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.

Construction of an integrated map of rose with AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers.

A high-density genetic map with a number of anchor markers has been created to be used as a tool to dissect genetic variation in rose. Linkage maps for the diploid 94/1 population consisting of 88 individuals were constructed using a total of 520 molecular markers including AFLP, SSR, PK, RGA, RFLP, SCAR and morphological markers. Seven linkage groups, putatively corresponding to the seven haploid rose chromosomes, were identified for each parent, spanning 487 cM and 490 cM, respectively. The average length of 70 cM may cover more than 90% of the rose genome. An integrated map was constructed by incorporating the homologous parental linkage groups, resulting in seven linkage groups with a total length of 545 cM. The present linkage map is currently the most advanced map in rose with regard to marker density, genome coverage and with robust markers, giving good perspectives for QTL mapping and marker-assisted breeding in rose. The SSR markers, together with RFLP markers, provide good anchor points for future map alignment studies in rose and related species. Codominantly scored AFLP markers were helpful in the integration of the parental maps.

Rpp1, a dominant gene providing race-specific resistance to rose powdery mildew ( Podosphaera pannosa): molecular mapping, SCAR development and confirmation of disease resistance data.

We have previously demonstrated that in the diploid rose population 97/9 resistance to the powdery mildew race 9 is controlled by a major dominant resistance gene, Rpp1. In the study reported here, we isolated several molecular markers closely linked to Rpp1 via bulked segregant analysis, with the gene being tagged in an interval of 5 cM between the two most adjacent markers. It was possible to convert the most closely linked amplified fragment length polymorphic (AFLP) marker into a sequence-characterised amplified region (SCAR) segregating in the same manner. Indirect mapping of Rpp1 in relation to the black spot resistance gene Rdr1 revealed no linkage between the two R genes. Furthermore, the genetic model based on a single dominant resistance gene was supported by the marker data.

Construction of a BAC library of Rosa rugosaThunb. and assembly of a contig spanning Rdr1, a gene that confers resistance to blackspot.

A BAC library to serve as a general tool for the physical mapping and positional cloning of rose genes has been constructed from Rosa rugosa DNA. With 27,264 clones the library contains 5.2 genome equivalents. The library was used to assemble a contig of BAC clones spanning Rdr1, a locus that confers resistance to blackspot. For this purpose fine-scale mapping of the target locus was achieved by bulked segregant analysis using 816 AFLP primer combinations. The target region around Rdr1 comprises about 400 kb and is covered by a minimum of six BAC clones. Furthermore, the detection of at least five resistance gene analogs of the TIR-NBS-LRR family on the contig indicates the presence of a cluster of resistance genes around Rdr1. These results will not only allow the isolation and identification of Rdr1 in the near future, but also provide the tools for the physical mapping and positional cloning of other horticulturally interesting genes in roses.

RFLP mapping on potato chromosomes of two genes controlling extreme resistance to potato virus X (PVX).

Two different chromosomal locations of major genes controlling extreme resistance to potato virus X (PVX) were found by restriction fragment length polymorphism (RFLP) analysis of two populations segregating for the resistance. The resistance gene Rx1 mapped to the distal end of chromosome XII, whereas Rx2 was located at an intermediate position on linkage group V in a region where reduced recombination and segregation distortion have also been observed. These linkage anomalies were due to abnormal behaviour of the chromosome contributed by the resistant parent P34. The results presented were obtained using two different strategies for mapping genes of unknown location. One approach was the use of probes revealing polymorphic loci spread throughout the genome and resulted in the mapping of Rx1. The second approach was based on the assumption of possible linkage between the resistance gene and clone-specific DNA fragments introduced from a wild potato species. Rx2 was mapped by adopting this strategy.

RFLP maps of potato and their alignment with the homoeologous tomato genome.

An RFLP linkage map of the potato is presented which comprises 304 loci derived from 230 DNA probes and one morphological marker (tuber skin color). The self-incompatibility locus of potato was mapped to chromosome I, which is homoeologous to tomato chromosome I. By mapping chromosome-specific tomato RFLP markers in potato and, vice versa, potato markers in tomato, the different potato and tomato RFLP maps were aligned to each other and the similarity of the potato and tomato genome was confirmed. The numbers given to the 12 potato chromosomes are now in accordance with the established tomato nomenclature. Comparisons between potato RFLP maps derived from different genetic backgrounds revealed conservation of marker order but differences in chromosome and total map length. In particular, significant reduction of map length was observed in interspecific compared to intraspecific crosses. The distribution of regions with distorted segregation ratios in the genome was analyzed for four potato parents. The most prominent distortion of recombination was found to be caused by the self-incompatibility locus.

Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis.

A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers. The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test. By linkage to nine RFLP markers the resistance locus Gro1 was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7. Deviations from the additivity of recombination frequencies between Gro1 and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots. Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus.

Phylogeny of wild and cultivatedSolanum species based on nuclear restriction fragment length polymorphisms (RFLPs).

Phylogenetic relationships between 14 wild and 3 cultivatedSolanum species, including the European potato,Solanum tuberosum ssp.tuberosum, were inferred using DNA restriction fragment length polymorphisms (RFLPs) as discriminating characters. Phenetic trees were obtained based on distance matrices as well as on parsimony methods, which were not significantly altered either by the computational method used, or by the individual plant genotypes or RFLP markers chosen for the analysis. The reliability of the tree topologies was assessed and, as expected, it increased with the number of polymorphic restriction fragments scored. The individual genotypes within each species, the different species themselves, and the main branches of the trees were clearly separated from each other. The least reliable parts in the trees were the positions of closely related species within the main clusters.S. tuberosum spp.tuberosum formed one group closely related withS. tuberosum spp.andigena,S. stenotomum, andS. canasense. This was well separated from a second group formed byS. sparsipilum,berthaultii,kurtzianum,gourlayi,vernei,spegazzinii,chacoense, andmegistacrolobum. Two further branches were formed byS. demissum andS. acaule, and byS. polyadenium,S. pinnatisectum,S. stoloniferum, andS. etuberosum. The phenetic trees presented here supported the description of relationships amongSolanum species based on biosystematic studies, with the exception of the placement ofS. chacoense andS. megistacrolobum.

Identification of 2n breeding lines and 4n varieties of potato (Solanum tuberosum, ssp. tuberosum) with RFLP-fingerprints.

The possibility of genotype identification with RFLP fingerprints was examined with 20 tetraploid potato varieties and 38 diploid potato lines. By using a sensitive detection system for small restriction fragment length differences and highly variable potato sequences as probes, all genotypes (diploids and tetraploids) were distinguished by a minimum of two probe/enzyme combinations. The best single probe/enzyme combination distinguished 19 out of 20 4n varieties and 33 out of 38 2n lines. Intravarietal variability was very small compared to the intervarietal variability, and patterns obtained with different DNA sources of the same genotype were identical.

RFLP analysis and linkage mapping in Solanum tuberosum.

A morphologically and agronomically heterogeneous collection of 38 diploid potato lines was analysed for restriction fragment length polymorphisms (RFLPs) with 168 potato probes, including random genomic and cDNA sequences as well as characterized potato genes of known function. The use of four cutter restriction enzymes and a fragment separation range from 250 to 2,000 bases on denaturing polyacrylamide gels allowed the detection of RFLPs of a few nucleotides. With this system, 90% of all probes tested showed useful polymorphism, and 95% of those were polymorphic with two or all three enzymes used. On the average, 80% of the probes were informative in all pairwise comparisons of the 38 lines with a minimum of 49% and a maximum of 95%. The percentage of heterozygosity was determined relative to each other for each line and indicated that direct segregation analysis in F1 populations should be feasible for most combinations. From a backcross involving one pair of the 38 lines, a RFLP linkage map with 141 loci was constructed, covering 690 cMorgan of the Solanum tuberosum genome.