Efficient analysis of ploidy levels in plant evolutionary ecology

Abstract

Flow cytometry (FCM) has provided an efficient method to determine DNA content in cells, thus providing a feasible alternative to microscopy for the estimation of ploidy for population level studies requiring large sample sizes. In cytological studies, searching for example chromosomal abnormalities, samples are commonly treated in separate tubes to ensure the capture of sufficient number of separate cells to meet the required resolution (nuclei number > 5000 and coefficient variation (CV) < 5%). In this study, we tested whether FCM can be expedited by using 96-well plate-based FCM sample preparation and automated data analysis using Flowing Software without compromising the resolution required for ploidy level detection in population biology. We estimated ploidy levels in red fescue (Festuca rubra L.) and viviparous sheep's-fescue (F. vivipara Sm.) plants. A tube-based procedure gave more tightly defined peaks compared to a plate-based procedure. However, our results suggest that the plate-based higher throughput FCM procedure gives equally reliable estimation of the number of sets of chromosomes (ploidy) in plant with three times less reagent consumed and over 20 times more time-efficiently compared to the tube-based FCM procedure. We propose that the integration of plate-based FCM with Flowing Software provides a time-and cost-efficient and less error-prone procedure to determine ploidy levels in taxonomy, evolutionary ecology and plant breeding.

Geographic Variation in Festuca rubra L. Ploidy Levels and Systemic Fungal Endophyte Frequencies

“…Ploidy levels of the plants were determined by flow cytometry (FCM) [49]. We used known chromosome counts of F .…”

“…We used known chromosome counts of F . rubra plants as references for the FCM results [49]. Plants from different populations (one to three plants for each population) were randomly chosen to microscopically determine cytotype.…”

“…DNA fluorochrome stock solution with 50 μl ml -1 propidium iodide (PI) and 50 μl ml -1 ribonuclease (RNase) was added and incubated on ice one hour before FCM analysis [51]. The 96 well plate-based FCM procedure [49] was carried out using LSR II (Bechton Dickinson San Jose, USA) flow cytometer at the Turku Centre for Biotechnology, Finland. Pisum sativum L. ‘Ctirad’ (2C DNA value = 9.09 pg) plants obtained from the Institute of Experimental Botany (Laboratory of Molecular Cytogenetics and Cytometry, Olomouc, Czech Republic), were used as an external reference to determine DNA quantity in pictograms (pg).…”

nQuire: A Statistical Framework For Ploidy Estimation Using Next Generation Sequencing

“…Although ploidy traditionally has been investigated by measuring DNA content using flow cytometry [8], it can also be inferred from next generation sequencing (NGS) data either by examining k-mer distributions, or by assessing the distribution of allele frequencies at biallelic single nucleotide polymorphisms (SNPs) [4]. This methodology has been used to estimate ploidy in newly assembled genomes in order to identify the number of likely collapsed haplotypes on a per-contig basis [9], as well as to detect intraspecific variation of ploidy in the oomycete Phytophthora infestans [4,6] and in the Baker's yeast Saccharomyces cerevisiae [5].…”

Development and characterization of chloroplast microsatellite markers in a fine‐leaved fescue, Festuca rubra (Poaceae)

“…clade ( F. rubra group, family Poaceae). Festuca rubra is perennial, rhizomatous, and highly interfertile with multiple ploidy levels ( Dirihan et al, 2013 ). It is one of the agriculturally most important turfgrasses, widely cultivated in temperate regions ( Gould and Shaw, 1983 ), with a number of commercial cultivars.…”