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Seminar: John Nix: Flow Cytometry for Estimating Plant Genome Size: Revisiting Assumptions, Sources of Variation, Confounding Factors, and Interpretations, Ploidy Manipulation of Cercis canadensis Seedlings, Use of ITS Region Markers to Confirm Putative Interspecific Hybrids
June 2, 2023 | 12:00 pm - 1:00 pm
Flow Cytometry for Estimating Plant Genome Size: Revisiting Assumptions, Sources of Variation, Confounding Factors, and Interpretations, Ploidy Manipulation of Cercis canadensis Seedlings, Use of ITS Region Markers to Confirm Putative Interspecific Hybrids
John Nix, MS Seminar
Friday, June 2, 2023, 12:00 pm
(Under the direction of Dr. Hsuan Chen, Chair)
Join Zoom Meeting:
https://ncsu.zoom.us/j/91488301512?pwd=bExsMjM3ZDFnR1pKd1BTdHd5OTZTZz09
Meeting ID: 914 8830 1512
Passcode: 902215
Abstract:
Flow cytometry has been widely used to estimate relative and absolute genome sizes (DNA contents) of plants for over 50 years. However, the accuracy of these estimates can vary widely due to many factors including errors in the genome size estimates of internal reference standards and various experimental methods. The objectives of this study were to reassess genome sizes of commonly used reference standards and to quantify sources of variation and error in estimating plant genome sizes that arise from extraction buffers, confounding plant tissues, tissue types, and plant reference standards using both DAPI (2-(4-amidinophenyl)-1H -indole-6-carboxamidine) and PI fluorochromes (propidium iodide). Five separate studies were completed to elucidate these objectives. Revised estimates of genome sizes of commonly used plant reference standards were determined using human male leukocytes, updated estimates of the genome size of human male leukocytes (6.15 pg, 12.14% lower than earlier studies), and both DAPI and PI fluorochromes. Comparison of six different extraction buffers (Glabraith’s, LB01, MB01, MgSO4, Otto’s, and Sysmex) resulted in variation in genome size estimates by as much as 18.1% depending on the buffer/fluorochrome combination. Addition of different confounding plants tissues (representing 10 diverse taxa and associated secondary metabolites) resulted in variation in genome size estimates by as much as 10.0%, depending on the tissue/fluorochrome combination. Different plant tissue types (leaf color/exposure and roots) resulted in a variation in genome size estimates of 10.7%, independent of the fluorochrome. The selection of different internal reference standards introduced additional variation in genome size estimates of 5.9% depending on the standard/fluorochrome combination. The choice of fluorochrome (DAPI vs. PI) had one of the greatest impacts on variation in genome size and differed by as much as 32.9% for Glycine max ‘Polanka’ when using human leucocytes as an internal standard. A portion of this variation (~10.8%) can be attributed to the base pair bias of DAPI and variation in AT:CG ratios between the sample and standard, leaving 22.1% of the variation in genome size estimate resulting from how effectively these fluorochromes stain and report the genome. The combined variation/error from these different factors, excluding variation from base pair bias for different fluorochromes, totaled 66.8%. Additional details of how selected factors impact accuracy, precision, and interaction of treatments are presented. Overall, it was found that flow cytometry can be very precise, repeatable, and extremely valuable for determining relative genome size and ploidy of closely related plants when using consistent methods. However, accurate determination of absolute genome size by flow cytometry remains elusive and estimates of genome size using flow cytometry should be considered approximations that may vary by ± 33% or more as a function of experimental methods. Additional recommendations on best practices are provided.
Cercis canadensis L., eastern redbuds, is a deciduous flowering tree species with a great ornamental value. In the US, Cercis had a total economic value of 28.4 million in 2019 and over 36 named cultivars exist today. Despite this horticultural value, no known polyploids of Cercis canadensis have been documented. The generation of polyploids through whole genome doubling has been a powerful tool for plant breeders and as such could have applications in C. canadensis such as developing seedless cultivars. With a need for tetraploid C. canadensis to pursue further plant improvement in this crop, experiments were designed to develop a protocol for generating polyploid genotypes. In this study, seedlings of C. canadensis were exposed to different chemistries, concentrations, and treatment duration of mitotic inhibitors via the agar drop technique to discover the optimal genome doubling method. Treatment effects for mitotic inhibitor chemistry were significant for survivorship and genome doubling, with oryzalin having fewer surviving seedlings but higher tetraploid conversion in surviving seedlings oryzalin concentration effect was not significant regarding the tested concentration levels. The duration treatment effect was significant in the seedling survivorship but not for tetraploid conversation rate, although a higher ratio of cytochimera plants was observed in the longest treatment duration. As a result, the treatment of oryzalin at 150uM concentrations and a duration of 9 days showed the optimal result that 15.38% and 34.07% of treated seedlings were tested tetraploid and mixoploids. The protocol of C. canadensis polyploidy manipulation generated through this research will be useful for plant breeders in developing cultivars possessing sterility and other novel traits for eastern redbuds and relative species.
In the horticultural trade and botanical gardens, there are many putative interspecific hybrids that are yet to be proven. For many of these hybrids, the parents may not long be available to investigate. A fast and accessible system where species–specific markers could give insight on an unknown hybrid genome would be useful for researchers and horticulturists alike. The Internal Transcribed Spacer (ITS) region is a highly conserved sequence with thousands of copies in the nuclear genome and has been useful for understanding phylogenetic relationships of plants. Theoretically, markers designed around this sequence could be species specific if any sequence polymorphism in the ITS is detected between species. In this research, an ITS marker approach is used to confirm the possible pedigrees of two groups of putative interspecific hybrid plants. GenBank ITS sequence vouchers of the pairs Cercis canadensis and C. chinensis, as well as Parrotia persica and Distlyium myricoides were compared to identify sequence polymorphisms for primer design. In the case of ×Distylparrotia, no informative polymorphism could be identified. Between the two Cercis species, multiple SNPs were observed, and primers were designed for testing. Species-specificity of the primers was tested using C. canadensis ‘NC 2016-2′, C. canadensis ‘Forest Pansy’, C. chinensis ‘Genpei’, C. chinensis ‘Kay’s Early Hope’, as well as two putative hybrids from the NCSU program and the putative hybrid C. ‘Wavecrest’. One primer pair was selected for its specificity in Cercis and the species-specific primers were able to support or refute hybrid claims. This is the first molecular evidence and report of the interspecific hybrid C. canadensis x chinensis. This also shows that the ITS region can be used to identify hybrids in Cercis, and this method could be applied to other groups of plants if the variation in sequence exists and is especially useful if the situation is appropriate, such as lost parents or an unknown but speculated pedigree.