The mitochondrial genome map of Nelumbo nucifera reveals ancient evolutionary features.

Nelumbo nucifera is an evolutionary relic from the Late Cretaceous period. Sequencing the N. nucifera mitochondrial genome is important for elucidating the evolutionary characteristics of basal eudicots. Here, the N. nucifera mitochondrial genome was sequenced using single molecule real-time sequencing technology (SMRT), and the mitochondrial genome map was constructed after de novo assembly and annotation. The results showed that the 524,797-bp N. nucifera mitochondrial genome has a total of 63 genes, including 40 protein-coding genes, three rRNA genes and 20 tRNA genes. Fifteen collinear gene clusters were conserved across different plant species. Approximately 700 RNA editing sites in the protein-coding genes were identified. Positively selected genes were identified with selection pressure analysis. Nineteen chloroplast-derived fragments were identified, and seven tRNAs were derived from the chloroplast. These results suggest that the N. nucifera mitochondrial genome retains evolutionarily conserved characteristics, including ancient gene content and gene clusters, high levels of RNA editing, and low levels of chloroplast-derived fragment insertions. As the first publicly available basal eudicot mitochondrial genome, the N. nucifera mitochondrial genome facilitates further analysis of the characteristics of basal eudicots and provides clues of the evolutionary trajectory from basal angiosperms to advanced eudicots.

Complete mitochondrial genome of the medicinal fungus Ophiocordyceps sinensis.

As part of a genome sequencing project for Ophiocordyceps sinensis, strain 1229, a complete mitochondrial (mt) genome was assembled as a single circular dsDNA of 157,510 bp, one of the largest reported for fungi. Conserved genes including the large and small rRNA subunits, 27 tRNA and 15 protein-coding genes, were identified. In addition, 58 non-conserved open reading frames (ncORFs) in the intergenic and intronic regions were also identified. Transcription analyses using RNA-Seq validated the expression of most conserved genes and ncORFs. Fifty-two introns (groups I and II) were found within conserved genes, accounting for 68.5% of the genome. Thirty-two homing endonucleases (HEs) with motif patterns LAGLIDADG (21) and GIY-YIG (11) were identified in group I introns. The ncORFs found in group II introns mostly encoded reverse transcriptases (RTs). As in other hypocrealean fungi, gene contents and order were found to be conserved in the mt genome of O. sinensis, but the genome size was enlarged by longer intergenic regions and numerous introns. Intergenic and intronic regions were composed of abundant repetitive sequences usually associated with mobile elements. It is likely that intronic ncORFs, which encode RTs and HEs, may have contributed to the enlarged mt genome of O. sinensis.

A precise chloroplast genome of Nelumbo nucifera (Nelumbonaceae) evaluated with Sanger, Illumina MiSeq, and PacBio RS II sequencing platforms: insight into the plastid evolution of basal eudicots.

BACKGROUND: The chloroplast genome is important for plant development and plant evolution. Nelumbo nucifera is one member of relict plants surviving from the late Cretaceous. Recently, a new sequencing platform PacBio RS II, known as 'SMRT (Single Molecule, Real-Time) sequencing', has been developed. Using the SMRT sequencing to investigate the chloroplast genome of N. nucifera will help to elucidate the plastid evolution of basal eudicots. RESULTS: The sizes of the de novo assembled complete chloroplast genome of N. nucifera were 163,307 bp, 163,747 bp and 163,600 bp with average depths of coverage of 7×, 712× and 105× sequenced by Sanger, Illumina MiSeq and PacBio RS II, respectively. The precise chloroplast genome of N. nucifera was obtained from PacBio RS II data proofread by Illumina MiSeq reads, with a quadripartite structure containing a large single copy region (91,846 bp) and a small single copy region (19,626 bp) separated by two inverted repeat regions (26,064 bp). The genome contains 113 different genes, including four distinct rRNAs, 30 distinct tRNAs and 79 distinct peptide-coding genes. A phylogenetic analysis of 133 taxa from 56 orders indicated that Nelumbo with an age of 177 million years is a sister clade to Platanus, which belongs to the basal eudicots. Basal eudicots began to emerge during the early Jurassic with estimated divergence times at 197 million years using MCMCTree. IR expansions/contractions within the basal eudicots seem to have occurred independently. CONCLUSIONS: Because of long reads and lack of bias in coverage of AT-rich regions, PacBio RS II showed a great promise for highly accurate 'finished' genomes, especially for a de novo assembly of genomes. N. nucifera is one member of basal eudicots, however, evolutionary analyses of IR structural variations of N. nucifera and other basal eudicots suggested that IR expansions/contractions occurred independently in these basal eudicots or were caused by independent insertions and deletions. The precise chloroplast genome of N. nucifera will present new information for structural variation of chloroplast genomes and provide new insight into the evolution of basal eudicots at the primary sequence and structural level.

DNA phosphorothioate modifications influence the global transcriptional response and protect DNA from double-stranded breaks.

The modification of DNA by phosphorothioate (PT) occurs when the non-bridging oxygen in the sugar-phosphate backbone of DNA is replaced with sulfur. This DNA backbone modification was recently discovered and is governed by the dndABCDE genes in a diverse group of bacteria and archaea. However, the biological function of DNA PT modifications is poorly understood. In this study, we employed the RNA-seq analysis to characterize the global transcriptional changes in response to PT modifications. Our results show that DNA without PT protection is susceptible to DNA damage caused by the dndFGHI gene products. The DNA double-stranded breaks then trigger the SOS response, cell filamentation and prophage induction. Heterologous expression of dndBCDE conferring DNA PT modifications at GPSA and GPST prevented the damage in Salmonella enterica. Our data provide insights into the physiological role of the DNA PT system.

An integrated tool to study MHC region: accurate SNV detection and HLA genes typing in human MHC region using targeted high-throughput sequencing.

The major histocompatibility complex (MHC) is one of the most variable and gene-dense regions of the human genome. Most studies of the MHC, and associated regions, focus on minor variants and HLA typing, many of which have been demonstrated to be associated with human disease susceptibility and metabolic pathways. However, the detection of variants in the MHC region, and diagnostic HLA typing, still lacks a coherent, standardized, cost effective and high coverage protocol of clinical quality and reliability. In this paper, we presented such a method for the accurate detection of minor variants and HLA types in the human MHC region, using high-throughput, high-coverage sequencing of target regions. A probe set was designed to template upon the 8 annotated human MHC haplotypes, and to encompass the 5 megabases (Mb) of the extended MHC region. We deployed our probes upon three, genetically diverse human samples for probe set evaluation, and sequencing data show that ∼97% of the MHC region, and over 99% of the genes in MHC region, are covered with sufficient depth and good evenness. 98% of genotypes called by this capture sequencing prove consistent with established HapMap genotypes. We have concurrently developed a one-step pipeline for calling any HLA type referenced in the IMGT/HLA database from this target capture sequencing data, which shows over 96% typing accuracy when deployed at 4 digital resolution. This cost-effective and highly accurate approach for variant detection and HLA typing in the MHC region may lend further insight into immune-mediated diseases studies, and may find clinical utility in transplantation medicine research. This one-step pipeline is released for general evaluation and use by the scientific community.

The draft genome of watermelon (Citrullus lanatus) and resequencing of 20 diverse accessions.

Watermelon, Citrullus lanatus, is an important cucurbit crop grown throughout the world. Here we report a high-quality draft genome sequence of the east Asia watermelon cultivar 97103 (2n = 2× = 22) containing 23,440 predicted protein-coding genes. Comparative genomics analysis provided an evolutionary scenario for the origin of the 11 watermelon chromosomes derived from a 7-chromosome paleohexaploid eudicot ancestor. Resequencing of 20 watermelon accessions representing three different C. lanatus subspecies produced numerous haplotypes and identified the extent of genetic diversity and population structure of watermelon germplasm. Genomic regions that were preferentially selected during domestication were identified. Many disease-resistance genes were also found to be lost during domestication. In addition, integrative genomic and transcriptomic analyses yielded important insights into aspects of phloem-based vascular signaling in common between watermelon and cucumber and identified genes crucial to valuable fruit-quality traits, including sugar accumulation and citrulline metabolism.