A Molecular Method To Identify Species Of Fine Roots And To Predict The Proportion Of A Species In Mixed Samples In Subtropical Forests

Plant roots are a central driver of ecosystem productivity, as plant investments belowground often comprise more than half of total plant biomass. Research on understanding of belowground interactions among tree species and the fine root (< 2mm in diameter) contribution of a species to forest ecosystem production are mostly important. Despite this general observation, almost nothing is known about plant fine root. Because roots of different species are morphologically indistinguishable restricts species identification.In this study, we use the trnL(UAA; i.e., tRNA-Leu-UAA) intron to identify and quantify the subtropical forest fine root speices. In Dashanchong Provincial Forest Park, the 11 tree species selected in this study consisted of three evergreen coniferous species (Cryptomeria japonica, Cunninghamia lanceolata, and Pinus massoniana), three deciduous broadleaved species(Choerospondias axillaris, Liquidambar formosana, and Quercus fabri), and six evergreen broadleaved species (Lithocarpus glaber, Adinandra hainanensis, Cyclobalanopsis glauca, Litsea coreana, Loropetalum chinense, and Symplocos bogotensis). Fine root samples of L. formosana and C. axillaris were mixed to form five different proportions of mixtures (1:0,1:3,1:1,3:1, and 0:1 based on the fresh weight). For the three species mixture, we used the fine root samples of L. formosana, C. axillaris, and C. glauca to form three different proportions of mixtures (1:2:3,2:3:1, and 3:1:2 based on the fresh weight). Through a DNA-sequence-based method, we quantitatively estimate the above mixture ratio of fine root. Field fine root soil samples (with unknown proportion of mixed fine root) were collected in nature forest field using a steel auger. Then all the fine roots were manually sorted out from the soil, through the DNA-sequence-based method for recognition and quantitative estimation of unknown proportion of mixed fine root. The results are presented as follows:(1) The plastid trnL(UAA) intron of the leaves and roots of 12 plant species were sequenced successfully. The sequences of leaf and fine root samples were BLAST searched through NCBI. We found that the sequences of the other four species (L. glaber, C. glauca, Q. fabri, and L. coreana) in this study aligned with different species (i.e., Quercus gilva, Quercus gilva, Quercus serrata, and Cinnamomum insularimontanum, respectively).(2) The DNA-sequence-based method can reliably estimate the ratio of each species in mixed fine root samples. 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). For the fine root mixtures of three species, the predicted relative proportions of each species exhibited a reasonable relationship with the actual proportion of fresh weight in the mixed samples (r2= 0.7012, p=0.0007 for L. formosana, r2= 0.6297, p=0.0021 for C. axillaris and r2= 0.6855,p= 0.0009 for C. glauca).(3) Fine roots in the soil in the field, through DNA-sequence-based method measured results show that the forest biomass above ground and underground part have no much relevant, and the diversity of the underground part is often higher than the ground part of diversity.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 and the first time in the world to directly identify and quantify the unknown proportion of mixed fine root in field forest. 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.