TrnL-trnF sequence data imply paraphyly of Aquilaria and Gyrinops (Thymelaeaceae) and provide new perspectives for agarwood identification

The genera Aquilaria and Gyrinops (Thymelaeaceae, Malvales) are well known for the production of agarwood which is a highly wanted forest product of substantial economic value. The taxonomic status of Aquilaria and Gyrinops as separate genera is doubted as they are only distinguished by the number of stamens. We investigated their status by conducting phylogenetic analyses of DNA sequences from the plastid trnL-trnF spacer. Control of international trade of agarwood is currently hampered by the failure of traditional methods such as microscopy to identify samples to species level. We therefore evaluated the potential of molecular identification of agarwood by searching for species- and region-specific plastid DNA polymorphisms. DNA sequences were obtained from 31 Thymelaeaceae accessions encompassing 20 different species in six genera. Aquilaria and Gyrinops appear to be paraphyletic. Success in sequencing wood samples demonstrates that molecular markers provide new perspectives for agarwood identification.     

六种沉香属植物叶片解剖结构研究

为比较沉香属不同种植物间的叶片形态解剖特征,将不同来源的六种沉香属植物在海南省兴隆南药园种植,运用石蜡切片法和撕片法对其成熟叶片的解剖特征进行观察,并对叶片的上下表皮,叶脉和叶横切面等12项数量性状进行统计分析。结果表明:六种沉香属植物叶片解剖结构基本一致,均为典型的异面叶,由表皮、叶肉和叶脉组成,表现出典型的旱生形态特点。表皮细胞单层,气孔微下陷,仅分布在下表皮,上下表皮上零星分布着表皮毛。叶肉组织发达,栅栏组织由1~2层排列紧密地圆柱状细胞组成,其间分布着大量的长方晶体,海绵组织内有一层排列较整齐,染色较深的异细胞组成的下皮层。主脉维管束双韧型,呈圆环状,内含大量异细胞。方差分析表明,除栅海比外,叶片厚度、叶脉条数、主脉厚度等其余11项数量指标在六种植物间差异均达到显著水平。聚类分析将这六种植物聚成3类,Aquilaria sinensis(白木香),A.crassna和A.banaensis聚为一类,A.baillonii和A.malaccensis聚为一类;A.yunnanensis(云南沉香)单独为一类。该研究结果为沉香属植物的物种鉴定提供了解剖学依据,同时对沉香属植物合理开发利用具有重要意义。     

Profiling of MicroRNAs under Wound Treatment in Aquilaria sinensis to Identify Possible MicroRNAs Involved in Agarwood Formation

Agarwood, a kind of highly valued non-timber product across Asia, is formed only when its resource trees -- the endangered genus Aquilaria are wounded or infected by some microbes. To promote the efficiency of agarwood production and protect the wild resource of Aquilaria species, we urgently need to reveal the regulation mechanism of agarwood formation. MicroRNAs (miRNAs) are a group of gene expression regulators with overwhelming effects on a large spectrum of biological processes. However, their roles in agarwood formation remain unknown. This work aimed at identifying possible miRNAs involved in the wound induced agarwood formation. In this study, the high-throughput sequencing was adopted to identify miRNAs and monitor their expression under wound treatment in the stems of A. sinensis. The miR171, miR390, miR394, miR2111, and miR3954 families remained at the reduced level two days after the treatment. 131 homologous miRNAs in the 0.5 h library showed over three-fold variation of read number compared with the control library, of which 12 exhibiting strong expression alterations were further confirmed by real-time quantitative PCR. Target prediction and annotation of the miRNAs demonstrated that the binding, metabolic process, catalytic activity, and cellular process are the most common functions of the predicted targets of these newly identified miRNAs in A.sinensis. The cleaveage sites of three newly predicted targets were verified by 5''RACE.     

A review of chemistry,quality and analysis of infected agarwood tree (<Emphasis Type="Italic">Aquilaria</Emphasis> sp.)

Agarwood is a valuable oleoresin obtained from wounded tree of various genus of Thymelaeaceae family especially from Aquilaria species. Agarwood is regarded as most treasured non-timber forest product used in fragrances as well as medicines. Quality of agarwood plays an important role to define its commercial value. Different countries use different grading pattern to define the quality of agarwood. More than 250 compounds have been identified so far, mostly sesquiterpenoids, chromones and volatile aromatic compounds. GC–MS is the considered as a best method for the identification of chemical profile of the agarwood by various authors. Present review discusses the various methods used to determine quality of agarwood and chemical constituents of infected agarwood tree.     

Artificial production of agarwood oil in <Emphasis Type="Italic">Aquilaria</Emphasis> sp. by fungi: a review

The resinous part called agarwood of genus Aquilaria Lam has been exploited as a source of incense by mankind since time immemorial besides its medicinal properties. In global market purest oud/agarwood oil enjoys an exorbitant price tag owing to its mystical fragrance. This has led to over-exploitation of Aquilaria sp. at worldwide level. All the Aquilaria sp. are protected under CITES since 2002. Agarwood production is considered to have a pathological relation, secreted actively in response to foreign body infection as a means of defense mechanism in Aquilaria sp. In order to fulfill the demand for sustainable production of agarwood, chemical, physical and biological methods of inducing artificial infection for agarwood enhancement/production have been formulated. In addition, artificial inoculation of fungi has been used to induce agarwood in Aquilaria trees. In this review, we have discussed the success and limitations of various methods employed for agarwood stimulation and provided the analysis of investigation conducted by various researchers to ascertain the contributory role of fungi (endophytic–pathogenic–epiphytic) on agar formation and chemical composition of oil from artificially infected agarwood. Based on available literature, Xylaria sp., Lasiodiplodia sp., Colletotrichum sp. and Botryosphaeria sp. are promising fungal isolates which deserved to be further studied and scaled up to a commercial level for production of agarwood oil.     

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.     

Hydrogen peroxide induces vessel occlusions and stimulates sesquiterpenes accumulation in stems of Aquilaria sinensis

Agarwood is highly valuable resinous and fragrant heartwood, produced principally from tropical tree species in the genus Aquilaria, which is used widely in countries of the Middle East, Southeast Asia and Japan. Generally, healthy trees will not produce agarwood, but wounding of the tree initiates the production of agarwood. In this study, the pruning of actively growing saplings of Aquilaria sinensis resulted in hydrogen peroxide (H₂O₂) burst, which was followed by formation of vessel occlusions and sesquiterpene biosynthesis in the pruned stems. Treatment of the pruned stems with scavenger of H₂O₂ (ascorbate, AsA) greatly reduced the amount of H₂O₂ released, the number of vessel occlusions, and the amount of sesquiterpenes produced. In addition, exogenous H₂O₂ also induced A. sinensis plants to form vessel occlusions and produce sesquiterpenes as pruning treatment. The results indicated that H₂O₂ may be an important post-wounding signal in A. sinensis that leads to the induction of vessel occlusions formation and sesquiterpene biosynthesis, and thus H₂O₂ might play a vital role in agarwood formation in pruned stems of A. sinensis.     

High‐contiguity genome assembly of the chemosynthetic gammaproteobacterial endosymbiont of the cold seep tubeworm Lamellibrachia barhami

Symbiotic relationships between vestimentiferan tubeworms and chemosynthetic Gammaproteobacteria build the foundations of many hydrothermal vent and hydrocarbon seep ecosystems in the deep sea. The association between the vent tubeworm Riftia pachyptila and its endosymbiont Candidatus Endoriftia persephone has become a model system for symbiosis research in deep‐sea vestimentiferans, while markedly fewer studies have investigated symbiotic relationships in other tubeworm species, especially at cold seeps. Here we sequenced the endosymbiont genome of the tubeworm Lamellibrachia barhami from a cold seep in the Gulf of California, using short‐ and long‐read sequencing technologies in combination with Hi‐C and Dovetail Chicago libraries. Our final assembly had a size of ~4.17 MB, a GC content of 54.54%, 137X coverage, 4153 coding sequences, and a CheckM completeness score of 97.19%. A single scaffold contained 99.51% of the genome. Comparative genomic analyses indicated that the L. barhami symbiont shares a set of core genes and many metabolic pathways with other vestimentiferan symbionts, while containing 433 unique gene clusters that comprised a variety of transposases, defence‐related genes and a lineage‐specific CRISPR/Cas3 system. This assembly represents the most contiguous tubeworm symbiont genome resource to date and will be particularly valuable for future comparative genomic studies investigating structural genome evolution, physiological adaptations and host‐symbiont communication in chemosynthetic animal‐microbe symbioses.     

Chemical constituents from the petioles and leaves of Aquilaria sinensis

Nineteen reported compounds (1–19) were obtained from the dried petioles and leaves of Aquilaria sinensis (Thymelaeacea) by phytochemical methods. Their structures were determined on the basis of spectroscopic methods. Among them, compounds 6–14 and 16 were firstly obtained from the genus Aquilaria, 2 and 3 were obtained from A. sinensis for the first time. In addition, the chemotaxonomic relationships between A. sinensis and some other plants were also discussed.     

Chromosome rearrangements during domestication of cucumber as revealed by high-density genetic mapping and draft genome assembly

Cucumber, Cucumis sativus L. is the only taxon with 2n = 2x = 14 chromosomes in the genus Cucumis. It consists of two cross‐compatible botanical varieties: the cultivated C. sativus var. sativus and the wild C. sativus var. hardwickii. There is no consensus on the evolutionary relationship between the two taxa. Whole‐genome sequencing of the cucumber genome provides a new opportunity to advance our understanding of chromosome evolution and the domestication history of cucumber. In this study, a high‐density genetic map for cultivated cucumber was developed that contained 735 marker loci in seven linkage groups spanning 707.8 cM. Integration of genetic and physical maps resulted in a chromosome‐level draft genome assembly comprising 193 Mbp, or 53% of the 367 Mbp cucumber genome. Strategically selected markers from the genetic map and draft genome assembly were employed to screen for fosmid clones for use as probes in comparative fluorescence in situ hybridization analysis of pachytene chromosomes to investigate genetic differentiation between wild and cultivated cucumbers. Significant differences in the amount and distribution of heterochromatins, as well as chromosomal rearrangements, were uncovered between the two taxa. In particular, six inversions, five paracentric and one pericentric, were revealed in chromosomes 4, 5 and 7. Comparison of the order of fosmid loci on chromosome 7 of cultivated and wild cucumbers, and the syntenic melon chromosome I suggested that the paracentric inversion in this chromosome occurred during domestication of cucumber. The results support the sub‐species status of these two cucumber taxa, and suggest that C. sativus var. hardwickii is the progenitor of cultivated cucumber.     

A chromosome‐level genome assembly of the parasitoid wasp Pteromalus puparum

Parasitoid wasps represent a large proportion of hymenopteran species. They have complex evolutionary histories and are important biocontrol agents. To advance parasitoid research, a combination of Illumina short‐read, PacBio long‐read and Hi‐C scaffolding technologies was used to develop a high‐quality chromosome‐level genome assembly for Pteromalus puparum, which is an important pupal endoparasitoid of caterpillar pests. The chromosome‐level assembly has aided in studies of venom and detoxification genes. The assembled genome size is 338 Mb with a contig N50 of 38.7 kb and a scaffold N50 of 1.16 Mb. Hi‐C analysis assembled scaffolds onto five chromosomes and raised the scaffold N50 to 65.8 Mb, with more than 96% of assembled bases located on chromosomes. Gene annotation was assisted by RNA sequencing for the two sexes and four different life stages. Analysis detected 98% of the BUSCO (Benchmarking Universal Single‐Copy Orthologs) gene set, supporting a high‐quality assembly and annotation. In total, 40.1% (135.6 Mb) of the assembly is composed of repetitive sequences, and 14,946 protein‐coding genes were identified. Although venom genes play important roles in parasitoid biology, their spatial distribution on chromosomes was poorly understood. Mapping has revealed venom gene tandem arrays for serine proteases, pancreatic lipase‐related proteins and kynurenine–oxoglutarate transaminases, which have amplified in the P. puparum lineage after divergence from its common ancestor with Nasonia vitripennis. In addition, there is a large expansion of P450 genes in P. puparum. These examples illustrate how chromosome‐level genome assembly can provide a valuable resource for molecular, evolutionary and biocontrol studies of parasitoid wasps.     

Sesquiterpenoids in two different kinds of agarwood

Sesquiterpenoids of an agarwood originating from Aquilaria agallocha and of the other kind of agarwood (Aquilaria sp.; probably Aquilaria malaccensis) were investigated by a combination of GLC and GC/MS. The differences in sesquiterpene composition between the two kinds of agarwood are discussed.     

An improved genome reference for the African cichlid,Metriaclima zebra

Background

Problems associated with using draft genome assemblies are well documented and have become more pronounced with the use of short read data for de novo genome assembly. We set out to improve the draft genome assembly of the African cichlid fish, Metriaclima zebra, using a set of Pacific Biosciences SMRT sequencing reads corresponding to 16.5× coverage of the genome. Here we characterize the improvements that these long reads allowed us to make to the state-of-the-art draft genome previously assembled from short read data.

Results

Our new assembly closed 68 % of the existing gaps and added 90.6Mbp of new non-gap sequence to the existing draft assembly of M. zebra. Comparison of the new assembly to the sequence of several bacterial artificial chromosome clones confirmed the accuracy of the new assembly. The closure of sequence gaps revealed thousands of new exons, allowing significant improvement in gene models. We corrected one known misassembly, and identified and fixed other likely misassemblies. 63.5 Mbp (70 %) of the new sequence was classified as repetitive and the new sequence allowed for the assembly of many more transposable elements.

Conclusions

Our improvements to the M. zebra draft genome suggest that a reasonable investment in long reads could greatly improve many comparable vertebrate draft genome assemblies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1930-5) contains supplementary material, which is available to authorized users.     

Epigenetic patterns within the haplotype phased fig (Ficus carica L.) genome

Due to DNA heterozygosity and repeat content, assembly of non‐model plant genomes is challenging. Herein, we report a high‐quality genome reference of one of the oldest known domesticated species, fig (Ficus carica L.), using Pacific Biosciences single‐molecule, real‐time sequencing. The fig genome is ~333 Mbp in size, of which 80% has been anchored to 13 chromosomes. Genome‐wide analysis of N⁶‐methyladenine and N⁴‐methylcytosine revealed high methylation levels in both genes and transposable elements, and a prevalence of methylated over non‐methylated genes. Furthermore, the characterization of N⁶‐methyladenine sites led to the identification of ANHGA, a species‐specific motif, which is prevalent for both genes and transposable elements. Finally, exploiting the contiguity of the 13 pseudomolecules, we identified 13 putative centromeric regions. The high‐quality reference genome and the characterization of methylation profiles, provides an important resource for both fig breeding and for fundamental research into the relationship between epigenetic changes and phenotype, using fig as a model species.     

The genome of cowpea (Vigna unguiculata [L.] Walp.)

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub‐Saharan Africa, that is resilient to hot and drought‐prone environments. An assembly of the single‐haplotype inbred genome of cowpea IT97K‐499‐35 was developed by exploiting the synergies between single‐molecule real‐time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination‐poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high‐recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS‐LRR and the SAUR‐like auxin superfamilies compared with other warm‐season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.     

Comparison of the mesophilic cellulosome‐producing Clostridium cellulovorans genome with other cellulosome‐related clostridial genomes

Clostridium cellulovorans, an anaerobic and mesophilic bacterium, degrades native substrates in soft biomass such as corn fibre and rice straw efficiently by producing an extracellular enzyme complex called the cellulosome. Recently, we have reported the whole‐genome sequence of C. cellulovorans comprising 4220 predicted genes in 5.10 Mbp [Y. Tamaru et al., (2010) J. Bacteriol., 192: 901–902]. As a result, the genome size of C. cellulovorans was about 1 Mbp larger than that of other cellulosome‐producing clostridia, mesophilic C. cellulolyticum and thermophilic C. thermocellum. A total of 57 cellulosomal genes were found in the C. cellulovorans genome, and they coded for not only carbohydrate‐degrading enzymes but also a lipase, peptidases and proteinase inhibitors. Interestingly, two novel genes encoding scaffolding proteins were found in the genome. According to KEGG metabolic pathways and their comparison with 11 Clostridial genomes, gene expansion in the C. cellulovorans genome indicated mainly non‐cellulosomal genes encoding hemicellulases and pectin‐degrading enzymes. Thus, by examining genome sequences from multiple Clostridium species, comparative genomics offers new insight into genome evolution and the way natural selection moulds functional DNA sequence evolution. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced cellulosome‐producing Clostridium strains for industrial applications such as biofuel production.     

猴欢喜叶绿体全基因组及杜英科系统地位分析

最新的分子系统发育(APG IV)研究中以猴欢喜属(Sloanea L.)为代表的杜英科(Elaeocarpaceae)所在的酢浆草目(Oxalidales)被置于豆类分支(Fabids),且与卫矛目(Celastrales)、金虎尾目(Malpighiales)组成一支(COM分支),但支持率较低.为提高COM分支支...     

Chromosome‐level genome assembly of a cyprinid fish Onychostoma macrolepis by integration of nanopore sequencing,Bionano and Hi‐C technology

Onychostoma macrolepis is an emerging commercial cyprinid fish species. It is a model system for studies of sexual dimorphism and genome evolution. Here, we report the chromosome‐level assembly of the O.macrolepis genome obtained from the integration of nanopore long‐read sequencing with physical maps produced using Bionano and Hi‐C technology. A total of 87.9 Gb of nanopore sequence provided approximately 100‐fold coverage of the genome. The preliminary genome assembly was 883.2 Mb in size with a contig N50 size of 11.2 Mb. The 969 corrected contigs obtained from Bionano optical mapping were assembled into 853 scaffolds and produced an assembly of 886.5 Mb with a scaffold N50 of 16.5 Mb. Finally, using the Hi‐C data, 881.3 Mb (99.4% of genome) in 526 scaffolds were anchored and oriented in 25 chromosomes ranging in size from 25.27 to 56.49 Mb. In total, 24,770 protein‐coding genes were predicted in the genome, and ~96.85% of the genes were functionally annotated. The annotated assembly contains 93.3% complete genes from the BUSCO reference set. In addition, we identified 409 Mb (46.23% of the genome) of repetitive sequence, and 11,213 non‐coding RNAs, in the genome. Evolutionary analysis revealed that O. macrolepis diverged from common carp approximately 24.25 million years ago. The chromosomes of O. macrolepis showed an unambiguous correspondence to the chromosomes of zebrafish. The high‐quality genome assembled in this work provides a valuable genomic resource for further biological and evolutionary studies of O. macrolepis.