A potato large-insert library for isolation of candidate loci for late blight resistance and studies on their genome organization

Abstract

Summary QTL mapping of quantitative resistance to P. infestans has been pursued in potato and several loci contributing to the resistance have been identified (Leonards-Schippers et al. 1994; Meyer et al. 1998; Ewing et al. 2000; Sandbrink et al. 2000 and Naess et al. 2000). Ghislain et al. (2001) detected two major QTL effects on linkage groups VIII and XII using a hybrid cross between S. phureja x dihaploid S. tuberosum. The strong QTL effect on linkage group XII was localized in a region where no major gene or QTL for P. infestans has been reported. So far, none of the QTLs for P. infestans resistance has been cloned and the genes behind the QTLs are still unknown. Map based cloning has proved to be a promising approach for cloning genes and QTLs. This approach requires DNA markers tightly linked to the trait in combination with large insert libraries. In this study, a large insert library was constructed from one of the resistant hybrids of the population analysed by Ghislain et al. (2001). The inserts have been cloned into the binary vector pCLD04541. The clones can be used for plant transformation via A. tumefaciens. The library contains approximately 50 000 clones with an average insert size of 80 kb. The coverage of the library has been calculated to be 3-4 times the haploid potato genome. The construction of a large insert library with this genetic material will facilitate the cloning and dissection of the genes underlying the QTL once a more precise map of the region is available. The library was used to clone members of three defense related gene families: PAL(Phenylalanine-Ammonia-Lyase), PR-5 (acidic and basic osmotin) and PPO(Polyphenol oxidase). 29, 7 and 10 BAC clones containing PAL, PR-5 and PPO genes, respectively were identified. The PR-5 BAC clones were further characterized. 6 BACs containing osmotin-like sequences were grouped in two small contigs: Contig A (100kb) and Contig B (120kb). DNA markers derived from the contigs identified two genetic loci on linkage groups VIII (Contig A) and XI (Contig B). Southern gel blot analysis of genomic and BAC DNA showed that potato has at least 5 copies of osmotin genes. Most of the osmotin genes are clustered on linkage group VIII in less than 90 kb whereas only one gene is present on linkage group XI. Interestingly, Trognitz et al. (2001) using the population of Ghislain et al. (2001) reported correlation between a QTL effect derived from S. tuberosum and an osmotin RFLP marker on linkage group VIII. On the other hand, one BAC with sequence similarity to acidic PR-5 showed that acidic members of PR-5 in potato are clustered on linkage group XII, where at least 3 copies are present in less than 35 kb. S. phureja and S. tuberosum are highly homozygous at these loci which made it difficult to develop specific markers. However, two CAPS markers were derived and mapped in the maternal line S. phureja, revealing that the position of the genes do not overlap with the QTL effect. Fragments of the PR-5 BACs inserts were subcloned into pBluescript. Sequence analysis of the subclones identified 6 osmotin-like genes and 4 acidic PR-5 genes, including a copy with a truncated sequence at the N-terminus. One of the acidic PR-5 genes had 100% identity to the partial sequence of potato Protein C (Pierpoint et al. 1990). All the PR-5 ORF did not contain introns and all except the one from linkage group XI had the 16 cysteine residues highly conserved in the PR-5 family. PCR and Southern gel blot analysis demonstrated that PR-5 genes are present in all members of the Solanaceae family tested. A phylogenetic analysis of PR-5 sequences of Solanaceae from the Genebank and the genes described in this study clustered the sequences in three main branches of acidic, neutral and basic genes