Genome assembly and annotation at the chromosomal level of first Pleuronectidae: Verasper variegatus provides a basis for phylogenetic study of Pleuronectiformes

High quality genome is of great significance for the mining of biological information resources of species. Up to now, the genomic information of several important economic flatfishes has been well explained. All these fishes are eyes on left side-type, and no high-quality genome of eyes on right side-type species has been reported. In this study, we applied a combined strategy involving stLFR and Hi-C technologies to generate sequencing data for constructing the chromosomal genome of Verasper variegates, which belongs to Pleuronectidae with characteristic of eyes on right side. The size of genome of V. variegatus is 556 Mb. More than 97.2% of BUSCO genes were detected, and N50 lengths of the contigs and scaffolds reached 79.8 Kb and 23.8 Mb, respectively, demonstrating the outstanding completeness and sequence continuity of the genome. A total of 22,199 protein-coding genes were predicted in the assembled genome, and more than 95% of those genes could be functionally annotated. Meanwhile, the genomic collinearity, gene family and phylogenetic analyses of similar species in Pleuronectiformes were also investigated and portrayed for metamorphosis and benthic adaptation. Sex related genes mapping has also been achieved at the chromosome level. This study is the first chromosomal level genome of a Pleuronectidae fish (V. variegatus). The chromosomal genome assembly constructed in this work will not only be valuable for conservation and aquaculture studies of the V. variegatus but will also be of general interest in the phylogenetic and taxonomic studies of Pleuronectiformes.     

De novo genome assembly of the bioluminescent mushroom Omphalotus guepiniiformis reveals an Omphalotus-specific lineage of the luciferase gene block

Omphalotus guepiniiformis, a bioluminescent mushroom species, is a source of the potentially valuable anticancer chemical. To provide genome information, we de novo assembled the high-quality O. guepiniiformis genome using two Next-Generation sequencing techniques, PacBio and Illumina sequencing. Our draft O. guepiniiformis genome comprises 42.5 Mbp of sequence with only 80 contigs and an N50 sequence length of over 1 Mbp. There were 15,554 predicted coding genes, and 7693 genes were functionally annotated with Gene Ontology terms. We performed a genomic study focusing on mushroom bioluminescent pathway cluster genes by comparing 17 luminescent and 23 non-luminescent Agaricales species belonging to 23 genera. Synteny analysis of genomic regions near the luminescent pathway cluster genes inferred that the Omphalotus lineage was genus-specific. In summary, our de novo assembled O. guepiniiformis genome provides significant biological insights into this organism, including the evolution of the luciferase gene block, and forms the basis for future analyses.     

The complete plastid genome of Cotinus coggygria and phylogenetic analysis of the Anacardiaceae

Cotinus coggygria Scop. (Anacardiaceae) is an important ornamental tree with beautiful characteristics that is grown in China. In this study, the complete plastid genome of C. coggygria was sequenced and assembled. This genome was 158,843 bp in size and presented a typical tetrad structure, consisting of a large single-copy region (87,121 bp), a pair of inverted repeat regions (26,829 bp), and a small single-copy region (18,064 bp). A total of 134 genes were annotated, including 88 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. We observed a deletion that caused the loss of the rpl32 gene, and a small expansion of IR regions resulted in the trnH gene accessing IR regions; two copies were obtained. Phylogenetic analysis showed that C. coggygria was most closely related to Pistacia, with 100% bootstrap support within Anacardiaceae. In this study, we report the plastid genome of Cotinus species for the first time, which provides insight into the evolution of the plastid genome in Anacardiaceae and promotes the understanding of Cotinus plants.     

Chromosome-level genome assembly of a xerophytic plant,Haloxylon ammodendron

Haloxylon ammodendron is a xerophytic perennial shrub or small tree that has a high ecological value in anti-desertification due to its high tolerance to drought and salt stress. Here, we report a high-quality, chromosome-level genome assembly of H. ammodendron by integrating PacBio’s high-fidelity sequencing and Hi-C technology. The assembled genome size was 685.4 Mb, of which 99.6% was assigned to nine pseudochromosomes with a contig N50 value of 23.6 Mb. Evolutionary analysis showed that both the recent substantial amplification of long terminal repeat retrotransposons and tandem gene duplication may have contributed to its genome size expansion and arid adaptation. An ample amount of low-GC genes was closely related to functions that may contribute to the desert adaptation of H. ammodendron. Gene family clustering together with gene expression analysis identified differentially expressed genes that may play important roles in the direct response of H. ammodendron to water-deficit stress. We also identified several genes possibly related to the degraded scaly leaves and well-developed root system of H. ammodendron. The reference-level genome assembly presented here will provide a valuable genomic resource for studying the genome evolution of xerophytic plants, as well as for further genetic breeding studies of H. ammodendron.     

Genome analysis of Candida subhashii reveals its hybrid nature and dual mitochondrial genome conformations

Candida subhashii belongs to the CUG-Ser clade, a group of phylogenetically closely related yeast species that includes some human opportunistic pathogens, such as Candida albicans. Despite being present in the environment, C. subhashii was initially described as the causative agent of a case of peritonitis. Considering the relevance of whole-genome sequencing and analysis for our understanding of genome evolution and pathogenicity, we sequenced, assembled and annotated the genome of C. subhashii type strain. Our results show that C. subhashii presents a highly heterozygous genome and other signatures that point to a hybrid ancestry. The presence of functional pathways for assimilation of hydroxyaromatic compounds goes in line with the affiliation of this yeast with soil microbial communities involved in lignin decomposition. Furthermore, we observed that different clones of this strain may present circular or linear mitochondrial DNA. Re-sequencing and comparison of strains with differential mitochondrial genome topology revealed five candidate genes potentially associated with this conformational change: MSK1, SSZ1, ALG5, MRPL9 and OYE32.     

Comprehensive Draft Genome Analyses of Three Rockfishes (Scorpaeniformes,Sebastiscus) via Genome Survey Sequencing

Sebastiscus species, marine rockfishes, are of essential economic value. However, the genomic data of this genus is lacking and incomplete. Here, whole genome sequencing of all species of Sebastiscus was conducted to provide fundamental genomic information. The genome sizes were estimated to be 802.49 Mb (S. albofasciatus), 786.79 Mb (S. tertius), and 776.00 Mb (S. marmoratus) by using k-mer analyses. The draft genome sequences were initially assembled, and genome-wide microsatellite motifs were identified. The heterozygosity, repeat ratios, and numbers of microsatellite motifs all suggested possibly that S. tertius is more closely related to S. albofasciatus than S. marmoratus at the genetic level. Moreover, the complete mitochondrial genome sequences were assembled from the whole genome data and the phylogenetic analyses genetically supported the validation of Sebastiscus species. This study provides an important genome resource for further studies of Sebastiscus species.     

High-quality genome assembly of Pseudocercospora ulei the main threat to natural rubber trees

Pseudocercospora ulei is the causal agent of South American Leaf Blight (SALB), the main disease affecting Hevea brasiliensis rubber tree, a native species to the Amazon. Rubber tree is a major crop in South American countries and SALB disease control strategies would benefit from the availability of genomic resources for the fungal pathogen. Here, we assembled and annotated the P. ulei genome. Shotgun sequencing was performed using second and third generation sequencing technologies. We present the first P. ulei high-quality genome assembly, the largest among Mycosphaerellaceae, with 93.8 Mbp, comprising 215 scaffolds, an N50 of 2.8 Mbp and a BUSCO gene completeness of 97.5%. We identified 12,745 protein-coding gene models in the P. ulei genome with 756 genes encoding secreted proteins and 113 genes encoding effector candidates. Most of the genome (80%) is composed of repetitive elements dominated by retrotransposons of the Gypsy superfamily. P. ulei has the largest genome size among Mycosphaerellaceae, with the highest TE content. In conclusion, we have established essential genomic resources for a wide range of studies on P. ulei and related species.     

Chromosome-level genome assembly of Japanese chestnut (Castanea crenata Sieb. et Zucc.) reveals conserved chromosomal segments in woody rosids

Japanese chestnut (Castanea crenata Sieb. et Zucc.), unlike other Castanea species, is resistant to most diseases and wasps. However, genomic data of Japanese chestnut that could be used to determine its biotic stress resistance mechanisms have not been reported to date. In this study, we employed long-read sequencing and genetic mapping to generate genome sequences of Japanese chestnut at the chromosome level. Long reads (47.7 Gb; 71.6× genome coverage) were assembled into 781 contigs, with a total length of 721.2 Mb and a contig N50 length of 1.6 Mb. Genome sequences were anchored to the chestnut genetic map, comprising 14,973 single nucleotide polymorphisms (SNPs) and covering 1,807.8 cM map distance, to establish a chromosome-level genome assembly (683.8 Mb), with 69,980 potential protein-encoding genes and 425.5 Mb repetitive sequences. Furthermore, comparative genome structure analysis revealed that Japanese chestnut shares conserved chromosomal segments with woody plants, but not with herbaceous plants, of rosids. Overall, the genome sequence data of Japanese chestnut generated in this study is expected to enhance not only its genetics and genomics but also the evolutionary genomics of woody rosids.     

Genome-Wide Analysis of Mitogen-Activated Protein Kinase Gene Family in Maize

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in plants. As the last component of the MAPK cascade (MAPKKK–MAPKK–MAPK), MAPK plays important roles in linking upstream kinases and downstream substrates. The MAPK proteins belong to a complex gene family in plants, with 20 MAPK genes in the Arabidopsis genome, 17 in the rice genome, and 21 in the poplar genome. Although the maize genome sequencing has been completed, no comprehensive study has been reported thus far for the MAPK gene family in maize. In this study, we identified 19 MAPK genes in maize. These ZmMPK genes belong to four groups (A–D) found in other plants. The phylogeny, chromosomal location, gene structure, and the functional relevancy of ZmMPK genes were analyzed. Moreover, we discuss the evolutionary divergence of MAPK genes in maize. Furthermore, we analyzed the expression profiles of ZmMPKs using the public microarray data and performed expression analyses in maize seedlings and adult plants. The data obtained from our study contribute to a better understanding of the complexity of MAPKs in plants and provide a useful reference for further functional analysis of MAPK genes in maize.     

A high quality draft consensus sequence of the genome of a heterozygous grapevine variety

Background

Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.

Principal Findings

We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).

Conclusions

Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.     

The genome of the Paleogene relic tree Bretschneidera sinensis: insights into trade-offs in gene family evolution,demographic history,and adaptive SNPs

Among relic species, genomic information may provide the key to inferring their long-term survival. Therefore, in this study, we investigated the genome of the Paleogene relic tree species, Bretschneidera sinensis, which is a rare endemic species within southeastern Asia. Specifically, we assembled a high-quality genome for B. sinensis using PacBio high-fidelity and high-throughput chromosome conformation capture reads and annotated it with long and short RNA sequencing reads. Using the genome, we then detected a trade-off between active and passive disease defences among the gene families. Gene families involved in salicylic acid and MAPK signalling pathways expanded as active defence mechanisms against disease, but families involved in terpene synthase activity as passive defences contracted. When inferring the long evolutionary history of B. sinensis, we detected population declines corresponding to historical climate change around the Eocene–Oligocene transition and to climatic fluctuations in the Quaternary. Additionally, based on this genome, we identified 388 single nucleotide polymorphisms (SNPs) that were likely under selection, and showed diverse functions in growth and stress responses. Among them, we further found 41 climate-associated SNPs. The genome of B. sinensis and the SNP dataset will be important resources for understanding extinction/diversification processes using comparative genomics in different lineages.     

Nuclear DNA content of Pongamia pinnata L. and genome size stability of in vitro-regenerated plantlets

Pongamia pinnata L. is a multipurpose versatile legume that is well known as a prospective feedstock biodiesel species. However, to date, there has been little genomic research aimed at the exploitation of the biotechnological potential of this species. Genetic characterization of any plant is a challenging task when there is no information about the genome size and organization of the species. Therefore, the genome size of P. pinnata was estimated by flow cytometry with respect to two standards (Zea mays and Pisum sativum), and compared with that of in vitro-raised plants (nodal segment, in vitro-rooted plantlets and acclimatized in vitro plants) to study the potential effect of somaclonal variation on genome size. This method can be used to support the establishment of true-to-type plants to encourage afforestation programs. Modified propidium iodide/hypotonic citrate buffer was used for isolation of the intact nuclei. The 2C DNA value of this species was estimated to be 2.51?±?0.01 pg. Statistically, there was no significant difference in the DNA content of the in vitro-grown plants and mother plant at α?=?0.05. As a result of the low genome size of P. pinnata, a species that has adapted itself to a wide range of edaphic and ecological condition, we can now proceed for its next generation sequencing and genomic diversity studies.     

Long-read sequence assembly: a technical evaluation in barley

Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.

A greatly improved reference genome sequence of barley was assembled from accurate long reads.     

The Genome Sequence of the Fungal Pathogen Fusarium virguliforme That Causes Sudden Death Syndrome in Soybean

Fusarium virguliforme causes sudden death syndrome (SDS) of soybean, a disease of serious concern throughout most of the soybean producing regions of the world. Despite the global importance, little is known about the pathogenesis mechanisms of F. virguliforme. Thus, we applied Next-Generation DNA Sequencing to reveal the draft F. virguliforme genome sequence and identified putative pathogenicity genes to facilitate discovering the mechanisms used by the pathogen to cause this disease.

Methodology/Principal Findings

We have generated the draft genome sequence of F. virguliforme by conducting whole-genome shotgun sequencing on a 454 GS-FLX Titanium sequencer. Initially, single-end reads of a 400-bp shotgun library were assembled using the PCAP program. Paired end sequences from 3 and 20 Kb DNA fragments and approximately 100 Kb inserts of 1,400 BAC clones were used to generate the assembled genome. The assembled genome sequence was 51 Mb. The N50 scaffold number was 11 with an N50 Scaffold length of 1,263 Kb. The AUGUSTUS gene prediction program predicted 14,845 putative genes, which were annotated with Pfam and GO databases. Gene distributions were uniform in all but one of the major scaffolds. Phylogenic analyses revealed that F. virguliforme was closely related to the pea pathogen, Nectria haematococca. Of the 14,845 F. virguliforme genes, 11,043 were conserved among five Fusarium species: F. virguliforme, F. graminearum, F. verticillioides, F. oxysporum and N. haematococca; and 1,332 F. virguliforme-specific genes, which may include pathogenicity genes. Additionally, searches for candidate F. virguliforme pathogenicity genes using gene sequences of the pathogen-host interaction database identified 358 genes.

Conclusions

The F. virguliforme genome sequence and putative pathogenicity genes presented here will facilitate identification of pathogenicity mechanisms involved in SDS development. Together, these resources will expedite our efforts towards discovering pathogenicity mechanisms in F. virguliforme. This will ultimately lead to improvement of SDS resistance in soybean.