A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits.

Rose is the world's most important ornamental plant, with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Roses are outbred and can have various ploidy levels. Our objectives were to develop a high-quality reference genome sequence for the genus Rosa by sequencing a doubled haploid, combining long and short reads, and anchoring to a high-density genetic map, and to study the genome structure and genetic basis of major ornamental traits. We produced a doubled haploid rose line ('HapOB') from Rosa chinensis 'Old Blush' and generated a rose genome assembly anchored to seven pseudo-chromosomes (512 Mb with N50 of 3.4 Mb and 564 contigs). The length of 512 Mb represents 90.1-96.1% of the estimated haploid genome size of rose. Of the assembly, 95% is contained in only 196 contigs. The anchoring was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features, including the pericentromeric regions, through annotation of transposable element families and positioned centromeric repeats using fluorescent in situ hybridization. The rose genome displays extensive synteny with the Fragaria vesca genome, and we delineated only two major rearrangements. Genetic diversity was analysed using resequencing data of seven diploid and one tetraploid Rosa species selected from various sections of the genus. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and the number of flower petals. A rose APETALA2/TOE homologue is proposed to be the major regulator of petal number in rose. This reference sequence is an important resource for studying polyploidization, meiosis and developmental processes, as we demonstrated for flower and prickle development. It will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny across Rosaceae.

Live imaging of DORNRÖSCHEN and DORNRÖSCHEN-LIKE promoter activity reveals dynamic changes in cell identity at the microcallus surface of Arabidopsis embryonic suspensions.

KEY MESSAGE : Transgenic DRN::erGFP and DRNL::erGFP reporters access the window from explanting Arabidopsis embryos to callus formation and provide evidence for the acquisition of shoot meristem cell fates at the microcalli surface. The DORNRÖSCHEN (DRN) and DORNRÖSCHEN-LIKE (DRNL) genes encode AP2-type transcription factors, which are activated shortly after fertilisation in the zygotic Arabidopsis embryo. We have monitored established transgenic DRN::erGFP and DRNL::erGFP reporter lines using live imaging, for expression in embryonic suspension cultures and our data show that transgenic fluorophore markers are suitable to resolve dynamic changes of cellular identity at the surface of microcalli and enable fluorescence-activated cell sorting. Although DRN::erGFP and DRNL::erGFP are both activated in surface cells, their promoter activity marks different cell identities based on real-time PCR experiments and whole transcriptome microarray data. These transcriptome analyses provide no evidence for the maintenance of embryogenic identity under callus-inducing high-auxin tissue culture conditions but are compatible with the acquisition of shoot meristem cell fates at the surface of suspension calli.

FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants.

A large collection of T-DNA insertion transformants of Arabidopsis thaliana has been generated at the Institute of Agronomic Research, Versailles, France. The molecular characterisation of the insertion sites is currently performed by sequencing genomic regions flanking the inserted T-DNA (FST). The almost complete sequence of the nuclear genome of A.thaliana provides the framework for organising FSTs in a genome oriented database, FLAGdb/FST (http://genoplante-info.infobiogen.fr). The main scope of FLAGdb/FST is to help biologists to find the FSTs that interrupt the genes in which they are interested. FSTs are anchored to the genome sequences of A.thaliana and positions of both predicted genes and FSTs are shown graphically on sequences. Requests to locate the genomic position of a query sequence are made using BLAST programs. The response delivered by FLAGdb/FST is a graphical representation of the putative FSTs and of predicted genes in a 20 kb region.

Activation of systemic acquired silencing by localised introduction of DNA.

BACKGROUND: In plants, post-transcriptional gene silencing results in RNA degradation after transcription. Among tobacco transformants carrying a nitrate reductase (Nia) construct under the control of the cauliflower mosaic virus 35S promoter (35S-Nia2), one class of transformants spontaneously triggers Nia post-transcriptional gene silencing (class II) whereas another class does not (class I). Non-silenced plants of both classes become silenced when grafted onto silenced stocks, indicating the existence of a systemic silencing signal. Graft-transmitted silencing is maintained in class II but not in class I plants when removed from silenced stocks, indicating similar requirements for spontaneous triggering and maintenance. RESULTS: Introduction of 35S-Nia2 DNA by the gene transfer method called biolistics led to localised acquired silencing (LAS) in bombarded leaves of wild-type, class I and class II plants, and to systemic acquired silencing (SAS) in class II plants. SAS occurred even if the targeted leaf was removed 2 days after bombardment, indicating that the systemic signal is produced, transmitted and amplified rapidly. SAS was activated by sense, antisense and promoterless Nia2 DNA constructs, indicating that transcription is not required although it does stimulate SAS. CONCLUSIONS: SAS was activated by biolistic introduction of promoterless constructs, indicating that the DNA itself is a potent activator of post-transcriptional gene silencing. The systemic silencing signal invaded the whole plant by cell-to-cell and long-distance propagation, and reamplification of the signal.

Arabidopsis mutants impaired in cosuppression.

Post-transcriptional gene silencing (cosuppression) results in the degradation of RNA after transcription. A transgenic Arabidopsis line showing post-transcriptional silencing of a 35S-uidA transgene and uidA-specific methylation was mutagenized using ethyl methanesulfonate. Six independent plants were isolated in which uidA mRNA accumulation and beta-glucuronidase activity were increased up to 3500-fold, whereas the transcription rate of the 35S-uidA transgene was increased only up to threefold. These plants each carried a recessive monogenic mutation that is responsible for the release of silencing. These mutations defined two genetic loci, called sgs1 and sgs2 (for suppressor of gene silencing). Transgene methylation was distinctly modified in sgs1 and sgs2 mutants. However, methylation of centromeric repeats was not affected, indicating that sgs mutants differ from ddm (for decrease in DNA methylation) and som (for somniferous) mutants. Indeed, unlike ddm and som mutations, sgs mutations were not able to release transcriptional silencing of a 35S-hpt transgene. Conversely, both sgs1 and sgs2 mutations were able to release cosuppression of host Nia genes and 35S-Nia2 transgenes. These results therefore indicate that sgs mutations act in trans to impede specifically transgene-induced post-transcriptional gene silencing.