Isolation of anthurium nuclei for molecular applications

Authors: Suzuki, Jon; AMORE, TERISITA - University Of Hawaii; Horrell, Jonathan; IMAMURA, JOANNE - University Of Hawaii; Matsumoto Brower, Tracie

Submitted to: Acta Horticulturae
Publication Type: Abstract Only
Publication Acceptance Date: 5/12/2017
Publication Date: 9/1/2017
Citation: Suzuki, J.Y., Amore, T.D., Horrell, J.R., Imamura, J.S., Matsumoto Brower, T.K. 2017. Isolation of anthurium nuclei for molecular applications. Acta Horticulturae. 52(9S):S372. https://doi.org/10.21273/HORTSCI.52.9S.S1.
DOI: https://doi.org/10.21273/HORTSCI.52.9S.S1

Interpretive Summary: The colorful tropical cut flower, Anthurium has a long and storied history in Hawaii. The Hawaii anthurium industry was founded by ardent local growers, breeders, and shippers, and was supported by the pioneering ornamental plant breeding and genetic research program of the University of Hawaii that produced novel cultivars and performed landmark basic research. Together, these activities contributed to worldwide populariza-tion of this floral crop. Recent research advances from numer-ous labs worldwide have improved the resources available to further cultivar improvement and basic research in Anthurium, non-model plants belonging to the most species-rich genus of the Araceae, or aroid family. Recent transcriptome analyses in particular have enabled a first insight into the gene content and expression patterns of large sets of Anthurium genes represent-ing different pathways. Despite advances in molecular research in Anthurium and the availability of improved protocols, reli-able and reproducible isolation of nucleic acids can still pose a challenge. These challenges may be attributed, in part, to the high polyphenolic content in various tissues, as well as the variability in tissue composition or integrity between cultivars and related species. Polyphenolics can damage nucleic acids or inhibit recovery and downstream applications. In this study, we combined components of several methods for isolation of plant nuclei as a means of obtaining the most intact and purest genomic DNA. Nuclei isolation is a method that enables gentle, physical separation of DNA from cellular contaminants, but to our knowledge has not been reported for the study of Anthurium. DNA intactness was evaluated from isolated nuclei prepared in gel plugs, based on migration patterns in pulse field gel electro-phoresis (PFGE) and conventional agarose gels, whereas DNA purity was assessed by 4',6-diamidino-2-phenylindole (DAPI) staining of nuclei, gel plug color, and DNA digestibility. Larger isolation buffer volume to sample mass ratios improved removal and lowered viscosity of cellular contaminants from Anthurium nuclei preparations. However, Percoll step gradient isolation of Anthurium nuclei was required in order to remove contaminants inhibiting DNA digestion. Among nuclei preparations from different tissue types, the spadix, a type of spike inflorescence typical of aroids, was found to be an efficient source, based on mass fresh tissue, of isolated nuclei. The findings of this work will be useful for isolating and evaluating HMW Anthurium and related plant DNA for large DNA fragment cloning, next gen sequencing and DNA blot applications.

Technical Abstract: The colorful tropical cut flower, Anthurium has a long and storied history in Hawaii. The Hawaii Anthurium industry was founded by ardent local growers, breeders, and shippers and was supported by the pioneering ornamental plant breeding and genetic research program of the University of Hawaii that produced novel cultivars and performed landmark basic research. Together, these activities contributed to worldwide popularization of this floral crop. Recent research advances from numerous labs worldwide has improved the resources available to further cultivar improvement and basic research in Anthurium, nonmodel plants belonging to the most species rich genus of the Aroid family. Transcriptome analyses in particular have enabled a first insight into the gene content and expression patterns of large sets of Anthurium genes representing different pathways. Despite advances in molecular research in Anthurium and the availability of improved protocols, reliable and reproducible isolation of nucleic acids can still pose a challenge. These challenges may be attributed in part to the high polyphenolic content in various tissues, as well as the variability in tissue composition or integrity between cultivars and related species. Polyphenolics can damage nucleic acids or inhibit recovery and downstream applications. In this study, we combined components of several methods for isolation of plant nuclei as a means of obtaining the most intact and purest genomic DNA. Nuclei isolation is a method that enables gentle, physical separation of DNA from cellular contaminants, but to our knowledge has not been reported for the study of Anthurium. DNA intactness was evaluated from isolated nuclei prepared in gel plugs based on migration patterns in PFGE and conventional agarose gels, whereas DNA purity was based on nuclei staining, gel plug color as well as DNA digestibility. Not surprisingly, we found that larger sample to isolation buffer ratios improved removal and lowered viscosity of cellular contaminants. However, we found that among nuclei preparations from different tissue types, spadices consistently yielded the most DNA per mass fresh tissue. During the course of evaluating DNA isolated using our modified methods for digestibility using various restriction endonucleases, the entire genomic DNA complement appeared to be digestible, but complete digestion required relatively longer incubation times and addition of restriction endonuclease enhancers such as spermidine. The findings of this work will be useful for isolating and evaluating HMW Anthurium and related plant DNA for large DNA fragment cloning, next gen sequencing and DNA blot applications.