Metabolomic and transcriptomic insights into how cotton fiber transitions to secondary wall synthesis, represses lignification, and prolongs elongation.

BACKGROUND: The morphogenesis of single-celled cotton fiber includes extreme elongation and staged cell wall differentiation. Designing strategies for improving cotton fiber for textiles and other uses relies on uncovering the related regulatory mechanisms. In this research we compared the transcriptomes and metabolomes of two Gossypium genotypes, Gossypium barbadense cv Phytogen 800 and G. hirsutum cv Deltapine 90. When grown in parallel, the two types of fiber developed similarly except for prolonged fiber elongation in the G. barbadense cultivar. The data were collected from isolated fibers between 10 to 28 days post anthesis (DPA) representing: primary wall synthesis to support elongation; transitional cell wall remodeling; and secondary wall cellulose synthesis, which was accompanied by continuing elongation only in G. barbadense fiber. RESULTS: Of 206 identified fiber metabolites, 205 were held in common between the two genotypes. Approximately 38,000 transcripts were expressed in the fiber of each genotype, and these were mapped to the reference set and interpreted by homology to known genes. The developmental changes in the transcriptomes and the metabolomes were compared within and across genotypes with several novel implications. Transitional cell wall remodeling is a distinct stable developmental stage lasting at least four days (18 to 21 DPA). Expression of selected cell wall related transcripts was similar between genotypes, but cellulose synthase gene expression patterns were more complex than expected. Lignification was transcriptionally repressed in both genotypes. Oxidative stress was lower in the fiber of G. barbadense cv Phytogen 800 as compared to G. hirsutum cv Deltapine 90. Correspondingly, the G. barbadense cultivar had enhanced capacity for management of reactive oxygen species during its prolonged elongation period, as indicated by a 138-fold increase in ascorbate concentration at 28 DPA. CONCLUSIONS: The parallel data on deep-sequencing transcriptomics and non-targeted metabolomics for two genotypes of single-celled cotton fiber showed that a discrete developmental stage of transitional cell wall remodeling occurs before secondary wall cellulose synthesis begins. The data showed how lignification can be transcriptionally repressed during secondary cell wall synthesis, and they implicated enhanced capacity to manage reactive oxygen species through the ascorbate-glutathione cycle as a positive contributor to fiber length.

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.

The complete chloroplast genome of Philodendron hederaceum (Jacq.) Schott 1829 (Alismatales: Araceae).

Philodendron hederaceum (Jacq.) Schott 1829, a species of the Araceae family, is a foliage plant of ornamental value. The complete chloroplast genome sequence of Philodendron hederaceum was obtained by the de novo assembly of NovaSeq 6000 (Illumina Co., San Diego, CA) paired-end short reads and Oxford Nanopore long reads. The complete chloroplast genome of P. hederaceum was 168,609 bp in length, with a large single-copy (LSC) region of 94,393 bp, a small single-copy (SSC) region of 25,774 bp, and a pair of identical inverted repeat regions (IRs) of 24,221 bp. The genome contained a total of 129 genes, including 85 protein-coding genes, 36 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The phylogenetic analysis of P. hederaceum with 19 related species and two outgroup species revealed the closest taxonomical relationship with Philodendron lanceolatum in the Araceae family.

The complete mitochondrial genome of Turbo cornutus (Trochida: Turbinidae) and its phylogeny analysis.

The complete mitochondrial genome sequence of Turbo cornutus, a species of the Turbinidae family, was characterized from the de novo assembly of Illumina paired-end sequencing data. The complete mitochondrial genome of T. cornutus was 17,297 bp in length and comprised of 13 protein-coding genes, 25 tRNAs, and two rRNAs. The base composition of T. cornutus exhibited a high A + T content of 71.17%. The phylogenetic analysis of T. cornutus with 14 species from GenBank revealed that the ancestor of Astralium haematragum and Bolma rugosa was diverged from T. cornutus.

The complete chloroplast genome of Echeveria lilacina Kimnach & Moran 1980 (Saxifragales: Crassulaceae).

Echeveria lilacina Kimnach & Moran 1980 is a succulent plant having ornamental and ecological importance. In this study, the first complete chloroplast genome of Echeveria lilacina, a species belonging to the Crassulaceae family, was characterized from the de novo assembly of Illumina NovaSeq 6000 paired-end sequencing data. The chloroplast genome of E. lilacina is 150,080 bp in length, which includes a large single-copy (LSC) region of 81,741 bp, a small single-copy (SSC) region of 16,747 bp, and a pair of identical inverted repeat regions (IRs) of 25,796 bp each. The genome annotation revealed a total of 138 genes, including 87 protein-coding genes, 41 transfer RNA (tRNA) genes, and 10 ribosomal RNA (rRNA) genes. The phylogenetic analysis with 15 complete chloroplast genome sequences including outgroup showed that E. lilacina formed the closest taxonomical relationship with Graptopetalum amethystinum in the Crassulaceae family.

Transcriptomic responses of Aspergillus flavus to temperature and oxidative stresses during aflatoxin production.

Aflatoxin is a group of polyketide-derived carcinogenic and mutagenic secondary metabolites produced by Aspergillus flavus that negatively impact global food security and threaten the health of both humans and livestock. Aflatoxin biosynthesis is strongly affected by the fungal developmental stage, cultivation conditions, and environmental stress. In this study, a novel float culture method was used to examine the direct responses of the A. flavus transcriptome to temperature stress, oxidative stress, and their dual effects during the aflatoxin production stage. The transcriptomic response of A. flavus illustrated that the co-regulation of different secondary metabolic pathways likely contributes to maintaining cellular homeostasis and promoting cell survival under stress conditions. In particular, aflatoxin biosynthetic gene expression was downregulated, while genes encoding secondary metabolites with antioxidant properties, such as kojic acid and imizoquins, were upregulated under stress conditions. Multiple mitochondrial function-related genes, including those encoding NADH:ubiquinone oxidoreductase, ubiquinol-cytochrome C reductase, and alternative oxidase, were differentially expressed. These data can provide insights into the important mechanisms through which secondary metabolism in A. flavus is co-regulated and facilitate the deployment of various approaches for the effective control and prevention of aflatoxin contamination in food crops.

Complete chloroplast genome of Gypsophila oldhamiana Miq. (Caryophyllales: Caryophyllaceae).

The complete chloroplast genome sequence of Gypsophila oldhamiana Miq., a species of the Caryophyllaceae family, was assembled and analyzed from the de novo assembly of Illumina paired-end sequencing data. The total length of the chloroplast genome of G. oldhamiana was 152,675 bp including a large single-copy (LSC) region of 83,552 bp, a small single-copy (SSC) region of 17,349 bp, and a pair of identical inverted repeat regions (IRs) of 25,887 bp. The genome possessed a total of 130 genes, including 85 protein-coding genes, 37 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. The phylogenetic analysis of G. oldhamiana with 14 related species discovered the closest taxonomical relationship with Gypsophila vaccaria voucher in the Caryophyllaceae family.

Revealing immune responses in the Mycobacterium avium subsp. paratuberculosis-infected THP-1 cells using single cell RNA-sequencing.

Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease, which is a chronic and debilitating disease in ruminants. MAP is also considered to be a possible cause of Crohn's disease in humans. However, few studies have focused on the interactions between MAP and human macrophages to elucidate the pathogenesis of Crohn's disease. We sought to determine the initial responses of human THP-1 cells against MAP infection using single-cell RNA-seq analysis. Clustering analysis showed that THP-1 cells were divided into seven different clusters in response to phorbol-12-myristate-13-acetate (PMA) treatment. The characteristics of each cluster were investigated by identifying cluster-specific marker genes. From the results, we found that classically differentiated cells express CD14, CD36, and TLR2, and that this cell type showed the most active responses against MAP infection. The responses included the expression of proinflammatory cytokines and chemokines such as CCL4, CCL3, IL1B, IL8, and CCL20. In addition, the Mreg cell type, a novel cell type differentiated from THP-1 cells, was discovered. Thus, it is suggested that different cell types arise even when the same cell line is treated under the same conditions. Overall, analyzing gene expression patterns via scRNA-seq classification allows a more detailed observation of the response to infection by each cell type.

Tandem duplication of the FLC locus and the origin of a new gene in Arabidopsis related species and their functional implications in allopolyploids.

Flowering time is an important adaptive trait and varies among Arabidopsis thaliana and its related species, including allopolyploids that are formed between A. thaliana and Arabidopsis arenosa. FLOWERING LOCUS C (FLC) inhibits early flowering in A. thaliana. A previous study showed that late-flowering A. arenosa contained two or more FLC alleles that were differentially expressed in Arabidopsis allotetraploids, but the genomic organization and evolution of FLC locus were unknown. Comparative sequence and evolutionary analyses were performed in FLC-containing genomic regions in A. thaliana, A. arenosa and Arabidopsis lyrata, and expression of FLC loci and alleles was examined in Arabidopsis allopolyploids. The FLC locus was tandemly duplicated in A. lyrata and triplicated in A. arenosa, and the tandem duplication event occurred after divergence from A. thaliana. Although FLC duplicates were highly conserved, their upstream sequences rapidly diverged. The third FLC copy in A. arenosa acquired a new splicing site through a point mutation in the intron and generated the new exon followed by an early stop codon, resulting in a novel MADS box gene. Flowering time variation in Arabidopsis allopolyploids is probably related to the expression diversity and/or copy number of multiple FLC loci. Moreover, exonization of intronic sequence is a mechanism for the origin of new genes.

Comparative sequence analysis of the SALT OVERLY SENSITIVE1 orthologous region in Thellungiella halophila and Arabidopsis thaliana.

To provide a framework for studies to understand the contribution of SALT OVERLY SENSITIVE1 (SOS1) to salt tolerance in Thellungiella halophila, we sequenced and annotated a 193-kb T. halophila BAC containing a putative SOS1 locus (ThSOS1) and compared the sequence to the orthologous 146-kb region of the genome of its salt-sensitive relative, Arabidopsis thaliana. Overall, the two sequences were colinear, but three major expansion/contraction regions in T. halophila were found to contain five Long Terminal Repeat retrotransposons, MuDR DNA transposons and intergenic sequences that contribute to the 47.8-kb size variation in this region of the genome. Twenty-seven genes were annotated in the T. halophila BAC including the putative ThSOS1 locus. ThSOS1 shares gene structure and sequence with A. thaliana SOS1 including 11 predicted transmembrane domains and a cyclic nucleotide-binding domain; however, different patterns of Simple Sequence Repeats were found within a 540-bp region upstream of SOS1 in the two species.

The complete chloroplast genome of Sinomenium acutum (Menispermaceae).

We generated the complete chloroplast genome sequence of Sinomenium acutum, a species of the Menispermaceae family, and characterized from the de novo assembly of Illumina HiSeq paired-end sequencing data. The total length of the chloroplast genome of S. acutum was 162,787 bp with a large single-copy (LSC) region of 91,430 bp, a small single-copy (SSC) region of 21,245 bp, and a pair of identical inverted repeat regions (IRs) of 25,056 bp. The total of 131 genes were annotated in the chloroplast genome of Sinomenium acutum, including 85 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. The phylogenetic analysis of S. acutum with 18 related species revealed the closest taxonomical relationship with Menispermum dauricum in the Menispermaceae family.

The complete chloroplast genome of Typhonium giganteum (Araceae).

The complete chloroplast genome sequence of Typhonium giganteum, a species of the Araceae family, was characterized from the de novo assembly of HiSeq (Illumina Co.) paired-end sequencing data. The chloroplast genome of T. giganteum was 165,289 bp in length, with a large single-copy (LSC) region of 91,747 bp, a small single-copy (SSC) region of 22,550 bp, and a pair of identical inverted repeat regions (IRs) of 25,496 bp. The genome contained a total of 132 genes, including 86 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. The phylogenetic analysis of T. giganteum with 12 related species revealed the closest taxonomical relationship with Pinellia pedatisecta in the Araceae.

The complete chloroplast genome of Hemerocallis Fulva.

We have sequenced the complete chloroplast genome of Hemerocallis fulva, a species of the Asphodelaceae family, through Illumina HiSeq paired-end sequencing. The total size of chloroplast genome of Hemerocallis fulva was 155,855 bp with a large single-copy (LSC) region of 84,607 bp, a small single-copy (SSC) region of 18,508 bp, and a pair of identical inverted repeat regions (IRs) of 26,370 bp. The genome contained a total of 112 genes, including 79 protein-coding genes, 29 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The phylogenetic analysis of Hemerocallis fulva with 10 related species exhibited the closest taxonomical relationship with Aloe species in the Asphodelaceae family.

The complete chloroplast genome of Codonopsis lanceolata (Campanulaceae).

The complete chloroplast genome sequence of Codonopsis lanceolata was determined by next generation sequencing. The total length of chloroplast genome of C. lanceolata was 169,447 bp long, including a large single-copy (LSC) region of 85,253 bp, a small single-copy (SSC) region of 8060 bp, and a pair of identical inverted repeat regions (IRs) of 38,067 bp. A total of 110 genes was annotated, resulting in 79 protein-coding genes, 27 tRNA genes, and 4 rRNA genes. The phylogenetic analysis of C. lanceolata with related chloroplast genome sequences in this study provided the taxonomical relationship of C. lanceolata in the genus Campanula.

The complete chloroplast genome of Caltha Palustris (Ranunculaceae).

The complete chloroplast genome sequence of Caltha palustris, a species of the Ranunculaceae family, was characterized from the de novo assembly of HiSeq (Illumina Co.) paired-end sequencing data. The chloroplast genome of C. palustris was 155,292 bp in length, with a large single-copy (LSC) region of 84,120 bp, a small single-copy (SSC) region of 18,342 bp, and a pair of identical inverted repeat regions (IRs) of 26,415 bp. The genome contained a total of 114 genes, including 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The phylogenetic analysis of C. palustris with 14 related species revealed the closest taxonomical relationship with Hydrastis canadensis in the Ranunculaceae family.

Assessment of potential environmental risks of transgene flow in smallholder farming systems in Asia: Brassica napus as a case study in Korea.

The cultivation of genetically modified (GM) crops has raised many questions regarding their environmental risks, particularly about their ecological impact on non-target organisms, such as their closely-related relative species. Although evaluations of transgene flow from GM crops to their conventional crops has been conducted under large-scale farming system worldwide, in particular in North America and Australia, few studies have been conducted under smallholder farming systems in Asia with diverse crops in co-existence. A two-year field study was conducted to assess the potential environmental risks of gene flow from glufosinate-ammonium resistant (GR) Brassica napus to its conventional relatives, B. napus, B. juncea, and Raphanus sativus under simulated smallholder field conditions in Korea. Herbicide resistance and simple sequence repeat (SSR) markers were used to identify the hybrids. Hybridization frequency of B. napus × GR B. napus was 2.33% at a 2 m distance, which decreased to 0.007% at 75 m. For B. juncea, it was 0.076% at 2 m and decreased to 0.025% at 16 m. No gene flow was observed to R. sativus. The log-logistic model described hybridization frequency with increasing distance from GR B. napus to B. napus and B. juncea and predicted that the effective isolation distances for 0.01% gene flow from GR B. napus to B. napus and B. juncea were 122.5 and 23.7 m, respectively. Results suggest that long-distance gene flow from GR B. napus to B. napus and B. juncea is unlikely, but gene flow can potentially occur between adjacent fields where the smallholder farming systems exist.

Evaluation of maximum potential gene flow from herbicide resistant Brassica napus to its male sterile relatives under open and wind pollination conditions.

Pollen-mediated gene flow (PMGF) from genetically modified (GM) Brassica napus to its wild relatives by wind and insects is a major ecological concern in agricultural ecosystems. This study conducted is to estimate maximum potential gene flow and differentiate between wind- and bee-mediated gene flows from herbicide resistant (HR) B. napus to its closely-related male sterile (MS) relatives, B. napus, B. juncea and Raphanus sativus. Various markers, including pods formation in MS plants, herbicide resistance, and SSR markers, were used to identify the hybrids. Our results revealed the following: 1) maximum potential gene flow (a maximum % of the progeny of pollen recipient confirmed hybrid) to MS B. napus ranged from 32.48 to 0.30% and from 14.69 to 0.26% at 2-128 m from HR B. napus under open and wind pollination conditions, respectively, and to MS B. juncea ranged from 21.95 to 0.24% and from 6.16 to 0.16%, respectively; 2) estimates of honeybee-mediated gene flow decreased with increasing distance from HR B. napus and ranged from 17.78 to 0.03% at 2-128 m for MS B. napus and from 15.33 to 0.08% for MS B. juncea; 3) a small-scale donor plots would strongly favour insect over wind pollination; 4) no gene flow occurred from HR B. napus to MS R. sativus. Our approach and findings are helpful in understanding the relative contribution of wind and bees to gene flow and useful for estimating maximum potential gene flow and managing environmental risks associated with gene flow.

Development of a Genetic Map for Onion (Allium cepa L.) Using Reference-Free Genotyping-by-Sequencing and SNP Assays.

Single nucleotide polymorphisms (SNPs) play important roles as molecular markers in plant genomics and breeding studies. Although onion (Allium cepa L.) is an important crop globally, relatively few molecular marker resources have been reported due to its large genome and high heterozygosity. Genotyping-by-sequencing (GBS) offers a greater degree of complexity reduction followed by concurrent SNP discovery and genotyping for species with complex genomes. In this study, GBS was employed for SNP mining in onion, which currently lacks a reference genome. A segregating F2 population, derived from a cross between 'NW-001' and 'NW-002,' as well as multiple parental lines were used for GBS analysis. A total of 56.15 Gbp of raw sequence data were generated and 1,851,428 SNPs were identified from the de novo assembled contigs. Stringent filtering resulted in 10,091 high-fidelity SNP markers. Robust SNPs that satisfied the segregation ratio criteria and with even distribution in the mapping population were used to construct an onion genetic map. The final map contained eight linkage groups and spanned a genetic length of 1,383 centiMorgans (cM), with an average marker interval of 8.08 cM. These robust SNPs were further analyzed using the high-throughput Fluidigm platform for marker validation. This is the first study in onion to develop genome-wide SNPs using GBS. The resulting SNP markers and developed linkage map will be valuable tools for genetic mapping of important agronomic traits and marker-assisted selection in onion breeding programs.

Transcriptome Analysis of Spartina pectinata in Response to Freezing Stress.

Prairie cordgrass (Spartina pectinata), a perennial C4 grass native to the North American prairie, has several distinctive characteristics that potentially make it a model crop for production in stressful environments. However, little is known about the transcriptome dynamics of prairie cordgrass despite its unique freezing stress tolerance. Therefore, the purpose of this work was to explore the transcriptome dynamics of prairie cordgrass in response to freezing stress at -5°C for 5 min and 30 min. We used a RNA-sequencing method to assemble the S. pectinata leaf transcriptome and performed gene-expression profiling of the transcripts under freezing treatment. Six differentially expressed gene (DEG) groups were categorized from the profiling. In addition, two major consecutive orders of gene expression were observed in response to freezing; the first being the acute up-regulation of genes involved in plasma membrane modification, calcium-mediated signaling, proteasome-related proteins, and transcription regulators (e.g., MYB and WRKY). The follow-up and second response was of genes involved in encoding the putative anti-freezing protein and the previously known DNA and cell-damage-repair proteins. Moreover, we identified the genes involved in epigenetic regulation and circadian-clock expression. Our results indicate that freezing response in S. pectinata reflects dynamic changes in rapid-time duration, as well as in metabolic, transcriptional, post-translational, and epigenetic regulation.

Complete chloroplast genomes of two Miscanthus species.

The complete chloroplast (cp) genomes of two Miscanthus species, M. sinensis and M. sacchariflorus, were sequenced and investigated for genes, genome size variation, and polymorphisms. There are 170 genes in both cp genomes, consisting of 122 mRNA genes (84 protein-coding genes and 38 hypothetical genes), 40 tRNA genes, and 8 rRNA genes. The cp genome contains two inverted repeat (IR) regions, separated by large single copy (LSC) region and small single copy (SSC) region. Indels were responsible for 40 bp difference in cp genome size in two species. In addition, we established phylogenetic relationship with other monocot cp genomes, and estimated divergence time. The two Miscanthus species clustered together among other C4 monocot species and the divergence time of two Miscanthus species was approximately 0.5 1-0.84 Mya.