Integrative approaches to a revision of the liverwort in genus Aneura (Aneuraceae, Marchantiophyta) from Thailand.

Background: The genus Aneura Dumort. is a simple thalloid liverwort with cosmopolitan distributions. Species circumscription is problematic in this genus due to a limited number of morphological traits. Two species are currently reported from Thailand, including A. maxima and A. pinguis. At the global scale, A. pinguis is considered a cryptic species, as the species contains several distinct genetic groups without clear morphological differentiation. At the same time, the identity of A. maxima remains unclear. In this work, we examined the level of diversity of Aneura species found in Thailand using both morphological and molecular data. Methods: We measured the morphological traits and generated the molecular data (four markers: trnL-F, trnH-psbA, rbcL, and ITS2) from the Thai specimens. The concatenated dataset was then used to reconstruct phylogeny. Species delimitation with GMYC, bPTP, ASAP, and ABGD methods was performed to estimate the number of putative species within the genus. Results: The samples of A. pinguis formed several clades, while A. maxima sequences from Poland were grouped in their clade and nested within another A. pinguis clade. We could not recover a sample of A. maxima from Thailand, even from the reported locality. Two putative species were detected among Thai Aneura samples. However, no morphological trait could distinguish the specimens from the two observed genetic groups. Discussion: The previously observed paraphyletic nature of A. pinguis globally was also found among Thai samples, including several putative species. However, we could not confirm the identity of A. maxima from Thai specimens. The previous report could result from misidentification and problematic species circumscription within Aneura. The results highlighted the need to include multiple lines of evidence for the future taxonomic investigation of the group.

Disruption of a DUF247 Containing Protein Alters Cell Wall Polysaccharides and Reduces Growth in Arabidopsis.

Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of At3g50120 (DUF247-1), a member of the DUF247 multigene family containing 28 genes in Arabidopsis, results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction. Furthermore, the polymer sizes of XyGs and GXs are reduced with concomitant increases in short-chain polymers, while those of HGs and mHGs are slightly increased. Complementation using 35S:DUF247-1 partially recovers the XyG and HG content, but not those of GX and HM, suggesting that DUF247-1 is more closely associated with XyGs and HGs. DUF247-1 is expressed throughout Arabidopsis, particularly in vascular and developing tissues, and its disruption affects the expression of other gene members, indicating a regulatory control role within the gene family. Our results demonstrate that DUF247-1 is required for normal cell wall composition and structure and Arabidopsis growth.

Dynamic Alteration of Microbial Communities of Duckweeds from Nature to Nutrient-Deficient Condition.

Duckweeds live with complex assemblages of microbes as holobionts that play an important role in duckweed growth and phytoremediation ability. In this study, the structure and diversity of duckweed-associated bacteria (DAB) among four duckweed subtypes under natural and nutrient-deficient conditions were investigated using V3-V4 16S rRNA amplicon sequencing. High throughput sequencing analysis indicated that phylum Proteobacteria was predominant in across duckweed samples. A total of 24 microbial genera were identified as a core microbiome that presented in high abundance with consistent proportions across all duckweed subtypes. The most abundant microbes belonged to the genus Rhodobacter, followed by other common DAB, including Acinetobacter, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, and Pseudomonas. After nutrient-deficient stress, diversity of microbial communities was significantly deceased. However, the relative abundance of Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Pelomonas, Roseateles and Novosphingobium were significantly enhanced in stressed duckweeds. Functional prediction of the metagenome data displayed the relative abundance of essential pathways involved in DAB colonization, such as bacterial motility and biofilm formation, as well as biodegradable ability, such as benzoate degradation and nitrogen metabolism, were significantly enriched under stress condition. The findings improve the understanding of the complexity of duckweed microbiomes and facilitate the establishment of a stable microbiome used for co-cultivation with duckweeds for enhancement of biomass and phytoremediation under environmental stress.

Anthranilic Acid Accumulation in Saccharomyces cerevisiae Induced by Expression of a Nonribosomal Peptide Synthetase Gene from Paecilomyces cinnamomeus BCC 9616.

Heterologous expression of nrps33, a nonribosomal peptide synthetase gene, from Paecilomyces cinnamomeus BCC 9616 in Saccharomyces cerevisiae unexpectedly resulted in the accumulation of anthranilic acid, an intermediate in tryptophan biosynthesis. Based on transcriptomic and real-time quantitative polymerase chain reaction (RT-qPCR) results, expression of nrps33 affected the transcription of tryptophan biosynthesis genes especially TRP1 which is also the selectable auxotrophic marker for the expression vector used in this work. The product of nrps33 could inhibit the activity of Trp4 involved in the conversion of anthranilate to N-(5'-phosphoribosyl)anthranilate and therefore caused the accumulation of anthranilic acid. This accumulation could in turn result in down-regulation of downstream tryptophan biosynthesis genes. Anthranilic acid is typically produced by chemical synthesis and has been used as a substrate for synthesising bioactive compounds including commercial drugs; our results could provide a new biological platform for production of this compound.

Gel purification of gDNA for next-generation sequencing applications.

We demonstrate that gDNA can be conveniently and efficiently isolated and purified using standard agarose gel electrophoresis, band excision and gel purification. This method yields a substantial amount at microgram levels of gDNA per gel cleanup with high purity. An RNase A treatment step can be omitted. The quality of gDNA is suitable for next-generation sequencing, resulting in >10 Mb reads and high-quality read data (Phred score >28 up to 100 of 150 base reads). Furthermore, the gDNA can be kept intact in a gel slice for several days. This method has been tested for dictyostelids, bacteria and plants.

Cassava root crown phenotyping using three-dimension (3D) multi-view stereo reconstruction.

Phenotypic analysis of cassava root crowns (CRCs) so far has been limited to visual inspection and very few measurements due to its laborious process in the field. Here, we developed a platform for acquiring 3D CRC models using close-range photogrammetry for phenotypic analysis. The state of the art is a low cost and easy to set up 3D acquisition requiring only a background sheet, a reference object and a camera, compatible with field experiments in remote areas. We tested different software with CRC samples, and Agisoft and Blender were the most suitable software for generating high-quality 3D models and data analysis, respectively. We optimized the workflow by testing different numbers of images for 3D reconstruction and found that a minimum of 25 images per CRC can provide high quality 3D models. Up to ten traits, including 3D crown volumes, 3D crown surface, root density, surface-to-volume ratio, root numbers, root angle, crown diameter, cylinder soil volume, CRC compactness and root length can be extracted providing novel parameters for studying cassava storage roots. We applied this platform to partial-inbred cassava populations and demonstrated that our platform provides reliable 3D CRC modelling for phenotypic analysis, analysis of genetic variances and supporting breeding selection.

Exploration of microbial communities in the guts and casts of Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida.

Earthworms and their casts have been widely used for organic waste degradation and plant growth promotion. The microbial communities that reside in the guts and casts of earthworms markedly influence both applications. In the present study, next-generation sequencing was applied to identify the microbial communities in the guts and casts of three epigeic earthworm species, Eudrilus eugeniae, Perionyx excavatus, and Eisenia fetida, reared under two different feeding conditions. A total of 580 genera belonging to 43 phyla were identified. By comparing bacterial diversity among samples divided into groups based on the earthworm species, sample types, and conditions, the beta diversity analysis supported the impact of the sample type and suggested that there was significant dissimilarity of the bacterial diversity between the gut and cast. Besides, bacterial Phylum compositions within the group were compared. The result showed that the top three high relative frequency phyla found in the casts were the same regardless of earthworm species, while those found in the gut depended on both the condition and earthworm species. Focusing on the cellulolytic and plant growth-promoting bacteria, certain cellulolytic bacteria, Paenibacillus, Comamonas, and Cytophaga, were found only in the cast. Citrobacter and Streptomyces aculeolatus were detected only in the guts of earthworms reared in the bedding containing vegetables and bedding alone, respectively. Besides, Actinomadura and Burkholderia were detected only in the gut of E. eugeniae and E. fetida, respectively. The results proved that the microbial composition was affected by sample type, condition, and earthworm species. In addition, the proportion of these beneficial bacteria was also influenced by these factors. Hence, the information from this study can be used as a guide for selecting earthworm species or their casts for more efficient organic waste decomposition and plant growth promotion.

The complete mitochondrial genome of Dictyostelium intermedium.

Dictyostelium intermedium is a member of dictyostelids, the unicellular eukaryotes with a unique life cycle, including a social cycle. Despite the high diversity of dictyostelids, only five species' complete mitochondrial genome sequences were reported. This study aimed to add the D. intermedium mitochondrial genome sequence to the list. The size of this genome is 58,627 bp, with 73.99% A/T, containing 62 genes located on one strand: 41 protein-coding genes, three ribosomal RNA genes, and 18 transfer RNA genes. The 41 protein-coding genes comprised 18 oxidative phosphorylation-related, 16 ribosomal, and seven hypothetical protein-coding genes. The cox1/2 and rnl gene contained introns, similar to other species of Dictyostelium. The phylogenetic tree built based on 34 protein sequences supported the monophyletic clade of Dictyostelium and the dictyostelids' ancestor's position between the two dictyostelids orders: Dictyosteliales and Acytosteliales.

Gel-permeation chromatography-enzyme-linked immunosorbent assay method for systematic mass distribution profiling of plant cell wall matrix polysaccharides.

Cell walls are dynamic and multi-component materials that play important roles in many areas of plant biology. The composition and interactions of the structural elements give rise to material properties, which are modulated by the activity of wall-related enzymes. Studies of the genes and enzymes that determine wall composition and function have made great progress, but rarely take account of potential compensatory changes in wall polymers that may accompany and accommodate changes in other components, particularly for specific polysaccharides. Here, we present a method that allows the simultaneous examination of the mass distributions and quantities of specific cell wall matrix components, allowing insight into direct and indirect consequences of cell wall manipulations. The method employs gel-permeation chromatography fractionation of cell wall polymers followed by enzyme-linked immunosorbent assay to identify polymer types. We demonstrate the potential of this method using glycan-directed monoclonal antibodies to detect epitopes representing xyloglucans, heteromannans, glucuronoxylans, homogalacturonans (HGs) and methyl-esterified HGs. The method was used to explore compositional diversity in different Arabidopsis organs and to examine the impacts of changing wall composition in a number of previously characterized cell wall mutants. As demonstrated in this article, this methodology allows a much deeper understanding of wall composition, its dynamism and plasticity to be obtained, furthering our knowledge of cell wall biology.

Phylogenetic and recombinant analyses of complete coding sequences of DENV-1 from field-caught mosquitoes in Thailand.

Dengue diseases are a group of infectious diseases that have been widespread throughout the world for several decades. Dengue outbreaks have occurred in many parts of the world, including Southeast Asia. The outbreak and the severity of the diseases depend on many factors including dengue genotype. Data on the genetic variation of dengue virus is highly informative for dengue protection plans and vaccine development. In this study, we focused on the analyses of genetic variation and amino acid changes of the whole coding sequences in two dengue strains isolated from Aedes mosquitoes in Bangkok, Thailand, an endemic area. The strains were identified as belonging to dengue virus serotype 1 (DENV-1) genotype I, and have unique nucleotide sequences. In the recombinant analysis, these strains were identified as recombinants derived from Chinese counterparts for both the major (DENV-1 genotype I) and the minor (DENV-1 genotype IV) parental strains. The recombination event occurred within the prM and E genes. This corresponded with the result of multiple alignments where several amino acid residues between the recombination breakpoints in the strains were identical to those in DENV-1 genotype IV. Several of the amino acid substitutions also have changed the amino acid properties, which might affect viral infection and antigenicity. These results provide insight into the genetic variation of DENV in this endemic area, which might have been involved in the dengue outbreak and high numbers of dengue haemorrhagic fever and dengue shock syndrome cases in Thailand in 2015.

RNA editing in the chloroplast of Asian Palmyra palm (Borassus flabellifer).

We have identified 46 RNA editing sites located in 20 chloroplast (cp) genes of Borassus flabellifer (Asian Palmyra palm), family Arecaceae, and tested these genes for supporting phylogenetic study among the commelinids. Among the 46 sites, 43 sites were found to cause amino acid alterations, which were predicted to increase the hydrophobicity and transmembrane regions of the proteins, and one site was to cause a premature stop codon. Analysis of these editing sites with data obtained from seed plants showed that a number of shared-editing sites depend on the evolutionary relationship between plants. We reconstructed a deep phylogenetic relationship among the commelinids using seven RNA edited genes that are orthologous among monocots. This tree could represent the relationship among subfamilies of Arecaceae family, but was insufficient to represent the relationship among the orders of the commelinid. After adding eight gene sequences with high parsimony-informative characters (PICs), the tree topology was improved and could support the topology for the commelinid orders ((Arecales,Dasypogenaceae) (Zingiberales+Commelinales,Poales)). The result provides support for inherent RNA editing along the evolution of seed plants, and we provide an alternative set of loci for the phylogenetic tree reconstruction of Arecaceae's subfamilies.

Portunus pelagicus mtDNA heteroplasmy inheritance and its effect on the use of mtCR and mtCOI sequence data.

Mitochondrial DNA (mtDNA) sequences, especially mitochondrial control region (mtCR) and mitochondrial cytochrome c oxidase subunit I (mtCOI), have been widely used in population and evolutionary genetic analyses of metazoan. The presence of mtDNA heteroplasmy - a mixture of mtDNA haplotypes - possibly affects these analyses. This study aimed to reveal mtDNA heteroplasmy in mtCR, mtCOI, and mtND2 (mitochondrial NADH dehydrogenase subunit 2) of Portunus pelagicus, and examine its effect on the use of mtCR and mtCOI sequences. The screening result showed that the probability of observing mtDNA heteroplasmy was approximately 8%. Across the three targeted regions, 92 heteroplasmic variants were observed from seven samples comprising three mothers and four offspring. Most inherited heteroplasmy presented transition and silence mutation. By comparing the proportion of shared variants among maternal relatives to that among non-relatives, the result suggested that most heteroplasmic variants observed in an individual are inherited. Statistical analyses carried out on the inter-generational differences suggested that random drift and purifying selection play roles in determining the offspring's heteroplasmy level. The size of the random shift varies according to the location of variants and the mothers. The phylogenetic analysis showed that the presence of mtDNA heteroplasmy in mtCR and mtCOI does not affect familial and species identification, respectively. This study firstly reported the mtDNA heteroplasmy in P. pelagicus, its inheritance pattern, and its effect on the use of mtDNA sequence data. This basic knowledge would be useful for the study based on mtDNA sequence data, especially in other invertebrates.

Towards sex identification of Asian Palmyra palm (Borassus flabellifer L.) by DNA fingerprinting, suppression subtractive hybridization and de novo transcriptome sequencing.

BACKGROUND: Asian Palmyra palm, the source of palm-sugar, is dioecious with a long juvenile period requiring at least 12 years to reach its maturity. To date, there is no reliable molecular marker for identifying sexes before the first bloom, limiting crop designs and utilization. We aimed to identify sex-linked markers for this palm using PCR-based DNA fingerprinting, suppression subtractive hybridization (SSH) and transcriptome sequencing. METHODS: DNA fingerprints were generated between males and females based on RAPD, AFLP, SCoT, modified SCoT, ILP, and SSR techniques. Large-scale cloning and screening of SSH libraries and de novo transcriptome sequencing of male and female cDNA from inflorescences were performed to identify sex-specific genes for developing sex-linked markers. RESULTS: Through extensive screening and re-testing of the DNA fingerprints (up to 1,204 primer pairs) and transcripts from SSH (>10,000 clones) and transcriptome data, however, no sex-linked marker was identified. Although de novo transcriptome sequencing of male and female inflorescences provided ∼32 million reads and 187,083 assembled transcripts, PCR analysis of selected sex-highly represented transcripts did not yield any sex-linked marker. This result may suggest the complexity and small sex-determining region of the Asian Palmyra palm. To this end, we provide the first global transcripts of male and female inflorescences of Asian Palmyra palm. Interestingly, sequence annotation revealed a large proportion of transcripts related to sucrose metabolism, which corresponds to the sucrose-rich sap produced in the inflorescences, and these transcripts will be useful for further understanding of sucrose production in sugar crop plants. Provided lists of sex-specific and differential-expressed transcripts would be beneficial to the further study of sexual development and sex-linked markers in palms and related species.

Origin of prehistoric cattle excavated from four archaeological sites in central and northeastern Thailand.

Cattle have been domesticated in Southeast Asia, including Thailand, for thousands of years, but the history of cattle domestication in the region remains unclear. To date, only genetic studies of modern Thai cattle DNA have been reported. To gain some insight into cattle domestication in the country, a total of 56 cattle remains excavated from four archaeological sites (dated to between 3550 and 1700 years before present (YBP)) in northeastern and central Thailand were analysed in this study. Of 56, the 157-bp D-loop fragment was successfully generated from 26 samples, all of which belonged to Bos taurus in haplogroup T/T3. One haplotype contained 19 members from all four archaeological sites and clustered with the ancient B. taurus from Iran, Turkey and China. Other haplotypes have not shared haplotype with B. taurus from other countries but they showed close relationship to those from China. This represents the first genetic evidence that B.taurus was domesticated in Thailand between 3550 and 1700 YBP. In addition, the close relationship among ancient Thai, Iranian and Chinese taurines suggests that cattle from the Near East were introduced into North China, and were subsequently brought into Thailand thousands of years ago.

Comparative mitochondrial genome analysis of the firefly, Inflata indica (Coleoptera: Lampyridae) and the first evidence of heteroplasmy in fireflies.

The first mitochondrial genome of the firefly genus Inflata Boontop, Inflata indica (Lampyridae, Luciolinae) is reported here. Thirteen protein coding genes, 22 tRNA, 2 rRNA genes and a 1570 bp non-coding region were annotated. The comparative analysis showed that the size, GC content, gene content and gene arrangement of this mitochondrial genome are similar to those of other Lampyridae. The mitochondrial phylogenomic analysis showed that I. indica is clustered with other fireflies belonging to the subfamily Luciolinae and most closely related to Pteroptyx maipo. The evolutionary relationship within group of Luciolinae fireflies presented by complete COI phylogeny differs from the relationship based on the mitochondrial phylogenomic tree. This individual firefly also carries mitochondrial DNA (mtDNA) heteroplasmy; however, no potential effect of heteroplasmic mutations could be observed in this study. Hence, this study is reporting not only the first mitochondrial genome of the genus Inflata and the comparative mitochondrial genome analysis of fireflies but also the first mitochondrial DNA heteroplasmy in Lampyridae.

Heterologous biosynthesis of a fungal macrocyclic polylactone requires only two iterative polyketide synthases.

Menisporopsin A is a bioactive macrocyclic polylactone produced by the fungus Menisporopsis theobromae BCC 4162. A scheme for the biosynthesis of this compound has been proposed, in which reducing (R) and non-reducing (NR) polyketide synthases (PKSs) would catalyze the formation of each menisporopsin A subunit, while an additional non-ribosomal peptide synthetase (NRPS)-like enzyme would be required to perform multiple esterification and cyclolactonization reactions. Transcriptome analysis of M. theobromae identified an R-PKS gene, men1, and an NR-PKS gene, men2, which both exhibited highest expression levels during the menisporopsin A production phase. These were cloned into separate vectors for heterologous expression in Aspergillus oryzae NSAR1. Unexpectedly, coexpression of the two PKSs alone was sufficient to catalyze the formation of the macrocyclic polylactone, ascotrichalactone A, a structural derivative of menisporopsin A. The unanticipated esterification and cyclolactonization activities could reside in the unusual thioesterase domain of the NR-PKS, which is similar to that of the NRPS catalyzing elongation and cyclization of trilactone in enterobactin biosynthesis and that of modular PKSs catalyzing macrodiolide formation in elaiophylin and conglobatin biosyntheses.

Phylogenetic analyses of DENV-3 isolated from field-caught mosquitoes in Thailand.

Dengue virus serotype 3 (DENV-3) can cause all forms of dengue diseases and is a predominant serotype in many countries. This serotype is classified into five genotypes: I-V. Genotypes I-III have widely spread throughout the world, whereas genotypes IV and V are rare. Despite the impact on the spread of dengue diseases, only a few studies have reported the characteristics of DENV present in mosquito vectors. Hence, this study aimed to identify DENV-3 genotypes and reveal genetic variation of this virus presented in field-caught mosquitoes collected from endemic areas in Thailand during 2011-2015. First, we examined the effectiveness of the E gene sequence on DENV-3 genotyping, with results supporting the use of this gene for genotype identification. Then, we sequenced this gene in ten DENV-3 strains isolated from mosquitoes. The results showed that eight and two samples were genotypes III and V, respectively, and that they are closely related to DENV-3 isolated from Southeast and East Asian samples. The translated E gene sequences showed 25 unique amino acid (AA) residues located at 23 positions. Eight out of 25 residues have different chemical properties compared to the conserved AAs that are distributed across the three domains functioning in virus-host interaction. Hence, our study reports the first DENV-3 genotype V in Thailand, with these viruses potentially influencing both the disease severity and epidemic potential of DENV-3.

The complete chloroplast genome sequence of Asian Palmyra palm (Borassus flabellifer).

OBJECTIVE: Borassus flabellifer or Asian Palmyra palm is widely distributed in South and Southeast Asia and is horticultural and economic importance for its fruit and palm sugar production. However, its population is in rapid decline, and only a few genetic data are available. We sequenced the complete chloroplast (cp) genome of B. flabellifer to provide its genetic data for further utilization. RESULTS: The cp genome was obtained by Illumina sequencing and manual gap fillings providing 160,021 bp in length containing a pair of inverted repeats (IRs) with 27,256 bp. These IRs divide the genome into a large single copy region 87,444 bp and a small single copy region 18,065 bp. In total, 113 unique genes, 134 SSRs and 47 large repeats were identified. This is the first complete cp genome reported in the genus Borassus. A comparative analysis among members of the Borasseae tribe revealed that the B. flabellifer cp genome is, so far, the largest and the cp genomes of this tribe have a similar structure, gene number and gene arrangement. A phylogenetic tree reconstructed based on 74 protein-coding genes from 70 monocots demonstrates short branch lengths indicating slow evolutionary rates of cp genomes in family Arecaceae.

Complete coding sequence of dengue virus serotype 4 isolated from field-caught mosquitoes in Thailand.

This report is the first to characterise the complete coding sequence of a dengue virus serotype 4 (DENV-4) genotype I that was isolated from field-caught mosquitoes from an endemic area in Thailand in June 2013. The sequence was assembled from high-throughput sequencing reads generated by Illumina HiSeq. Three out of four observed intra-sample variants caused an amino acid variation in C, NS2B, and NS5 genes. The C4279T variant located in the NS2B gene can indirectly affect the proteolytic activity of the NS3 protein. The sequence provided in this study might be useful for the epidemiological study of DENV-4.

Mitochondrial DNA sequence characteristics modulate the size of the genetic bottleneck.

With a combined carrier frequency of 1:200, heteroplasmic mitochondrial DNA (mtDNA) mutations cause human disease in ∼1:5000 of the population. Rapid shifts in the level of heteroplasmy seen within a single generation contribute to the wide range in the severity of clinical phenotypes seen in families transmitting mtDNA disease, consistent with a genetic bottleneck during transmission. Although preliminary evidence from human pedigrees points towards a random drift process underlying the shifting heteroplasmy, some reports describe differences in segregation pattern between different mtDNA mutations. However, based on limited observations and with no direct comparisons, it is not clear whether these observations simply reflect pedigree ascertainment and publication bias. To address this issue, we studied 577 mother-child pairs transmitting the m.11778G>A, m.3460G>A, m.8344A>G, m.8993T>G/C and m.3243A>G mtDNA mutations. Our analysis controlled for inter-assay differences, inter-laboratory variation and ascertainment bias. We found no evidence of selection during transmission but show that different mtDNA mutations segregate at different rates in human pedigrees. m.8993T>G/C segregated significantly faster than m.11778G>A, m.8344A>G and m.3243A>G, consistent with a tighter mtDNA genetic bottleneck in m.8993T>G/C pedigrees. Our observations support the existence of different genetic bottlenecks primarily determined by the underlying mtDNA mutation, explaining the different inheritance patterns observed in human pedigrees transmitting pathogenic mtDNA mutations.