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April 21, 2020

Closing the Yield Gap for Cannabis: A Meta-Analysis of Factors Determining Cannabis Yield.

Until recently, the commercial production of Cannabis sativa was restricted to varieties that yielded high-quality fiber while producing low levels of the psychoactive cannabinoid tetrahydrocannabinol (THC). In the last few years, a number of jurisdictions have legalized the production of medical and/or recreational cannabis with higher levels of THC, and other jurisdictions seem poised to follow suit. Consequently, demand for industrial-scale production of high yield cannabis with consistent cannabinoid profiles is expected to increase. In this paper we highlight that currently, projected annual production of cannabis is based largely on facility size, not yield per square meter. This meta-analysis of cannabis yields reported in scientific literature aimed to identify the main factors contributing to cannabis yield per plant, per square meter, and per W of lighting electricity. In line with previous research we found that variety, plant density, light intensity and fertilization influence cannabis yield and cannabinoid content; we also identified pot size, light type and duration of the flowering period as predictors of yield and THC accumulation. We provide insight into the critical role of light intensity, quality, and photoperiod in determining cannabis yields, with particular focus on the potential for light-emitting diodes (LEDs) to improve growth and reduce energy requirements. We propose that the vast amount of genomics data currently available for cannabis can be used to better understand the effect of genotype on yield. Finally, we describe diversification that is likely to emerge in cannabis growing systems and examine the potential role of plant-growth promoting rhizobacteria (PGPR) for growth promotion, regulation of cannabinoid biosynthesis, and biocontrol.

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April 21, 2020

Long-Read Sequencing Emerging in Medical Genetics

The wide implementation of next-generation sequencing (NGS) technologies has revolutionized the field of medical genetics. However, the short read lengths of currently used sequencing approaches pose a limitation for identification of structural variants, sequencing repetitive regions, phasing alleles and distinguishing highly homologous genomic regions. These limitations may significantly contribute to the diagnostic gap in patients with genetic disorders who have undergone standard NGS, like whole exome or even genome sequencing. Now, the emerging long-read sequencing (LRS) technologies may offer improvements in the characterization of genetic variation and regions that are difficult to assess with the currently prevailing NGS approaches. LRS has so far mainly been used to investigate genetic disorders with previously known or strongly suspected disease loci. While these targeted approaches already show the potential of LRS, it remains to be seen whether LRS technologies can soon enable true whole genome sequencing routinely. Ultimately, this could allow the de novo assembly of individual whole genomes used as a generic test for genetic disorders. In this article, we summarize the current LRS-based research on human genetic disorders and discuss the potential of these technologies to facilitate the next major advancements in medical genetics.

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April 21, 2020

Divergent evolutionary trajectories following speciation in two ectoparasitic honey bee mites.

Multispecies host-parasite evolution is common, but how parasites evolve after speciating remains poorly understood. Shared evolutionary history and physiology may propel species along similar evolutionary trajectories whereas pursuing different strategies can reduce competition. We test these scenarios in the economically important association between honey bees and ectoparasitic mites by sequencing the genomes of the sister mite species Varroa destructor and Varroa jacobsoni. These genomes were closely related, with 99.7% sequence identity. Among the 9,628 orthologous genes, 4.8% showed signs of positive selection in at least one species. Divergent selective trajectories were discovered in conserved chemosensory gene families (IGR, SNMP), and Halloween genes (CYP) involved in moulting and reproduction. However, there was little overlap in these gene sets and associated GO terms, indicating different selective regimes operating on each of the parasites. Based on our findings, we suggest that species-specific strategies may be needed to combat evolving parasite communities. © The Author(s) 2019.

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April 21, 2020

Characterization of mcr-1-Harboring Plasmids from Pan Drug-Resistant Escherichia coli Strains Isolated from Retail Raw Chicken in South Korea

A number of studies from different countries have characterized mcr-1-harboring plasmids isolated from food; however, nothing has been reported about it in South Korea. In this study, we report the characterization of mcr-1 plasmids from pan drug-resistant (PDR) Escherichia coli strains isolated from retail food in the country. Colistin-resistant E. coli strains were isolated from retail raw chicken, and PCR was carried out to detect the mcr-1 gene. Whole genome sequencing of the mcr-1-positive strains was performed for further characterization. The results of whole genome sequencing revealed that all mcr-1 plasmids belonged to the IncI2 type. In addition to the mcr-1 plasmids, all of the isolates also carried additional plasmids possessing multiple antibiotic resistance genes, and the PDR was mediated by resistant plasmids except for fluoroquinolone resistance resulting from mutations in gyrA and parC. Interestingly, the mcr-1 plasmids were transferred by conjugation to other pathogenic strains including enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), Salmonella, and Klebsiella at the frequencies of 10−3−10−6, 10−2−10−5, 10−4−10−5, 10−4−10−6, and 10−5−10−6, respectively. The results showed that mcr-1 plasmids can be easily transmitted to pathogenic bacteria by conjugation.

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April 21, 2020

A hybrid de novo genome assembly of the honeybee, Apis mellifera, with chromosome-length scaffolds.

The ability to generate long sequencing reads and access long-range linkage information is revolutionizing the quality and completeness of genome assemblies. Here we use a hybrid approach that combines data from four genome sequencing and mapping technologies to generate a new genome assembly of the honeybee Apis mellifera. We first generated contigs based on PacBio sequencing libraries, which were then merged with linked-read 10x Chromium data followed by scaffolding using a BioNano optical genome map and a Hi-C chromatin interaction map, complemented by a genetic linkage map.Each of the assembly steps reduced the number of gaps and incorporated a substantial amount of additional sequence into scaffolds. The new assembly (Amel_HAv3) is significantly more contiguous and complete than the previous one (Amel_4.5), based mainly on Sanger sequencing reads. N50 of contigs is 120-fold higher (5.381 Mbp compared to 0.053 Mbp) and we anchor >?98% of the sequence to chromosomes. All of the 16 chromosomes are represented as single scaffolds with an average of three sequence gaps per chromosome. The improvements are largely due to the inclusion of repetitive sequence that was unplaced in previous assemblies. In particular, our assembly is highly contiguous across centromeres and telomeres and includes hundreds of AvaI and AluI repeats associated with these features.The improved assembly will be of utility for refining gene models, studying genome function, mapping functional genetic variation, identification of structural variants, and comparative genomics.

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April 21, 2020

Hybrid-Transcriptome Sequencing and Associated Metabolite Analysis Reveal Putative Genes Involved in Flower Color Difference in Rose Mutants.

Gene mutation is a common phenomenon in nature that often leads to phenotype differences, such as the variations in flower color that frequently occur in roses. With the aim of revealing the genomic information and inner mechanisms, the differences in the levels of both transcription and secondary metabolism between a pair of natural rose mutants were investigated by using hybrid RNA-sequencing and metabolite analysis. Metabolite analysis showed that glycosylated derivatives of pelargonidin, e.g., pelargonidin 3,5 diglucoside and pelargonidin 3-glucoside, which were not detected in white flowers (Rosa ‘Whilte Mrago Koster’), constituted the major pigments in pink flowers. Conversely, the flavonol contents of petal, such as kaempferol-3-glucoside, quercetin 3-glucoside, and rutin, were higher in white flowers. Hybrid RNA-sequencing obtained a total of 107,280 full-length transcripts in rose petal which were annotated in major databases. Differentially expressed gene (DEG) analysis showed that the expression of genes involved in the flavonoid biosynthesis pathway was significantly different, e.g., CHS, FLS, DFR, LDOX, which was verified by qRT-PCR during flowering. Additionally, two MYB transcription factors were found and named RmMYBAN2 and RmMYBPA1, and their expression patterns during flowering were also analyzed. These findings indicate that these genes may be involved in the flower color difference in the rose mutants, and competition between anthocyanin and flavonol biosynthesis is a primary cause of flower color variation, with its regulation reflected by transcriptional and secondary metabolite levels.

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April 21, 2020

Large Plasmid Complement Resolved: Complete Genome Sequencing of Lactobacillus plantarum MF1298, a Candidate Probiotic Strain Associated with Unfavorable Effect.

Considerable attention has been given to the species Lactobacillus plantarum regarding its probiotic potential. L. plantarum strains have shown health benefits in several studies, and even nonstrain-specific claims are allowed in certain markets. L. plantarum strain MF1298 was considered a candidate probiotic, demonstrating in vitro probiotic properties and the ability to survive passage through the human intestinal tract. However, the strain showed an unfavorable effect on symptoms in subjects with irritable bowel syndrome in a clinical trial. The properties and the genome of this strain are thus of general interest. Obtaining the complete genome of strain MF1298 proved difficult due to its large plasmid complement. Here, we exploit a combination of sequencing approaches to obtain the complete chromosome and plasmid assemblies of MF1298. The Oxford Nanopore Technologies MinION long-read sequencer was particularly useful in resolving the unusually large number of plasmids in the strain, 14 in total. The complete genome sequence of 3,576,440 basepairs contains 3272 protein-encoding genes, of which 315 are located on plasmids. Few unique regions were found in comparison with other L. plantarum genomes. Notably, however, one of the plasmids contains genes related to vitamin B12 (cobalamin) turnover and genes encoding bacterial reverse transcriptases, features not previously reported for L. plantarum. The extensive plasmid information will be important for future studies with this strain.

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April 21, 2020

Iso-Seq analysis of the Taxus cuspidata transcriptome reveals the complexity of Taxol biosynthesis.

Taxus cuspidata is well known worldwide for its ability to produce Taxol, one of the top-selling natural anticancer drugs. However, current Taxol production cannot match the increasing needs of the market, and novel strategies should be considered to increase the supply of Taxol. Since the biosynthetic mechanism of Taxol remains largely unknown, elucidating this pathway in detail will be very helpful in exploring alternative methods for Taxol production.Here, we sequenced Taxus cuspidata transcriptomes with next-generation sequencing (NGS) and third-generation sequencing (TGS) platforms. After correction with Illumina reads and removal of redundant reads, more than 180,000 nonredundant transcripts were generated from the raw Iso-Seq data. Using Cogent software and an alignment-based method, we identified a total of 139 cytochrome P450s (CYP450s), 31 BAHD acyltransferases (ACTs) and 1940 transcription factors (TFs). Based on phylogenetic and coexpression analysis, we identified 9 CYP450s and 7 BAHD ACTs as potential lead candidates for Taxol biosynthesis and 6 TFs that are possibly involved in the regulation of this process. Using coexpression analysis of genes known to be involved in Taxol biosynthesis, we elucidated the stem biosynthetic pathway. In addition, we analyzed the expression patterns of 12 characterized genes in the Taxol pathway and speculated that the isoprene precursors for Taxol biosynthesis were mainly synthesized via the MEP pathway. In addition, we found and confirmed that the alternative splicing patterns of some genes varied in different tissues, which may be an important tissue-specific method of posttranscriptional regulation.A strategy was developed to generate corrected full-length or nearly full-length transcripts without assembly to ensure sequence accuracy, thus greatly improving the reliability of coexpression and phylogenetic analysis and greatly facilitating gene cloning and characterization. This strategy was successfully utilized to elucidate the Taxol biosynthetic pathway, which will greatly contribute to the goals of improving the Taxol content in Taxus spp. using molecular breeding or plant management strategies and synthesizing Taxol in microorganisms using synthetic biological technology.

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April 21, 2020

Improving the sensitivity of long read overlap detection using grouped short k-mer matches.

Single-molecule, real-time sequencing (SMRT) developed by Pacific BioSciences produces longer reads than second-generation sequencing technologies such as Illumina. The increased read length enables PacBio sequencing to close gaps in genome assembly, reveal structural variations, and characterize the intra-species variations. It also holds the promise to decipher the community structure in complex microbial communities because long reads help metagenomic assembly. One key step in genome assembly using long reads is to quickly identify reads forming overlaps. Because PacBio data has higher sequencing error rate and lower coverage than popular short read sequencing technologies (such as Illumina), efficient detection of true overlaps requires specially designed algorithms. In particular, there is still a need to improve the sensitivity of detecting small overlaps or overlaps with high error rates in both reads. Addressing this need will enable better assembly for metagenomic data produced by third-generation sequencing technologies.In this work, we designed and implemented an overlap detection program named GroupK, for third-generation sequencing reads based on grouped k-mer hits. While using k-mer hits for detecting reads’ overlaps has been adopted by several existing programs, our method uses a group of short k-mer hits satisfying statistically derived distance constraints to increase the sensitivity of small overlap detection. Grouped k-mer hit was originally designed for homology search. We are the first to apply group hit for long read overlap detection. The experimental results of applying our pipeline to both simulated and real third-generation sequencing data showed that GroupK enables more sensitive overlap detection, especially for datasets of low sequencing coverage.GroupK is best used for detecting small overlaps for third-generation sequencing data. It provides a useful supplementary tool to existing ones for more sensitive and accurate overlap detection. The source code is freely available at https://github.com/Strideradu/GroupK .

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April 21, 2020

The sequencing and de novo assembly of the Larimichthys crocea genome using PacBio and Hi-C technologies.

Larimichthys crocea is an endemic marine fish in East Asia that belongs to Sciaenidae in Perciformes. L. crocea has now been recognized as an “iconic” marine fish species in China because not only is it a popular food fish in China, it is a representative victim of overfishing and still provides high value fish products supported by the modern large-scale mariculture industry. Here, we report a chromosome-level reference genome of L. crocea generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The genome sequences were assembled into 1,591 contigs with a total length of 723.86?Mb and a contig N50 length of 2.83?Mb. After chromosome-level scaffolding, 24 scaffolds were constructed with a total length of 668.67?Mb (92.48% of the total length). Genome annotation identified 23,657 protein-coding genes and 7262 ncRNAs. This highly accurate, chromosome-level reference genome of L. crocea provides an essential genome resource to support the development of genome-scale selective breeding and restocking strategies of L. crocea.

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April 21, 2020

The sequence and de novo assembly of Takifugu bimaculatus genome using PacBio and Hi-C technologies.

Takifugu bimaculatus is a native teleost species of the southeast coast of China where it has been cultivated as an important edible fish in the last decade. Genetic breeding programs, which have been recently initiated for improving the aquaculture performance of T. bimaculatus, urgently require a high-quality reference genome to facilitate genome selection and related genetic studies. To address this need, we produced a chromosome-level reference genome of T. bimaculatus using the PacBio single molecule sequencing technique (SMRT) and High-through chromosome conformation capture (Hi-C) technologies. The genome was assembled into 2,193 contigs with a total length of 404.21?Mb and a contig N50 length of 1.31?Mb. After chromosome-level scaffolding, 22 chromosomes with a total length of 371.68?Mb were constructed. Moreover, a total of 21,117 protein-coding genes and 3,471 ncRNAs were annotated in the reference genome. The highly accurate, chromosome-level reference genome of T. bimaculatus provides an essential genome resource for not only the genome-scale selective breeding of T. bimaculatus but also the exploration of the evolutionary basis of the speciation and local adaptation of the Takifugu genus.

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April 21, 2020

Metaepigenomic analysis reveals the unexplored diversity of DNA methylation in an environmental prokaryotic community.

DNA methylation plays important roles in prokaryotes, and their genomic landscapes-prokaryotic epigenomes-have recently begun to be disclosed. However, our knowledge of prokaryotic methylation systems is focused on those of culturable microbes, which are rare in nature. Here, we used single-molecule real-time and circular consensus sequencing techniques to reveal the ‘metaepigenomes’ of a microbial community in the largest lake in Japan, Lake Biwa. We reconstructed 19 draft genomes from diverse bacterial and archaeal groups, most of which are yet to be cultured. The analysis of DNA chemical modifications in those genomes revealed 22 methylated motifs, nine of which were novel. We identified methyltransferase genes likely responsible for methylation of the novel motifs, and confirmed the catalytic specificities of four of them via transformation experiments using synthetic genes. Our study highlights metaepigenomics as a powerful approach for identification of the vast unexplored variety of prokaryotic DNA methylation systems in nature.

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April 21, 2020

Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei.

In Trypanosoma brucei, most mitochondrial mRNAs undergo editing, and 3′ adenylation and uridylation. The internal sequence changes and terminal extensions are coordinated: pre-editing addition of the short (A) tail protects the edited transcript against 3′-5′ degradation, while post-editing A/U-tailing renders mRNA competent for translation. Participation of a poly(A) binding protein (PABP) in coupling of editing and 3′ modification processes has been inferred, but its identity and mechanism of action remained elusive. We report identification of KPAF4, a pentatricopeptide repeat-containing PABP which sequesters the A-tail and impedes mRNA degradation. Conversely, KPAF4 inhibits uridylation of A-tailed transcripts and, therefore, premature A/U-tailing of partially-edited mRNAs. This quality check point likely prevents translation of incompletely edited mRNAs. We also find that RNA editing substrate binding complex (RESC) mediates the interaction between the 5′ end-bound pyrophosphohydrolase MERS1 and 3′ end-associated KPAF4 to enable mRNA circularization. This event appears to be critical for edited mRNA stability.

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April 21, 2020

Long-read based de novo assembly of low-complexity metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system.

Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii.Two NWCs from Swiss Gruyère producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats.Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities.

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April 21, 2020

Valinomycin, produced by Streptomyces sp. S8, a key antifungal metabolite in large patch disease suppressiveness.

Large patch disease, caused by Rhizoctonia solani AG2-2, is the most devastating disease in Zoysiagrass (Zoysia japonica). Current large patch disease control strategies rely primarily upon the use of chemical pesticides. Streptomyces sp. S8 is known to possess exceptional antagonistic properties that could potentially suppress the large patch pathogen found at turfgrass plantations. This study aims to demonstrate the feasibility of using the strain as a biological control mechanism. Sequencing of the S8 strain genome revealed a valinomycin biosynthesis gene cluster. This cluster is composed of the vlm1 and vlm2 genes, which are known to produce antifungal compounds. In order to verify this finding for the large patch pathogen, a valinomycin biosynthesis knockout mutant was created via the CRISPR/Cas9 system. The mutant lost antifungal activity against the large patch pathogen. Consequently, it is anticipated that eco-friendly microbial preparations derived from the S8 strain can be utilized to biologically control large patch disease.

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