STSM Scientific Report: Subject: Sweet cherry evaluation methods and techniques. Processing new cultivars and hybrids. STSM Reference number: COST-STSM-FA1104-14180 Reference code: COST-STSM-ECOST-STSM-FA1104-100713-034251 Period : 10-July-2013 to 24-July-2013 COST Action number: FA1104 STSM Applicant: Fedon Makris Cyprus University of Technology Anexartisias 33, Cyprus fedonasm@gmail.com Purpose of the STSM Objectives of the STSM: Work Plan: To provide an insight into the new methods and techniques used at INRA Bordeaux for characterizing sweet cherry germplasm To participate to a study of quantification of the most important fruit metabolites of two segregating mapping progenies To participate to QTL detection analyses of sweet cherry fruit quality traits Phenotyping of the late-maturity cherry materials Sample preparation Collecting data regarding metabolomics analyses Data analyses (software MultiQTL) Description of the work carried during the STSM 1. Visit at the experimental fields of the institute, Unité Expérimentale Arboricole, located at Toulenne, 40 kms away from Bordeaux: there is cultivation of sunflower, peaches, apples, walnuts, forest trees but the main culture is cherries. The area is divided into different plots: nursery where the seedlings stay for one or two years and evaluation plots from which the
fruits are either analysed or sold. Concerning the evaluation plots, three different types of plots exist: hybrid plots used for genetic analyses purposes (QTL detection trials), varietal evaluation plots with two clones per hybrid (multiplied by grafting) and a plot devoted to DHS (Distinction, Homogeneity, Stability) activities. For the commercial plot traps for the flies were used and generally to avoid the problem of the birds canons are used. Some important problems of the cherry tree culture in the area are the flies (Drosophila suzukii and Rhagoletis cerasi), the birds, aphids, botrytis, monilia and fruit cracking. Picture 1: traps for Drosophila suzukii on cherry trees 2. Last harvest of cherries and observation of the fruits: type and variety, shape, homogeneity, attractiveness, peduncle length, flesh, juice and peel color, flavor, total soluble solids, acidity, ph, tolerance at stress, sensibility to Monilia and three types of fruit cracking : circular or semicircular cracks around the stem end, similar cracks at the apical end and long, irregular splits on the sides of the fruit. 3. Preparation of cherry embryos for stratification : Over 400 fruits were collected from a late self-fertile variety. The flowers had been protected with paper bags to avoid crosspollination. The following protocol was used: Break the stones with a hammer and take out the embryos free of hits and undamaged. This tedious procedure improves and speeds up germination by removing the pericarp, which is one of the main germination barriers.
Wash the embryos with 20g CaCl2O2/ 500ml H2O for 20 min and then wash 3 times the embryos for 5 minutes with distillated water. Stratification of the embryos in vermiculite for 3 months because this process helps overcome embryo dormancy by providing conditions which allow imbibition of water by the embryo, expose to chilling temperatures (2 C) and provide embryos with sufficient oxygen. The stratification is used for the varieties that produce their fruits late in the season. Picture 2: starting from the left to right the first picture is the cracking of stones to take out the embryo, in the middle is the embryos in vermiculite and the right picture is the embryos mixed in vermiculite and closed in a bag to retain high moisture, oxygen, and dark. For the cultivars that produce cherries early, embryo-rescue through in vitro culture is used. Embryos are sterilized and put into MS medium (Murashige and Skoog medium) for their germination. Picture 3: the embryos in tubes with media (MS) 4. Statistic analysis of the data from the observation of the fruits:
First, the phenotypic data from the latest hybrids of two mapping progenies (Regina x Lapins and Regina x Garnet) were entered into an Excel spreadsheet. Phenotypic files were then prepared to be used in QTL detection analyses. Analyses were carried out with the software MultiQTL in order to detect the QTL for eight quantitative agronomic traits: fruit weight, fruit firmness, percentage of cracked fruits at three different areas (pistillar end, stem end and fruit side), titrable acidity, ph and refractometry index (for sugar content). Two tests were conducted, either to detect one QTL per LG or two linked QTL per LG. Also, multi-year analyses were performed in order to increase the statistical power of QTL detection. The main conclusion is that QTL could be identified for all traits with the exception of sugar content, although they were much more significant for fruit weight, fruit firmness and acidity/ph than for fruit cracking tolerance/susceptibility. 5. Observation of preparation of the samples for the determination of metabolites: 18-22mg ground FW in a 1.5 ml screw-tube or in a Micronics screw type. 6. Determination of metabolites: protocol for extraction and determination of chlorophylls, soluble carbohydrates (glucose, fructose, sucrose), amino acids and major organic acids (malic acid, citric acid). 7. Test the effect of 3 genes (FT, TFL1, CEN) in cherry trees blossom (Cultivars: Regina, Lapins, Garnet) : RNA extraction from different organs of cherry trees (leaves, flowers, buds, fruits) Preparation RNA samples for Q-PCR Q-PCR 8. Visit to the fields and greenhouses of INRA : At the site of INRA-Bordeaux, in Villenave d Ornnon, there are cultures of cherry, peach, plums, strawberries and vineyard. All Prunus species and strawberries are planted in pots either outside or in closed structures such as tunnels or greenhouses. Cherry trees planted in pots are used to conduct controlled crosses with bumble bees.
Main results Picture 4: Cherry tree under tunnel and covered by nets for controlled pollination purposes Concerning the objectives of the STSM, the main result concerns the analyses of QTL detection. Analyses dealing with the quantification of metabolites could not be conducted since the methodology is still being optimized and the analyses will be finalized during the months of September-October. Concerning QTL detection analyses, they were successfully conducted on all the traits analysed. These results will be integrated in future publications. Future collaborations Cyprus University of Technology has sound experience in the characterization of fruit characteristics, in particular those dealing with post-harvest technology aspects. One very interesting possibility of collaboration would be to characterize for these post-harvest technology properties the mapping progenies present at INRA-Bordeaux. QTLs for these traits have not yet been reported in sweet cherry. On the other hand, Cyprus has very interesting climatic conditions to study the adaptation of cherry trees to mild autumns and winters, and thus, to low-chilling conditions. Promising lowchilling varieties could be tested in Cyprus conditions and mapping progenies could be installed there in order to have a better idea of the QTL x environment interactions.