Characterization of black spot resistance in diploid roses with QTL detection, meta-analysis and candidate-gene identification.

KEY MESSAGE: Two environmentally stable QTLs linked to black spot disease resistance in the Rosa wichurana genetic background were detected, in different connected populations, on linkage groups 3 and 5. Co-localization between R-genes and defense response genes was revealed via meta-analysis. The widespread rose black spot disease (BSD) caused by the hemibiotrophic fungus Diplocarpon rosae Wolf. is efficiently controlled with fungicides. However, in the actual context of reducing agrochemical use, the demand for rose bushes with higher levels of resistance has increased. Qualitative resistance conferred by major genes (Rdr genes) has been widely studied but quantitative resistance to BSD requires further investigation. In this study, segregating populations connected through the BSD resistant Rosa wichurana male parent were phenotyped for disease resistance over several years and locations. A pseudo-testcross approach was used, resulting in six parental maps across three populations. A total of 45 individual QTLs with significant effect on BSD resistance were mapped on the male maps (on linkage groups (LG) B3, B4, B5 and B6), and 12 on the female maps (on LG A1, A2, A3, A4 and A5). Two major regions linked to BSD resistance were identified on LG B3 and B5 of the male maps and were integrated into a consensus map built from all three of the male maps. A meta-analysis was used to narrow down the confidence intervals of individual QTLs from three populations by generating meta-QTLs. Two 'hot spots' or meta-QTLs were found per LG, enabling reduction of the confidence interval to 10.42 cM for B3 and 11.47 cM for B5. An expert annotation of NBS-LRR encoding genes of the genome assembly of Hibrand et al. was performed and used to explore potential co-localization with R-genes. Co-localization with defense response genes was also investigated.

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.

Genotype of FLOWERING LOCUS T homologue contributes to flowering time differences in wild and cultivated roses.

Rose flowers have long delighted humans as ornamental plants. To improve the ornamental value of roses it is necessary to understand the regulatory mechanisms of flowering. We previously found that flowering time is controlled by three minor quantitative trait loci (QTLs) and a major QTL co-localised with RoFT. In this study, we isolated three RoFT alleles encoding completely identical amino acid sequences from the parents of a mapping population. Correlation analysis of the RoFT genotypes and flowering time phenotypes in the mapping population showed that the RoFT_f and RoFT_g alleles contribute to the early-flowering phenotype, while the RoFT_e allele contributes to the late-flowering phenotype. We developed two novel cleaved amplified polymorphic sequence (CAPS) markers based on the genomic sequences of the RoFT alleles and clearly showed that the relationship between RoFT genotype and flowering time was applicable to 12 of 13 cultivated roses grown at the Higashiyama Botanical Gardens, Japan. Allele-specific expression analysis using a reverse transcription CAPS assay suggested that these RoFT alleles are regulated differentially at the transcription level. Furthermore, transgenic Arabidopsis thaliana plants ectopically expressing the RoFT gene showed an early-flowering phenotype. Conversely, in roses, RoFT was continuously expressed after floral bud formation, and RoFT transcript accumulation reached its peak after that of the floral meristem identity gene RoAP1b. These data suggest that RoFT may be essential not only for floral transition but also for normal floral development and flowering in roses.

Overexpression of RoDELLA impacts the height, branching, and flowering behaviour of Pelargonium × domesticum transgenic plants.

KEY MESSAGE : We reported the cloning of a rose DELLA gene. We obtained transgenic Pelargonium lines overexpressing this gene which presented several phenotypes in plant growth, root growth, flowering time and number of inflorescences. Control of development is an important issue for production of ornamental plant. The plant growth regulator, gibberellins (GAs), plays a pivotal role in regulating plant growth and development. DELLA proteins are nuclear negative regulator of GA signalling. Our objective was to study the role of GA in the plant architecture and in the blooming of ornamentals. We cloned a rose DELLA homologous gene, RoDELLA, and studied its function by genetic transformation of pelargonium. Several transgenic pelargonium (Pelargonium × domesticum 'Autum Haze') lines were produced that ectopically expressed RoDELLA under the control of the 35S promoter. These transgenic plants exhibited a range of phenotypes which could be related to the reduction in GA response. Most of transgenic plants showed reduced growth associated to an increase of the node and branch number. Moreover, overexpression of RoDELLA blocked or delayed flowering in transgenic pelargonium and exhibited defects in the root formation. We demonstrated that pelargonium could be used to validate ornamental gene as the rose DELLA gene. RoDELLA overexpression modified many aspects of plant developmental pathways, as the plant growth, the transition of vegetative to floral stage and the ability of rooting.

Differential recruitment of T- and IgA B-lymphocytes in the developing mammary gland in relation to homing receptors and vascular addressins.

The mammary gland (MG) develops new vasculature and is colonized by lymphocytes, primarily T-cells, during pregnancy. In contrast, during lactation it is colonized primarily by IgA-containing B-cells (c-IgA cells). To explain this difference, we analyzed the spatiotemporal relationships between lymphocytes that expressed peripheral or mucosal homing receptors (HR) and the location of their vascular counterreceptors using quantitative immunohistochemical techniques. We observed that the density of beta(7+)/CD3(+) T-cells varied with the amount of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-stained area. Both increased during pregnancy to peak at delivery, decreased rapidly in early lactation to a steady level in mid- and late lactation, and returned to resting values after weaning. Although 60% of these beta(7+)/CD3(+) T-cells scattered in the epithelium co-expressed alpha(E)beta(7), whereas the remaining 40% in association with blood vessels were alpha(4)beta(7), these results are consistent with a role of MAdCAM-1 in the localization of alpha(4)beta(7+) T-cells. In contrast to T-cells, beta(7+)/c-IgA(+) B plasmablasts (approximately 30% of total c-IgA cells) were located at the alveolar confluence, and their numbers increased in mid- and late lactation when MAdCAM-1 density plateaued. However, both T-and B-cells decreased after weaning. These results show an association between MAdCAM-1 expression level and recruitment of T-cells that does not hold for c-IgA B cells. Furthermore, the recruitment and accumulation of alpha(4)beta(7+) c-IgA cells are reminiscent of locally produced chemoattractants. (J Histochem Cytochem 47:1581-1592, 1999)