Diversity of Frankia Populations in Root Nodules of Geographically Isolated Arizona Alder Trees in Central Arizona (United States)

Abstract

The diversity of uncultured Frankia populations in root nodules of Alnus oblongifolia trees geographically isolated on mountaintops of central Arizona was analyzed by comparative sequence analyses of nifH gene fragments. Sequences were retrieved from Frankia populations in nodules of four trees from each of three mountaintops (n ‫؍‬ 162) and their levels of diversity compared using spatial genetic clustering methods and single-nucleotide or 1, 3, or 5% sequence divergence thresholds. With the single-nucleotide threshold level, 45 different sequences with significant differences between the mountaintops were retrieved, with the southern site partitioning in a separate population from the two other sites. Some of these sequences were identical in nodules from different mountaintops and to those of strains isolated from around the world. A high level of diversity that resulted in the assignment of 14 clusters of sequences was also found on the 1% divergence level. Single-nucleotide and 1% divergence levels thus demonstrate microdiversity of frankiae in root nodules of A. oblongifolia trees and suggest a partitioning of diversity by site. At the 3 and 5% divergence levels, however, diversity was reduced to three clusters or one cluster, respectively, with no differentiation by mountaintop. Only at the 5% threshold level do all Frankia strains previously assigned to one genomic group cluster together.

Diversity of Frankia in root nodules of six Morella sp. from the Cape flora of South Africa

“…Cluster I sequences displayed low local diversity, a finding often encountered in studies of this host infection group (Huguet et al 2001;Welsh et al 2009b;Kennedy et al 2010). Cluster I strains are typically associated with specific hosts, as demonstrated in sympatric Alnus stands, and where Alnus and Myrica gale grow together (Huguet et al 2001;Pokharel et al 2011).…”

Frankia bacteria in Alnus rubra forests: genetic diversity and determinants of assemblage structure

“…Total genomic DNA was extracted from individual root nodules using the DNeasy® Blood and Tissue Kit (Qiagen, Carlsbad, CA). A 606 bp nifH gene fragment was amplified with the Frankia-specific primers nifHf 1 and nifHr (Welsh et al 2009a). For samples that initially did not amplify, PCR reactions were rerun at 1:20, 1:100, or 1:1000 dilutions of DNA extract.…”

“…For this dataset, 1%, 3%, and 5% cutoffs resulted 15, 5, and 3 genotypes, respectively. The cutoff of 3% was used in the analyses below because this level of similarity has been shown to place Frankia genotypes into their proper genomic groups (Mirza et al 2009;Welsh et al 2009a). Representatives of all non-identical sequences for each genotype were submitted to GenBank with the accession numbers GU810473-GU810477 and HM031938-HM031960.…”

“…A second more detailed analysis was conducted to examine the relationships between the A. rubra genotypes and representatives of the five Alnus sub-groups designated by Welsh et al (2009a). For that analysis, all non-identical sequences of each A. rubra Frankia genotype, three divergent representatives of each Alnus sub-group, and two additional sequences (one from a Frankia strain isolated from A. rubra and one from the genome sequence of Frankia ACN14a) were aligned using MUSCLE (Edgar 2004), along with an outgroup sequence from a Datisca cannabina Frankia nodule.…”

“…Previous research on the A. rubra-Frankia symbiosis has examined various aspects of nodulation potential (Markham and Chanway 1996;Martin et al 2003a;, seedling growth (Martin et al 2003b;Markham 2008), spore type distribution (Markham and Chanway 1998) and nitrogen fixation (Markham and Chanway 1999;Rojas et al 2002). Little work has, however, examined the diversity of Frankia genotypes in A. rubra forests using molecular methods, which typically capture greater genotypic diversity than culture and spore type characterization methods (Welsh et al 2009a). In a recent study, found only two Frankia genotypes associated with A. rubra in Olympic National Rainforest, WA, USA.…”