title: A molecular method to identify species of fine roots and to predict the proportion of a species in mixed samples in subtropical forests.
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.
authors.affiliation: Faculty of Life Science and Technology, Central South University of Forestry and Technology Changsha, China ; National Engineering Laboratory of Applied Technology for Forestry and Ecology in Southern China Changsha, China.
authors.forename: Weixian
authors.initials: W
authors.lastname: Zeng
authors.affiliation: Faculty of Life Science and Technology, Central South University of Forestry and Technology Changsha, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhou
authors.affiliation: Faculty of Life Science and Technology, Central South University of Forestry and Technology Changsha, China ; National Engineering Laboratory of Applied Technology for Forestry and Ecology in Southern China Changsha, China.
authors.forename: Pifeng
authors.initials: P
authors.lastname: Lei
authors.affiliation: Faculty of Life Science and Technology, Central South University of Forestry and Technology Changsha, China ; Huitong National Field Station for Scientific Observation and Research of Chinese Fir Plantation Ecosystem in Hunan Province Huitong, China.
authors.forename: Yeling
authors.initials: Y
authors.lastname: Zeng
authors.affiliation: Faculty of Life Science and Technology, Central South University of Forestry and Technology Changsha, China ; National Engineering Laboratory of Applied Technology for Forestry and Ecology in Southern China Changsha, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Faculty of Life Science and Technology, Central South University of Forestry and Technology Changsha, China ; National Engineering Laboratory of Applied Technology for Forestry and Ecology in Southern China Changsha, China.
authors.forename: Cong
authors.initials: C
authors.lastname: Liu
authors.affiliation: Faculty of Life Science and Technology, Central South University of Forestry and Technology Changsha, China ; Huitong National Field Station for Scientific Observation and Research of Chinese Fir Plantation Ecosystem in Hunan Province Huitong, China.
authors.forename: Wenhua
authors.initials: W
authors.lastname: Xiang
keywords: DNA-sequence-based method; PCR; belowground species abundance; fine root; intron; subtropical forest; trnL(UAA)