A cDNA-AFLP based strategy to identify transcripts associated with avirulence in Phytophthora infestans.
Expression profiling using cDNA-AFLP is commonly used to display the transcriptome of a specific tissue or developmental stage. Here, cDNA-AFLP was used to identify transcripts in a segregating F1 population of Phytophthora infestans, the oomycete pathogen that causes late blight. To find transcripts derived from putative avirulence (Avr) genes germinated cyst cDNA from F1 progeny with defined avirulence phenotypes was pooled and used in a bulked segregant analysis (BSA). Over 30,000 transcript derived fragments (TDFs) were screened resulting in 99 Avr-associated TDFs as well as TDFs with opposite pattern. With 142 TDF sequences homology searches and database mining was carried out. cDNA-AFLP analysis on individual F1 progeny revealed 100% co-segregation of four TDFs with particular AVR phenotypes and this was confirmed by RT-PCR. Two match the same P. infestans EST with unknown sequence and this is a likely candidate for Avr4. The other two are associated with the Avr3b-Avr10-Avr11 locus. This combined cDNA-AFLP/BSA strategy is an efficient approach to identify Avr-associated transcriptome markers that can complement positional cloning.
Guo
Jun
J
Plant Sciences Group, Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, NL-6709 PD Wageningen, The Netherlands.
Jiang
Rays H Y
RH
Kamphuis
Lars G
LG
Govers
Francine
F
D002874:Chromosome Mapping; D019295:Computational Biology / Q000379:methods; D003433:Crosses, Genetic; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020869:Gene Expression Profiling; D005819:Genetic Markers; D008969:Molecular Sequence Data; D010641:Phenotype; D010838:Phytophthora / Q000235:genetics / Q000254:growth & development / Q000378:metabolism / Q000472:pathogenicity*; D012150:Polymorphism, Restriction Fragment Length; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014774:Virulence
D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D005819:Genetic Markers; D012331:RNA, Fungal