Sequencing of 6.7 Mb of the melon genome using a BAC pooling strategy

<p>Abstract</p> <p>Background</p> <p><it>Cucumis melo </it>(melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has a high intra-specific genetic variation, morphologic diversity and a small genome size (454 Mb), which make it suitable for a great variety of molecular and genetic studies. A number of genetic and genomic resources have already been developed, such as several genetic maps, BAC genomic libraries, a BAC-based physical map and EST collections. Sequence information would be invaluable to complete the picture of the melon genomic landscape, furthering our understanding of this species' evolution from its relatives and providing an important genetic tool. However, to this day there is little sequence data available, only a few melon genes and genomic regions are deposited in public databases. The development of massively parallel sequencing methods allows envisaging new strategies to obtain long fragments of genomic sequence at higher speed and lower cost than previous Sanger-based methods.</p> <p>Results</p> <p>In order to gain insight into the structure of a significant portion of the melon genome we set out to perform massive sequencing of pools of BAC clones. For this, a set of 57 BAC clones from a double haploid line was sequenced in two pools with the 454 system using both shotgun and paired-end approaches. The final assembly consists of an estimated 95% of the actual size of the melon BAC clones, with most likely complete sequences for 50 of the BACs, and a total sequence coverage of 39x. The accuracy of the assembly was assessed by comparing the previously available Sanger sequence of one of the BACs against its 454 sequence, and the polymorphisms found involved only 1.7 differences every 10,000 bp that were localized in 15 homopolymeric regions and two dinucleotide tandem repeats. Overall, the study provides approximately 6.7 Mb or 1.5% of the melon genome. The analysis of this new data has allowed us to gain further insight into characteristics of the melon genome such as gene density, average protein length, or microsatellite and transposon content. The annotation of the BAC sequences revealed a high degree of collinearity and protein sequence identity between melon and its close relative <it>Cucumis sativus </it>(cucumber). Transposon content analysis of the syntenic regions suggests that transposition activity after the split of both cucurbit species has been low in cucumber but very high in melon.</p> <p>Conclusions</p> <p>The results presented here show that the strategy followed, which combines shotgun and BAC-end sequencing together with anchored marker information, is an excellent method for sequencing specific genomic regions, especially from relatively compact genomes such as that of melon. However, in agreement with other results, this map-based, BAC approach is confirmed to be an expensive way of sequencing a whole plant genome. Our results also provide a partial description of the melon genome's structure. Namely, our analysis shows that the melon genome is highly collinear with the smaller one of cucumber, the size difference being mainly due to the expansion of intergenic regions and proliferation of transposable elements.</p

Retrotransposons are specified as DNA replication origins in the gene-poor regions of Arabidopsis heterochromatin

Genomic stability depends on faithful genome replication. This is achieved by the concerted activity of thousands of DNA replication origins (ORIs) scattered throughout the genome. The DNA and chromatin features determining ORI specification are not presently known. We have generated a high-resolution genome-wide map of 3230 ORIs in cultured Arabidopsis thaliana cells. Here, we focused on defining the features associated with ORIs in heterochromatin. In pericentromeric gene-poor domains ORIs associate almost exclusively with the retrotransposon class of transposable elements (TEs), in particular of the Gypsy family. ORI activity in retrotransposons occurs independently of TE expression and while maintaining high levels of H3K9me2 and H3K27me1, typical marks of repressed heterochromatin. ORI-TEs largely colocalize with chromatin signatures defining GC-rich heterochromatin. Importantly, TEs with active ORIs contain a local GC content higher than the TEs lacking them. Our results lead us to conclude that ORI colocalization with retrotransposons is determined by their transposition mechanism based on transcription, and a specific chromatin landscape. Our detailed analysis of ORIs responsible for heterochromatin replication has implications on the mechanisms of ORI specification in other multicellular organisms in which retrotransposons are major components of heterochromatin and of the entire genome

Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics

The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of citie

Next-generation sequencing, FISH mapping and synteny-based modeling reveal mechanisms of decreasing dysploidy in Cucumis

In the large Cucurbitaceae genus Cucumis, cucumber (C. sativus) is the only species with 2n = 2x = 14 chromosomes. The majority of the remaining species, including melon (C. melo) and the sister species of cucumber, C. hystrix, have 2n = 2x = 24 chromosomes, implying a reduction from n = 12 to n = 7. To understand the underlying mechanisms, we investigated chromosome synteny among cucumber, C. hystrix and melon using integrated and complementary approaches. We identified 14 inversions and a C. hystrix lineage-specific reciprocal inversion between C. hystrix and melon. The results reveal the location and orientation of 53 C. hystrix syntenic blocks on the seven cucumber chromosomes, and allow us to infer at least 59 chromosome rearrangement events that led to the seven cucumber chromosomes, including five fusions, four translocations, and 50 inversions. The 12 inferred chromosomes (AK1–AK12) of an ancestor similar to melon and C. hystrix had strikingly different evolutionary fates, with cucumber chromosome C1 apparently resulting from insertion of chromosome AK12 into the centromeric region of translocated AK2/AK8, cucumber chromosome C3 originating from a Robertsonian-like translocation between AK4 and AK6, and cucumber chromosome C5 originating from fusion of AK9 and AK10. Chromosomes C2, C4 and C6 were the result of complex reshuffling of syntenic blocks from three (AK3, AK5 and AK11), three (AK5, AK7 and AK8) and five (AK2, AK3, AK5, AK8 and AK11) ancestral chromosomes, respectively, through 33 fusion, translocation and inversion events. Previous results (Huang, S., Li, R., Zhang, Z. et al., 2009, Nat. Genet. 41, 1275–1281; Li, D., Cuevas, H.E., Yang, L., Li, Y., Garcia-Mas, J., Zalapa, J., Staub, J.E., Luan, F., Reddy, U., He, X., Gong, Z., Weng, Y. 2011a, BMC Genomics, 12, 396) showing that cucumber C7 stayed largely intact during the entire evolution of Cucumis are supported. Results from this study allow a fine-scale understanding of the mechanisms of dysploid chromosome reduction that has not been achieved previously.This research was supported by US Department of Agriculture Current Research Information System Project 3655-21000-048-00D and a US Department of Agriculture Specialty Crop Research Initiative grant (project number 2011-51181-30661) to Y.W.Peer reviewe

Ocean observations in support of studies and forecasts of tropical and extratropical cyclones

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Domingues, R., Kuwano-Yoshida, A., Chardon-Maldonado, P., Todd, R. E., Halliwell, G., Kim, H., Lin, I., Sato, K., Narazaki, T., Shay, L. K., Miles, T., Glenn, S., Zhang, J. A., Jayne, S. R., Centurioni, L., Le Henaff, M., Foltz, G. R., Bringas, F., Ali, M. M., DiMarco, S. F., Hosoda, S., Fukuoka, T., LaCour, B., Mehra, A., Sanabia, E. R., Gyakum, J. R., Dong, J., Knaff, J. A., & Goni, G. Ocean observations in support of studies and forecasts of tropical and extratropical cyclones. Frontiers in Marine Science, 6, (2019): 446, doi:10.3389/fmars.2019.00446.Over the past decade, measurements from the climate-oriented ocean observing system have been key to advancing the understanding of extreme weather events that originate and intensify over the ocean, such as tropical cyclones (TCs) and extratropical bomb cyclones (ECs). In order to foster further advancements to predict and better understand these extreme weather events, a need for a dedicated observing system component specifically to support studies and forecasts of TCs and ECs has been identified, but such a system has not yet been implemented. New technologies, pilot networks, targeted deployments of instruments, and state-of-the art coupled numerical models have enabled advances in research and forecast capabilities and illustrate a potential framework for future development. Here, applications and key results made possible by the different ocean observing efforts in support of studies and forecasts of TCs and ECs, as well as recent advances in observing technologies and strategies are reviewed. Then a vision and specific recommendations for the next decade are discussed.This study was supported by the National Oceanic and Atmospheric Administration and JSPS KAKENHI (Grant Numbers: JP17K19093, JP16K12591, and JP16H01846)

The Tnt1 Retrotransposon Escapes Silencing in Tobacco, Its Natural Host

Retrotransposons' high capacity for mutagenesis is a threat that genomes need to control tightly. Transcriptional gene silencing is a general and highly effective control of retrotransposon expression. Yet, some retrotransposons manage to transpose and proliferate in plant genomes, suggesting that, as shown for plant viruses, retrotransposons can escape silencing. However no evidence of retrotransposon silencing escape has been reported. Here we analyze the silencing control of the tobacco Tnt1 retrotransposon and report that even though constructs driven by the Tnt1 promoter become silenced when stably integrated in tobacco, the endogenous Tnt1 elements remain active. Silencing of Tnt1-containing transgenes correlates with high DNA methylation and the inability to incorporate H2A.Z into their promoters, whereas the endogenous Tnt1 elements remain partially methylated at asymmetrical positions and incorporate H2A.Z upon induction. Our results show that the promoter of Tnt1 is a target of silencing in tobacco, but also that endogenous Tnt1 elements can escape this control and be expressed in their natural host

Genome-wide transposon analyses: annotation, movement and impact on plant function and evolution

[eng]The diversity of life forms around us is astounding: a walk in the woods, or even down the street, shows us organisms of different morphologies: two legs, four legs, wings; different capacity of interaction with our environment: plants photosynthesizing while bacteria break down our garbage. How can life take on so many forms? While there is some increase of genes when comparing the most simple eukaryotes to the most complex ones it is clear that organism complexity is not the result of the number of genes. Therefore is has been postulated that the complexity of an organism arises from the complexity of its gene regulation, rather than the number of genes. This regulation must come then from the non-gene part of the genome. We now know that genes constitute but a small portion of genomes, (about 5% of the human genome). The advent of whole-genome sequencing has enabled us to get a more complete picture of what is in a genome, and with that has come the surprise that a significant part of all genomes characterized is constituted of transposable elements (TEs). Transposable elements are mobile genetic sequences, meaning that they have the capacity to change their position within the genome of a single cell. The goal of my dissertation has been to investigate the role of TEs in plants and their impact on gene and genome evolution. For this I have taken two approaches. The first is a study in the newly sequenced genome of Cucumis melo, an important crop plant in Spain. In the context of this project I have characterized the transposon landscape in the genome, and identified TE related polymorphisms between seven different varieties. This project has of interest the fact that this is an important plant for agriculture and domestication is a particularly relevant evolutionary context in which to study the impact of transposons, as the lines analyzed come from different geographic and selection backgrounds. In the context of this project I have developed a pipeline for genome annotation, and a software for detection of polymorphisms using next-generation paired-end sequencing data. This yielded insight into the dynamics of transposon evolution in this genome, and the selective forces that have shaped the transposon landscape. To our knowledge this is the first analysis that uses polymorphic TEs to investigate differential chromosomal distribution of recent and old transposons, and thus revealing at an intra-species scale the timeline of selection. The results obtained are promising for studying the contribution of mobile elements to the evolution of two genetically similar yet phenotypically different cultivated varieties. In order to study the impact of transposition on gene regulation, I investigated MITE families which have amplified a transciption factor binding site (TF BS) in the model plant Arabidopsis thaliana. This project focuses on the potential impact on gene regulation networks of the redistribution of this TFBS, a phenomenon that has been described for various master TF in animals but not yet to my knowledge in plants. This study combines in silico analysis with molecular data such as microarrays and ChIP, and is a striking example of one of the many manners in which TEs can impact gene regulation. The work in this dissertation highlights the contradictory nature of transposable elements: on one hand, they are invasive, and on the other, are the source of essential innovations. Here we provide insight as to the functions they play in plant genome evolutio

MITEs, miniature elements with a major role in plant genome evolution

Miniature Inverted-repeat Transposable Elements (MITEs) are a particular type of class II transposons found in genomes in high copy numbers. Most MITEs are deletion derivatives of class II transposons whose transposases have been shown to mobilize them by a typical cut-and-paste mechanism. However, unlike class II transposons, MITEs can amplify rapidly and dramatically and attain very high copy numbers, in particular, in plant genomes. This high copy number, together with their close association with genes, endows MITEs with a high potential to generate variability, and impact gene and genome evolution.Peer reviewe

Jitterbug: somatic and germline transposon insertion detection at single-nucleotide resolution

Background: Transposable elements are major players in genome evolution. Transposon insertion polymorphisms can/ntranslate into phenotypic differences in plants and animals and are linked to different diseases including human cancer, making their characterization highly relevant to the study of genome evolution and genetic diseases./nResults: Here we present Jitterbug, a novel tool that identifies transposable element insertion sites at single-nucleotide resolution based on the pairedend mapping and clipped-read signatures produced by NGS alignments. Jitterbug can be easily integrated into existing NGS analysis pipelines, using the standard BAM format produced by frequently applied alignment tools (e.g. bwa, bowtie2), with no need to realign reads to a set of consensus transposon sequences. Jitterbug is highly sensitive and able to recall transposon insertions with a very high specificity, as demonstrated by benchmarks in the human and Arabidopsis genomes, and validation using long PacBio reads. In addition, Jitterbug estimates the zygosity of transposon insertions with high accuracy and can also identify somatic insertions. Conclusions: We demonstrate that Jitterbug can identify mosaic somatic transposon movement using sequenced tumor-normal sample pairs and allows for estimating the cancer cell fraction of clones containing a somatic TE insertion. We suggest that the independent methods we use to evaluate performance are a step towards creating a gold standard dataset for benchmarking structural variant prediction tools