CONSTRUCTION OF KIWIFRUIT BAC CONTIG MAPS BY OVERGO HYBRIDIZATION AND THEIR USE FOR MAPPING THE SEX LOCUS

The physical location of gene clusters among closely related organisms is often conserved and is known as synteny. Overlapping oligonucleotides (overgos) are useful molecular markers for determining synteny among organisms. Our reference genome is Arabidopsis thaliana, and since there is no information on the genome structure of Actinidia chinensis, our extensive collection of expressed sequence tags (ESTs) was used as query for the overgo design pairwise comparisons. Overgo probes consist in two self-annealing oligonucleotides 40 bp long, annealing at regular intervals to conserved regions between genomes. We developed a non-radioactive method for labelling the 1006 overgo probes designed to screen the Bacterial Artificial Chromosome (BAC) library constructed from genomic DNA of the A. chinensis female parent of the kiwifruit mapping population. Distribution and clone assignment were examined. This project will enhance the link between our breeding and genomics programmes by assigning the corresponding BAC contigs to hundreds of expressed sequence tags (ESTs) deposited in our database and speed up positional cloning. The sex-linked sequenced-characterized amplified regions SCAR marker SMX, derived from a RAPD marker at HortResearch, is located on the X chromosome of the male parent of the A. chinensis mapping population. A non-polymorphic marker derived from SCAR-SMX was developed to identify the SMX region of the female parent. The SMX non-polymorphic marker was amplified in both parents of the mapping population and all progeny genotypes. A non-radioactive SMX non-polymorphic probe used for screening the BAC library identified two overlapping BAC clones. Neighbouring clones were isolated by developing DNA probes to their 5’- and 3’- insert ends. Sequencing of these clones is underway to develop new markers able to predict sex across species boundaries. The BAC contig around the sex locus will associate the genetic distances from the linkage map to physical distances.