Rethinking Plant Genome Size: Flow Cytometry Guide

Mills River, NC - A new study released by researchers at North Carolina State University offers new insights and guidelines for the accurate estimation of plant genome size using flow cytometry. Flow cytometry has been widely used to estimate relative and absolute genome sizes (DNA contents) of plants for more than 50 years. However, the accuracy of these estimates can vary widely because of many factors, including errors in the genome size estimates of reference standards and various experimental methods.

This latest research revisits long-held assumptions, identifies sources of variation, and establishes best practices and reference standards, with updated genome sizes, to enhance the precision and reliability of genome size estimation. It also critically examines the methodologies used and provides guidelines to address inconsistencies and improve accuracy.

The specific objectives of this study were to reassess genome sizes of commonly used reference standards and quantify sources of variation and error in estimating plant genome sizes that arise from buffers, confounding plant tissues, tissue types, and plant reference standards using both 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) fluorochromes. Five separate studies were performed to elucidate these objectives

Fluorochrome Effects. A variety of fluorochromes are used in plant flow cytometry, but their effectiveness in staining the entire genome, the impact of chromatin on staining efficacy, and the influence of buffers and plant metabolites on dye fluorescence remain largely unknown. Significant variations in estimated plant genome sizes have been observed with different fluorochromes, due to differences in staining, base pair biases, and incubation times. Additionally, factors like the efficacy of staining densely packed chromatin and the sensitivity of fluorescence to interfering metabolites also affect the accuracy of genome size estimation.

Buffer Effects. Buffers are crucial for nuclei isolation and staining in flow cytometry sample preparation. These buffers, which can be separate or combined, often include surfactants, isotonic agents, pH buffers, phenolic binding agents (like polyvinylpyrrolidone), DNA-preserving compounds (such as spermine), and RNA or protein-degrading enzymes. Due to the variety of tissue types, morphology, and metabolite compositions, buffers are optimized for specific applications, with up to 28 different buffers commonly used.

Metabolite effects. Diverse secondary metabolites in plants can significantly vary among taxa and tissues, leading to errors in genome size estimation using flow cytometry. Compounds such as anthocyanins, caffeine, chlorogenic acid, coumarins, ellagic acid, tannic acid, and others can interact with fluorochromes and nuclear DNA, causing underestimation or overestimation of genome size.

Tissue type effects. The choice of plant tissue significantly affects genome size estimation using flow cytometry. Factors influencing this include the organ of origin, the state of the tissue (alive, dead, or fixed), and the presence of cytosolic compounds. Different secondary metabolites in varying types and amounts across tissues can cause tissue-specific interference.

Reference Standard Effects. In flow cytometry, including a reference standard with a known genome size is crucial for accurately calculating the genome size of an unknown sample. Revised estimates of genome sizes of commonly used plant reference standards were determined using human male leukocytes as a primary standard with an updated genome size (6.15 pg; 12.14% lower than that of earlier studies) using both DAPI and PI fluorochromes.

The results of this study show that flow cytometry can precisely and repeatedly determine relative plant genome sizes and ploidy, making it valuable for closely related plants when consistent methods are used. However, accurate determination of absolute genome size remains challenging and should be considered approximate, with potential errors of ±29% or more. Factors influencing accuracy include the fluorochrome,

extraction buffer, tissue type, reference standard, and plant metabolites, which can affect fluorochrome binding and fluorescence.

Overall, flow cytometry can be precise, repeatable, and extremely valuable for determining the relative genome size and ploidy of closely related plants when using consistent methods, regardless of fluorochrome. However, accurate determination of the absolute genome size by flow cytometry remains elusive, and estimates of genome size using flow cytometry should be considered gross approximations that may vary by ±29% or more as a function of experimental methods and plant environments. Additional recommendations of best practices are provided.

The implications of this study are significant for researchers, breeders, and biotechnologists. Accurate genome size estimation is crucial for a variety of applications, including plant breeding, genetic mapping, and biodiversity studies. The new guidelines and reference standards established by this study are expected to further improve the accuracy and precision of flow cytometry for estimating plant gnome size.

According to the author, With our labs being actively involved in plant breeding, genetics, cytogenetics, and crop improvement, flow cytometry is an extremely valuable tool for quickly estimating plant ploidy and relative genome size. However, having utilized this technology for decades, it became clear how subject these estimates are to variation in methodology. Because flow cytometry is precise and repeatable when using consistent methods, it is often incorrectly assumed and stated that it is "accurate". In this study, we were able to reassess genome sizes of commonly used reference standards and quantify sources of variation and error in estimating plant genome sizes that arise from using different tissues and methods that can result in errors of ±29% or more. Furthermore, use of reference standards with outdated genome size estimates contributed additional errors of 12% or more. This research provides improved estimates of genome sizes of reference standards, quantified sources of error, and recommended best practices for improved estimates of plant genome size."

Dr. Ranney is the JC Raulston Distinguished Professor, Department of Horticulture, North Carolina State University.

The full story can be found on the Journal of the American Society for Horticultural Science electronic journal website at: https://doi.org/10.21273/JASHS05376-24

Established in 1903, the American Society for Horticultural Science is recognized around the world as one of the most respected and influential professional societies for horticultural scientists. ASHS is committed to promoting and encouraging national and international interest in scientific research and education in all branches of horticulture.

Comprised of thousands of members worldwide, ASHS represents a broad cross-section of the horticultural community - scientists, educators, students, landscape and turf managers, government, extension agents and industry professionals. ASHS members focus on practices and problems in horticulture: breeding, propagation, production and management, harvesting, handling and storage, processing, marketing and use of horticultural plants and products. To learn more, visit ashs.org.