Lentil (Lens culinaris Medik.) is usually grown under rainfed environments and often encounters drought stress from limited rainfall. Little information is available about shoot and root traits in association with drought tolerance. We studied variability for root and shoot traits related to drought tolerance using an F6–8 population of 133 recombinant inbred lines (RILs) from the cross ILL6002 × ILL5888. We found important variation between genotypes and also high variation in heritability values for root and shoot traits at 38 days after sowing the parents and RILs under both well-watered and drought-stressed treatments during two consecutive seasons in the greenhouse. The higher heritability values were obtained under drought stress treatment and suggest that selection in water-limited environments would be more effective in achieving genetic gains. Drought had reduced trait values, except root–shoot ratio that was likely to be enhanced underlying the importance of this trait for drought tolerance. The quantitative and continuous distributions of variation are the evidence for polygenic control of these traits and the possibility of mapping the quantitative trait loci (QTL). Statistically significant associations between root and shoot traits such as dry shoot biomass and chlorophyll content were noted, highlighting the reliability of indirect selection for underground traits (root) based on these aboveground traits in breeding programs. Significant correlations and regressions were demonstrated between dry root biomass, lateral root number, root surface area, dry shoot biomass, root–shoot ratio, chlorophyll content and drought tolerance as estimated by wilting severity from limited water supply. This shows the importance of a well-developed root system and early biomass development for drought tolerance. Identification and mapping of QTL related to studied traits in this population would be a first step for starting marker-assisted selection

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BackgroundFlower 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.ResultsAn 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.ConclusionsCurrently 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.

BackgroundAzalea (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.ResultsAn 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.ConclusionsThis 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.

The application of amplified fragment length polymorphism (AFLP), sequence tagged microsatellite site (STMS) and expressed sequence tag (EST) markers to study the genetic relationships in an evergreen azalea gene pool was investigated. STMS and EST markers revealed a higher genetic distance detection capacity than AFLPs, which, nevertheless, were the most efficient marker system due to their highest polymorphism detection capacity. Similarity matrices showed weak, yet significant, correlations when Mantel's test was applied. To assess the usefulness of the overall information provided by these marker data for establishing phylogenetic relationships and horticultural classification, cluster analysis was performed. The joint AFLP, STMS and EST data were demonstrated to be remarkably effective for group discrimination and phylogenetic studies. The use of these polymerase chain reaction marker systems is discussed in terms of the choice of appropriate marker techniques for different aspects of evergreen azalea germplasm evaluation.

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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.

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.