Chromosomal‐level genomes of three rice planthoppers provide new insights into sex chromosome evolution

The brown planthopper Nilaparvata lugens, white‐backed planthopper Sogatella furcifera, and small brown planthopper Laodelphax striatellus are three major insect pests of rice. They are genetically close; however, they differ in several ecological traits such as host range, migration capacity, and in their sex chromosomes. Though the draft genome of these three planthoppers have been previously released, the quality of genome assemblies need to be improved. The absence of chromosome‐level genome resources has hindered in‐depth research of these three species. Here, we performed a de novo genome assembly for N. lugens to increase its genome assembly quality with PacBio and Illumina platforms, increasing the contig N50 to 589.46 Kb. Then, with the new N. lugens genome and previously reported S. furcifera and L. striatellus genome assemblies, we generated chromosome‐level scaffold assemblies of these three planthopper species using HiC scaffolding technique. The scaffold N50s significantly increased to 77.63 Mb, 43.36 Mb and 29.24 Mb for N. lugens, S. furcifera and L. striatellus, respectively. To identify sex chromosomes of these three planthopper species, we carried out genome re‐sequencing of males and females and successfully determined the X and Y chromosomes for N. lugens, and X chromosome for S. furcifera and L. striatellus. The gene content of the sex chromosomes showed high diversity among these three planthoppers suggesting the rapid evolution of sex‐linked genes, and all chromosomes showed high synteny. The chromosome‐level genome assemblies of three planthoppers would provide a valuable resource for a broad range of future research in molecular ecology, and subsequently benefits development of modern pest control strategies.     

Finding Nemo’s Genes: A chromosome‐scale reference assembly of the genome of the orange clownfish Amphiprion percula

The iconic orange clownfish, Amphiprion percula, is a model organism for studying the ecology and evolution of reef fishes, including patterns of population connectivity, sex change, social organization, habitat selection and adaptation to climate change. Notably, the orange clownfish is the only reef fish for which a complete larval dispersal kernel has been established and was the first fish species for which it was demonstrated that antipredator responses of reef fishes could be impaired by ocean acidification. Despite its importance, molecular resources for this species remain scarce and until now it lacked a reference genome assembly. Here, we present a de novo chromosome‐scale assembly of the genome of the orange clownfish Amphiprion percula. We utilized single‐molecule real‐time sequencing technology from Pacific Biosciences to produce an initial polished assembly comprised of 1,414 contigs, with a contig N50 length of 1.86 Mb. Using Hi‐C‐based chromatin contact maps, 98% of the genome assembly were placed into 24 chromosomes, resulting in a final assembly of 908.8 Mb in length with contig and scaffold N50s of 3.12 and 38.4 Mb, respectively. This makes it one of the most contiguous and complete fish genome assemblies currently available. The genome was annotated with 26,597 protein‐coding genes and contains 96% of the core set of conserved actinopterygian orthologs. The availability of this reference genome assembly as a community resource will further strengthen the role of the orange clownfish as a model species for research on the ecology and evolution of reef fishes.     

Draft genome of the American Eel (Anguilla rostrata)

Freshwater eels (Anguilla sp.) have large economic, cultural, ecological and aesthetic importance worldwide, but they suffered more than 90% decline in global stocks over the past few decades. Proper genetic resources, such as sequenced, assembled and annotated genomes, are essential to help plan sustainable recoveries by identifying physiological, biochemical and genetic mechanisms that caused the declines or that may lead to recoveries. Here, we present the first sequenced genome of the American eel. This genome contained 305 043 contigs (N50 = 7397) and 79 209 scaffolds (N50 = 86 641) for a total size of 1.41 Gb, which is in the middle of the range of previous estimations for this species. In addition, protein‐coding regions, including introns and flanking regions, are very well represented in the genome, as 95.2% of the 458 core eukaryotic genes and 98.8% of the 248 ultra‐conserved subset were represented in the assembly and a total of 26 564 genes were annotated for future functional genomics studies. We performed a candidate gene analysis to compare three genes among all three freshwater eel species and, congruent with the phylogenetic relationships, Japanese eel (A. japanica) exhibited the most divergence. Overall, the sequenced genome presented in this study is a crucial addition to the presently available genetic tools to help guide future conservation efforts of freshwater eels.     

Chromosome‐level genome assembly of the predator Propylea japonica to understand its tolerance to insecticides and high temperatures

The ladybird beetle Propylea japonica is an important natural enemy in agro‐ecological systems. Studies on the strong tolerance of P. japonica to high temperatures and insecticides, and its population and phenotype diversity have recently increased. However, abundant genome resources for obtaining insights into stress‐resistance mechanisms and genetic intra‐species diversity for P. japonica are lacking. Here, we constructed the P. japonica genome maps using Pacific Bioscience (PacBio) and Illumina sequencing technologies. The genome size was 850.90 Mb with a contig N50 of 813.13 kb. The Hi‐C sequence data were used to upgrade draft genome assemblies; 4,777 contigs were assembled to 10 chromosomes; and the final draft genome assembly was 803.93 Mb with a contig N50 of 813.98 kb and a scaffold N50 of 100.34 Mb. Approximately 495.38 Mb of repeated sequences was annotated. The 18,018 protein‐coding genes were predicted, of which 95.78% were functionally annotated, and 1,407 genes were species‐specific. The phylogenetic analysis showed that P. japonica diverged from the ancestor of Anoplophora glabripennis and Tribolium castaneum ~ 236.21 million years ago. We detected that some important gene families involved in detoxification of pesticides and tolerance to heat stress were expanded in P. japonica, especially cytochrome P450 and Hsp70 genes. Overall, the high‐quality draft genome sequence of P. japonica will provide invaluable resource for understanding the molecular mechanisms of stress resistance and will facilitate the research on population genetics, evolution and phylogeny of Coccinellidae. This genome will also provide new avenues for conserving the diversity of predator insects.     

The pomegranate (Punica granatum L.) draft genome dissects genetic divergence between soft‐ and hard‐seeded cultivars

Complete and highly accurate reference genomes and gene annotations are indispensable for basic biological research and trait improvement of woody tree species. In this study, we integrated single‐molecule sequencing and high‐throughput chromosome conformation capture techniques to produce a high‐quality and long‐range contiguity chromosome‐scale genome assembly of the soft‐seeded pomegranate cultivar ‘Tunisia’. The genome covers 320.31 Mb (scaffold N50 = 39.96 Mb; contig N50 = 4.49 Mb) and includes 33 594 protein‐coding genes. We also resequenced 26 pomegranate varieties that varied regarding seed hardness. Comparative genomic analyses revealed many genetic differences between soft‐ and hard‐seeded pomegranate varieties. A set of selective loci containing SUC8‐like, SUC6, FoxO and MAPK were identified by the selective sweep analysis between hard‐ and soft‐seeded populations. An exceptionally large selective region (26.2 Mb) was identified on chromosome 1. Our assembled pomegranate genome is more complete than other currently available genome assemblies. Our results indicate that genomic variations and selective genes may have contributed to the genetic divergence between soft‐ and hard‐seeded pomegranate varieties.     

Improving Illumina assemblies with Hi‐C and long reads: An example with the North African dromedary

Researchers have assembled thousands of eukaryotic genomes using Illumina reads, but traditional mate‐pair libraries cannot span all repetitive elements, resulting in highly fragmented assemblies. However, both chromosome conformation capture techniques, such as Hi‐C and Dovetail Genomics Chicago libraries and long‐read sequencing, such as Pacific Biosciences and Oxford Nanopore, help span and resolve repetitive regions and therefore improve genome assemblies. One important livestock species of arid regions that does not have a high‐quality contiguous reference genome is the dromedary (Camelus dromedarius). Draft genomes exist but are highly fragmented, and a high‐quality reference genome is needed to understand adaptation to desert environments and artificial selection during domestication. Dromedaries are among the last livestock species to have been domesticated, and together with wild and domestic Bactrian camels, they are the only representatives of the Camelini tribe, which highlights their evolutionary significance. Here we describe our efforts to improve the North African dromedary genome. We used Chicago and Hi‐C sequencing libraries from Dovetail Genomics to resolve the order of previously assembled contigs, producing almost chromosome‐level scaffolds. Remaining gaps were filled with Pacific Biosciences long reads, and then scaffolds were comparatively mapped to chromosomes. Long reads added 99.32 Mbp to the total length of the new assembly. Dovetail Chicago and Hi‐C libraries increased the longest scaffold over 12‐fold, from 9.71 Mbp to 124.99 Mbp and the scaffold N50 over 50‐fold, from 1.48 Mbp to 75.02 Mbp. We demonstrate that Illumina de novo assemblies can be substantially upgraded by combining chromosome conformation capture and long‐read sequencing.     

SNP discovery using Paired‐End RAD‐tag sequencing on pooled genomic DNA of Sisymbrium austriacum (Brassicaceae)

Single nucleotide polymorphisms SNPs are rapidly replacing anonymous markers in population genomic studies, but their use in non model organisms is hampered by the scarcity of cost‐effective approaches to uncover genome‐wide variation in a comprehensive subset of individuals. The screening of one or only a few individuals induces ascertainment bias. To discover SNPs for a population genomic study of the Pyrenean rocket (Sisymbrium austriacum subsp. chrysanthum), we undertook a pooled RAD‐PE (Restriction site Associated DNA Paired‐End sequencing) approach. RAD tags were generated from the PstI‐digested pooled genomic DNA of 12 individuals sampled across the species distribution range and paired‐end sequenced using Illumina technology to produce ~24.5 Mb of sequences, covering ~7% of the specie's genome. Sequences were assembled into ~76 000 contigs with a mean length of 323 bp (N₅₀ = 357 bp, sequencing depth = 24x). In all, >15 000 SNPs were called, of which 47% were annotated in putative genic regions based on homology with the Arabidopsis thaliana genome. Gene ontology (GO) slim categorization demonstrated that the identified SNPs covered extant genic variation well. The validation of 300 SNPs on a larger set of individuals using a KASPar assay underpinned the utility of pooled RAD‐PE as an inexpensive genome‐wide SNP discovery technique (success rate: 87%). In addition to SNPs, we discovered >600 putative SSR markers.     

Identification and validation of single nucleotide polymorphisms as tools to detect hybridization and population structure in freshwater stingrays

Single nucleotide polymorphism (SNP) markers were identified and validated for two stingrays species, Potamotrygon motoro and Potamotrygon falkneri, using double digest restriction‐site associated DNA (ddRAD) reads using 454‐Roche technology. A total of 226 774 reads (65.5 Mb) were obtained (mean read length 289 ± 183 bp) detecting a total of 5399 contigs (mean contig length: 396 ± 91 bp). Mining this data set, a panel of 143 in silico SNPs was selected. Eighty‐two of these SNPs were successfully validated and 61 were polymorphic: 14 in P. falkneri, 21 in P. motoro, 3 in both species and 26 fixed for alternative variants in both species, thus being useful for population analyses and hybrid detection.     

Chromosome-level genome assembly of Scapharca kagoshimensis reveals the expanded molecular basis of heme biosynthesis in ark shells

Ark shells are commercially important clam species that inhabit in muddy sediments of shallow coasts in East Asia. For a long time, the lack of genome resources has hindered scientific research of ark shells. Here, we report a high-quality chromosome-level genome assembly of Scapharca kagoshimensis, with an aim to unravel the molecular basis of heme biosynthesis, and develop genomic resources for genetic breeding and population genetics in ark shells. Nineteen scaffolds corresponding to 19 chromosomes were constructed from 938 contigs (contig N50 = 2.01 Mb) to produce a final high-quality assembly with a total length of 1.11 Gb and scaffold N50 around 60.64 Mb. The genome assembly represents 93.4% completeness via matching 303 eukaryota core conserved genes. A total of 24,908 protein-coding genes were predicted and 24,551 genes (98.56%) of which were functionally annotated. The enrichment analyses suggested that genes in heme biosynthesis pathways were expanded and positive selection of the haemoglobin genes was also found in the genome of S. kagoshimensis, which gives important insights into the molecular mechanisms and evolution of the heme biosynthesis in mollusca. The valuable genome assembly of S. kagoshimensis would provide a solid foundation for investigating the molecular mechanisms that underlie the diverse biological functions and evolutionary adaptations of S. kagoshimensis.     

Design of Mini‐barcode for Catfishes for assessment of archival biodiversity

Recovery of DNA barcode sequences is often challenging from the archived specimens. However, short fragments of DNA may be recovered, which would significantly improve many unresolved taxonomic conflicts. Here, we designed a mini‐barcode for catfishes comprising several species and many cryptic taxa. We analysed a data set of 3048 publicly available COI barcode sequences representing 547 worldwide catfish species and performed 152 628 interspecies comparisons. A significantly more positively correlated interspecies distance was detected with transversion (0.78, P < 0.001) than with transition (0.70, P < 0.001). This suggested that transversions were better diagnostics for species identification. In the aligned data set, two transversion‐rich fragments (53 bp and 119 bp) were identified. Transition/transversion bias value was 1.04 in 53‐bp fragment, 1.23 in 119‐bp fragment and 1.50 in full‐length barcode. The interspecies distance with full‐length barcode was 0.212 ± 0.037, while that with 53‐bp and 119‐bp fragments was 0.325 ± 0.039 and 0.218 ± 0.045, respectively. Survey of 53‐bp fragment showed a possibility of only 1144 barcodes, while that of 119‐bp fragment showed >4 million barcodes. Thus, the 119‐bp fragment is a viable mini‐barcode for catfishes comprising >3000 extant species. Experiment with 82 archived catfishes showed successful recovery of this mini‐barcode using the designed primer. The mini‐barcode sequences showed species‐specific similarity in the range of 98‐100% with the global database. Therefore, survey of a transversion‐rich fragment within the full‐length barcode would be an ideal approach of mini‐barcode design for biodiversity assessment.     

Single‐nucleotide polymorphism discovery in Leptographium longiclavatum,a mountain pine beetle‐associated symbiotic fungus,using whole‐genome resequencing

Single‐nucleotide polymorphisms (SNPs) are rapidly becoming the standard markers in population genomics studies; however, their use in nonmodel organisms is limited due to the lack of cost‐effective approaches to uncover genome‐wide variation, and the large number of individuals needed in the screening process to reduce ascertainment bias. To discover SNPs for population genomics studies in the fungal symbionts of the mountain pine beetle (MPB), we developed a road map to discover SNPs and to produce a genotyping platform. We undertook a whole‐genome sequencing approach of Leptographium longiclavatum in combination with available genomics resources of another MPB symbiont, Grosmannia clavigera. We sequenced 71 individuals pooled into four groups using the Illumina sequencing technology. We generated between 27 and 30 million reads of 75 bp that resulted in a total of 1, 181 contigs longer than 2 kb and an assembled genome size of 28.9 Mb (N₅₀ = 48 kb, average depth = 125x). A total of 9052 proteins were annotated, and between 9531 and 17 266 SNPs were identified in the four pools. A subset of 206 genes (containing 574 SNPs, 11% false positives) was used to develop a genotyping platform for this species. Using this roadmap, we developed a genotyping assay with a total of 147 SNPs located in 121 genes using the Illumina^® Sequenom iPLEX Gold. Our preliminary genotyping (success rate = 85%) of 304 individuals from 36 populations supports the utility of this approach for population genomics studies in other MPB fungal symbionts and other fungal nonmodel species.     

The first complete mitochondrial genome sequence of Nanorana parkeri and Nanorana ventripunctata (Amphibia: Anura: Dicroglossidae), with related phylogenetic analyses

Members of the Nanorana genus (family Dicroglossidae) are often referred to as excellent model species with which to study amphibian adaptations to extreme environments and also as excellent keystone taxa for providing insights into the evolution of the Dicroglossidae. However, a complete mitochondrial genome is currently only available for Nanorana pleskei. Thus, we analyzed the complete mitochondrial genomes of Nanorana parkeri and Nanorana ventripunctata to investigate their evolutionary relationships within Nanorana and their phylogenetic position in the family Dicroglossidae. Our results showed that the genomes of N. parkeri (17,837 bp) and N. ventripunctata (18,373 bp) encode 13 protein‐coding genes (PCGs), two ribosomal RNA genes, 23 transfer RNA (tRNA) genes, and a noncoding control region. Overall sequences and genome structure of the two species showed high degree of similarity with N. pleskei, although the motif structures and repeat sequences of the putative control region showed clear differences among these three Nanorana species. In addition, a tandem repeat of the tRNA‐Met gene was found located between the tRNA‐Gln and ND2 genes. On both the 5′ and 3′‐sides, the control region possessed distinct repeat regions; however, the CSB‐2 motif was not found in N. pleskei. Based on the nucleotide sequences of 13 PCGs, our phylogenetic analyses, using Bayesian inference and maximum‐likelihood methods, illustrate the taxonomic status of Nanorana with robust support showing that N. ventripunctata and N. pleskei are more closely related than they are to N. parkeri. In conclusion, our analyses provide a more robust and reliable perspective on the evolutionary history of Dicroglossidae than earlier analyses, which used only a single species (N. pleskei).     

Identification of SNP markers for inferring phylogeny in temperate bamboos (Poaceae: Bambusoideae) using RAD sequencing

Phylogenetic relationships among temperate species of bamboo are difficult to resolve, owing to both the challenge of detecting sufficiently variable markers and their polyploid history. Here, we use restriction site–associated DNA sequencing to identify candidate loci with fixed allelic differences segregating between and within two temperate species of bamboos: Arundinaria faberi and Yushania brevipaniculata. Approximately 27 million paired‐end sequencing reads were generated across four samples. From pooled data, we assembled 67 685 and 70 668 de novo contigs from partial overlap among paired‐end reads, with an average length of 240 and 241 bp for the two species, respectively, which were used to investigate functional classification of RAD tags in a blastx search. Analysed separately by population, we recovered 29 443 putatively orthologous RAD tags shared across the four sampled populations, containing 28 023 sequence variants, of which c. 13 000 are segregating between species, and c. 3000 segregating between populations within each species. Analyses based on these RAD tags yielded robust phylogenetic inferences, even with data set constructed from surprisingly few loci. This study illustrates the potential for reduced‐representation genome data to resolve difficult phylogenetic relationships in temperate bamboos.     

Assessing fine-scale genetic structure and relatedness in the micro-endemic Florida bog frog

The Florida bog frog (Rana okaloosae) is restricted to approximately 25 seepage drainages on the Florida Panhandle, southeastern USA. We evaluated fine-scale (<1–10 km) genetic structure among 80 samples from a long-term study area in one portion of its range. We also included co-distributed samples (N = 48) from bronze frogs (R. clamitans). Individual R. okaloosae were significantly more related to one another than expected under panmixia, though significant patterns of isolation-by-distance were detected reflecting limited dispersal. Bayesian clustering failed to identify discrete genetic clusters within species. Subtle, though important differences in genetic structuring between R. okaloosae and R. clamitans suggests that future efforts to predict the impact of landscape changes on Rana okaloosae, should focus on the species itself rather than R. clamitans as a surrogate species.