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.

Genetic diversity of the dengue virus population in dengue fever and dengue hemorrhagic fever patients.

BACKGROUND: The error-prone replication of dengue virus (DENV) in host results in the highly diverse viral population. Together with the host factor, intra-host diversity may influence the disease severity. Therefore, it is worth investigating whether there is a correlation between intra-host genetic diversity and disease severity. OBJECTIVE: To investigate the genetic diversity in DENV for four serotypes of the dengue population from patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) using next-generation sequencing (NGS) technology. METHODS: Forty RNA samples categorized into eight groups by severity and serotypes were sequenced and analyzed for genetic variation. Analysis on the hot-cold genomic regions, selection pressure and correlation between genotype and disease severity were performed in this study. RESULTS: Comparison between the NGS data of the DF and DHF specimens showed conservation between their major populations with the consensus sequences for DF and DHF sharing 99% similarity. However, the minor populations in DF and DHF were more diverse. Many genes in DF had an #NS/#S ratio higher than in DHF. Only NS4B of DENV1 DF has #NS/#S ratio higher than one. Hot regions of the DF were detected in NS3 of DENV1, DENV2 and Envelope of DENV3, whereas the hot regions of the DHF samples were detected in the small region in 3'UTR of DENV2 and DENV3. CONCLUSIONS: Various explorations of the variations of DF and DHF were performed in this study. However, we have not yet found any specific characteristics of intra-host diversity associated with disease severity.

A draft chromosome-scale genome assembly of a commercial sugarcane.

Sugarcane accounts for a large portion of the worlds sugar production. Modern commercial cultivars are complex hybrids of S. officinarum, S. spontaneum, and several other Saccharum species, resulting in an auto-allopolyploid with 8-12 copies of each chromosome. The current genome assembly gold standard is to generate a long read assembly followed by chromatin conformation capture sequencing to scaffold. We used the PacBio RSII and chromatin conformation capture sequencing to sequence and assemble the genome of a South East Asian commercial sugarcane cultivar, known as Khon Kaen 3. The Khon Kaen 3 genome assembled into 104,477 contigs totalling 7 Gb, which scaffolded into 56 pseudochromosomes containing 5.2 Gb of sequence. Genome annotation produced 242,406 genes from 30,927 orthogroups. Aligning the Khon Kaen 3 genome sequence to S. officinarum and S. spontaneum revealed a high level of apparent recombination, indicating a chimeric assembly. This assembly error is explained by high nucleotide identity between S. officinarum and S. spontaneum, where 91.8% of S. spontaneum aligns to S. officinarum at 94% identity. Thus, the subgenomes of commercial sugarcane are so similar that using short reads to correct long PacBio reads produced chimeric long reads. Future attempts to sequence sugarcane must take this information into account.

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.

Assessment of the Genetic Diversity and Population Structure of Rhizophora apiculata Blume (Rhizophoraceae) in Thailand.

Rhizophora apiculata is one of the most widespread and economically important mangrove trees in the Indo-West Pacific region. Knowledge of the genetic variation of R. apiculata in Thailand is limited. Here, we generated a whole-genome sequence of R. apiculata using the 10× Genomics technology. R. apiculata genome assembly was 230.47 Mb. Based on its genome, 2640 loci of high-quality biallelic SNPs were identified from 82 R. apiculata accessions collected from 17 natural mangrove forests in Thailand to assess the genetic diversity and population structure among them. A moderate level of genetic diversity of R. apiculata was observed. The average observed heterozygosity (Ho = 0.48) was higher than the average expected heterozygosity (He = 0.36). Two subpopulations were observed and confirmed from three approaches: population structure, PCA, and phylogenetic analyses. They corresponded to the Gulf of Thailand and the Andaman Sea separated by the Malay Peninsula. AMOVA analyses indicated that genetic variation was attributable to 76.22% within populations and 23.78% among populations. A high level of genetic differentiation between the two subpopulations (FST = 0.24, p < 0.001) was observed. This study evaluated the genetic diversity and population structure of R. apiculata, providing useful information for sustainable mangrove management in Thailand.

Chromosome-level genome assembly of Indian mangrove (Ceriops tagal) revealed a genome-wide duplication event predating the divergence of Rhizophoraceae mangrove species.

Mangrove ecosystems are unique, highly diverse, provide benefits to humans, and aid in coastal protection. The Indian mangrove, or spurred mangrove, [Ceriops tagal (Perr.) C. B. Rob.] is a member of the Rhizophoraceae family and is commonly found along the intertidal zones in tropical regions in Southeast Asia, southern Asia, and Africa. Here, we present the first high-quality reference genome assembly of the Ceriops species. A preliminary draft assembly, generated from the 10× Genomics linked-read library, was scaffolded using the proximity ligation chromatin contact mapping technique (Hi-C) to obtain a chromosome-scale assembly of 231,919,005 bases with an N50 length of 11,408,429 bases. The benchmarking universal single-copy orthologs (BUSCO) analysis revealed that C. tagal gene predictions recovered 95.8% of the highly conserved orthologs. Phylogenetic analyses suggested that C. tagal diverged from the last common ancestor of flat-leaf spurred mangrove [C. decandra (Griff.) Ding Hou] and C. zippeliana Blume ∼10.4 million yr ago (MYA), and the last common ancestor of genera Ceriops, Kandelia, and Rhizophora diverged from that of genus Bruguiera ∼49.4 MYA. In addition, our analysis of the transversion rate at fourfold-degenerate sites from orthologous gene pairs provided evidence supporting a recent whole-genome duplication in C. tagal. The STRUCTURE and principal component analyses illustrated that C. tagal individuals investigated in this study were the admixture of two subpopulations, the genetic background of which was influenced primarily by location. The availability of genomic and transcriptomic resources and biodiversity data reported in this work will be useful for future studies that may shed light on adaptive evolutions of mangrove species.

A SNP variation in an expansin (EgExp4) gene affects height in oil palm.

Oil palm (Elaeis guineensis Jacq.), an Aracaceae family plant, is utilized for both consumable and non-consumable products, including cooking oil, cosmetics and biodiesel production. Oil palm is a perennial tree with 25 years of optimal harvesting time and a height of up to 18 m. However, harvesting of oil palm fruit bunches with heights of more than 2-3 meters is challenging for oil palm farmers. Thus, understanding the genetic control of height would be beneficial for using gene-based markers to speed up oil palm breeding programs to select semi-dwarf oil palm varieties. This study aims to identify Insertion/Deletions (InDels) and single nucleotide polymorphisms (SNPs) of five height-related genes, including EgDELLA1, EgGRF1, EgGA20ox1, EgAPG1 and EgExp4, in short and tall oil palm groups by PacBio SMRT sequencing technology. Then, the SNP variation's association with height was validated in the Golden Tenera (GT) population. All targeted genes were successfully amplified by two rounds of PCR amplification with expected sizes that ranged from 2,516 to 3,015 base pair (bp), covering 5' UTR, gene sequences and 3' UTR from 20 short and 20 tall oil palm trees. As a result, 1,166, 909, 1,494, 387 and 5,384 full-length genomic DNA sequences were revealed by PacBio SMRT sequencing technology, from EgDELLA1, EgGRF1, EgGA20ox1, EgAPG1 and EgExp4 genes, respectively. Twelve variations, including eight InDels and four SNPs, were identified from EgDELLA1, EgGRF1, EgGA20ox1 and EgExp4. No variation was found for EgAPG1. After SNP through-put genotyping of 4 targeted SNP markers was done by PACE™ SNP genotyping, the association with height was determined in the GT population. Only the mEgExp4_SNP118 marker, designed from EgExp4 gene, was found to associate with height in 2 of 4 height-recordings, with p values of 0.0383 for height (HT)-1 and 0.0263 for HT-4. In conclusion, this marker is a potential gene-based marker that may be used in oil palm breeding programs for selecting semi-dwarf oil palm varieties in the near future.

A chromosome-scale reference genome assembly of yellow mangrove (Bruguiera parviflora) reveals a whole genome duplication event associated with the Rhizophoraceae lineage.

Mangrove forest ecosystems support a diverse flora and fauna of marine and terrestrial species and have important direct and indirect economic, ecological and social values to mankind. Yellow mangrove (Bruguiera parviflora) belongs to the Rhizophoraceae family and is widely distributed in the intertidal zones along sheltered coastal areas in tropical latitudes. Here, we present a high-quality, chromosome-level assembly of the B. parviflora genome. We employed the 10x Genomics linked-read technology to obtain a preliminary assembly, which was subsequently scaffolded using the long-range chromatin contact mapping technique (HiC) to obtain a final assembly containing 213,026,782 bases in 10,045 scaffolds with an N50 length of 10,906,948 bases. Our gene prediction recovered 96.5% of the highly conserved orthologues in the Embryophyta lineage based on the Benchmarking Universal Single-Copy Orthologues (BUSCO) analysis. We analysed the transversion rate at fourfold-degenerate sites from orthologous gene pairs and discovered evidence supporting a recent whole-genome duplication event in B. parviflora and other Rhizophoreae members. Comparative studies based on single-copy orthologous genes indicated that B. parviflora and Bruguiera gymnorrhiza diverged approximately 24.1 million years ago. The population structure analysis revealed that 63 B. parviflora accessions from different geographical regions in Thailand were an admixture of two subpopulations. The examination of alternative splicing events in B. parviflora showed that the most prevalent splicing mechanism was intron retention. This high-quality genome assembly together with the genetic diversity information obtained from the germplasm provide useful genomic resources for future studies on comparative phylogenetics and evolution of adaptive traits in mangrove species.

Genome assemblies of Vigna reflexo-pilosa (créole bean) and its progenitors, Vigna hirtella and Vigna trinervia, revealed homoeolog expression bias and expression-level dominance in the allotetraploid.

Vigna reflexo-pilosa (créole bean) is a wild legume belonging to the subgenus Ceratoropis and is widely distributed in Asia. Créole bean is the only tetraploid species in the genus Vigna, and it has been shown to derive from the hybridization of Vigna hirtella and Vigna trinervia. In this study, we combined the long-read PacBio technology with the chromatin contact mapping (Hi-C) technique to obtain a chromosome-level assembly of V. reflexo-pilosa. The final assembly contained 998,724,903 bases with an N50 length of 42,545,650 bases. Our gene prediction recovered 99.4% of the highly conserved orthologs based on the BUSCO analysis. To investigate homoeolog expression bias and expression level dominance in the tetraploid, we also sequenced and assembled the genomes of its progenitors. Overall, the majority of the homoeolog pairs (72.9%) displayed no expression bias, and among those that exhibited biased expression, 16.3% showed unbalanced homoeolog expression bias toward the V. trinervia subgenome. Moreover, 41.2% and 36.2% of the expressed gene pairs exhibited transgressive expression and expression level dominance, respectively. Interestingly, the genome-wide expression level dominance in the tetraploid was biased toward the V. trinervia subgenome. The analysis of methylation patterns also revealed that the average methylation levels in coding regions were higher in the V. hirtella subgenome than those in the V. trinervia subgenome. The genomic/transcriptomic resources for these three species are useful not only for the development of elite cultivars in Vigna breeding programs but also to researchers studying comparative genomics and investigating genomic/epigenomic changes following polyploid events.

The complete mitochondrial genome of the Hipposideros pendleburyi (Pendlebury's leaf-nosed bat) an endemic species in Thailand.

This study presents the first complete mitochondrial genome of the Hipposideros pendleburyi (Pendlebury's leaf-nosed bat), an endemic species in Thailand. The mitochondrial genome was 16,820 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region. The overall base composition was 31.5% A, 26.2% T, 28.3% C, and 14.0% G. A maximum-likelihood tree revealed that H. pendleburyi was grouped with Hipposideros armiger within the Hipposideridae clade, which has Rhinolophidae as a sister clade.

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.

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.

The complete mitochondrial genome sequence of the mountain crab Indochinamon bhumibol.

Indochinamon bhumibol has been found as the biggest freshwater crab in Thailand. In this study, we report the first complete sequence of mitochondrial genome from I. bhumibol encoding 13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. The nucleotide composition of I. bhumibol mitogenome showed a strong AT bias (70.4%) with a low GC content (29.6%). Comparative phylogenetic analysis with 28 crustaceans based on nine conserved genes demonstrated that I. bhumibol was closely related to members of the Potamidae family.

A chromosome-level assembly of the black tiger shrimp (Penaeus monodon) genome facilitates the identification of growth-associated genes.

To salvage marine ecosystems from fishery overexploitation, sustainable and efficient aquaculture must be emphasized. The knowledge obtained from available genome sequence of marine organisms has accelerated marine aquaculture in many cases. The black tiger shrimp (Penaeus monodon) is one of the most prominent cultured penaeid shrimps (Crustacean) with an average annual global production of half a million tons in the last decade. However, its currently available genome assemblies lack the contiguity and completeness required for accurate genome annotation due to the highly repetitive nature of the genome and technical difficulty in extracting high-quality, high-molecular weight DNA. Here, we report the first chromosome-level whole-genome assembly of P. monodon. The combination of long-read Pacific Biosciences (PacBio) and long-range Chicago and Hi-C technologies enabled a successful assembly of this first high-quality genome sequence. The final assembly covered 2.39 Gb (92.3% of the estimated genome size) and contained 44 pseudomolecules, corresponding to the haploid chromosome number. Repetitive elements occupied a substantial portion of the assembly (62.5%), the highest of the figures reported among crustacean species. The availability of this high-quality genome assembly enabled the identification of genes associated with rapid growth in the black tiger shrimp through the comparison of hepatopancreas transcriptome of slow-growing and fast-growing shrimps. The results highlighted several growth-associated genes. Our high-quality genome assembly provides an invaluable resource for genetic improvement and breeding penaeid shrimp in aquaculture. The availability of P. monodon genome enables analyses of ecological impact, environment adaptation and evolution, as well as the role of the genome to protect the ecological resources by promoting sustainable shrimp farming.

De novo assemblies of Luffa acutangula and Luffa cylindrica genomes reveal an expansion associated with substantial accumulation of transposable elements.

Luffa spp. (sponge gourd or ridge gourd) is an economically important vegetable crop widely cultivated in China, India and Southeast Asia. Here, we employed PacBio long-read single-molecule real-time (SMRT) sequencing to perform de novo genome assemblies of two commonly cultivated Luffa species, L. acutangula and L. cylindrica. We obtained preliminary draft genomes of 734.6 Mb and 689.8 Mb with scaffold N50 of 786,130 and 578,616 bases for L. acutangula and L. cylindrica, respectively. We also applied long-range Chicago and HiC techniques to obtain the first chromosome-scale whole-genome assembly of L. acutangula. The final assembly contained 13 pseudomolecules, corresponding to the haploid chromosome number in Luffa spp. (1n = 13, 2n = 26). The sizes of the assembled Luffa genomes are approximately twice as large as the genome assemblies of related Cucurbitaceae. A large proportion of L. acutangula (62.17%; 456.69 Mb) and L. cylindrica (56.78%; 391.65 Mb) genome assemblies contained repetitive elements. Phylogenetic analyses revealed that the substantial accumulation of transposable elements likely contributed to the expansion of the Luffa genomes. We also investigated alternative splicing events in Luffa using full-length transcript sequences obtained from PacBio Isoform Sequencing (Iso-seq). While the predominant form of alternative splicing in most plant species examined was intron retention, alternative 3' acceptor site selection appeared to be a major event observed in Luffa. High-quality genome assemblies for L. acutangula and L. cylindrica reported here provide valuable resources for Luffa breeding and future genetics and comparative genomics studies in Cucurbitaceae.

The Fungus Metarhizium sp. BCC 4849 Is an Effective and Safe Mycoinsecticide for the Management of Spider Mites and Other Insect Pests.

Five isolates of Metarhizium sp. were evaluated for their pathogenicity against the spider mite (Tetranychus truncatus Ehara) (Acari: Tetranychidae) and Metarhizium sp. BCC 4849 resulted in the highest mortality (82%) on the 5th day post-inoculation (DPI). Subsequent insect bioassay data indicated similar high virulence against five other insects: African red mites (Eutetranychus africanus Tucker) (Acari: Tetranychidae), bean aphid (Aphis craccivora Koch) (Hemiptera: Aphididae), cassava mealybug (Phenacoccus manihoti Matile-Ferrero) (Hemiptera: Pseudococcidae), sweet potato weevil (Cylas formicarius Fabricius) (Coleoptera: Brentidae), and oriental fruit fly (Bactrocera dorsalis Hendel) (Diptera: Tephritidae), at mortalities of 92-99%, on 3rd-6th DPI, and in laboratory conditions. The pathogenicity assay against E. africanus in hemp plants under greenhouse conditions indicated 85-100% insect mortality on 10th DPI using the fungus alone or in combination with synthetic acaricide. Genome sequencing of Metarhizium sp. BCC 4849 revealed the high abundance of proteins associated with zinc-, heme-, and iron-binding; oxidation-reduction; and transmembrane transport, implicating its versatile mode of interaction with the environment and adaptation to various ion homeostasis. The light and scanning electron microscopy indicated that at 24 h post inoculation (PI), adhesion and appressorial formation occurred, notably near the setae. Most infected mites had stopped moving and started dying by 48-72 h PI. Elongated hyphal bodies and oval blastospores were detected in the legs. At 96-120 h PI or longer, dense mycelia and conidial mass had colonized the interior and exterior of dead mites, primarily at the bottom than the upper part. The shelf-life study also indicated that conidial formulation combined with an oxygen-moisture absorber markedly enhanced the viability and germination after storage at 35 °C for four months. The fungus was tested as safe for humans and animals, according to our toxicological assays.

A chromosome-scale assembly of the black gram (Vigna mungo) genome.

Black gram (Vigna mungo) is an important short duration grain legume crop. Black gram seeds provide an inexpensive source of dietary protein. Here, we applied the 10X Genomics linked-read technology to obtain a de novo whole genome assembly of V. mungo cultivated variety Chai Nat 80 (CN80). The preliminary assembly contained 12,228 contigs and had an N50 length of 5.2 Mb. Subsequent scaffolding using the long-range Chicago and HiC techniques yielded the first high-quality, chromosome-level assembly of 499 Mb comprising 11 pseudomolecules. Comparative genomics analyses based on sequence information from single-copy orthologous genes revealed that black gram and mungbean (Vigna radiata) diverged about 2.7 million years ago . The transversion rate (4DTv) analysis in V. mungo revealed no evidence supporting a recent genome-wide duplication event observed in the tetraploid créole bean (Vigna reflexo-pilosa). The proportion of repetitive elements in the black gram genome is slightly lower than the numbers reported for related Vigna species. The majority of long terminal repeat retrotransposons appeared to integrate into the genome within the last five million years. We also examined alternative splicing events in V. mungo using full-length transcript sequences. While intron retention was the most prevalent mode of alternative splicing in several plant species, alternative 3' acceptor site selection represented the majority of events in black gram. Our high-quality genome assembly along with the genomic variation information from the germplasm provides valuable resources for accelerating the development of elite varieties through marker-assisted breeding and for future comparative genomics and phylogenetic studies in legume species.

Chloroplast genome data of Luffa acutangula and Luffa aegyptiaca and their phylogenetic relationships.

Luffa acutangula and Luffa aegyptiaca are domesticated plants in the family Cucurbitaceae. They are mainly cultivated in the tropical and subtropical regions of Asia. The chloroplast genomes of many Cucurbitaceae species were sequenced to examine gene content and evolution. However, the chloroplast genome sequences of L. acutangula and L. aegyptiaca have not been reported. We report the first complete sequences of L. acutangula and L. aegyptiaca chloroplast genomes obtained from Pacific Biosciences sequencing and use them to infer evolutionary relationships. The chloroplast genomes of L. acutangula and L. aegyptiaca are 157,202 and 157,275 bp, respectively. Both genomes possessed the typical quadripartite structure and contained 131 genes, including 87 coding genes, 36 tRNA genes and 8 rRNA genes. We identified simple sequence repeats (SSR) and single nucleotide polymorphisms (SNP) from both chloroplast genomes. Polycistronic mRNA was examined in L. acutangula and L. aegyptiaca using RNA sequences from Isoform sequencing to identify co-transcribed genes. IR size and locations were compared to other species and found to be relatively unchanged. Phylogenetic analysis confirmed the close relationship between L. acutangula and L. aegyptiaca in the Cucurbitaceae lineage and showed separation of the Luffa monophyletic clade from other species in the subtribe Sicyocae. The results obtained from this study can be useful for studying the evolution of Cucurbitaceae plants.

Differential expression between drought-tolerant and drought-sensitive sugarcane under mild and moderate water stress as revealed by a comparative analysis of leaf transcriptome.

Sugarcane contributes 80% of global sugar production and to bioethanol generation for the bioenergy industry. Its productivity is threatened by drought that can cause up to 60% yield loss. This study used RNA-Seq to gain a better understanding of the underlying mechanism by which drought-tolerant sugarcane copes with water stress. We compared gene expression in KPS01-12 (drought-tolerant genotype) and UT12 (drought-sensitive genotype) that have significantly different yield loss rates under drought conditions. We treated KPS01-12 and UT12 with mild and moderate water stress and found differentially expressed genes in various biological processes. KPS01-12 had higher expression of genes that were involved in water retention, antioxidant secondary metabolite biosynthesis, and oxidative and osmotic stress response than UT12. In contrast, the sensitive genotype had more down-regulated genes that were involved in photosynthesis, carbon fixation and Calvin cycle than the tolerant genotype. Our obtained expression profiles suggest that the tolerant sugarcane has a more effective genetic response than the sensitive genotype at the initiation of drought stress. The knowledge gained from this study may be applied in breeding programs to improve sugarcane production in drought conditions.

Optimization of high molecular weight DNA extraction methods in shrimp for a long-read sequencing platform.

Marine organisms are important to global food security as they are the largest source of animal proteins feeding mankind. Genomics-assisted aquaculture can increase yield while preserving the environment to ensure sufficient and sustainable production for global food security. However, only few high-quality genome sequences of marine organisms, especially shellfish, are available to the public partly because of the difficulty in the sequence assembly due to the complex nature of their genomes. A key step for a successful genome sequencing is the preparation of high-quality high molecular weight (HMW) genomic DNA. This study evaluated the effectiveness of five DNA extraction protocols (CTAB, Genomic-tip, Mollusc DNA, TIANamp Marine Animals DNA, and Sbeadex livestock kits) in obtaining shrimp HMW DNA for a long-read sequencing platform. DNA samples were assessed for quality and quantity using a Qubit fluorometer, NanoDrop spectrophotometer and pulsed-field gel electrophoresis. Among the five extraction methods examined without further optimization, the Genomic-tip kit yielded genomic DNA with the highest quality. However, further modifications of these established protocols might yield even better DNA quality and quantity. To further investigate whether the obtained genomic DNA could be used in a long-read sequencing application, DNA samples from the top three extraction methods (CTAB method, Genomic-tip and Mollusc DNA kits) were used for Pacific Biosciences (PacBio) library construction and sequencing. Genomic DNA obtained from Genomic-tip and Mollusc DNA kits allowed successful library construction, while the DNA obtained from the CTAB method did not. Genomic DNA isolated using the Genomic-tip kit yielded a higher number of long reads (N50 of 14.57 Kb) than those obtained from Mollusc DNA kits (N50 of 9.74 Kb). Thus, this study identified an effective extraction method for high-quality HMW genomic DNA of shrimp that can be applied to other marine organisms for a long-read sequencing platform.