DNA Sequence Identification of Tree Root Tips and Their Mutualist Fungi Using Landscape-Harvested Tissues




Tim Kerin

Introduction

Plant-fungal mycorrhizal symbioses play an important role in the health of forest trees and the functioning of forest communities and ecosystems. Tree species composing the forest community shift through time, and along with them, their mycorrhizal fungal symbionts. Therefore, understanding the makeup of both the tree and fungal members of a community can be important in understanding forest succession. The focus of my research is the eastern hemlock (Tsuga canadensis), a foundation tree species of our eastern forests that is presently experiencing a range reduction due to insect herbivory and climate warming. My hypothesis is that remaining hemlock trees will associate with a somewhat altered mycorrhizal fungal community due to isolation in predominantly oak hardwood forests, and that this will have a bearing on the health of the trees.

Testing this hypothesis requires taking soil cores around focal hemlock trees in both dense hemlock stands and mixed forests, and examining the hemlock root tips and their adherent mycorrhizal fungi using DNA sequence analysis. Mycorrhizal fungi are often identified through DNA sequencing but since the roots of tree species other than hemlock are present in soil cores from mixed forests, a technique to distinguish between root tips of hemlock and other tree species is also needed.

The specific aim of the present study was to develop a reliable molecular technique for identifying the symbionts of a fungal/tree association from a single root tip DNA extract, using PCR with primers for fungal ribosomal DNA and plant chloroplast DNA, followed by DNA sequencing. I achieved this using single root tips from seven known, potted specimens representing the various tree species found in my outdoor study plots. The rDNA primers anneal to the conserved 18S and 28S subunits of ribosomal DNA and amplify the variable length/sequence internal transcribed spacers (ITS A and ITS B) along with the conserved 5.8S subunit (figure 1). The cDNA primers anneal to conserved regions of the chloroplast trnL (UAA) intron that flank the variable length/sequence P6 loop (figure 1). Both members of each plant/fungal pair were identified by comparing their sequence with those in the GenBank database, using the BLAST program.

In addition to sequencing the DNA from both the fungal and plant components of single root tips, I wanted to differentiate between hemlock cDNA and cDNA from neighboring trees by band position on an agarose gel. This would reduce the time and cost associated with sequencing both the fungal and tree DNA from each root tip.

Figures


Figure 1-Locations for rDNA and cDNA primers (a and b), primer sequences and amplicon lengths (c) and a photomicrograph of a mycorrhizal root tip (d).


Figure 2-2% agarose gel of DNA amplified from four hemlock root tips (1-4) and two different species of oak (5 and 6). Actual length of tree cDNA amplicons are in parentheses along with the common names.


Figure 3-2% agarose gel of both plant and fungal DNA amplified from single root tips of seven known potted trees. Double bands were excised and separated for sequencing. Actual length of tree cDNA amplicons are in parentheses along with the common names.


Summary

Both aims of this study were achieved. First, tree and fungal species were identified using DNA sequencing and comparison with published GenBank sequences. Sequence homologies were 100% for the plant species (except for Quercus prinus (97%), which is not yet in GenBank), and 95-100% for the various fungal species. Second, hemlock DNA bands can be easily distinguished from oak, birch and beech bands by their greater migration distance on a gel (figures 2 and 3). The technique presented here can conceivably be adapted to any target species or forest community.

Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin for his insight and guidance, and for keeping the project focused. I would like to offer extra special thanks to Bo Liu and Leleesha Samaraweera, who made themselves available for countless questions, kept me supplied with necessary materials and gave much valuable advice.


This document was last modified 05/11/2009.
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