Untangling the hedge: Genetic diversity in clonally and sexually transmitted genomes of European wild roses, Rosa L.

While European wild roses are abundant and widely distributed, their morphological taxonomy is complicated and ambiguous. In particular, the polyploid Rosa section Caninae (dogroses) is characterised by its unusual meiosis, causing simultaneous clonal and sexual transmission of sub-genomes. This hemisexual reproduction, which often co-occurs with vegetative reproduction, defies the standard definition of species boundaries. We analysed seven highly polymorphic microsatellite loci, scored for over 2 600 Rosa samples of differing ploidy, collected across Europe within three independent research projects. Based on their morphology, these samples had been identified as belonging to 21 dogrose and five other native rose species. We quantified the degree of clonality within species and at individual sampling sites. We then compared the genetic structure within our data to current rose morpho-systematics and searched for hemisexually co-inherited sets of alleles at individual loci. We found considerably fewer copies of identical multi-locus genotypes in dogroses than in roses with regular meiosis, with some variation recorded among species. While clonality showed no detectable geographic pattern, some genotypes appeared to be more widespread. Microsatellite data confirmed the current classification of subsections, but they did not support most of the generally accepted dogrose microspecies. Under canina meiosis, we found co-inherited sets of alleles as expected, but could not distinguish between sexually and clonally inherited sub-genomes, with only some of the detected allele combinations being lineage-specific.

Can Knowledge of Genetic Distances, Genome Sizes and Chromosome Numbers Support Breeding Programs in Hardy Geraniums?

Breeding programs in ornamentals can be facilitated by integrating knowledge of phylogenetic relatedness of potential parents along with other genomic information. Using AFLP, genetic distances were determined for 59 Geranium genotypes, comprising 55 commercial cultivars of the three subgenera of a total collection of 61 Geranium genotypes. A subgroup of 45 genotypes, including intragroup and intergroup hybrids, were selected and further characterized for genome sizes and chromosome numbers. The variation in genome size ranged from 1.51 ± 0.01 pg/2C to 12.94 ± 0.07 pg/2C. The chromosome numbers ranged from 26 to 108-110 with some hybrids showing an aberrant number of chromosomes based on their parents' constitution. All chromosome numbers of Geranium are an even number, which presumes that unreduced gametes occur in some cross combinations. Overall, parental difference in genome size and chromosome number were not limiting for cross compatibility. Good crossing compatibility was correlated to a Jaccard similarity coefficient as parameter for parental relatedness of about 0.5. Additionally, parent combinations with high differences in the DNA/chromosome value could not result in a successful cross. We expect that our results will enable breeding programs to overcome crossing barriers and support further breeding initiatives.

A General Protocol for Accurate Gene Expression Analysis in Plants.

Gene expression analysis by means of RT-qPCR is a highly sensitive technique. However, this requires an accurate protocol for the whole procedure from sampling to data analysis. We have optimized this protocol specifically for the analysis of plant tissues. Special attention is paid to RNA quality and integrity and to the appropriate setup of the assays in order to be compliant with the MIQE guidelines. This protocol was already successfully applied in ten different plant species.

Do New Breeding Techniques in Ornamentals and Fruits Lead to Essentially Derived Varieties?

Do new breeding techniques (NBT) lead to essentially derived varieties (EDV)? It depends! It depends on the definition of EDV in the plant variety right (PVR) laws and their interpretation by the courts. This paper aims at providing an overview of the EDV concept and an analysis of the question whether NBT lead to EDV on the basis of the UPOV 1991 Act, the most recent UPOV Explanatory Notes on EDV of 2017 as well as some selected PVR laws. Almost 30 years ago, the concept of EDV has been incorporated into the UPOV 1991 Act. In order to strengthen the rights of breeders, in particular to provide breeders of original genotypes an additional source of remuneration, a system of "Plant Variety Right specific dependency," based on "essential derivation," was developed. Only a very limited number of court cases have been concerned with EDV. However, an escalation in EDV-related conflicts can be expected in the future due to increased competition in the ornamental and fruit breeding business as well as to the application of more sophisticated NBT.

Genetic diversity, population structure, and traditional culture of Camellia reticulata.

Camellia reticulata is an arbor tree that has been cultivated in southwestern China by various sociolinguistic groups for esthetic purposes as well as to derive an edible seed oil. This study examined the influence of management, socio-economic factors, and religion on the genetic diversity patterns of Camellia reticulata utilizing a combination of ethnobotanical and molecular genetic approaches. Semi-structured interviews and key informant interviews were carried out with local communities in China's Yunnan Province. We collected plant material (n = 190 individuals) from five populations at study sites using single-dose AFLP markers in order to access the genetic diversity within and between populations. A total of 387 DNA fragments were produced by four AFLP primer sets. All DNA fragments were found to be polymorphic (100%). A relatively high level of genetic diversity was revealed in C. reticulata samples at both the species (Hsp = 0.3397, Isp = 0.5236) and population (percentage of polymorphic loci = 85.63%, Hpop = 0.2937, Ipop = 0.4421) levels. Findings further revealed a relatively high degree of genetic diversity within C. reticulata populations (Analysis of Molecular Variance = 96.31%). The higher genetic diversity within populations than among populations of C. reticulata from different geographies is likely due to the cultural and social influences associated with its long cultivation history for esthetic and culinary purposes by diverse sociolinguistic groups. This study highlights the influence of human management, socio-economic factors, and other cultural variables on the genetic and morphological diversity of C. reticulata at a regional level. Findings emphasize the important role of traditional culture on the conservation and utilization of plant genetic diversity.

Identification of Quantitative Trait Loci Controlling Root and Shoot Traits Associated with Drought Tolerance in a Lentil (Lens culinaris Medik.) Recombinant Inbred Line Population.

Drought is one of the major abiotic stresses limiting lentil productivity in rainfed production systems. Specific rooting patterns can be associated with drought avoidance mechanisms that can be used in lentil breeding programs. In all, 252 co-dominant and dominant markers were used for Quantitative Trait Loci (QTL) analysis on 132 lentil recombinant inbred lines based on greenhouse experiments for root and shoot traits during two seasons under progressive drought-stressed conditions. Eighteen QTLs controlling a total of 14 root and shoot traits were identified. A QTL-hotspot genomic region related to a number of root and shoot characteristics associated with drought tolerance such as dry root biomass, root surface area, lateral root number, dry shoot biomass and shoot length was identified. Interestingly, a QTL (QRSratioIX-2.30) related to root-shoot ratio, an important trait for drought avoidance, explaining the highest phenotypic variance of 27.6 and 28.9% for the two consecutive seasons, respectively, was detected. This QTL was closed to the co-dominant SNP marker TP6337 and also flanked by the two SNP TP518 and TP1280. An important QTL (QLRNIII-98.64) related to lateral root number was found close to TP3371 and flanked by TP5093 and TP6072 SNP markers. Also, a QTL (QSRLIV-61.63) associated with specific root length was identified close to TP1873 and flanked by F7XEM6b SRAP marker and TP1035 SNP marker. These two QTLs were detected in both seasons. Our results could be used for marker-assisted selection in lentil breeding programs targeting root and shoot characteristics conferring drought avoidance as an efficient alternative to slow and labor-intensive conventional breeding methods.

Change in Auxin and Cytokinin Levels Coincides with Altered Expression of Branching Genes during Axillary Bud Outgrowth in Chrysanthemum.

In the production and breeding of Chrysanthemum sp., shoot branching is an important quality aspect as the outgrowth of axillary buds determines the final plant shape. Bud outgrowth is mainly controlled by apical dominance and the crosstalk between the plant hormones auxin, cytokinin and strigolactone. In this work the hormonal and genetic regulation of axillary bud outgrowth was studied in two differently branching cut flower Chrysanthemum morifolium (Ramat) genotypes. C17 is a split-type which forms an inflorescence meristem after a certain vegetative period, while C18 remains vegetative under long day conditions. Plant growth of both genotypes was monitored during 5 subsequent weeks starting one week before flower initiation occurred in C17. Axillary bud outgrowth was measured weekly and samples of shoot apex, stem and axillary buds were taken during the first two weeks. We combined auxin and cytokinin measurements by UPLC-MS/MS with RT-qPCR expression analysis of genes involved in shoot branching regulation pathways in chrysanthemum. These included bud development genes (CmBRC1, CmDRM1, CmSTM, CmLsL), auxin pathway genes (CmPIN1, CmTIR3, CmTIR1, CmAXR1, CmAXR6, CmAXR2, CmIAA16, CmIAA12), cytokinin pathway genes (CmIPT3, CmHK3, CmRR1) and strigolactone genes (CmMAX1 and CmMAX2). Genotype C17 showed a release from apical dominance after floral transition coinciding with decreased auxin and increased cytokinin levels in the subapical axillary buds. As opposed to C17, C18 maintained strong apical dominance with vegetative growth throughout the experiment. Here high auxin levels and decreasing cytokinin levels in axillary buds and stem were measured. A differential expression of several branching genes accompanied the different hormonal change and bud outgrowth in C17 and C18. This was clear for the strigolactone biosynthesis gene CmMAX1, the transcription factor CmBRC1 and the dormancy associated gene CmDRM1, that all showed a decreased expression in C17 at floral transition and an increased expression in C18 with continuous vegetative growth. These results offer a case study for Chrysanthemum, showing an altered cytokinin to auxin balance and differential gene expression between vegetative growth with apical dominance and transition to generative growth with loss of apical dominance and axillary bud outgrowth. This suggests a conservation of several aspects of the hormonal and genetical regulation of bud outgrowth in Chrysanthemum. Furthermore, 15 previously uncharacterised genes in chrysanthemum, were described in this study. Of those genes involved in axillary bud outgrowth we identified CmDRM1, CmBRC1 and CmMAX1 as having an altered expression preceding axillary bud outgrowth, which could be useful as markers for bud activity.

Suboptimal Light Conditions Influence Source-Sink Metabolism during Flowering.

Reliance on carbohydrates during flower forcing was investigated in one early and one late flowering cultivar of azalea (Rhododendron simsii hybrids). Carbohydrate accumulation, invertase activity, and expression of a purported sucrose synthase gene (RsSUS) was monitored during flower forcing under suboptimal (natural) and optimal (supplemental light) light conditions, after a cold treatment (7°C + dark) to break flower bud dormancy. Post-production sucrose metabolism and flowering quality was also assessed. Glucose and fructose concentrations and invertase activity increased in petals during flowering, while sucrose decreased. In suboptimal light conditions RsSUS expression in leaves increased as compared to optimal light conditions, indicating that plants in suboptimal light conditions have a strong demand for carbohydrates. However, carbohydrates in leaves were markedly lower in suboptimal light conditions compared to optimal light conditions. This resulted in poor flowering of plants in suboptimal light conditions. Post-production flowering relied on the stored leaf carbon, which could be accumulated under optimal light conditions in the greenhouse. These results show that flower opening in azalea relies on carbohydrates imported from leaves and is source-limiting under suboptimal light conditions.

Impact of traditional culture on Camellia reticulata in Yunnan, China.

BACKGROUND: Cha-hua (Camellia reticulata) is one of China's traditional ornamental flowers developed by the local people of Yunnan Province. Today, more than 500 cultivars and hybrids are recognized. Many ancient camellia trees still survive and are managed by local peopl. A few records on cha-hua culture exist, but no studies expound the interaction between C. reticulata and traditional culture of ethnic groups. The contribution of traditional culture of different nationalities and regions to the diversity of Camellia reticulate is discussed. METHODS: Ethnobotanical surveys were conducted throughout Central and Western Yunnan to investigate and document the traditional culture related to Camellia reticulata. Five sites were selected to carry out the field investigation. Information was collected using participatory observation, semi-structured interviews, key informant interviews, focus group discussions, and participatory rural appraisal (PRA). RESULTS: Most of the ancient camellia trees were preserved or saved in the courtyards of old buildings and cultural or religious sites. Religion-associated culture plays an important role in C. reticulata protection. In every site we investigated, we found extensive traditional culture on C. reticulata and its management. These traditional cultures have not only protected the germplasm resources of C. reticulata, but also improved the diversity of Camellia cultivars. CONCLUSIONS: There are abundant and diverse genetic resources of cha-hua, Camellia reticulata in Yunnan. Cha-hua is not only an ornamental flower but also has been endowed with rich spiritual connotation. The influence of traditional culture had improved the introduction and domestication of wild plants, breeding and selection of different varieties, and the propagation and dissemination of the tree in Yunnan. However, either some ancient cha-hua trees or their associated traditional culture are facing various threats. The old cha-hua trees and the ethnic camellia culture should be respected and protected since they have made great contributions in the history, and will make more contributions in the future.

Quantitative Trait Loci for Light Sensitivity, Body Weight, Body Size, and Morphological Eye Parameters in the Bumblebee, Bombus terrestris.

Bumblebees such as Bombus terrestris are essential pollinators in natural and managed ecosystems. In addition, this species is intensively used in agriculture for its pollination services, for instance in tomato and pepper greenhouses. Here we performed a quantitative trait loci (QTL) analysis on B. terrestris using 136 microsatellite DNA markers to identify genes linked with 20 traits including light sensitivity, body size and mass, and eye and hind leg measures. By composite interval mapping (IM), we found 83 and 34 suggestive QTLs for 19 of the 20 traits at the linkage group wide significance levels of p = 0.05 and 0.01, respectively. Furthermore, we also found five significant QTLs at the genome wide significant level of p = 0.05. Individual QTLs accounted for 7.5-53.3% of the phenotypic variation. For 15 traits, at least one QTL was confirmed with multiple QTL model mapping. Multivariate principal components analysis confirmed 11 univariate suggestive QTLs but revealed three suggestive QTLs not identified by the individual traits. We also identified several candidate genes linked with light sensitivity, in particular the Phosrestin-1-like gene is a primary candidate for its phototransduction function. In conclusion, we believe that the suggestive and significant QTLs, and markers identified here, can be of use in marker-assisted breeding to improve selection towards light sensitive bumblebees, and thus also the pollination service of bumblebees.

Cold hardiness of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae).

The cold hardiness of the broad mite, Polyphagotarsonemus latus, a key pest in Rhododendron simsii hybrid production in northwestern Europe, was investigated in the laboratory. Survival of eggs, larvae and female adults and reproduction capacity of female P. latus were evaluated following cold exposure at 7 °C. Adult females were also exposed to temperatures of 2 and -3 °C. Further, the supercooling point and lower lethal times of adult females were determined. No eggs survived exposure to 7 °C for 17 or more days. Larval survival upon the cold treatment decreased from 53 to 13% when exposed to 7 °C for 14 and 49 days, respectively. Two-day-old adult females exposed to 7 °C for up to 42 days did not suffer significant mortality, but when returned to 25 °C their oviposition rates were lower than those of mites maintained at 25 °C. Less than 40% of females exposed for 13 days to 2 °C survived; only 20% of these females was able to reproduce upon recovery. Subzero temperatures dramatically decreased survival and reproduction capacity of adult females. The supercooling point of female adults was -16.5 °C. Median lethal times averaged 61.2 h and 9.3 days at -3 and 2 °C, respectively. In conclusion, a long term exposure (up to 6 weeks) of R. simsii plants infested with P. latus to a temperature of 7 °C, which is required for breaking dormancy of the flowers, is not expected to have detrimental effects on the survival and reproductive performance of the female mites.

Anchoring linkage groups of the Rosa genetic map to physical chromosomes with tyramide-FISH and EST-SNP markers.

In order to anchor Rosa linkage groups to physical chromosomes, a combination of the Tyramide-FISH technology and the modern molecular marker system based on High Resolution Melting (HRM) is an efficient approach. Although, Tyramide-FISH is a very promising technique for the visualization of short DNA probes, it is very challenging for plant species with small chromosomes such as Rosa. In this study, we successfully applied the Tyramide-FISH technique for Rosa and compared different detection systems. An indirect detection system exploiting biotinylated tyramides was shown to be the most suitable technique for reliable signal detection. Three gene fragments with a size of 1100 pb-1700 bp (Phenylalanine Ammonia Lyase, Pyrroline-5-Carboxylate Synthase and Orcinol O-Methyl Transferase) have been physically mapped on chromosomes 7, 4 and 1, respectively, of Rosa wichurana. The signal frequency was between 25% and 40%. HRM markers of these 3 gene fragments were used to include the gene fragments on the existing genetic linkage map of Rosa wichurana. As a result, three linkage groups could be anchored to their physical chromosomes. The information was used to check for synteny between the Rosa chromosomes and Fragaria.

Temperature-dependent development of the broad mite Polyphagotarsonemus latus (Acari: Tarsonemidae) on Rhododendron simsii.

The broad mite, Polyphagotarsonemus latus (Banks), is one of the major pests causing severe economic damage in Rhododendron simsii Planch hybrid production in Belgium. In order to optimize biological control programs and to parameterize warning programs, we studied the effect of environmental temperature on the development of P. latus on R. simsii leaves. In combination with a photoperiod of 16:8 h (L:D) and a relative humidity of 80 ± 5 %, six constant temperatures (15, 17, 20, 25, 30 and 33 ± 1 °C), were studied. Total developmental times of 13.3, 10.5, 6.6, 4.2, 3.5 and 4.0 days were measured, respective to each of the aforementioned temperatures. Development of females took significantly longer than that of males at 15, 17, 20 and 30 °C. Survival rates observed between 17 and 30 °C varied between 43.5 and 96.9 %. Lower survival rates were found at 15 and 33 °C, i.e. 31.8 and 23.6 %, respectively. The lower, optimal and upper developmental threshold (t min , t opt and t max , respectively) and thermal constant (K) of the pest were estimated for each life stage by a linear and two non-linear models. Based on measurements of total development of P. latus thermal thresholds of 10.0, 30.1 and 36.0 °C were calculated for t min , t opt and t max , respectively. The number of degree-days needed to complete immature development when feeding on R. simsii was 66.7.

How to perform RT-qPCR accurately in plant species? A case study on flower colour gene expression in an azalea (Rhododendron simsii hybrids) mapping population.

BACKGROUND: Flower colour variation is one of the most crucial selection criteria in the breeding of a flowering pot plant, as is also the case for azalea (Rhododendron simsii hybrids). Flavonoid biosynthesis was studied intensively in several species. In azalea, flower colour can be described by means of a 3-gene model. However, this model does not clarify pink-coloration. The last decade gene expression studies have been implemented widely for studying flower colour. However, the methods used were often only semi-quantitative or quantification was not done according to the MIQE-guidelines. We aimed to develop an accurate protocol for RT-qPCR and to validate the protocol to study flower colour in an azalea mapping population. RESULTS: An accurate RT-qPCR protocol had to be established. RNA quality was evaluated in a combined approach by means of different techniques e.g. SPUD-assay and Experion-analysis. We demonstrated the importance of testing noRT-samples for all genes under study to detect contaminating DNA. In spite of the limited sequence information available, we prepared a set of 11 reference genes which was validated in flower petals; a combination of three reference genes was most optimal. Finally we also used plasmids for the construction of standard curves. This allowed us to calculate gene-specific PCR efficiencies for every gene to assure an accurate quantification. The validity of the protocol was demonstrated by means of the study of six genes of the flavonoid biosynthesis pathway. No correlations were found between flower colour and the individual expression profiles. However, the combination of early pathway genes (CHS, F3H, F3'H and FLS) is clearly related to co-pigmentation with flavonols. The late pathway genes DFR and ANS are to a minor extent involved in differentiating between coloured and white flowers. Concerning pink coloration, we could demonstrate that the lower intensity in this type of flowers is correlated to the expression of F3'H. CONCLUSIONS: Currently in plant research, validated and qualitative RT-qPCR protocols are still rare. The protocol in this study can be implemented on all plant species to assure accurate quantification of gene expression. We have been able to correlate flower colour to the combined regulation of structural genes, both in the early and late branch of the pathway. This allowed us to differentiate between flower colours in a broader genetic background as was done so far in flower colour studies. These data will now be used for eQTL mapping to comprehend even more the regulation of this pathway.

AFLP-based population structure analysis as a means to validate the complex taxonomy of dogroses (Rosa section Caninae).

Within the genus Rosa numerous species have been described. Circumscription of the dogrose section Caninae is straightforward, but the delineation of species and subsections within this section is less clear, partly due to hybridisation between species. We have investigated the extent to which DNA marker-based information of wild populations corroborates present-day dogrose taxonomy and hypotheses about the origination of taxa. Sampling was conducted in a transect across Europe, collecting over 900 specimens of all encountered dogrose taxa. For comparison, we also included more than 200 samples of species belonging to other sections. Two lines of statistical analyses were used to investigate the genetic structure based on AFLP data: (1) an unstructured model with principal coordinate analysis and hierarchical clustering, and (2) a model with a superimposed taxonomic structure based on analysis of genetic diversity using a novel approach combining assignment tests with canonical discriminant analysis. Support was found for five of the seven subsections, whereas R. balsamica apparently belongs to subsection Caninae thus omitting the need for recognising subsection Tomentellae. For R. stylosa, a hybridogenic origin with a non-dogrose section member has been suggested, and it can be treated either as a separate subsection or within subsection Caninae. Within the subsection Rubigineae, a species cluster with low support for the taxa R. micrantha, R. rubiginosa and the putatively hybridogenous R. gremlii was identified. Similarly, several species in the subsection Caninae overlapped considerably, and are best regarded as one common species complex. This population genetic approach provides a general method to validate the taxonomic system in complex and polyploid taxa.

Characterisation of sugar beet (Beta vulgaris L. ssp. vulgaris) varieties using microsatellite markers.

BACKGROUND: Sugar beet is an obligate outcrossing species. Varieties consist of mixtures of plants from various parental combinations. As the number of informative morphological characteristics is limited, this leads to some problems in variety registration research. RESULTS: We have developed 25 new microsatellite markers for sugar beet. A selection of 12 markers with high quality patterns was used to characterise 40 diploid and triploid varieties. For each variety 30 individual plants were genotyped. The markers amplified 3-21 different alleles. Varieties had up to 7 different alleles at one marker locus. All varieties could be distinguished. For the diploid varieties, the expected heterozygosity ranged from 0.458 to 0.744. The average inbreeding coefficient F(is) was 0.282 +/- 0.124, but it varied widely among marker loci, from F(is) = +0.876 (heterozygote deficiency) to F(is) = -0.350 (excess of heterozygotes). The genetic differentiation among diploid varieties was relatively constant among markers (F(st) = 0.232 +/- 0.027). Among triploid varieties the genetic differentiation was much lower (F(st) = 0.100 +/- 0.010). The overall genetic differentiation between diploid and triploid varieties was F(st) = 0.133 across all loci. Part of this differentiation may coincide with the differentiation among breeders' gene pools, which was Fst = 0.063. CONCLUSIONS: Based on a combination of scores for individual plants all varieties can be distinguished using the 12 markers developed here. The markers may also be used for mapping and in molecular breeding. In addition, they may be employed in studying gene flow from crop to wild populations.

Micropropagation of Rhododendron.

Methods for in vitro initiation and multiplication and general culture practices of Rhododendron are presented. Also acclimatization procedures are described. Protocols for callus, shoot, and root induction are described. Several protocols for breeding applications are highlighted in more detail.

Azalea phylogeny reconstructed by means of molecular techniques.

Plants belonging to the Rhododendron subgenera Pentanthera (deciduous) and Tsutsusi and Azaleastrum (evergreen) are called azaleas. Concerning their mutual phylogenetic positions, the Pentanthera subgenus is closer to evergreen rhododendrons (subgenera Rhododendron and Hymenanthes) than to the Tsutsusi subgenus. Both azalea types are important ornamentals with a long breeding tradition. Different hybrid groups are often named after the supposed principal ancestor species. Molecular techniques for phylogenetic and kinship research have been evaluated to a great extent. First, some studies using comparative gene sequencing are presented; this approach was then widened to the use of molecular markers to reveal more detailed genetic relationships. Finally, the use of candidate genes as functional markers for the assessment of genetic diversity is presented. This opens new research lines to the genetic mapping of plant traits and azalea genomics.

Multipoint-likelihood maximization mapping on 4 segregating populations to achieve an integrated framework map for QTL analysis in pot azalea (Rhododendron simsii hybrids).

BACKGROUND: Azalea (Rhododendron simsii hybrids) is the most important flowering pot plant produced in Belgium, being exported world-wide. In the breeding program, flower color is the main feature for selection, only in later stages cultivation related plant quality traits are evaluated. As a result, plants with attractive flowering are kept too long in the breeding cycle. The inheritance of flower color has been well studied; information on the heritability of cultivation related quality traits is lacking. For this purpose, QTL mapping in diverse genetic backgrounds appeared to be a must and therefore 4 mapping populations were made and analyzed. RESULTS: An integrated framework map on four individual linkage maps in Rhododendron simsii hybrids was constructed. For genotyping, mainly dominant scored AFLP (on average 364 per population) and MYB-based markers (15) were combined with co-dominant SSR (23) and EST markers (12). Linkage groups were estimated in JoinMap. A consensus grouping for the 4 mapping populations was made and applied in each individual mapping population. Finally, 16 stable linkage groups were set for the 4 populations; the azalea chromosome number being 13. A combination of regression mapping (JoinMap) and multipoint-likelihood maximization (Carthagène) enabled the construction of 4 maps and their alignment. A large portion of loci (43%) was common to at least two populations and could therefore serve as bridging markers. The different steps taken for map optimization and integration into a reference framework map for QTL mapping are discussed. CONCLUSIONS: This is the first map of azalea up to our knowledge. AFLP and SSR markers are used as a reference backbone and functional markers (EST and MYB) were added as candidate genes for QTL analysis. The alignment of the 4 maps on the basis of framework markers will facilitate in turn the alignment of QTL regions detected in each of the populations. The approach we took is thoroughly different than the recently published integrated maps and well-suited for mapping in a non-model crop.

AFLP markers as a tool to reconstruct complex relationships: A case study in Rosa (Rosaceae).

The genus Rosa has a complex evolutionary history caused by several factors, often in conjunction: extensive hybridization, recent radiation, incomplete lineage sorting, and multiple events of polyploidy. We examined the applicability of AFLP markers for reconstructing (species) relationships in Rosa, using UPGMA clustering, Wagner parsimony, and Bayesian inference. All trees were well resolved, but many of the deeper branches were weakly supported. The cluster analysis showed that the rose cultivars can be separated into a European and an Oriental cluster, each being related to different wild species. The phylogenetic analyses showed that (1) two of the four subgenera (Hulthemia and Platyrhodon) do not deserve subgeneric status; (2) section Carolinae should be merged with sect. Cinnamomeae; (3) subsection Rubigineae is a monophyletic group within sect. Caninae, making sect. Caninae paraphyletic; and (4) there is little support for the distinction of the five other subsections within sect. Caninae. Comparison of the trees with morphological classifications and with previous molecular studies showed that all methods yielded reliable trees. Bayesian inference proved to be a useful alternative to parsimony analysis of AFLP data. Because of their genome-wide sampling, AFLPs are the markers of choice to reconstruct (species) relationships in evolutionary complex groups.