CONSTRUCTION OF A PHYSICAL MAP OF CHROMOSOME 3B FROM HEXAPLOID WHEAT: AN UPDATE

Paux E¹, Sourdille P¹, Salse J¹, Leroy P¹, Bartos J³, Dolezel J³, Chalhoub B², Bernard M¹, Feuillet C*¹

¹Genetics and Plant Breeding, UMR INRA-UBP, Clermont-Ferrand, France. ²Organization and Evolution of plant genomes, (INRA-URGV), 2 rue Gaston Crémieux, CP 5708, F-91057 Évry Cedex, France. ³Laboratory of Molecular Cytogenetics and Cytometry, IEB, Sokolovska 6, CZ-77200 Olomouc, Czech Republic.

*Corresponding author: CF: +33473624684; E-mail: catherine.feuillet@clermont.inra.fr

To test the feasibility of a chromosome by chromosome-based approach to establish a physical map and sequence the hexaploid wheat genome, we have recently initiated the construction of a physical map of chromosome 3B. The 67,968 BAC clones of the 3B chromosome-specific BAC library from cv. Chinese Spring (Safar et al., 2004; Plant J 39: 960-968) have been fingerprinted using an improved SNaPshot protocol (modified from Luo et al. 2003; Genomics 82:378-389) and high-throughput facilities at the French National Sequencing Center (Genoscope). A first phase of BAC assembly and contig construction has been recently achieved leading to about 3’000 contigs (after DQing at e-70). The finishing phase of contig assembly is currently underway and the anchoring of the physical contigs to the genetic map has been initiated. About 2’000 SSR, EST, RFLP markers will be used to PCR screen the 3B BAC library three-dimensional pools and will be mapped onto the ITMI genetic map. In addition, 20’000 BAC ends sequences (BES) selected from BACs which are distributed across and at the end of the BAC physical contigs established by FPC are currently being produced. These 10 Mb of sequence will be used to (i) control the BAC assembly, (2) study sequence composition and genome organisation of chromosome 3B and (3) design additional probes for genetic and physical mapping. BAC contigs orientation and assembly will also be supported by cytogenetic analysis using BAC FISH on DNA fibers or stretched 3B flow sorted chromosomes. The availability of the 3B physical map should greatly accelerate map-based cloning of genes of interest from 3B and homoelogous chromosomes. This will be illustrated with the rapid identification of a ~1Mb physical contig at the “Rph7” locus by combining plate pool screening and physical contig information.