The use of flow cytometry for fungal nuclear DNA quantification

Abstract

Genome size information is sparse across fungi, with information being available for less than 2000 species. So far, most records have been obtained using static, microscopebased cytometry methods or derived from genome sequencing projects. Flow cytometry is now considered the state-of-the-art method for obtaining genome size measurements, and appropriate methods and DNA standards are available, enabling the analysis of most genome size ranges in a rapid, robust and inexpensive way. The average fungal genome size is 60 Mbp, but sizes vary across phylogeny, ranging from 2.2 (Encephalitozoon romaleae) to 3706 Mbp (Jafnea semitosta). In several fungal clades, genome size expansion seems to accompany evolution either to plant mutualism or to plant parasitism (particularly biotrophy), and fungi that interact with plants seem to have larger genomes than saprobes and those that interact with animals. Whereas flow cytometry for nuclear DNA quantification is routinely employed in plant sciences for genome size and ploidy studies, its use in fungal biology is still infrequent. Appropriate standards, methods and best practices are described here, with the aim of stimulating a more generalized and widespread use of flow cytometry for fungal genome size measurement.

Meet the challenges of analyzing small genomes using flow cytometry

“…Moreover, the DNA content of other fungal species is generally not much higher (with a median value <40 Mbp; [3]). In the study by Talhinhas et al [4], the authors nicely summarized the currently used methods for fungal genome size estimation using FCM and addressed the potential pitfalls. Interestingly, these pitfalls are widely shared with many other groups of microorganisms with small genomes.…”

“…For the uncultivated microorganisms in trophic interactions, another approach could be taken in simultaneous analysis of studied microorganism and its symbiont/host/prey and then to analyze these partners separately to correctly distinguish peaks of each organisms. Such approach seems especially suitable for parasites as is nicely illustrated by Talhinhas and colleagues [4] for pathogenic fungi and its host plant. However, simultaneous analysis might not be suitable for organisms substantially differing in their genome size.…”

“…Another possibility is to test different isolation buffers. For example, the Woody Plant Buffer or Tris-MgCl 2 buffer seems to work with fungal samples and LB01 buffer found wide application in FCM of microalgae (Talhinhas et al [4]; Čertnerová [5]. However, new lysis buffers reflecting the specifics of particular groups of microorganisms still need to be developed.…”

“…Talhinhas and colleagues [4] further discussed the lack of appropriate FCM standards, which is yet another important issue accompanying analysis of small genomes. In recent years, the number of newly introduced FCM standards is slowly rising up, with, for example, Saccharomyces cerevisiae, Aspergillus fumigatus or Chlamydomonas reinhardtii possessing very small genome sizes (1C values of 24.1, 29.2, and 0.12 pg, respectively; [8,9]).…”

“…In contrast to other groups of microorganisms, fungal genome size data are listed in their own database [3]. Talhinhas and colleagues [4] analyzed these data from many different angles. They highlighted that the majority of genomes size data were obtained using WGS or static microscope-based cytometry methods, and only less than 5% were obtained with FCM.…”