Publications by authors named "Wirulda Pootakham"

34 Publications

The complete mitochondrial genome sequence of the mountain crab .

Mitochondrial DNA B Resour 2021 Feb 17;6(2):634-635. Epub 2021 Feb 17.

National Omics Center, National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.

has been found as the biggest freshwater crab in Thailand. In this study, we report the first complete sequence of mitochondrial genome from encoding 13 protein-coding genes, 22 transfer RNAs, and 2 ribosomal RNAs. The nucleotide composition of 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 was closely related to members of the Potamidae family.
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http://dx.doi.org/10.1080/23802359.2021.1877203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894412PMC
February 2021

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

Mol Ecol Resour 2021 Feb 15. Epub 2021 Feb 15.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, 12120, Thailand.

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.
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http://dx.doi.org/10.1111/1755-0998.13357DOI Listing
February 2021

The complete mitochondrial genome of .

Mitochondrial DNA B Resour 2020 Aug 26;5(3):3208-3209. Epub 2020 Aug 26.

National Omics Center, National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand.

Based on PacBio assembly, we report the first complete mitochondrial genome of (460,333 bp) containing nine large chloroplast-derived sequences (1.9-17.3 kb) across the mitogenome. The base composition of the mitogenome in descending order is A: 28.02%, C: 22.04%, G: 21.83% and T: 28.10%, and the G + C content is 43.87%. There are 63 mitochondrial genes including 40 protein-coding genes, 3 rRNA genes and 20 tRNA genes. Additionally, a total of 288 repeats ranging from 31 to 5,301 bp were identified, accounting for 5.7% of the mitogenome. Two large direct repeats (5,301 and 405 bp) within the mitogenome were found for the formation of four subgenomic molecules. A phylogenetic analysis showed that was closely related to other species in Cucurbiaceae. This mitogenome provides useful genetic information for evolutionary studies.
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http://dx.doi.org/10.1080/23802359.2020.1810165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781975PMC
August 2020

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

PeerJ 2020 13;8:e10340. Epub 2020 Nov 13.

Microarray Research Team, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand.

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.
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http://dx.doi.org/10.7717/peerj.10340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7668203PMC
November 2020

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

PeerJ 2020 28;8:e9608. Epub 2020 Jul 28.

National Omics Center (NOC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathum Thani, Thailand.

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.
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http://dx.doi.org/10.7717/peerj.9608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7676377PMC
July 2020

Chloroplast genome data of and and their phylogenetic relationships.

Data Brief 2020 Dec 28;33:106470. Epub 2020 Oct 28.

National Omics Center, National Science and Technology Development Agency, 111 Thailand Science Park, Paholyothin Road, Khlong Nueng, Khlong Luang, Pathum Thani, 12120, Thailand.

and 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 and have not been reported. We report the first complete sequences of and chloroplast genomes obtained from Pacific Biosciences sequencing and use them to infer evolutionary relationships. The chloroplast genomes of and 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 and 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 and in the Cucurbitaceae lineage and showed separation of the 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.
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http://dx.doi.org/10.1016/j.dib.2020.106470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644877PMC
December 2020

Assembly of the durian chloroplast genome using long PacBio reads.

Sci Rep 2020 10 7;10(1):15980. Epub 2020 Oct 7.

National Omics Center, National Science and Technology Development Agency, 111 Thailand Science Park, Paholyothin Road, Khlong Nueng, Khlong Luang, Pathumthani, 12120, Thailand.

We have assembled the complete sequence of the Durio zibethinus chloroplast genome using long PacBio reads. Durian is a valuable commercial tree that produces durian fruit, which is popular in Southeast Asia. The chloroplast genome assembled into a single 143 kb cyclic contig that contained 111 genes. There were 46 short direct repeats (45 to 586 bp) and five short inverted repeats (63 to 169 bp). The long reads that were used for the assembly span the entire chloroplast with > 10 kb overlaps and multiple long reads join the start of the contig to the end of the contig. The durian chloroplast was found to lack the large inverted repeat that is common in chloroplast genomes. An additional 24 durian varieties were sequenced and compared to the assembly and found to also lack the large inverted repeat. There were nine SNPs among the varieties.
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http://dx.doi.org/10.1038/s41598-020-73549-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541610PMC
October 2020

The Genome and Transcriptome Analysis of the Chloroplast.

Plants (Basel) 2020 Sep 21;9(9). Epub 2020 Sep 21.

National Omics Center (NOC), National Science and Technology Development Agency, 111 Thailand Science Park, Khlong Nueng, Khlong Luang, Pathum Thani 12120, Thailand.

is cultivated in approximately 5 million hectares worldwide. The chloroplast genome of this species has not been previously reported. In this study, we sequenced the genome and transcriptome of the chloroplast. We identified many positively selected genes in the photosynthetic pathway (e.g., , , and ) and RNA polymerase genes (e.g., ) from the comparison of the chloroplast genome of , temperate legume species, and tropical legume species. Our transcriptome data from PacBio isoform sequencing showed that the 51-kb DNA inversion could affect the transcriptional regulation of polycistronic. Using Illumina deep RNA sequencing, we found RNA editing of in the leaf, shoot, flower, fruit, and root tissues of . We also found three G-to-A RNA editing events that change guanine to adenine in the transcripts transcribed from the adenine-rich regions of the gene. The edited guanine bases were found particularly in the chloroplast genome of the species. These G-to-A RNA editing events were likely to provide a mechanism for correcting DNA base mutations. The chloroplast genome sequence and the analysis results obtained in this study can apply to phylogenetic studies and chloroplast genome engineering.
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http://dx.doi.org/10.3390/plants9091247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570002PMC
September 2020

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

Mol Ecol Resour 2021 Jan 25;21(1):212-225. Epub 2020 Aug 25.

National Omics Center, National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.

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.
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http://dx.doi.org/10.1111/1755-0998.13240DOI Listing
January 2021

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

Mol Ecol Resour 2021 Jan 8;21(1):238-250. Epub 2020 Sep 8.

National Omics Center, National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.

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.
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http://dx.doi.org/10.1111/1755-0998.13243DOI Listing
January 2021

Author Correction: Transcriptome analyses reveal the synergistic effects of feeding and eyestalk ablation on ovarian maturation in black tiger shrimp.

Sci Rep 2020 Mar 20;10(1):5481. Epub 2020 Mar 20.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, 12120, Thailand.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-62221-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7083951PMC
March 2020

Transcriptome analyses reveal the synergistic effects of feeding and eyestalk ablation on ovarian maturation in black tiger shrimp.

Sci Rep 2020 02 24;10(1):3239. Epub 2020 Feb 24.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, 12120, Thailand.

Unilateral eyestalk ablation in the female black tiger shrimp Penaeus monodon is commonly employed to induce ovarian maturation. However, the importance of complementing this practice with the provision of live feed supplement (such as polychaetes) has not been emphasized in previous studies. Indeed, it has been less emphasized that female broodstock must be fed with live feeds such as polychaetes for this practice to be effective. While the effects of eyestalk ablation have been thoroughly studied in various aspects, the synergistic effects of feeding with live feeds and the ablation have never been elucidated at a transcriptome-wide level. With recent advances in the next-generation sequencing platforms, it is now possible to investigate the effects of eyestalk ablation and live feeds at the transcriptomic levels. This study employed both short-read Illumina RNA sequencing and long-read Pacific Biosciences (PacBio) isoform sequencing (Iso-seq) to generate the first high-quality ovarian reference transcriptome in P. monodon. This novel assembly allowed us to dissect the effects of feeds and eyestalk ablation and reveal their synergistic effects at the transcriptomic level through the regulation of important genes involved in fatty acid regulation, energy production, and hormone-mediated oocyte maturation pathways. The synergistic effects between the polychaete feeding and the eyestalk ablation in the process of ovarian maturation in black tiger shrimp suggest that without having proper nutrients from the polychaetes, female broodstock might not be ready to develop its ovary. However, even with proper nutrients, the eyestalk ablation is still necessary to perhaps manipulate the female endocrine of the black tiger shrimp. These findings shed the light on molecular mechanisms and key molecular pathways that lead to successful ovarian maturation.
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http://dx.doi.org/10.1038/s41598-020-60192-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7040003PMC
February 2020

Heat-induced shift in coral microbiome reveals several members of the Rhodobacteraceae family as indicator species for thermal stress in Porites lutea.

Microbiologyopen 2019 12 23;8(12):e935. Epub 2019 Sep 23.

National Omics Center, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.

The coral holobiont is a complex ecosystem consisting of coral animals and a highly diverse consortium of associated microorganisms including algae, fungi, and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their potential roles in promoting the host fitness and survival. Recently, dynamics of coral-associated microbiomes have been demonstrated to be linked to patterns of coral heat tolerance. Here, we examined the effect of elevated seawater temperature on the structure and diversity of bacterial populations associated with Porites lutea, using full-length 16S rRNA sequences obtained from Pacific Biosciences circular consensus sequencing. We observed a significant increase in alpha diversity indices and a distinct shift in microbiome composition during thermal stress. There was a marked decline in the apparent relative abundance of Gammaproteobacteria family Endozoicomonadaceae after P. lutea had been exposed to elevated seawater temperature. Concomitantly, the bacterial community structure shifted toward the predominance of Alphaproteobacteria family Rhodobacteraceae. Interestingly, we did not observe an increase in relative abundance of Vibrio-related sequences in our heat-stressed samples even though the appearance of Vibrio spp. has often been detected in parallel with the increase in the relative abundance of Rhodobacteraceae during thermal bleaching in other coral species. The ability of full-length 16S rRNA sequences in resolving taxonomic uncertainty of associated bacteria at a species level enabled us to identify 24 robust indicator bacterial species for thermally stressed corals. It is worth noting that the majority of those indicator species were members of the family Rhodobacteraceae. The comparison of bacterial community structure and diversity between corals in ambient water temperature and thermally stressed corals may provide a better understanding on how bacteria symbionts contribute to the resilience of their coral hosts to ocean warming.
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http://dx.doi.org/10.1002/mbo3.935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925168PMC
December 2019

Transcriptome analysis of oil palm inflorescences revealed candidate genes for an auxin signaling pathway involved in parthenocarpy.

PeerJ 2018 17;6:e5975. Epub 2018 Dec 17.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand.

Oil palm parthenocarpic fruits, which are produced without fertilization, can be targeted to increase oil content because the majority of the fruit is occupied by mesocarp, the part in which palm oil is stored. Consequently, gaining an understanding of the parthenocarpic mechanism would be instrumental for producing parthenocarpic oil palm. This study aims to determine effects of auxin treatment and analyze differentially expressed genes in oil palm pistils at the pollination/anthesis stage, using an RNA sequencing (RNA seq) approach. The auxin treatment caused 100% parthenocarpy when auxin was sprayed before stigmas opened. The parthenocarpy decreased to 55%, 8% and 5% when the auxin was sprayed 1, 2 and 3 days after the opening of stigmas, respectively. Oil palm plants used for RNA seq were plants untreated with auxin as controls and auxin-treated plants on the day before pollination and 1 day after pollination. The number of raw reads ranged from 8,425,859 to 11,811,166 reads, with an average size ranging from 99 to 137 base pairs (bp). When compared with the oil palm transcriptome, the mapped reads ranged from 8,179,948 to 11,320,799 reads, representing 95.85-98.01% of the oil palm matching. Based on five comparisons between RNA seq of treatments and controls, and confirmation using reverse transcription polymerase chain reaction and quantitative real-time RT-PCR expression, five candidate genes, including probable indole-3-acetic acid (IAA)-amido synthetase GH3.8 (), IAA-amido synthetase GH3.1 (), IAA induced ARG7 like (), tryptophan amino transferase-related protein 3-like () and flavin-containing monooxygenase 1 (), were differentially expressed between auxin-treated and untreated samples. This evidence suggests a pathway of parthenocarpic fruit development at the beginning of fruit development. However, more research is needed to identify which genes are definitely involved in parthenocarpy.
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http://dx.doi.org/10.7717/peerj.5975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301279PMC
December 2018

Uncovering full-length transcript isoforms of sugarcane cultivar Khon Kaen 3 using single-molecule long-read sequencing.

PeerJ 2018 30;6:e5818. Epub 2018 Oct 30.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani, Thailand.

Background: Sugarcane is an important global food crop and energy resource. To facilitate the sugarcane improvement program, genome and gene information are important for studying traits at the molecular level. Most currently available transcriptome data for sugarcane were generated using second-generation sequencing platforms, which provide short reads. The assembled transcripts from these data are limited in length, and hence may be incomplete and inaccurate, especially for long RNAs.

Methods: We generated a transcriptome dataset of leaf tissue from a commercial Thai sugarcane cultivar Khon Kaen 3 (KK3) using PacBio RS II single-molecule long-read sequencing by the Iso-Seq method. Short-read RNA-Seq data were generated from the same RNA sample using the Ion Proton platform for reducing base calling errors.

Results: A total of 119,339 error-corrected transcripts were generated with the N50 length of 3,611 bp, which is on average longer than any previously reported sugarcane transcriptome dataset. 110,253 sequences (92.4%) contain an open reading frame (ORF) of at least 300 bp long with ORF N50 of 1,416 bp. The mean lengths of 5' and 3' untranslated regions in 73,795 sequences with complete ORFs are 1,249 and 1,187 bp, respectively. 4,774 transcripts are putatively novel full-length transcripts which do not match with a previous Iso-Seq study of sugarcane. We annotated the functions of 68,962 putative full-length transcripts with at least 90% coverage when compared with homologous protein coding sequences in other plants.

Discussion: The new catalog of transcripts will be useful for genome annotation, identification of splicing variants, SNP identification, and other research pertaining to the sugarcane improvement program. The putatively novel transcripts suggest unique features of KK3, although more data from different tissues and stages of development are needed to establish a reference transcriptome of this cultivar.
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http://dx.doi.org/10.7717/peerj.5818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214230PMC
October 2018

Genome-wide association mapping of virulence gene in rice blast fungus Magnaporthe oryzae using a genotyping by sequencing approach.

Genomics 2019 07 15;111(4):661-668. Epub 2018 May 15.

National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Pahonyothin Road, Khlong Nueng, Khlong Luang, PathumThani 12120, Thailand. Electronic address:

Magnaporthe oryzae is a fungal pathogen causing blast disease in many plant species. In this study, seventy three isolates of M. oryzae collected from rice (Oryza sativa) in 1996-2014 were genotyped using a genotyping-by-sequencing approach to detect genetic variation. An association study was performed to identify single nucleotide polymorphisms (SNPs) associated with virulence genes using 831 selected SNP and infection phenotypes on local and improved rice varieties. Population structure analysis revealed eight subpopulations. The division into eight groups was not related to the degree of virulence. Association mapping showed five SNPs associated with fungal virulence on chromosome 1, 2, 3, 4 and 7. The SNP on chromosome 1 was associated with virulence against RD6-Pi7 and IRBL7-M which might be linked to the previously reported AvrPi7.
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http://dx.doi.org/10.1016/j.ygeno.2018.05.011DOI Listing
July 2019

Dynamics of coral-associated microbiomes during a thermal bleaching event.

Microbiologyopen 2018 10 23;7(5):e00604. Epub 2018 Mar 23.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand.

Coral-associated microorganisms play an important role in their host fitness and survival. A number of studies have demonstrated connections between thermal tolerance in corals and the type/relative abundance of Symbiodinium they harbor. More recently, the shifts in coral-associated bacterial profiles were also shown to be linked to the patterns of coral heat tolerance. Here, we investigated the dynamics of Porites lutea-associated bacterial and algal communities throughout a natural bleaching event, using full-length 16S rRNA and internal transcribed spacer sequences (ITS) obtained from PacBio circular consensus sequencing. We provided evidence of significant changes in the structure and diversity of coral-associated microbiomes during thermal stress. The balance of the symbiosis shifted from a predominant association between corals and Gammaproteobacteria to a predominance of Alphaproteobacteria and to a lesser extent Betaproteobacteria following the bleaching event. On the contrary, the composition and diversity of Symbiodinium communities remained unaltered throughout the bleaching event. It appears that the switching and/or shuffling of Symbiodinium types may not be the primary mechanism used by P. lutea to cope with increasing seawater temperature. The shifts in the structure and diversity of associated bacterial communities may contribute more to the survival of the coral holobiont under heat stress.
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http://dx.doi.org/10.1002/mbo3.604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6182559PMC
October 2018

High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system.

Sci Rep 2017 06 5;7(1):2774. Epub 2017 Jun 5.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand.

Coral reefs are a complex ecosystem consisting of coral animals and a vast array of associated symbionts including the dinoflagellate Symbiodinium, fungi, viruses and bacteria. Several studies have highlighted the importance of coral-associated bacteria and their fundamental roles in fitness and survival of the host animal. The scleractinian coral Porites lutea is one of the dominant reef-builders in the Indo-West Pacific. Currently, very little is known about the composition and structure of bacterial communities across P. lutea reefs. The purpose of this study is twofold: to demonstrate the advantages of using PacBio circular consensus sequencing technology in microbial community studies and to investigate the diversity and structure of P. lutea-associated microbiome in the Indo-Pacific. This is the first metagenomic study of marine environmental samples that utilises the PacBio sequencing system to capture full-length 16S rRNA sequences. We observed geographically distinct coral-associated microbial profiles between samples from the Gulf of Thailand and Andaman Sea. Despite the geographical and environmental impacts on the coral-host interactions, we identified a conserved community of bacteria that were present consistently across diverse reef habitats. Finally, we demonstrated the superior performance of full-length 16S rRNA sequences in resolving taxonomic uncertainty of coral associates at the species level.
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http://dx.doi.org/10.1038/s41598-017-03139-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459821PMC
June 2017

De novo hybrid assembly of the rubber tree genome reveals evidence of paleotetraploidy in Hevea species.

Sci Rep 2017 02 2;7:41457. Epub 2017 Feb 2.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathum Thani, Thailand.

Para rubber tree (Hevea brasiliensis) is an important economic species as it is the sole commercial producer of high-quality natural rubber. Here, we report a de novo hybrid assembly of BPM24 accession, which exhibits resistance to major fungal pathogens in Southeast Asia. Deep-coverage 454/Illumina short-read and Pacific Biosciences (PacBio) long-read sequence data were acquired to generate a preliminary draft, which was subsequently scaffolded using a long-range "Chicago" technique to obtain a final assembly of 1.26 Gb (N50 = 96.8 kb). The assembled genome contains 69.2% repetitive sequences and has a GC content of 34.31%. Using a high-density SNP-based genetic map, we were able to anchor 28.9% of the genome assembly (363 Mb) associated with over two thirds of the predicted protein-coding genes into rubber tree's 18 linkage groups. These genetically anchored sequences allowed comparative analyses of the intragenomic homeologous synteny, providing the first concrete evidence to demonstrate the presence of paleotetraploidy in Hevea species. Additionally, the degree of macrosynteny conservation observed between rubber tree and cassava strongly supports the hypothesis that the paleotetraploidization event took place prior to the divergence of the Hevea and Manihot species.
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http://dx.doi.org/10.1038/srep41457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288721PMC
February 2017

Effects of methylation-sensitive enzymes on the enrichment of genic SNPs and the degree of genome complexity reduction in a two-enzyme genotyping-by-sequencing (GBS) approach: a case study in oil palm ().

Mol Breed 2016 10;36(11):154. Epub 2016 Nov 10.

National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Pathum Thani, 12120 Thailand.

Advances in next generation sequencing have facilitated a large-scale single nucleotide polymorphism (SNP) discovery in many crop species. Genotyping-by-sequencing (GBS) approach couples next generation sequencing with genome complexity reduction techniques to simultaneously identify and genotype SNPs. Choice of enzymes used in GBS library preparation depends on several factors including the number of markers required, the desired level of multiplexing, and whether the enrichment of genic SNP is preferred. We evaluated various combinations of methylation-sensitive (II, I, I) and methylation-insensitive (I, I) enzymes for their effectiveness in genome complexity reduction and enrichment of genic SNPs. We discovered that the use of two methylation-sensitive enzymes effectively reduced genome complexity and did not require a size selection step. On the contrary, the genome coverage of libraries constructed with methylation-insensitive enzymes was quite high, and the additional size selection step may be required to increase the overall read depth. We also demonstrated the effectiveness of methylation-sensitive enzymes in enriching for SNPs located in genic regions. When two methylation-insensitive enzymes were used, only 16% of SNPs identified were located in genes and 18% in the vicinity (± 5 kb) of the genic regions, while most SNPs resided in the intergenic regions. In contrast, a remarkable degree of enrichment was observed when two methylation-sensitive enzymes were employed. Almost two thirds of the SNPs were located either inside (32-36%) or in the vicinity (28-31%) of the genic regions. These results provide useful information to help researchers choose appropriate GBS enzymes in oil palm and other crop species.
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http://dx.doi.org/10.1007/s11032-016-0572-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104780PMC
November 2016

The two chromosomes of the mitochondrial genome of a sugarcane cultivar: assembly and recombination analysis using long PacBio reads.

Sci Rep 2016 08 17;6:31533. Epub 2016 Aug 17.

National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Paholyothin Road, Khlong Nueng, Khlong Luang, Pathumthani, 12120, Thailand.

Sugarcane accounts for a large portion of the worlds sugar production. Modern commercial cultivars are complex hybrids of S. officinarum and several other Saccharum species. Historical records identify New Guinea as the origin of S. officinarum and that a small number of plants originating from there were used to generate all modern commercial cultivars. The mitochondrial genome can be a useful way to identify the maternal origin of commercial cultivars. We have used the PacBio RSII to sequence and assemble the mitochondrial genome of a South East Asian commercial cultivar, known as Khon Kaen 3. The long read length of this sequencing technology allowed for the mitochondrial genome to be assembled into two distinct circular chromosomes with all repeat sequences spanned by individual reads. Comparison of five commercial hybrids, two S. officinarum and one S. spontaneum to our assembly reveals no structural rearrangements between our assembly, the commercial hybrids and an S. officinarum from New Guinea. The S. spontaneum, from India, and one sample of S. officinarum (unknown origin) are substantially rearranged and have a large number of homozygous variants. This supports the record that S. officinarum plants from New Guinea are the maternal source of all modern commercial hybrids.
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http://dx.doi.org/10.1038/srep31533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987617PMC
August 2016

Construction of a high-density integrated genetic linkage map of rubber tree (Hevea brasiliensis) using genotyping-by-sequencing (GBS).

Front Plant Sci 2015 27;6:367. Epub 2015 May 27.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency Pathum Thani, Thailand.

Construction of linkage maps is crucial for genetic studies and marker-assisted breeding programs. Recent advances in next generation sequencing technologies allow for the generation of high-density linkage maps, especially in non-model species lacking extensive genomic resources. Here, we constructed a high-density integrated genetic linkage map of rubber tree (Hevea brasiliensis), the sole commercial producer of high-quality natural rubber. We applied a genotyping-by-sequencing (GBS) technique to simultaneously discover and genotype single nucleotide polymorphism (SNP) markers in two rubber tree populations. A total of 21,353 single nucleotide substitutions were identified, 55% of which represented transition events. GBS-based genetic maps of populations P and C comprised 1704 and 1719 markers and encompassed 2041 cM and 1874 cM, respectively. The average marker densities of these two maps were one SNP in 1.23-1.25 cM. A total of 1114 shared SNP markers were used to merge the two component maps. An integrated linkage map consisted of 2321 markers and spanned the cumulative length of 2052 cM. The composite map showed a substantial improvement in marker density, with one SNP marker in every 0.89 cM. To our knowledge, this is the most saturated genetic map in rubber tree to date. This integrated map allowed us to anchor 28,965 contigs, covering 135 Mb or 12% of the published rubber tree genome. We demonstrated that GBS is a robust and cost-effective approach for generating a common set of genome-wide SNP data suitable for constructing integrated linkage maps from multiple populations in a highly heterozygous agricultural species.
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http://dx.doi.org/10.3389/fpls.2015.00367DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444744PMC
June 2015

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

Genomics 2015 May 20;105(5-6):288-95. Epub 2015 Feb 20.

National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Pathum Thani 12120, Thailand. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ygeno.2015.02.002DOI Listing
May 2015

Large-scale SNP discovery through RNA sequencing and SNP genotyping by targeted enrichment sequencing in cassava (Manihot esculenta Crantz).

PLoS One 2014 31;9(12):e116028. Epub 2014 Dec 31.

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Pathum Thani, Thailand.

Cassava (Manihot esculenta Crantz) is one of the most important crop species being the main source of dietary energy in several countries. Marker-assisted selection has become an essential tool in plant breeding. Single nucleotide polymorphism (SNP) discovery via transcriptome sequencing is an attractive strategy for genome complexity reduction in organisms with large genomes. We sequenced the transcriptome of 16 cassava accessions using the Illumina HiSeq platform and identified 675,559 EST-derived SNP markers. A subset of those markers was subsequently genotyped by capture-based targeted enrichment sequencing in 100 F1 progeny segregating for starch viscosity phenotypes. A total of 2,110 non-redundant SNP markers were used to construct a genetic map. This map encompasses 1,785 cM and consists of 19 linkage groups. A major quantitative trait locus (QTL) controlling starch pasting properties was identified and shown to coincide with the QTL previously reported for this trait. With a high-density SNP-based linkage map presented here, we also uncovered a novel QTL associated with starch pasting time on LG 10.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116028PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4281258PMC
October 2015

Critical function of a Chlamydomonas reinhardtii putative polyphosphate polymerase subunit during nutrient deprivation.

Plant Cell 2014 Oct 3;26(10):4214-29. Epub 2014 Oct 3.

The Carnegie Institution for Science, Department of Plant Biology, Stanford, California 94305.

Forward genetics was used to isolate Chlamydomonas reinhardtii mutants with altered abilities to acclimate to sulfur (S) deficiency. The ars76 mutant has a deletion that eliminates several genes, including VACUOLAR TRANSPORTER CHAPERONE1 (VTC1), which encodes a component of a polyphosphate polymerase complex. The ars76 mutant cannot accumulate arylsulfatase protein or mRNA and shows marked alterations in levels of many transcripts encoded by genes induced during S deprivation. The mutant also shows little acidocalcisome formation compared with wild-type, S-deprived cells and dies more rapidly than wild-type cells following exposure to S-, phosphorus-, or nitrogen (N)-deficient conditions. Furthermore, the mutant does not accumulate periplasmic L-amino acid oxidase during N deprivation. Introduction of the VTC1 gene specifically complements the ars76 phenotypes, suggesting that normal acidocalcisome formation in cells deprived of S requires VTC1. Our data also indicate that a deficiency in acidocalcisome function impacts trafficking of periplasmic proteins, which can then feed back on the transcription of the genes encoding these proteins. These results and the reported function of vacuoles in degradation processes suggest a major role of the acidocalcisome in reshaping the cell during acclimation to changing environmental conditions.
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http://dx.doi.org/10.1105/tpc.114.129270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4247568PMC
October 2014

Tiered regulation of sulfur deprivation responses in Chlamydomonas reinhardtii and identification of an associated regulatory factor.

Plant Physiol 2013 May 12;162(1):195-211. Epub 2013 Mar 12.

Department of Plant Biology, Carnegie Institution for Science, Stanford, California 94305, USA.

During sulfur (S) deprivation, the unicellular alga Chlamydomonas reinhardtii exhibits increased expression of numerous genes. These genes encode proteins associated with sulfate (SO4(2-)) acquisition and assimilation, alterations in cellular metabolism, and internal S recycling. Administration of the cytoplasmic translational inhibitor cycloheximide prevents S deprivation-triggered accumulation of transcripts encoding arylsulfatases (ARS), an extracellular polypeptide that may be important for cell wall biosynthesis (ECP76), a light-harvesting protein (LHCBM9), the selenium-binding protein, and the haloperoxidase (HAP2). In contrast, the rapid accumulation of transcripts encoding high-affinity SO4(2-) transporters is not affected. These results suggest that there are two tiers of transcriptional regulation associated with S deprivation responses: the first is protein synthesis independent, while the second requires de novo protein synthesis. A mutant designated ars73a exhibited low ARS activity and failed to show increases in ECP76, LHCBM9, and HAP2 transcripts (among others) in response to S deprivation; increases in transcripts encoding the SO4(2-) transporters were not affected. These results suggest that the ARS73a protein, which has no known activity but might be a transcriptional regulator, is required for the expression of genes associated with the second tier of transcriptional regulation. Analysis of the ars73a strain has helped us generate a model that incorporates a number of complexities associated with S deprivation responses in C. reinhardtii.
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http://dx.doi.org/10.1104/pp.113.214593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3641202PMC
May 2013

Single nucleotide polymorphism marker development in the rubber tree, Hevea brasiliensis (Euphorbiaceae).

Am J Bot 2011 Nov 24;98(11):e337-8. Epub 2011 Oct 24.

National Center for Genetic Engineering and Biotechnology, 113 Phaholyothin Rd., Klong 1, Klong Luang, Pathumthani 12120 Thailand.

Premise Of The Study: We demonstrated the application of high-throughput 454 sequencing technology in the identification of single nucleotide polymorphism (SNP) markers in Hevea brasiliensis.

Methods And Results: A total of 5883 putative SNP positions were discovered in silico, and 10 biallelic SNP markers were validated from 454-derived EST sequences. The polymorphism information content (PIC) and the observed heterozygosity (H(o)) ranged from 0.0963-0.5135 and 0.1071-0.4643, respectively.

Conclusions: These markers can be useful for the construction of genetic maps, the identification of quantitative trait loci linked to commercially desirable traits, and the study of genetic structure in H. brasiliensis.
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http://dx.doi.org/10.3732/ajb.1100228DOI Listing
November 2011

Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants.

Plant Methods 2011 Jul 27;7:24. Epub 2011 Jul 27.

Department of Plant Biology, The Carnegie Institution for Science, Stanford, CA 94305, USA.

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants.
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http://dx.doi.org/10.1186/1746-4811-7-24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161022PMC
July 2011

Identification and regulation of plasma membrane sulfate transporters in Chlamydomonas.

Plant Physiol 2010 Aug 24;153(4):1653-68. Epub 2010 May 24.

Department of Biology, Stanford University, Stanford, California 94305-5020, USA.

Chlamydomonas (Chlamydomonas reinhardtii) exhibits several responses following exposure to sulfur (S)-deprivation conditions, including an increased efficiency of import and assimilation of the sulfate anion (SO(4)(2-)). Aspects of SO(4)(2-) transport during S-replete and S-depleted conditions were previously studied, although the transporters had not been functionally identified. We employed a reverse genetics approach to identify putative SO(4)(2-) transporters, examine their regulation, establish their biogenesis and subcellular locations, and explore their functionality. Upon S starvation of wild-type Chlamydomonas cells, the accumulation of transcripts encoding the putative SO(4)(2-) transporters SLT1 (for SAC1-like transporter 1), SLT2, and SULTR2 markedly increased, suggesting that these proteins function in high-affinity SO(4)(2-) transport. The Chlamydomonas sac1 and snrk2.1 mutants (defective for acclimation to S deprivation) exhibited much less of an increase in the levels of SLT1, SLT2, and SULTR2 transcripts and their encoded proteins in response to S deprivation compared with wild-type cells. All three transporters were localized to the plasma membrane, and their rates of turnover were significantly impacted by S availability; the turnover of SLT1 and SLT2 was proteasome dependent, while that of SULTR2 was proteasome independent. Finally, mutants identified for each of the S-deprivation-responsive transporters were used to establish their critical role in the transport of SO(4)(2-) into S-deprived cells.
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http://dx.doi.org/10.1104/pp.110.157875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923900PMC
August 2010