Abstract
BackgroundThe specific associations between plant roots and the soil microbial community are key to understanding nutrient cycling in grasslands, but grass roots can be difficult to identify using morphology alone. A molecular technique to identify plant species from root DNA would greatly facilitate investigations of the root rhizosphere.ResultsWe show that trnL PCR product length heterogeneity and a maximum of two restriction digests can separate 14 common grassland species. The RFLP key was used to identify root fragments at least to genus level in a field study of upland grassland community diversity. Roots which could not be matched to known types were putatively identified by comparison of the nuclear ribosomal ITS sequence to the GenBank database. Ten taxa were identified among almost 600 root fragments. Additionally, we have employed capillary electrophoresis of fluorescent trnL PCR products (fluorescent fragment length polymorphism, FFLP) to discriminate all taxa identified at the field site.ConclusionWe have developed a molecular database for the identification of some common grassland species based on PCR-RFLP of the plastid transfer RNA leucine (trnL) UAA gene intron. This technique will allow fine-scale studies of the rhizosphere, where root identification by morphology is unrealistic and high throughput is desirable.
Highlights
The specific associations between plant roots and the soil microbial community are key to understanding nutrient cycling in grasslands, but grass roots can be difficult to identify using morphology alone
To understand nutrient cycling in an upland grassland we are studying the spatial relationship between co-occurring plant roots and their associated microbes, both endomycorrhizal fungi and rhizoplane bacteria
Our aims were firstly to extend the molecular data for grasses so that we could identify, to species level, small (< 1 cm) root fragments collected from soil cores sampled from an upland grassland, and secondly, to develop a high-throughput method of plant root identification utilising capillary electrophoresis of fluorescently labelled PCR products
Summary
The specific associations between plant roots and the soil microbial community are key to understanding nutrient cycling in grasslands, but grass roots can be difficult to identify using morphology alone. A molecular technique to identify plant species from root DNA would greatly facilitate investigations of the root rhizosphere. Studies of of below-ground processes are currently hindered by the inability to identify fine roots in soil [1]. To understand nutrient cycling in an upland grassland we are studying the spatial relationship between co-occurring plant roots and their associated microbes, both endomycorrhizal fungi and rhizoplane bacteria. A molecular method for identification must satisfy requirements for specific amplification of plant DNA and reveal enough variability to distinguish species. The trnL UAA intron from plastid DNA shows variability comparable to that of nuclear intergenic regions and can resolve taxa at the genus/species level in RFLP-based studies[2]. Sequence variation in the trnL (page number not for citation purposes)
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