A molecular method to identify species of fine roots and to predict the proportion of a species in mixed samples in subtropical forests

Weixian Zeng,Yan Liu,Bo Zhou,Yeling Zeng,Pifeng Lei,Wenhua Xiang,Cong Liu

doi:10.3389/fpls.2015.00313

Abstract

Understanding of belowground interactions among tree species and the fine root (≤2 mm in diameter) contribution of a species to forest ecosystem production are mostly restricted by experimental difficulties in the quantification of the species composition. The available approaches have various defects. By contrast, DNA-based methods can avoid these drawbacks. Quantitative real-time polymerase chain reaction (PCR) is an advanced molecular technology, but it is difficult to develop specific primer sets. The method of next-generation sequencing has several limitations, such as inaccurate sequencing of homopolymer regions, as well as being time-consuming, and requiring special knowledge for data analysis. This study evaluated the potential of the DNA-sequence-based method to identify tree species and to quantify the relative proportion of each species in mixed fine root samples. We discriminated the species by isolating DNA from individual fine roots and amplifying the plastid trnL(UAA; i.e., tRNA-Leu-UAA) intron using the PCR. To estimate relative proportions, we extracted DNA from fine root mixtures. After the plastid trnL(UAA) intron amplification and TA-cloning, we sequenced the positive clones from each mixture. Our results indicated that the plastid trnL(UAA) intron spacer successfully distinguished tree species of fine roots in subtropical forests. In addition, the DNA-sequence-based approach could reliably estimate the relative proportion of each species in mixed fine root samples. To our knowledge, this is the first time that the DNA-sequence-based method has been used to quantify tree species proportions in mixed fine root samples in Chinese subtropical forests. As the cost of DNA-sequencing declines and DNA-sequence-based methods improve, the molecular method will be more widely used to determine fine root species and abundance.

Highlights

Fine roots (≤2 mm in diameter) are the primary organ used by plants to acquire soil water and nutrients for growth (Jackson et al, 1997; de Kroon, 2007)
We found that eight species (C. japonica, C. lanceolata, P. massoniana, C. axillaries, L. formosana, A. hainanensis, L. chinense, and S. bogotensis) were consistent with their corresponding species names in NCBI
For the fine root mixtures of two species, we found a good fitness between the predicted relative proportion based on the DNA sequence method and the actual fresh weight proportion in the mixed samples (r2 = 0.8157, p < 0.0001 for L. formosana and r2 = 0.8157, p < 0.0001 for C. axillaris; Figure 5)

Summary

Introduction

Fine roots (≤2 mm in diameter) are the primary organ used by plants to acquire soil water and nutrients for growth (Jackson et al, 1997; de Kroon, 2007). Fine root species composition quantification ecosystem production and the nutrient cycle in forests (Silver et al, 2005; Børja et al, 2008). Fine root turnover contributes considerably to net primary productivity (NPP) in forest ecosystems (Gill and Jackson, 2000). The foremost challenge to understand belowground functions in forests is the ability to identify the fine root tree species. In subtropical areas, natural forests have more diverse tree species whose roots intermingle and intertwine so that the fine roots are more difficult to identify

Objectives

Methods

Results

Discussion

Conclusion