPY), and autosomal single nucleotide polymorphism (SNP) derived from Restriction Site Associated DNA Sequencing (RADseq) as genetic markers. The findings in this study illustrated the influence of the early-middle Pleistocene transition (EMPT) on the split of M. f. aurea lineage through the southward migration from Myanmar/Bangladesh to southwestern Thailand. This study confirmed the previous hypothesis of the male-mediated intraspecific hybridization from M. f. aurea to M. f. fascicularis populations. In addition, the analysis of RADseq-derived SNPs indicated an asymmetrically (or unidirectionally) genetic introgression of M. f. aurea to M. f. fascicularis populations that occurred far beyond the previously reported intraspecific hybrid zone. The analysis of an association between the identified morphological subspecies or identified genetic subspecies and the stone-assisted behaviors, that were categorized into 4 levels; stone-tool use behavior, food pounding behavior, stone-play behavior and none, was performed. The statistically significant association between the morphologically identified M. f. aurea and hybrids and the complex stone-associated behaviors was detected, while it was no association between the identified genetic subspecies (based on mtDNA, SRY and TSPY) and the stone-associated behaviors. However, the SNP markers that indicate the degree of genetic admixture in hybrid populations can associate the high degree of M. f. aurea ancestry in M. fascicularis with stone-play and stone-tool use behaviors and the low degree of M. f. aurea ancestry in M. fascicularis with non-stone use behavior. Thus, these results imply the possibility of genetic influences on the emergence, the prevalence and the restriction of stone-tool use behavior in M. f. aurea and hybrids.