CONSTRUCTION AND DNA SEQUENCING OF A BAC CONTIG SPANNING THE PHYTOPHTHORA SOJAE GENOME
F ARREDONDO1, WX SHAN1, A CHAN1, P HRABER2, M WAUGH2, B SOBRAL2 and BM TYLER1
1Department of Plant Pathology, University of California, Davis, CA 95616, USA; 2National Center for Genome Resources, Santa Fe, NM 87505, USA
More than 40 species of the oomycete Phytophthora cause serious diseases of a huge range of crop and ornamental plants. We are characterizing genes that control recognition, host specificity and pathogenicity in the soybean pathogen Phytophthora sojae. To facilitate isolation of such genes from P. sojae by map-based cloning, we are constructing a BAC contig of the entire genome of this organism, using a novel hybridization fingerprinting strategy. We have constructed a library of 7680 BACs of average size 55 kb, spanning the 62 Mb genome seven times. We are hybridizing the BACs with unique mixtures of random probes, most of them repetitive. We will use the subset of probes hybridizing to each BAC to identify overlapping BACs.
Computer software has been developed to collect, simulate and analyse the data. At present, we have probed the library with 49 of the mixtures. Each mixture hybridized to around 300-500 BACs resulting in 20,200 hybridization hits to BACs in the library. So far, 19% of the BACs have received the minimum number of hits needed to establish statistically significant overlaps (five each). Of these BACS, 34% have been placed into contigs, which at present average eight BACs per contig. These results suggest about 180 probe mixtures will be sufficient to enable us to assemble the BACs into less than 100 contigs spanning >95% of the genome. We have tested the authenticity of three of the contigs by HindIll digestion. The contigs contained 30, 13 and 7 BACs and spanned 640, 250 and 160 kb, respectively. All three contigs proved to be bona fide contigs, validating our strategy.
With the long-term goal of sequencing the entire genome of P. sojae and selected sequences from other Phytophthora species, such as P. infestans, we have established the Phytophthora genome initiative (PGI) in collaboration with P. infestans researchers. We have begun preliminary sequencing of a 200-kb BAC contig spanning two avirulence genes from P. sojae. Sequencing of the first 60-kb BAC is nearly complete, and software has been developed for automatic processing, annotation and publishing of the sequence data via the web. In the region sequenced so far, the gene density is extremely high. Sixteen firm matches to the sequence databases have been obtained, even though only 50% of all P. sojae genes are expected to show sufficient conservation to obtain matches. Four pairs of genes are less than 600 bp apart, including one pair of overlapping genes. These observations prompt the speculation that in P. sojae, functional genes are located in high-density gene islands dispersed among clusters of repetitive sequences, which are known to constitute around 50% of the genome.