Researchers at North Carolina State University have unveiled a groundbreaking guide for accurately estimating plant genome size using flow cytometry. This flow cytometry technique has been widely used for over 50 years, but the new study addresses long-standing issues and provides updated best practices and reference standards to enhance the precision and reliability of genome size estimation. The findings have significant implications for researchers, breeders, and biotechnologists working in plant genetics and breeding.
Unconfounding the Conundrum of Estimating Plant Genome Size
Precise plant genome size estimation is essential for a broad spectrum of studies, including applications in plant breeding, genetic mapping, and biodiversity; yet it remains a laborious task in cases where suitable methods are lacking. But the system has been rife with inconsistencies and errors for generations. In a new study from North Carolina State University, Richard Akresh and coauthors took on this challenge directly by rethinking old assumptions as well as accounting for sources of variation in genome size estimates.
Here, the researchers present five case studies designed to identify error and variation sources in flow cytometric experiments for estimating plant genome sizes. That included investigating the influence of various fluorochromes, buffers, plant metabolites, tissue types and reference standards — all potentially contributing to how accurate such measurements are. The study quantifies these modulating factors for the first time, providing important insight and a guideline for how researchers can work to overcome some of the challenges encountered when estimating plant genome sizes.
Unveiling the Complexity of Fluorochromes, Buffers and Metabolites
One of the main take home messages from our study is the major influence on genome size estimation exerted by the fluorescent dyes. They revealed that staining efficacies, base pair biases and incubation times have a strong impact on the outcomes of rRNA-based FISH with different fluorochromes. Furthermore, limitations such as the capacity to stain tightly packed chromatin and the fluorescence interference from helper metabolites enable further inaccuracies.
The investigation of the importance of buffers in flow cytometry was another aspect done in this study. These buffers could be because of various combination components like surfactants, osmotic pressure protectors, pH regulators and enzyme-based compounds; these formulations are usually optimized for specific applications. Nonetheless, our results indicate that the performance of these buffers can be significantly impacted by diversity of plant tissue types, morphology and metabolite compositions leading to genome size measurement error.
Body Matters Types of Tissues, Standards of Reference, and the Search for Precision
One other important conclusion of the study is related to plant tissue and genome size estimation. Factors including the organ, the tissue state (dead or alive), and the presence of cytosolic compounds are all shown to affect measurement accuracy, according to discoveries by researchers.
Additionally, the paper examined relevance of reference standards in flow cytometry. The team recalculated the standard genome sizes for man plant references, in terms of human male leukocytes as the reference standard with an updated genome size. Therefore, the reference genomes need to be validated and updated continuously at regular intervals for estimating the plant genome sizes reliably.
In broad, our study shows that flow cytometry although precise and repeatable method for relative genome size or ploidy determination it still suffers from problems of a great lack of precision when deciding about the absolute genome sizes. Here, the researchers offer detailed instructions and best practices for researchers, breeders and biotechnologists when estimating plant genome sizes to avoid common pitfalls and obtain accurate results.
