Identifying Terpenoid Biosynthesis Genes in Euphorbia maculata via Full-Length cDNA Sequencing.

The annual herb Euphorbia maculata L. produces anti-inflammatory and biologically active substances such as triterpenoids, tannins, and polyphenols, and it is used in traditional Chinese medicine. Of these bioactive compounds, terpenoids, also called isoprenoids, are major secondary metabolites in E. maculata. Full-length cDNA sequencing was carried out to characterize the transcripts of terpenoid biosynthesis reference genes and determine the copy numbers of their isoforms using PacBio SMRT sequencing technology. The Illumina short-read sequencing platform was also employed to identify differentially expressed genes (DEGs) in the secondary metabolite pathways from leaves, roots, and stems. PacBio generated 62 million polymerase reads, resulting in 81,433 high-quality reads. From these high-quality reads, we reconstructed a genome of 20,722 genes, in which 20,246 genes (97.8%) did not have paralogs. About 33% of the identified genes had two or more isoforms. DEG analysis revealed that the expression level differed among gene paralogs in the leaf, stem, and root. Whole sets of paralogs and isoforms were identified in the mevalonic acid (MVA), methylerythritol phosphate (MEP), and terpenoid biosynthesis pathways in the E. maculata L. The nucleotide information will be useful for identifying orthologous genes in other terpenoid-producing medicinal plants.

Application of Upstream Open Reading Frames (uORFs) Editing for the Development of Stress-Tolerant Crops.

Global population growth and climate change are posing increasing challenges to the production of a stable crop supply using current agricultural practices. The generation of genetically modified (GM) crops has contributed to improving crop stress tolerance and productivity; however, many regulations are still in place that limit their commercialization. Recently, alternative biotechnology-based strategies, such as gene-edited (GE) crops, have been in the spotlight. Gene-editing technology, based on the clustered regularly interspaced short palindromic repeats (CRISPR) platform, has emerged as a revolutionary tool for targeted gene mutation, and has received attention as a game changer in the global biotechnology market. Here, we briefly introduce the concept of upstream open reading frames (uORFs) editing, which allows for control of the translation of downstream ORFs, and outline the potential for enhancing target gene expression by mutating uORFs. We discuss the current status of developing stress-tolerant crops, and discuss uORF targets associated with salt stress-responsive genes in rice that have already been verified by transgenic research. Finally, we overview the strategy for developing GE crops using uORF editing via the CRISPR-Cas9 system. A case is therefore made that the mutation of uORFs represents an efficient method for developing GE crops and an expansion of the scope of application of genome editing technology.

Genome-wide transcriptome profiling of the medicinal plant Zanthoxylum planispinum using a single-molecule direct RNA sequencing approach.

High-throughput RNA sequencing has revolutionized transcriptome-based studies of candidate genes, key pathways and gene regulation in non-model organisms. We analyzed full-length cDNA sequences in Zanthoxylum planispinum (Z. planispinum), a medicinal herb in major parts of East Asia. The full-length mRNA derived from tissues of leaf, early fruit and maturing fruit stage were sequenced using PacBio RSII platform to identify isoform transcriptome. We obtained 51,402 unigenes, with average 1781 bp per gene in 82.473 Mb gene lengths. Among 51,402, 3963 unigenes showed variety of isoform. By selection of one representative gene among each of the various isoforms, we finalized 46,306 unique gene set for this herb. We identified 76 cytochrome P450 (CYP450) and related isoforms that are of the wide diversity in the molecular function and biological process. These transcriptome data of Z. planispinum will provide a good resource to study metabolic engineering for the production of valuable medicinal drugs and phytochemicals.

A bioinformatics approach for identifying transgene insertion sites using whole genome sequencing data.

BACKGROUND: Genetically modified crops (GM crops) have been developed to improve the agricultural traits of modern crop cultivars. Safety assessments of GM crops are of paramount importance in research at developmental stages and before releasing transgenic plants into the marketplace. Sequencing technology is developing rapidly, with higher output and labor efficiencies, and will eventually replace existing methods for the molecular characterization of genetically modified organisms. METHODS: To detect the transgenic insertion locations in the three GM rice gnomes, Illumina sequencing reads are mapped and classified to the rice genome and plasmid sequence. The both mapped reads are classified to characterize the junction site between plant and transgene sequence by sequence alignment. RESULTS: Herein, we present a next generation sequencing (NGS)-based molecular characterization method, using transgenic rice plants SNU-Bt9-5, SNU-Bt9-30, and SNU-Bt9-109. Specifically, using bioinformatics tools, we detected the precise insertion locations and copy numbers of transfer DNA, genetic rearrangements, and the absence of backbone sequences, which were equivalent to results obtained from Southern blot analyses. CONCLUSION: NGS methods have been suggested as an effective means of characterizing and detecting transgenic insertion locations in genomes. Our results demonstrate the use of a combination of NGS technology and bioinformatics approaches that offers cost- and time-effective methods for assessing the safety of transgenic plants.

Uncovering the novel characteristics of Asian honey bee, Apis cerana, by whole genome sequencing.

BACKGROUND: The honey bee is an important model system for increasing understanding of molecular and neural mechanisms underlying social behaviors relevant to the agricultural industry and basic science. The western honey bee, Apis mellifera, has served as a model species, and its genome sequence has been published. In contrast, the genome of the Asian honey bee, Apis cerana, has not yet been sequenced. A. cerana has been raised in Asian countries for thousands of years and has brought considerable economic benefits to the apicultural industry. A cerana has divergent biological traits compared to A. mellifera and it has played a key role in maintaining biodiversity in eastern and southern Asia. Here we report the first whole genome sequence of A. cerana. RESULTS: Using de novo assembly methods, we produced a 238 Mbp draft of the A. cerana genome and generated 10,651 genes. A.cerana-specific genes were analyzed to better understand the novel characteristics of this honey bee species. Seventy-two percent of the A. cerana-specific genes had more than one GO term, and 1,696 enzymes were categorized into 125 pathways. Genes involved in chemoreception and immunity were carefully identified and compared to those from other sequenced insect models. These included 10 gustatory receptors, 119 odorant receptors, 10 ionotropic receptors, and 160 immune-related genes. CONCLUSIONS: This first report of the whole genome sequence of A. cerana provides resources for comparative sociogenomics, especially in the field of social insect communication. These important tools will contribute to a better understanding of the complex behaviors and natural biology of the Asian honey bee and to anticipate its future evolutionary trajectory.

Precision genome engineering with programmable DNA-nicking enzymes.

Zinc finger nucleases (ZFNs) are powerful tools of genome engineering but are limited by their inevitable reliance on error-prone nonhomologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs), which gives rise to randomly generated, unwanted small insertions or deletions (indels) at both on-target and off-target sites. Here, we present programmable DNA-nicking enzymes (nickases) that produce single-strand breaks (SSBs) or nicks, instead of DSBs, which are repaired by error-free homologous recombination (HR) rather than mutagenic NHEJ. Unlike their corresponding nucleases, zinc finger nickases allow site-specific genome modifications only at the on-target site, without the induction of unwanted indels. We propose that programmable nickases will be of broad utility in research, medicine, and biotechnology, enabling precision genome engineering in any cell or organism.

A Genome-wide Scan for Selective Sweeps in Racing Horses.

Using next-generation sequencing, we conducted a genome-wide scan of selective sweeps associated with selection toward genetic improvement in Thoroughbreds. We investigated potential phenotypic consequence of putative candidate loci by candidate gene association mapping for the finishing time in 240 Thoroughbred horses. We found a significant association with the trait for Ral GApase alpha 2 (RALGAP2) that regulates a variety of cellular processes of signal trafficking. Neighboring genes around RALGAP2 included insulinoma-associated 1 (INSM1), pallid (PLDN), and Ras and Rab interactor 2 (RIN2) genes have similar roles in signal trafficking, suggesting that a co-evolving gene cluster located on the chromosome 22 is under strong artificial selection in racehorses.

Multiple Genes Related to Muscle Identified through a Joint Analysis of a Two-stage Genome-wide Association Study for Racing Performance of 1,156 Thoroughbreds.

Thoroughbred, a relatively recent horse breed, is best known for its use in horse racing. Although myostatin (MSTN) variants have been reported to be highly associated with horse racing performance, the trait is more likely to be polygenic in nature. The purpose of this study was to identify genetic variants strongly associated with racing performance by using estimated breeding value (EBV) for race time as a phenotype. We conducted a two-stage genome-wide association study to search for genetic variants associated with the EBV. In the first stage of genome-wide association study, a relatively large number of markers (~54,000 single-nucleotide polymorphisms, SNPs) were evaluated in a small number of samples (240 horses). In the second stage, a relatively small number of markers identified to have large effects (170 SNPs) were evaluated in a much larger number of samples (1,156 horses). We also validated the SNPs related to MSTN known to have large effects on racing performance and found significant associations in the stage two analysis, but not in stage one. We identified 28 significant SNPs related to 17 genes. Among these, six genes have a function related to myogenesis and five genes are involved in muscle maintenance. To our knowledge, these genes are newly reported for the genetic association with racing performance of Thoroughbreds. It complements a recent horse genome-wide association studies of racing performance that identified other SNPs and genes as the most significant variants. These results will help to expand our knowledge of the polygenic nature of racing performance in Thoroughbreds.

Variation block-based genomics method for crop plants.

BACKGROUND: In contrast with wild species, cultivated crop genomes consist of reshuffled recombination blocks, which occurred by crossing and selection processes. Accordingly, recombination block-based genomics analysis can be an effective approach for the screening of target loci for agricultural traits. RESULTS: We propose the variation block method, which is a three-step process for recombination block detection and comparison. The first step is to detect variations by comparing the short-read DNA sequences of the cultivar to the reference genome of the target crop. Next, sequence blocks with variation patterns are examined and defined. The boundaries between the variation-containing sequence blocks are regarded as recombination sites. All the assumed recombination sites in the cultivar set are used to split the genomes, and the resulting sequence regions are termed variation blocks. Finally, the genomes are compared using the variation blocks. The variation block method identified recurring recombination blocks accurately and successfully represented block-level diversities in the publicly available genomes of 31 soybean and 23 rice accessions. The practicality of this approach was demonstrated by the identification of a putative locus determining soybean hilum color. CONCLUSIONS: We suggest that the variation block method is an efficient genomics method for the recombination block-level comparison of crop genomes. We expect that this method will facilitate the development of crop genomics by bringing genomics technologies to the field of crop breeding.

O- and N-Methyltransferases in benzylisoquinoline alkaloid producing plants.

BACKGROUND: Secondary metabolites such as benzylisoquinoline alkaloids (BIA) have attracted considerable attention because of their pharmacological properties and potential therapeutic applications. Methyltransferases (MTs) can add methyl groups to alkaloid molecules, altering their physicochemical properties and bioactivity, stability, solubility, and recognition by other cellular components. Five types of O-methyltransferases and two types of N-methyltransferases are involved in BIA biosynthesis. OBJECTIVE: Since MTs may be the source for the discovery and development of novel biomedical, agricultural, and industrial compounds, we performed extensive molecular and phylogenetic analyses of O- and N-methyltransferases in BIA-producing plants. METHODS: MTs involved in BIA biosynthesis were isolated from transcriptomes of Berberis koreana and Caulophyllum robustum. We also mined the methyltransferases of Coptis japonica, Papaver somniferum, and Nelumbo nucifera from the National Center for Biotechnology Information protein database. Then, we analyzed the functional motifs and phylogenetic analysis. RESULT: We mined 42 O-methyltransferases and 8 N-methyltransferases from the five BIA-producing plants. Functional motifs for S-adenosyl-L-methionine-dependent methyltransferases were retained in most methyltransferases, except for the three O-methyltransferases from N. nucifera. Phylogenetic analysis revealed that the methyltransferases were grouped into four clades, I, II, III and IV. The clustering patterns in the phylogenetic analysis suggested a monophyletic origin of methyltransferases and gene duplication within species. The coexistence of different O-methyltransferases in the deep branch subclade might support some cases of substrate promiscuity. CONCLUSIONS: Methyltransferases may be a source for the discovery and development of novel biomedical, agricultural, and industrial compounds. Our results contribute to further understanding of their structure and reaction mechanisms, which will require future functional studies.

The MYB-CC Transcription Factor PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) Functions in Phosphate Homeostasis and Affects Salt Stress Tolerance in Rice.

Inorganic phosphate (Pi) homeostasis plays an important role in plant growth and abiotic stress tolerance. Several MYB-CC transcription factors involved in Pi homeostasis have been identified in rice (Oryza sativa). PHOSPHATE STARVATION RESPONSE-LIKE 7 (PHL7) is a class II MYC-CC protein, in which the MYC-CC domain is located at the N terminus. In this study, we established that OsPHL7 is localized to the nucleus and that the encoding gene is induced by Pi deficiency. The Pi-responsive genes and Pi transporter genes are positively regulated by OsPHL7. The overexpression of OsPHL7 enhanced the tolerance of rice plants to Pi starvation, whereas the RNA interference-based knockdown of this gene resulted in increased sensitivity to Pi deficiency. Transgenic rice plants overexpressing OsPHL7 produced more roots than wild-type plants under both Pi-sufficient and Pi-deficient conditions and accumulated more Pi in the shoots and roots. In addition, the overexpression of OsPHL7 enhanced rice tolerance to salt stress. Together, these results demonstrate that OsPHL7 is involved in the maintenance of Pi homeostasis and enhances tolerance to Pi deficiency and salt stress in rice.

Dynamic genetic features of chromosomes revealed by comparison of soybean genetic and sequence-based physical maps.

Despite the intensive soybean [Glycine max (L.) Merrill] genome studies, the high chromosome number (20) of the soybean plant relative to many other major crops has hindered the development of a high-resolution genomewide genetic map derived from a single population. Here, we report such a map, which was constructed in an F15 population derived from a cross between G. max and G. soja lines using indel polymorphisms detected via a G. soja genome resequencing. By targeting novel indel markers to marker-poor regions, all marker intervals were reduced to under 6 cM on a genome scale. Comparison of the Williams 82 soybean reference genome sequence and our genetic map indicated that marker orders of 26 regions were discrepant with each other. In addition, our comparison showed seven misplaced and two absent markers in the current Williams 82 assembly and six markers placed on the scaffolds that were not incorporated into the pseudomolecules. Then, we showed that, by determining the missing sequences located at the presumed beginning points of the five major discordant segments, these observed discordant regions are mostly errors in the Williams 82 assembly. Distributions of the recombination rates along the chromosomes were similar to those of other organisms. Genotyping of indel markers and genome resequencing of the two parental lines suggested that some marker-poor chromosomal regions may represent introgression regions, which appear to be prevalent in soybean. Given the even and dense distribution of markers, our genetic map can serve as a bridge between genomics research and breeding programs.

Whole spectrum of cytochrome P450 genes and molecular responses to water-accommodated fractions exposure in the marine medaka.

Water-accommodated fractions (WAFs) of crude oil include chemicals that are potent toxicants in fish. Increasing oil pollution thus demands a better understanding of molecular mechanisms for detoxification, metabolism, toxicity, and adaptation of fish. Previous studies with fish show modulation of expression of key genes in relation to stress response against WAF exposure, but there is still a lack of studies on responses of cytochrome P450 (CYP) genes and changes in biotransformation upon WAF exposure. In this study, we used the full spectrum of CYP genes of the marine medaka, Oryzias melastigma, to understand their potential mode of action on WAFs-triggered molecular mechanisms. We also analyzed further CYP-involved detoxification and endogenous steroidogenic metabolism after exposure to different concentrations of WAFs over different time courses in the marine medaka. Also, detoxification- and antioxidant-related enzymes' activities were analyzed with different concentrations of WAFs. As a result, the WAF exposure induced CYP-involved detoxification mechanism but reduced CYP-involved steroidogenic metabolism in the marine medaka. These data suggest that whole CYP profiling can be a way of understanding and uncovering the mode of action particularly with respect to emerging chemicals such as WAF exposure with the new finding that WAFs have dual functions on CYP-involved metabolisms.

Whole-genome sequencing and intensive analysis of the undomesticated soybean (Glycine soja Sieb. and Zucc.) genome.

The genome of soybean (Glycine max), a commercially important crop, has recently been sequenced and is one of six crop species to have been sequenced. Here we report the genome sequence of G. soja, the undomesticated ancestor of G. max (in particular, G. soja var. IT182932). The 48.8-Gb Illumina Genome Analyzer (Illumina-GA) short DNA reads were aligned to the G. max reference genome and a consensus was determined for G. soja. This consensus sequence spanned 915.4 Mb, representing a coverage of 97.65% of the G. max published genome sequence and an average mapping depth of 43-fold. The nucleotide sequence of the G. soja genome, which contains 2.5 Mb of substituted bases and 406 kb of small insertions/deletions relative to G. max, is ∼0.31% different from that of G. max. In addition to the mapped 915.4-Mb consensus sequence, 32.4 Mb of large deletions and 8.3 Mb of novel sequence contigs in the G. soja genome were also detected. Nucleotide variants of G. soja versus G. max confirmed by Roche Genome Sequencer FLX sequencing showed a 99.99% concordance in single-nucleotide polymorphism and a 98.82% agreement in insertion/deletion calls on Illumina-GA reads. Data presented in this study suggest that the G. soja/G. max complex may be at least 0.27 million y old, appearing before the relatively recent event of domestication (6,000∼9,000 y ago). This suggests that soybean domestication is complicated and that more in-depth study of population genetics is needed. In any case, genome comparison of domesticated and undomesticated forms of soybean can facilitate its improvement.

Cellular Morphology and Transcriptome Comparative Analysis of Astragalus membranaceus Bunge Sprouts Cultured In Vitro under Different LED Light.

Astragalus membranaceus, the major components of which are saponins, flavonoids, and polysaccharides, has been established to have excellent pharmacological activity. After ginseng, it is the second most used medicinal plant. To examine the utility of A. membranaceus as a sprout crop for plant factory cultivation, we sought to establish a functional substance control model by comparing the transcriptomes of sprouts grown in sterile, in vitro culture using LED light sources. Having sown the seeds of A. membranaceus, these were exposed to white LED light (continuous spectrum), red LED light (632 nm, 1.58 µmol/m2/s), or blue LED light (465 nm, 1.44 µmol/m2/s) and grown for 6 weeks; after which, the samples were collected for transcriptome analysis. Scanning electron microscopy analysis of cell morphology in plants exposed to the three light sources revealed that leaf cell size was largest in those plants exposed to red light, where the thickest stem was observed in plants exposed to white light. The total number of genes in A. membranaceus spouts determined via de novo assembly was 45,667. Analysis of differentially expressed genes revealed that for the comparisons of blue LED vs. red LED, blue LED vs. white LED, and red LED vs. white LED, the numbers of upregulated genes were 132, 148, and 144, respectively. Binding, DNA integration, transport, phosphorylation, DNA biosynthetic process, membrane, and plant-type secondary cell wall biogenesis were the most enriched in the comparative analysis of blue LED vs. red LED, whereas Binding, RNA-templated DNA biosynthetic process, DNA metabolic process, and DNA integration were the most enriched in the comparative analysis of blue vs. white LED, and DNA integration and resolution of meiotic recombination intermediates were the most enrichment in the comparison between red LED vs. white LED. The GO term associated with flavonoid biosynthesis, implying the functionality of A. membranaceus, was the flavonoid biosynthetic process, which was enriched in the white LED vs. red LED comparison. The findings of this study thus indicate that different LED light sources can differentially influence the transcriptome expression pattern of A. membranaceus sprouts, which can provide a basis for establishing a flavonoid biosynthesis regulation model and thus, the cultivation of high-functional Astragalus sprouts.

Isolation and Characterization of the Genes Involved in the Berberine Synthesis Pathway in Asian Blue Cohosh, Caulophyllum robustum.

Caulophyllum robustum, commonly named Asian blue cohosh, is a perennial herb in the family Berberidaceae. It has traditionally been used for folk medicine in China. We isolated berberine from the leaves, stem, roots, and fruits of C. robustum, and this is the first report on berberine in this species. Transcriptome analysis was conducted for the characterization of berberine biosynthesis genes in C. robustum, in which, all the genes for berberine biosynthesis were identified. From 40,094 transcripts, using gene ontology (GO) analysis, 26,750 transcripts were assigned their functions in the categories of biological process, molecular function, and cellular component. In the analysis of genes expressed in different tissues, the numbers of genes in the categories of intrinsic component of membrane and transferase activity were up-regulated in leaves versus stem. The berberine synthesis genes in C. robustum were characterized by phylogenetic analysis with corresponding genes from other berberine-producing species. The co-existence of genes from different plant families in the deepest branch subclade implies that the differentiation of berberine synthesis genes occurred early in the evolution of berberine-producing plants. Furthermore, the copy number increment of the berberine synthesis genes was detected at the species level.

Genome sequence of Pectobacterium carotovorum subsp. carotovorum strain PCC21, a pathogen causing soft rot in Chinese cabbage.

Pectobacterium carotovorum is a plant-pathogenic enterobacterium responsible for soft rot in various commercially important plants. Here we report the complete genome sequence and automatic annotation of strain PCC21.

Complete genome sequence of the fenitrothion-degrading Burkholderia sp. strain YI23.

Burkholderia species are ubiquitous in soil environments. Many Burkholderia species isolated from various environments have the potential to biodegrade man-made chemicals. Burkholderia sp. strain YI23 was isolated from a golf course soil and identified as a fenitrothion-degrading bacterium. In this study, we report the complete genome sequence of Burkholderia sp. strain YI23.

Complete genome sequence of Mycobacterium intracellulare clinical strain MOTT-36Y, belonging to the INT5 genotype.

Here we report the complete genome sequence of the Mycobacterium intracellulare clinical strain MOTT-36Y, previously grouped into the INT5 genotype among the 5 genotypes of M. intracellulare. This genome sequence will serve as a valuable reference for understanding the disparity in virulence and epidemiologic traits between M. intracellulare-related strains.

Complete genome sequence of Mycobacterium intracellulare strain ATCC 13950(T).

Here we report the first complete genome sequence of Mycobacterium intracellulare ATCC 13950(T), a Mycobacterium avium complex (MAC) strain. This genome sequence will serve as a valuable reference for understanding the epidemiologic, biological, and pathogenic aspects of the disparity between MAC members.