DNA barcoding of arbuscular mycorrhizal fungi

Plant beneficial microorganisms, such as arbuscular mycorrhiza
fungi (AMF), increasingly attract scientific and agronomic
attention due to their capacity to increase nutrient accessibility
for plants and to reduce inorganic fertilizer requirements. AMF are
thought to form symbioses with most land plants, obtaining carbon
from the autotrophic host whilst enhancing uptake of poorly
available nutrients. The species of AMF are mainly identified by
spore morphology, which is time consuming, requires expertise and
is rarely applicable to AMF identification in roots. Molecular
tools such as analysis of standardized DNA fragment sequences may
allow the recognition of species through a ‘DNA barcode’, which may
partly overcome this problem. The focus of this study was to
evaluate different regions of widely used rDNA repeats for their
use as DNA barcodes for AMF including the small subunit rRNA gene
(SSU), the internal transcribed spacer (ITS) and the large subunit
rRNA gene (LSU). Closely related species in the genus Ambispora,
members of which have dimorphic spores, could not be separated by
analysis of the SSU region, but of the ITS region. Consequently,
the SSU was not used for subsequent analysis, but a DNA fragment
covering a small part of the SSU, the entire ITS region and about
800 bp of the LSU (SSUmCf-LSUmBr fragment) was analysed, providing
phylogenetic resolution to species. New AMF specific primers for
these potential barcoding regions were developed and can be
applied, without amplification of non-target organisms, for AMF
species determination, including identification from field and root
samples. Analyses based on the application of the SSUmCf-LSUmBr
fragment showed that the widely used AMF model organism Glomus sp.
DAOM197198 (formerly called Glomus intraradices) is not conspecific
with Gl. intraradices. The SSUmCf-LSUmBr fragment clearly provides
a much higher species resolution capacity when compared with the
formerly preferred ITS and LSU regions. Further study of several
groups of AMF species using different regions of the SSUmCf-LSUmBr
fragment revealed that only the complete SSUmCf-LSUmBr fragment
allowed separation of all analysed species. Based on these results,
an extended DNA barcode covering the ITS region and parts of the
LSU region is suggested as a DNA barcode for AMF. The complete
SSUmCf-LSUmBr fragment sequences can serve as a database backbone
for also using smaller rDNA fragments as barcodes. Although the
smallest fragment (approximately 400 bp) analysed in this study was
not able to discriminate among AMF species completely, such short
regions covering the ITS2 or LSU D2 regions, respectively, would
most likely be suitable for community analyses with 454 GS-FLX
Titanium sequencing, providing that the analyses is based on the
longer DNA sequences.