1. Fine genetic characterization of the Co locus responsible for the columnar habit in apple (Malus x domestica Borkh.) BALDI P. 1, KOMJANC M. 1, CASTELLETTI S.. 1, MAGNAGO P. 1, VELASCO R. 1, SALVI S. 1 1 FEM-IASMA Research Centre, Via E. Mach, 1 – 38010 – San Michele all’Adige, Italy BALDI P. 1, KOMJANC M. 1, CASTELLETTI S.. 1, MAGNAGO P. 1, VELASCO R. 1, SALVI S. 1 1 FEM-IASMA Research Centre, Via E. Mach, 1 – 38010 – San Michele all’Adige, Italy Fondazione E. Mach Istituto Agrario S. Michele all’Adige Fondazione E. Mach Istituto Agrario S. Michele all’Adige Background. Apple ( Malus x domestica Borkh.) is one of the more widely cultivated fruit tree. It is therefore not surprising that a lot of efforts are being made worldwide to obtain new varieties with improved characteristics especially regarding fruit quality, pathogen resistance and tree architecture. In case of fruit trees the plant architecture is considered a very important trait, because it can influence fruit quality, plant density, production and labour requirements. The columnar growth habit in apple was discovered in 1970 as a dominant mutation of the cultivar McIntosh (Fisher 1970). It is characterized by short internodes, a thick stem with little difference in diameter between the base and the top and a reduced plant height and branching (Hemmat et al. 1997). The axillary buds grow mostly into spurs, resulting in a relatively sturdy tree with few and mainly short side shoots. These characteristics are considered useful for the minimal pruning interventions required and for the possibility of high-density cultivation. At present different apple cultivars carrying the mutated allele ( Co ) responsible of the columnar phenotype have been released such as Wijcick, Telamon, Tuscan, Trajan, Maple and Charlotte. Results. As a starting point for fine mapping of the genomic region of interest, one SSR marker available from literature was chosen on one side of Co (HI01b01), mapping 4 cM from the gene, while on the opposite side a new SSR marker was developed (Co01L02) approximately at the same distance from Co, as shown in figure 1A. In order to reduce the genomic region of interest new SSR markers were developed using data available at FEM-IASMA from the apple sequencing project and seven were chosen (Co04R03, Co02R10, Co04R09, Co04R10, Co04R11 and Co04R12 Co04R13) defining a region of 1 cM on each side of Co (Fig 1A). Within this region five SSR markers (Co02R10, Co04R09, Co04R10, Co04R11 and Co04R12) showed complete linkage with Co. The next step was to anchor the genetic map of the Co region to the real genome sequence available from the apple sequencing project, going from Co01L02 to HI01b01. All the SSR markers under analysis were located on three distinct and consecutive clusters of sequences (Fig. 1B), delimiting a genomic region of approximately 4 Mb. In particular all the seven SSR markers delimiting the more inner 2 cM region were all placed on the same genomic cluster. According to the latest genomic data available the physical distance between Co04R03 and Co04R13 was estimated to be 820 kb (Fig.1B). At this point the main limiting factor was due to the little size of the segregating population available (340 plants). In order to overcome this problem new crosses have been made both in 2008 and 2009. The goal was to obtain at least 1000-1500 segregating individuals in order to increase the resolution level to less than 0.1 cM on each side of the gene. Genetic analysis of the new segregating populations was performed and new recombinants were identified. Unfortunately it was not yet possible to identify univocally the phenotype of all the plants, especially those deriving from the last round of crosses (2009), that at the moment underwent only the first growing season. In figure 2 the genetic recombinants of a population of 419 individuals deriving from the 2008 crosses are shown. Nevertheless all the phenotype are still putative and must be confirmed the next year. According to these preliminary phenotypes the genomic region containing the Co gene should be comprised between Co04R11 and Co04R13 for an estimated length of approximately 370 kb. 3 cM LG 10 Hi01b01 Co01L02 1 cM Clusters Cluster 1 Cluster 2 Cluster 3 AB Co04R03 1 cM Co04R13 3 cM Co02R10 Co04R09 Co04R10 Co04R11 Co04R12 Co gene 200kb 100kb 50kb 200kb 170kb 3A Early phenotyping of apple seedlings. One of the most time-consuming parts of the work is to obtain a segregating population wide enough to allow a good genetic resolution. Apple, as all the long living woody trees, grows relatively slowly and during the first years does not show a well defined crown shape. It is therefore particularly difficult to phenotype apple seedlings correctly, especially during the first year of growth. None the less, in map-based cloning approaches, the phenotyping work provides crucial information that, together with reliable genotyping, will Columnar trees at FEM-IASMA Figure 3. Apple seedlings at FEM- IASMA A – Standard plant during the first growing season. B - Standard plant during the second growing season. C - Columnar plant during the first growing season. D Columnar plant during the second growing season. Figure 1. A - Fine mapping of the Co region. Five SSR markers (Co02R10, Co04R09, Co04R10, Co04R11, Co04R12) showed complete linkage with Co. The distances are expressed in centimorgans (cM). B - Genomic clusters and physical position of the marker used. Lengths are expressed in thousands of base pairs (kb) References Fisher, DV (1970) Spur strains of “McIntosh” discovered in BritishColombia. Can Fruit Var Hort Digest 24: 27–32. Hemmat M, Weeden NF, Conner PJ, Brown SK (1997) A DNA marker for columnar growth habit in apple contains a simple sequence repeat. J Am Soc Hort Sci122:347–349. Co04R03Co02R10Co09R04Co10R04Co11R04Co12R04Co13R04Phenotype GW09_06bcbcbcbcababac S GW09_24bcbcacaaac S GW09_33aabcbcababbcbcbcbcbcbcbcbcC GW09_89ababbcbcababac S GW09_138ababbcbcaaac S GW09_236aabcbcababbcbcbcbcbcbcbcbcC GW09_364bcbcbcbcababbcbcbcbcac S GW09_412ac aaac bcbcS Figure 2. Seedlings of the segregating population (419 individuals) obtained from 2008 crosses showing a recombination event within the genomic region putatively containing the Co gene. On the left the plant code is reported, on top the SSR markers used for the analysis are shown. The phenotypes indicated on the right (S=Standard plant; C=Columnar plant) are only putative and need to be confirmed. For all the markers the b allele is always associated to the columnar phenotype. 3B finally allow to narrow the genetic interval where the gene of interest can be located. At FEM-IASMA new crosses between Golden delicious and Wijcick have been made during 2008 and 2009, and the seedling are currently under analysis. A typical standard apple seedling usually develops an elongated stem with well spaced internodes (Fig. 3A and 3B). On the contrary a typical columnar seedling shows a shorter and thick stem with internodes very close to each other (Fig. 3C and 3D). Unfortunately not always the phenotype is clearly noticeable, and probably many minor modifiers can contribute to alter the aspect of the young seedlings. 3C 3D 5.19 Objective: The main goal of the present work is to provide a fine and physical map of the genomic region carrying Co in order to allow the development of closely-associated molecular markers and the subsequent identification and cloning of the gene responsible of the columnar habit.