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.

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.

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.

Rice height QTLs in KDML105 chromosome segment substitution lines.

Rice is an important crop that is consumed by approximately half of the world's population on a regular basis. Plant height is an important characteristic with shorter rice often having higher lodging resistance and better soil nutrient utilization allowing for lower fertilizer use. We used a Chromosome Segment Substitution Line (CSSL) population generated by introgressing segments of CT9993 and IR62266 into KDML 105. We identified height QTLs on chromosomes 1 and 4. We performed whole genome sequencing of the parental lines and found that IR62266 has the deletion in Gibberellin 20-oxidase 2 corresponding to the semi-dwarf 1 locus. However, short height on chromosome 1 came from CT9993 with no mutation in Gibberellin 20-oxidase 2, or any known height genes. The height QTL on chromosome 4 contains mutations in Peroxisome biogenesis protein 6, which has been linked to a reduced growth phenotype in A. thaliana, making this a good candidate height gene.

A single segment substitution line population for identifying traits relevant to drought tolerance and avoidance.

A population of chromosome segment substitution lines was developed using KDML105 as the recurrent parent and one of DH212 (IR68586-F2-CA-143) or DH103 (IR68586-F2-CA-31) as the donor parent. The donor parents are part of a doubled haploid population from a cross between CT9993, an upland japonica accession, and IR62266, a lowland indica accession. Multiple QTL that are relevant to drought avoidance, drought tolerance and yield traits under drought stress were mapped in this doubled haploid population and the segments selected for the chromosome segment substitution lines were chosen to capture these QTL. The chromosome segment substitution line population was phenotyped under irrigated and mild drought stress conditions, which identified that many yield traits under drought stress had been introduced into the chromosome segment substitution lines.

Complete chloroplast genome sequences of five Bruguiera species (Rhizophoraceae): comparative analysis and phylogenetic relationships.

Bruguiera is a genus of true mangroves that are mostly distributed in the Indo-West Pacific region. However, the number of published whole chloroplast genome sequences of Bruguiera species are limited. Here, the complete chloroplast sequences of five Bruguiera species were sequenced and assembled using Illumina data. The chloroplast genomes of B. gymnorhiza, B. hainesii, B. cylindrica, B. parviflora and B. sexangula were assembled into 161,195, 164,295, 164,297, 163,228 and 164,170 bp, respectively. All chloroplast genomes contain 37 tRNA and eight rRNA genes, with either 84 or 85 protein-coding genes. A comparative analysis of these genomes revealed high similarity in gene structure, gene order and boundary position of the LSC, SSC and two IR regions. Interestingly, B. gymnorhiza lost a rpl32 gene in the SSC region. In addition, a ndhF gene in B. parviflora straddles both the SSC and IRB boundary regions. These genes reveal differences in chloroplast evolution among Bruguiera species. Repeats and SSRs in the chloroplast genome sequences were found to be highly conserved between B. cylindrica and B. hainesii as well as B. gymnorhiza and B. sexangula indicating close genetic relationships based on maternal inheritance. Notably, B. hainesii, which is considered a hybrid between B. gymnorhiza and B. cylindrica, appears to have inherited the chloroplast from B. cylindrica. Investigating the effects of selection events on shared protein-coding genes showed a positive selection in rps7 and rpl36 genes in all species compared to land-plant species. A phylogenetic analysis, based on 59 conserved chloroplast protein-coding genes, showed strong support that all Bruguiera species are in the clade Rhizophoraceae. This study provides valuable genetic information for the study of evolutionary relationships and population genetics in Bruguiera and other mangrove species.

Population Structure of Nation-Wide Rice in Thailand.

BACKGROUND: Thailand is a country with large diversity in rice varieties due to its rich and diverse ecology. In this paper, 300 rice accessions from all across Thailand were sequenced to identify SNP variants allowing for the population structure to be explored. RESULTS: The result of inferred population structure from admixture and clustering analysis illustrated strong evidence of substructure in each geographical region. The results of phylogenetic tree, PCA analysis, and machine learning on population identifying SNPs also supported the inferred population structure. CONCLUSION: The population structure inferred in this study contains five subpopulations that tend to group individuals based on location. So, each subpopulation has unique genetic patterns, agronomic traits, as well as different environmental conditions. This study can serve as a reference point of the nation-wide population structure for supporting breeders and researchers who are interested in Thai rice.

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.

Assembly of the durian chloroplast genome using long PacBio reads.

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.

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

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.

SNP identification from RNA sequencing and linkage map construction of rubber tree for anchoring the draft genome.

Hevea brasiliensis, or rubber tree, is an important crop species that accounts for the majority of natural latex production. The rubber tree nuclear genome consists of 18 chromosomes and is roughly 2.15 Gb. The current rubber tree reference genome assembly consists of 1,150,326 scaffolds ranging from 200 to 531,465 bp and totalling 1.1 Gb. Only 143 scaffolds, totalling 7.6 Mb, have been placed into linkage groups. We have performed RNA-seq on 6 varieties of rubber tree to identify SNPs and InDels and used this information to perform target sequence enrichment and high throughput sequencing to genotype a set of SNPs in 149 rubber tree offspring from a cross between RRIM 600 and RRII 105 rubber tree varieties. We used this information to generate a linkage map allowing for the anchoring of 24,424 contigs from 3,009 scaffolds, totalling 115 Mb or 10.4% of the published sequence, into 18 linkage groups. Each linkage group contains between 319 and 1367 SNPs, or 60 to 194 non-redundant marker positions, and ranges from 156 to 336 cM in length. This linkage map includes 20,143 of the 69,300 predicted genes from rubber tree and will be useful for mapping studies and improving the reference genome assembly.

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.

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.

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

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.

Transcriptome analysis of normal and mantled developing oil palm flower and fruit.

Elaeis guineensis (oil palm) accounts for a large and increasing proportion of the world's cooking oil production. Cloning via somatic embryogenesis results in a somaclonal variant known as mantled which produce fruit with little to no oil yield. The mantled phenotype is believed to be epigenetic in nature. We performed RNA-Seq on developing flower and fruit samples of normal and mantled oil palm to characterize their transcriptomes. We present expression data for all transcripts in normal and mantled flower and fruit samples. Many genes are differentially expressed, including several from pathways that may be the cause of the mantled phenotype if disrupted, such as genes involved in primary hormone responses, DNA replication and repair, chromatin remodeling and a gene involved in RNA mediated DNA methylation. In addition, the gene expression data for developing flower and fruit will serve as a valuable resource for oil palm genetics and genomic studies.

A canine model of Cohen syndrome: Trapped Neutrophil Syndrome.

BACKGROUND: Trapped Neutrophil Syndrome (TNS) is a common autosomal recessive neutropenia in Border collie dogs. RESULTS: We used a candidate gene approach and linkage analysis to show that the causative gene for TNS is VPS13B. We chose VPS13B as a candidate because of similarities in clinical signs between TNS and Cohen syndrome, in human, such as neutropenia and a typical facial dysmorphism. Linkage analysis using microsatellites close to VPS13B showed positive linkage of the region to TNS. We sequenced each of the 63 exons of VPS13B in affected and control dogs and found that the causative mutation in Border collies is a 4 bp deletion in exon 19 of the largest transcript that results in premature truncation of the protein. Cohen syndrome patients present with mental retardation in 99% of cases, but learning disabilities featured in less than half of TNS affected dogs. It has been implied that loss of the alternate transcript of VPS13B in the human brain utilising an alternate exon, 28, may cause mental retardation. Mice cannot be used to test this hypothesis as they do not express the alternate exon. We show that dogs do express alternate transcripts in the brain utilising an alternate exon homologous to human exon 28. CONCLUSION: Dogs can be used as a model organism to explore the function of the alternately spliced transcript of VPS13B in the brain. TNS in Border collies is the first animal model for Cohen syndrome and can be used to study the disease aetiology.

Origins of the domestic dog and the rich potential for gene mapping.

The unique breeding structure of the domestic dog makes canine genetics a useful tool to further the understanding of inherited diseases and gene function. Answers to the questions of when and where the dog was domesticated from the wolf are uncertain, but how the modern diversity of dog breeds was developed is documented. Breed development has resulted in many genetically isolated populations which are segregating for different alleles for disease and morphological and behavioral traits. Many genetic tools are available for dog research allowing investigation into the genetic basis of these phenotypes. Research into causes of diseases in dogs is relevant to humans and other species; comparative genomics is being used to transfer genetic information to them, including some studies on morphological and behavioral phenotypes. Because of the unique breed structure and well-maintained pedigrees, dogs represent a model organism containing a wealth of genetic information.