Guo Jun, Jiang Rays H Y, Kamphuis Lars G, Govers Francine
Plant Sciences Group, Laboratory of Phytopathology, Wageningen University, Binnenhaven 5, NL-6709 PD Wageningen, The Netherlands.
Fungal Genet Biol. 2006 Feb;43(2):111-23. doi: 10.1016/j.fgb.2005.11.001. Epub 2006 Feb 7.
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.
使用cDNA-AFLP进行表达谱分析通常用于展示特定组织或发育阶段的转录组。在此，cDNA-AFLP被用于在致病疫霉（引起晚疫病的卵菌病原体）的分离F1群体中鉴定转录本。为了找到源自假定无毒（Avr）基因的转录本，将具有明确无毒表型的F1后代的萌发囊肿cDNA汇集起来，并用于混合分组分析法（BSA）。筛选了超过30,000个转录衍生片段（TDF），得到了99个与Avr相关的TDF以及具有相反模式的TDF。对142个TDF序列进行了同源性搜索和数据库挖掘。对单个F1后代进行的cDNA-AFLP分析揭示了四个TDF与特定AVR表型的100%共分离，这通过RT-PCR得到了证实。其中两个与序列未知的相同致病疫霉EST匹配，这可能是Avr4的候选基因。另外两个与Avr3b-Avr10-Avr11位点相关。这种cDNA-AFLP/BSA联合策略是鉴定可补充定位克隆的与Avr相关的转录组标记的有效方法。
