Developing a DNA barcode to distinguish between species of Shorea




Adam Essene

Introduction

Shorea is a genus of tropical hardwood trees found throughout Southeast Asia. All species of Shorea form symbiotic root associations with ectomycorrhizal fungi (ECM). At Lambir Hills National Park in Malaysian Borneo, there are over 50 different species of Shorea, many with overlapping distributions. This means that any root core samples used to study ECM assembly will contain a mix of ECM roots from many different host trees. I am interested in characterizing differences between the ECM communities associated with three species of Shorea; S. acuta, S. almon and S. inappendiculata (fig. 1). In order to ensure that the ECM root samples collected in the vicinity of these target trees are actually associated with them, I need to develop a DNA barcode that can distinguish between species of Shorea.

The internal transcribed spacers (ITS) 1 and 2 are two non-functional regions of ribosomal DNA flanked by sequences that code for the large and small subunits of the ribosome. These regions have been proposed as a barcode for plant species identification because they are short, highly variable, and have many genomic copies (Chen et al 2010). In this study, I assess the utility of these loci to differentiate between my three target Shorea species.

Methods

Leaf tissue was collected from three individuals of each species of Shorea at Lambir Hills National Park, Malaysia. DNA was extracted from each sample using the FastDNA Spin Kit (MP Biomedicals).

Primers used to amplify ITS1 and ITS2 were designed to target the order Malvales (contains Shorea) and prevent fungal amplification. Target regions were amplified with PCR and run on a 1% agarose gel to visualize the products.

All samples that were successfully amplified were PCR purified and sent out for sequencing. Sequences were aligned with Clustal Omega and pairwise similarity comparisons were made using BLAST.

Results

Successful amplification for each species of Shorea was achieved for ITS1 (Fig. 2A) and ITS2 (Fig. 2B). All PCR products were purified except for S. inappendiculata sample 1, ITS2, and sequenced. Sequencing failed for S. almon sample 2, ITS2.

Alignment of ITS1 sequences revealed 2 insertion/deletions and 8 base pair differences that distinguish S. inappendiculata from S. almon and S. acuta (Fig. 3A). Differences between S. almon and S. acuta sequences were inconsistent with species designations. Pairwise similarity comparisons between samples showed that S. acuta 1, S. almon 2 and S. almon 3 are nearly identical, while S. acuta 3 and S. almon 1 have relatively low inter- and intraspecific similarity (Table 1).

Alignment of ITS2 sequences revealed 5 insertion/deletions and 13 base pair differences that distinguish S. inappendiculata from S. almon/S. acuta (Fig. 3B). There were no insertion/deletions or base pair differences that distinguish between S. almon and S. acuta at this locus.

Discussion

Primer pairs designed for ITS1 and ITS2 were successful at amplifying DNA from all three species of Shorea. Both of these regions provide enough variation to identify S. inappendiculata, but neither could adequately distinguish between S. acuta and S. almon.

The pairwise similarity comparisons between S. acuta and S. almon ITS1 sequences illustrate that there is variation at this locus, but it is inconsistent with species identification. This inconsistency could be due to hybridization, which has been observed in other species of Shorea (Kamiya et al. 2011). It may also be a product of species misidentification; all of the leaf samples used in this study were collected from morphologically similar saplings identified during the large scale tree census at Lambir Hills. In order to address these questions and enable the identification of Shorea root tips, an additional barcoding locus will be necessary in combination with ITS1.


Figures


Figure 1


Figure 2-PCR amplification results from primers designed for ITS1 (A) and ITS2 (B). Amplicons were loaded on a 1% agarose gel. Specimens of each species of Shorea are labeled 1-3, NC denotes no template, negative control.


Figure 3-Sections of ITS1 (A) and ITS2 (B) alignments showing base pair changes and insertion/deletions that differentiate between S. inappendiculata and S. almon/S. acuta


Figure 4-Pairwise similarity (%) of S. acuta and S. almon ITS1 sequences


Abstract

Shorea is a genus of tropical hardwood trees found throughout Southeast Asia. The purpose of this study was to develop a DNA barcode for three species of Shorea found in Malaysian Borneo: S. acuta, S. almon and S. inappendiculata. In order to distinguish between these three species, primers were designed to target internal transcribed spacers (ITS) 1 and 2, two non-functional regions of ribosomal DNA. ITS1 and ITS2 were successfully amplified and sequenced for each species. Both loci contained variation that could identify S. inappendiculata, but neither were able to distinguish between S. acuta and S. almon. An additional barcoding locus is necessary in order to genetically identify these species

Works Cited
Chen, Shilin, H. Yao, J. Han, C. Liu, J. Song, L. Shi, Y. Zhu, X. Ma, T. Gao, X. Pang, K. Luo, Y. Li, X. Li, X. Jia, Y. Lin, C. Leon. 2010. “Validation of the ITS2 region as a novel DNA barcode for identifying medicinal plants. PLoS ONE 5(1): e8613.

Kamiya, Koichi, Y. Gan, S. Lum, M. S. Khoo, S. C. Chua, N. Faizu. 2011. Morphological and molecular evidence of natural hybridization in Shorea. Tree Genetics & Genomes, 7(2): 297-306.

Full Paper

Acknowledgments

Dr. Krista McGuire provided the impetus and direction for this project. Dr. Kabir Peay was extremely helpful negotiating my permission to conduct research at Lambir Hills National Park. I would like to thank Dr. Stuart Davies for providing tree distribution data for Lambir Hills, and Krystal Diaz and Amos Lim for giving me so much help in the field. Catharina Grubaugh and Kate Reid deserve a medal for their endless patience throughout this project and beyond. I would also like to thank Dr. Berish Rubin for his valuable guidance and constant support


This document was last modified 05/16/2014.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University
Biotechniques.org Home