Heterokaryotic nuclear conditions and a heterogeneous nuclear population are observed by flow cytometry in Phytophthora infestans

Abstract

A simple and reliable method for preparation of whole nuclei of a common oomycete, Phytophthora infestans, is described for laser flow cytometry. The ease of preparation, the absence of detectable debris and aggregates, and the precision in determinations of DNA content per nucleus improve interpretation and understanding of the genetics of the organism. Phytophthora infestans is the pathogen that causes potato and tomato late blight. The genetic flexibility of P. infestans and other oomycete pathogens has complicated understanding of the mechanisms of variation contributing to shifts in race structure and virulence profiles on important agricultural crops. Significant phenotypic and genotypic changes are being reported in the apparent absence of sexual recombination in the field. Laser flow cytometry with propidium iodide is useful in investigating the nuclear condition of the somatic colony of field strains of P. infestans. The majority of the studied strains contain a single population of nuclei in nonreplicated diplophase. However, mean DNA content per nucleus varies considerably among isolates confirming the heterogeneity of the nuclear population in regard to C-value, for field isolates. Nuclear DNA content varies from 1.753 to 0.753 that of nuclei in a standard strain from central Mexico. Some strains contain two to three populations of nuclei with differing DNA contents in the mycelium and are heterokaryons. Such a range in DNA content suggests DNA-aneuploidy, but direct confirmation of aneuploidy will require microscopy of chromosomes. Heterokaryosis and populations of nuclei of differing DNA content necessarily confound standardized assays used worldwide in crop breeding programs for determination of race profiles and virulence phenotypes of this important pathogen. '

The rise and fall of the Phytophthora infestans lineage that triggered the Irish potato famine

“…Genetic diversity can be increased by polyploidy, which has been reported in isolates of P. infestans (e.g., Daggett et al, 1995; Catal et al, 2010), and which has major evolutionary implications for asexual organisms. To estimate ploidy level in our specimens, we investigated the distribution of read counts at biallelic SNPs for high-coverage genomes.…”

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

“…(2002). FCM measurement of Phytophthora species revealed complex nuclear conditions, such as heterokaryosis and indications of aneuploidy (Catal et al ., 2010; Vercauteren et al ., 2011).…”

Aberrant genome size and instability of Phytophthora ramorum oospore progenies

“…The technique is based on the simultaneous analysis of propidium iodidestained nuclear suspensions, together with an internal DNA reference standard of known genome size. This optimizes the opportunity to determine the parental genome sizes and perform highresolution genome size analysis of the progenies (Catal et al, 2010;Galbraith, 2009;Si-Ammour, 2002). Karyotyping by light microscopy (cytological) or with pulse field gel electrophoresis can be useful to give additional information on chromosome number and size in the progenies and their parents, but was not crucial here.…”