De novo assembly and analysis of Sonneratia ovata genome and population analysis.

Mangroves are an important part of coastal and estuarine ecosystems where they serve as nurseries for marine species and prevent coastal erosion. Here we report the genome of Sonneratia ovata, which is a true mangrove that grows in estuarine environments and can tolerate moderate salt exposure. We sequenced the S. ovata genome and assembled it into chromosome-level scaffolds through the use of Hi-C. The genome is 212.3 Mb and contains 12 chromosomes that range in size from 12.2 to 23.2 Mb. Annotation identified 29,829 genes with a BUSCO completeness of 95.9%. We identified salt genes and found copy number expansion of salt genes such as ADP-ribosylation factor 1, and elongation factor 1-alpha. Population analysis identified a low level of genetic variation and a lack of population structure within S. ovata.

Identification of genes associated with the high-temperature fermentation trait in the Saccharomyces cerevisiae natural isolate BCC39850.

The fermentative model yeast Saccharomyces cerevisiae has been extensively used to study the genetic basis of stress response and homeostasis. In this study, we performed quantitative trait loci (QTL) analysis of the high-temperature fermentation trait of the progeny from the mating of the S. cerevisiae natural isolate BCC39850 (haploid#17) and the laboratory strain CEN.PK2-1C. A single QTL on chromosome X was identified, encompassing six candidate genes (GEA1, PTK2, NTA1, NPA3, IRT1, and IML1). The functions of these candidates were tested by reverse genetic experiments. Deletion mutants of PTK2, NTA1, and IML1 showed growth defects at 42 °C. The PTK2 knock-out mutant also showed significantly reduced ethanol production and plasma membrane H+ ATPase activity and increased sensitivity to acetic acid, ethanol, amphotericin B (AMB), and ß-1,3-glucanase treatment. The CRISPR-Cas9 system was used to construct knock-in mutants by replacement of PTK2, NTA1, IML1, and NPA3 genes with BCC39850 alleles. The PTK2 and NTA1 knock-in mutants showed increased growth and ethanol production titers at 42 °C. These findings suggest an important role for the PTK2 serine/threonine protein kinase in regulating plasma membrane H+ ATPase activity and the NTA1 N-terminal amidase in protein degradation via the ubiquitin-proteasome system machinery, which affects tolerance to heat stress in S. cerevisiae.

Genes controlling hydrolysate toxin tolerance identified by QTL analysis of the natural Saccharomyces cerevisiae BCC39850.

Lignocellulosic material can be converted to valorized products such as fuels. Pretreatment is an essential step in conversion, which is needed to increase the digestibility of the raw material for microbial fermentation. However, pretreatment generates by-products (hydrolysate toxins) that are detrimental to microbial growth. In this study, natural Saccharomyces strains isolated from habitats in Thailand were screened for their tolerance to synthetic hydrolysate toxins (synHTs). The Saccharomyces cerevisiae natural strain BCC39850 (toxin-tolerant) was crossed with the laboratory strain CEN.PK2-1C (toxin-sensitive), and quantitative trait locus (QTL) analysis was performed on the segregants using phenotypic scores of growth (OD600) and glucose consumption. VMS1, DET1, KCS1, MRH1, YOS9, SYO1, and YDR042C were identified from QTLs as candidate genes associated with the tolerance trait. CEN.PK2-1C knockouts of the VMS1, YOS9, KCS1, and MRH1 genes exhibited significantly greater hydrolysate toxin sensitivity to growth, whereas CEN.PK2-1C knock-ins with replacement of VMS1 and MRH1 genes from the BCC39850 alleles showed significant increased ethanol production titers compared with the CEN.PK2-1C parental strain in the presence of synHTs. The discovery of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin tolerance in S. cerevisiae indicates the roles of the endoplasmic-reticulum-associated protein degradation pathway, plasma membrane protein association, and the phosphatidylinositol signaling system in this trait. KEY POINTS: • QTL analysis was conducted using a hydrolysate toxin-tolerant S. cerevisiae natural strain • Deletion of VMS1, YOS9, MRH1, and KCS1 genes associated with hydrolysate toxin-sensitivity • Replacement of VMS1 and MRH1 with natural strain alleles increased ethanol production titers in the presence of hydrolysate toxins.

Assembly of a hybrid mangrove, Bruguiera hainesii, and its two ancestral contributors, Bruguiera cylindrica and Bruguiera gymnorhiza.

Mangroves are plants that live in tropical and subtropical coastal regions of the world, they are adapted to high salt environments and cyclic tidal flooding. Mangroves play important ecological roles, including acting as breeding grounds for many fish species and to prevent coastal erosion. The genomes of three mangrove species, Bruguiera gymnorhiza, Bruguiera cylindrica, and a hybrid of the two, Bruguiera hainesii were sequenced, assembled and annotated. The two progenitor species, B. gymnorhiza and B. cylindrica, were found to be highly similar to each other and sufficiently similar to B. parviflora to allow it to be used for reference based scaffolding to generate chromosome level scaffolds. The two subgenomes of B. hainesii were independently assembled and scaffolded. Analysis of B. hainesii confirms that it is a hybrid and the hybridisation event was estimated at 2.4 to 3.5 million years ago using a Bayesian Relaxed Molecular Clock approach.

De novo chromosome-level assembly of the Centella asiatica genome.

Centella asiatica is a herbaceous, perennial species indigenous to India and Southeast Asia. C. asiatica possesses several medicinal properties: anti-aging, anti-inflammatory, wound healing and memory enhancing. The lack of available genomics resources significantly impedes the improvement of C. asiatica varieties through molecular breeding. Here, we combined the 10× Genomics linked-read technology and the long-range HiC technique to obtain the genome assembly. The final assembly contained nine pseudomolecules, corresponding to the haploid chromosome number in C. asiatica. These nine chromosomes covered 402,536,584 bases or 93.6% of the 430-Mb assembly. Comparative genomics analyses based on single-copy orthologous genes showed that C. asiatica and the common ancestor of Coriandrum sativum (coriander) and Daucus carota (carrot) diverged about 48 million years ago. This assembly provides a valuable reference genome for future molecular studies, varietal development through marker-assisted breeding and comparative genomics studies in C. asiatica.

Taxonomic profiling of Symbiodiniaceae and bacterial communities associated with Indo-Pacific corals in the Gulf of Thailand using PacBio sequencing of full-length ITS and 16S rRNA genes.

Corals live with complex assemblages of microbes including bacteria, the dinoflagellate Symbiodiniaceae, fungi and viruses in a coral holobiont. These coral-associated microorganisms play an important role in their host fitness and survival. Here, we investigated the structure and diversity of algal and bacterial communities associated with five Indo-Pacific coral species, using full-length 16S rRNA and internal transcribed spacer sequences. While the dinoflagellate communities associated with Poriteslutea were dominated with Symbiodiniaceae genus Cladocopium, the other four coral hosts were associated mainly with members of the Durusdinium genus, suggesting that host species was one of the underlying factors influencing the structure and composition of dinoflagellate communities associated with corals in the Gulf of Thailand. Alphaproteobacteria dominated the microbiomes of Pocillopora spp. while Pavonafrondifera and P. lutea were associated primarily with Gammaproteobacteria. Finally, we demonstrated a superior performance of full-length 16S rRNA sequences in achieving species-resolution taxonomic classification of coral-associated microbiota.

Genome-wide SNP discovery and identification of QTL associated with agronomic traits in oil palm using genotyping-by-sequencing (GBS).

Oil palm has become one of the most important oil crops in the world. Marker-assisted selections have played a pivotal role in oil palm breeding programs. Here, we report the use of genotyping-by-sequencing (GBS) approach for a large-scale SNP discovery and genotyping of a mapping population. Reduced representation libraries of 108 F2 progeny were sequenced and a total of 524 million reads were obtained. We detected 21,471 single nucleotide substitutions, most of which (62.6%) represented transition events. Of 3417 fully informative SNP markers, we were able to place 1085 on a linkage map, which spanned 1429.6 cM and had an average of one marker every 1.26 cM. Three QTL affecting trunk height were detected on LG 10, 14 and 15, whereas a single QTL associated with fruit bunch weight was identified on LG 3. The use of GBS approach proved to be rapid, cost-effective and highly reproducible in this species.

The AKR gene family and modifying sex ratios in palms through abiotic stress responsiveness.

Sex ratio (SR), the ratio of female inflorescences to total inflorescences, is one of the main yield components of oil palm (Elaeis guineensis Jacq). The SR quantitative trait locus (QTL) was recently identified on linkage (LG) 8 with a phenotype variance explained (PVE) of 11.3 %. The use of both genetic and physical mapping is one strategy for uncovering the genetic basis of the traits. Here, we report the construction of bacterial artificial chromosome (BAC) and fosmid libraries, and their use for physical mapping in oil palm. Combined, the libraries consist of more than 200,000 clones, representing 6.35 genome equivalents. Physical mapping at the SR locus was implemented by incorporating the published oil palm genome sequence and positive BAC/fosmid clones as identified by colony PCR screening. Based on the previously published sequences, the interval (about 184 kb) was comprised of 19 contigs of the known sequences (~117 kb, 64 %). After, combining the 454 pyrosequences of 15 positive clones and the previously published sequences, the known sequences were revealed to cover about 82 % of the interval (~150 kb), and were used for identifying the new markers by designing 35 gene-based and 23 simple sequence repeat (SSR)-amplified primers. As a result, a putative aldo-keto reductase gene (named EgAKR1) was revealed to be a promising candidate for sex ratio determination, via controlling female inflorescence number (11 % of PVE). This was predicted from the two newly identified polymorphic marker loci (mEgSSRsr8-21LB and mEgAKR1-9) designing from EgAKR1. The functions of AKR gene families in other plant species and our promoter analysis suggested that EgAKR1 may contribute to the sex ratio through abiotic stress responsiveness.

Assembly and analysis of a male sterile rubber tree mitochondrial genome reveals DNA rearrangement events and a novel transcript.

BACKGROUND: The rubber tree, Hevea brasiliensis, is an important plant species that is commercially grown to produce latex rubber in many countries. The rubber tree variety BPM 24 exhibits cytoplasmic male sterility, inherited from the variety GT 1. RESULTS: We constructed the rubber tree mitochondrial genome of a cytoplasmic male sterile variety, BPM 24, using 454 sequencing, including 8 kb paired-end libraries, plus Illumina paired-end sequencing. We annotated this mitochondrial genome with the aid of Illumina RNA-seq data and performed comparative analysis. We then compared the sequence of BPM 24 to the contigs of the published rubber tree, variety RRIM 600, and identified a rearrangement that is unique to BPM 24 resulting in a novel transcript containing a portion of atp9. CONCLUSIONS: The novel transcript is consistent with changes that cause cytoplasmic male sterility through a slight reduction to ATP production efficiency. The exhaustive nature of the search rules out alternative causes and supports previous findings of novel transcripts causing cytoplasmic male sterility.

Comparative Proteomic Analysis of Ridge Gourd Seed (Luffa acutangula (L.) Roxb.) during Artificial Aging.

Seed aging is a complicated process influenced by environmental conditions, impacting biochemical processes in seeds and causing deterioration that results in reduced viability and vigor. In this study, we investigated the seed aging process of ridge gourd, which is one of the most exported commercial seeds in Thailand using sequential window acquisition of all theoretical fragment ion spectra mass spectrometry. A total of 855 proteins were identified among the two groups (0 d/15 d and 0 d/30 d). The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses of differentially expressed proteins revealed that in ridge gourd seeds, the aging process altered the abundance of proteins related to the oxidative stress response, nutrient reservoir, and metabolism pathway. The most identified DEPs were mitochondrial proteins, ubiquitin-proteasome system proteins, ribosomal proteins, carbohydrate metabolism-related proteins, and stress response-related proteins. This study also presented the involvement of aconitase and glutathione pathway-associated enzymes in seed aging, with aconitase and total glutathione being determined as possible suggestive biomarkers for aged ridge gourd seeds. This acquired knowledge has the potential to considerably improve growing methods and seed preservation techniques, enhancing seed storage and maintenance.

A chromosome-level reference genome assembly and a full-length transcriptome assembly of the giant freshwater prawn (Macrobrachium rosenbergii).

The giant freshwater prawn (Macrobrachium rosenbergii) is a key species in the aquaculture industry in several Asian, African, and South American countries. Despite a considerable growth in its production worldwide, the genetic complexities of M. rosenbergii various morphotypes pose challenges in cultivation. This study reports the first chromosome-scale reference genome and a high-quality full-length transcriptome assembly for M. rosenbergii. We employed the PacBio High Fidelity (HiFi) sequencing to obtain an initial draft assembly and further scaffolded it with the chromatin contact mapping (Hi-C) technique to achieve a final assembly of 3.73-Gb with an N50 scaffold length of 33.6 Mb. Repetitive elements constituted nearly 60% of the genome assembly, with simple sequence repeats and retrotransposons being the most abundant. The availability of both the chromosome-scale assembly and the full-length transcriptome assembly enabled us to thoroughly probe alternative splicing events in M. rosenbergii. Among the 2,041 events investigated, exon skipping represented the most prevalent class, followed by intron retention. Interestingly, specific isoforms were observed across multiple tissues. Additionally, within a single tissue type, transcripts could undergo alternative splicing, yielding multiple isoforms. We believe that the availability of a chromosome-level reference genome for M. rosenbergii, along with its full-length transcriptome, will be instrumental in advancing our understanding of the giant freshwater prawn biology and enhancing its molecular breeding programs, paving the way for the development of M. rosenbergii with valuable traits in commercial aquaculture.

A de novo chromosome-scale assembly of the Lablab purpureus genome.

Introduction: Lablab (Lablab purpureus (L.) Sweet), an underutilized tropical legume crop, plays a crucial role in global food and nutritional security. To enhance our understanding of its genetic makeup towards developing elite cultivars, we sequenced and assembled a draft genome of L. purpureus accession PK2022T020 using a single tube long fragment read (stLFR) technique. Results and discussion: The preliminary assembly encompassed 367 Mb with a scaffold N50 of 4.3 Mb. To improve the contiguity of our draft genome, we employed a chromatin contact mapping (Hi-C) approach to obtain a pseudochromosome-level assembly containing 366 Mb with an N50 length of 31.1 Mb. A total of 327.4 Mb had successfully been anchored into 11 pseudomolecules, corresponding to the haploid chromosome number in lablab. Our gene prediction recovered 98.4% of the highly conserved orthologs based on the Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis. Comparative analyses utilizing sequence information from single-copy orthologous genes demonstrated that L. purpureus diverged from the last common ancestor of the Phaseolus/Vigna species approximately 27.7 million years ago. A gene family expansion analysis revealed a significant expansion of genes involved in responses to biotic and abiotic stresses. Our high-quality chromosome-scale reference assembly provides an invaluable genomic resource for lablab genetic improvement and future comparative genomics studies among legume species.

Adaptation of African swine fever virus to MA-104 cells: Implications of unique genetic variations.

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal, contagious disease specifically in pigs. However, prevention and control of ASFV outbreaks have been hampered by the lack of an effective vaccine or antiviral treatment for ASFV. Although ASFV has been reported to adapt to a variety of continuous cell lines, the phenotypic and genetic changes associated with ASFV adaptation to MA-104 cells remain poorly understood. Here, we adapted ASFV field isolates to efficiently propagate through serial viral passages in MA-104 cells. The adapted ASFV strain developed a pronounced cytopathic effect and robust infection in MA-104 cells. Interestingly, the adapted variant maintained its tropism in primary porcine kidney macrophages. Whole genome analysis of the adapted virus revealed unique gene deletions in the left and right variable regions of the viral genome compared to other previously reported cell culture-adapted ASFV strains. Notably, gene duplications at the 5' and 3' ends of the viral genome were in reverse complementary alignment with their paralogs. Single point mutations in protein-coding genes and intergenic regions were also observed in the viral genome. Collectively, our results shed light on the significance of these unique genetic changes during adaptation, which facilitate the growth of ASFV in MA-104 cells.

microRNA profiling of exosomes derived from plasma and their potential as biomarkers for Opisthorchis viverrini-associated cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a life-threatening disease that impacts patients worldwide. In Southeast Asian countries, the liver fluke Opisthorchis viverrini plays a major role in inducing carcinogenesis of the bile ducts. Due to its asymptomatic nature, O. viverrini infections are rarely treated, consequently leading to the development of advanced stages of CCA before diagnosis. Despite the current use of exosomal microRNAs (miRNA) as diagnostic biomarkers for the early detection of many types of cancer, the applications for miRNA remain limited with CCA. Circulating exosomes, membranous vesicles essential for intercellular communication, were found to contain unique miRNA. In this study, we conducted next-generation sequencing (Ion Torrent PGM) and bioinformatics to characterize and compare the contents of exosomal miRNA derived from the plasma of CCA patients, O. viverrini-infected patients, and healthy individuals, as well as to identify and validate key molecules as markers for screening the diagnosis of CCA and O. viverrini infection. The obtained results showed the success of using NGS technology in discovering exosomal miRNAs, specifically miR-194-5p and miR-192-5p, both of which were upregulated in the O. viverrini-infected group. Interestingly, miR-192-5p was upregulated while miR-194-5p was downregulated in CCA, suggesting their potential use as biomarkers for screening CCA and O. viverrini infection, especially in O. viverrini-endemic areas.

Integrative taxonomy of Metarhizium anisopliae species complex, based on phylogenomics combined with morphometrics, metabolomics, and virulence data.

Metarhizium anisopliae (Clavicipitaceae, Hypocreales) is a globally distributed entomopathogenic fungus, which has been largely studied and used in agriculture for its potent entomopathogenicity. Since its taxonomic establishment as a member of Metarhizium, many closely related taxa have been described with highly similar morphology (cryptic species). A holotype specimen of M. anisopliae is not extant, and the ex-neotype strain (CBS 130.71) does not form a monophyletic clade with other strains, up to now, recognized as M. anisopliae sensu stricto. In this study, we have conducted an integrative taxonomic treatment of M. anisopliae sensu lato by including the ex-neotype strain of M. anisopliae, other unknown strains from our collections identified as M. anisopliae s. lat., as well as other known species that have been previously delimited as closely related but distinct to M. anisopliae. By including whole-genome sequencing, morphometric analysis, LC-MS based metabolomics, and virulence assays, we have demonstrated that M. anisopliae s. str. should also include M. lepidiotae (synonym), and that M. anisopliae s. str. differentiates from the other species of the complex by its metabolome and less severe entomopathogenicity. New taxa, namely M. hybridum, M. neoanisopliae and M. parapingshaense spp. nov., are proposed. The novel taxa proposed here have strong phylogenomics support, corroborated by fine-scale differences in the length/width of conidia/phialides, while the metabolomics and virulence data still largely overlap. We have also demonstrated via population genomics data the existence of local clonal lineages, particularly the one corresponding to the persistence of a biocontrol candidate strain that has been used in the field application for three years. This study showcases the utility of combining various data sources for accurate delimitation of species within an important group of fungal biocontrol agents against pest insects.

Comprehensive genome assembly reveals genetic diversity and carcass consumption insights in critically endangered Asian king vultures.

The Asian king vulture (AKV), a vital forest scavenger, is facing globally critical endangerment. This study aimed to construct a reference genome to unveil the mechanisms underlying its scavenger abilities and to assess the genetic relatedness of the captive population in Thailand. A reference genome of a female AKV was assembled from sequencing reads obtained from both PacBio long-read and MGI short-read sequencing platforms. Comparative genomics with New World vultures (NWVs) and other birds in the Family Accipitridae revealed unique gene families in AKV associated with retroviral genome integration and feather keratin, contrasting with NWVs' genes related to olfactory reception. Expanded gene families in AKV were linked to inflammatory response, iron regulation and spermatogenesis. Positively selected genes included those associated with anti-apoptosis, immune response and muscle cell development, shedding light on adaptations for carcass consumption and high-altitude soaring. Using restriction site-associated DNA sequencing (RADseq)-based genome-wide single nucleotide polymorphisms (SNPs), genetic relatedness and inbreeding status of five captive AKVs were determined, revealing high genomic inbreeding in two females. In conclusion, the AKV reference genome was established, providing insights into its unique characteristics. Additionally, the potential of RADseq-based genome-wide SNPs for selecting AKV breeders was demonstrated.

The complete chloroplast genome sequence and phylogenetic analysis of Heritiera fomes Buch.-Ham. (Malvales: Sterculiaceae).

Heritiera fomes Buch.-Ham. (1800) is a species of mangrove in the family Malvaceae, widely distributed in the Indo-Pacific and listed as 'endangered' (EN) on the International Union for Conservation of Nature's (IUCN) red list. We reported the complete chloroplast genome sequence of H. fomes. The genome was 168,521 bp in length and included two inverted repeats (IRs) of 34,496 bp, separated by a large single-copy (LSC) region of 88,604 bp and a small single-copy (SSC) region of 10,925 bp, respectively. The genome contained 87 protein-coding genes (PCGs), 8 rRNA genes, and 37 tRNA genes. The maximum-likelihood (ML) phylogenetic tree suggested that H. fomes is closely related to Heritiera angustata and Heritiera parvifolia with relatively high support bootstrap values of 86% and 100% with other species (Heritiera littoralis and Heritiera javanica), suggesting a relatively close genetic relationship between the five Heritiera plants. The chloroplast genome sequence provided a useful resource for conservation genetics studies of H. fomes and for phylogenetic studies of Heritiera.

Comparative genomics and genome-wide SNPs of endangered Eld's deer provide breeder selection for inbreeding avoidance.

Eld's deer, a conserved wildlife species of Thailand, is facing inbreeding depression, particularly in the captive Siamese Eld's deer (SED) subspecies. In this study, we constructed genomes of a male SED and a male Burmese Eld's deer (BED), and used genome-wide single nucleotide polymorphisms to evaluate the genetic purity and the inbreeding status of 35 SED and 49 BED with limited pedigree information. The results show that these subspecies diverged approximately 1.26 million years ago. All SED were found to be purebred. A low proportion of admixed SED genetic material was observed in some BED individuals. Six potential breeders from male SED with no genetic relation to any female SED and three purebred male BED with no relation to more than 10 purebred female BED were identified. This study provides valuable insights about Eld's deer populations and appropriate breeder selection in efforts to repopulate this endangered species while avoiding inbreeding.

The complete chloroplast genome of Sonneratia griffithii Kurz (Lythraceae).

Sonneratia griffithii Kurz is a critically endangered mangrove species that can be found along the western coast of Thailand. In this study, we reported the complete chloroplast genome of S. griffithii. The chloroplast genome is 152,730 bp, consisting of one large single-copy (LSC) region, one small single-copy (SSC) region and a pair of inverted repeats (IRs). The LSC, SSC, and IR lengths are 87,226, 17,764, and 23,870 bp, respectively. The genome contains 113 unique genes, including 79 protein-coding, 30 tRNA, and 4 rRNA genes. The GC content of the chloroplast genome is 37.31%. The phylogenetic analysis based on 76 protein-coding genes showed a monophyletic group of S. griffithii and other Sonneratia species.

Genome assembly of the Pendlebury's roundleaf bat, Hipposideros pendleburyi, revealed the expansion of Tc1/Mariner DNA transposons in Rhinolophoidea.

Bats (Chiroptera) constitute the second largest order of mammals and have several distinctive features, such as true self-powered flight and strong immunity. The Pendlebury's roundleaf bat, Hipposideros pendleburyi, is endemic to Thailand and listed as a vulnerable species. We employed the 10× Genomics linked-read technology to obtain a genome assembly of H. pendleburyi. The assembly size was 2.17 Gb with a scaffold N50 length of 15,398,518 bases. Our phylogenetic analysis placed H. pendleburyi within the rhinolophoid clade of the suborder Yinpterochiroptera. A synteny analysis showed that H. pendleburyi shared conserved chromosome segments (up to 105 Mb) with Rhinolophus ferrumequinum and Phyllostomus discolor albeit having different chromosome numbers and belonging different families. We found positive selection signals in genes involved in inflammation, spermatogenesis and Wnt signalling. The analyses of transposable elements suggested the contraction of short interspersed nuclear elements (SINEs) and the accumulation of young mariner DNA transposons in the analysed hipposiderids. Distinct mariners were likely horizontally transferred to hipposiderid genomes over the evolution of this family. The lineage-specific profiles of SINEs and mariners might involve in the evolution of hipposiderids and be associated with the phylogenetic separations of these bats from other bat families.