Properties of group-specific primers and their coverage for phylogenetic and environmental groupings in the nifH database.

aData indicate primer binding to all nifH sequences in the database with 0, 1, and 2 mismatches allowed. In some cases highly degenerate primers bind to multiple positions in the sequence generating coverage values that exceed 100%.bData indicate primer binding to specific groupings in the nifH phylogeny. Abbreviations are as follows: Alpha-, Beta-, and Gammaproteobacteria (Pr); Cyanobacteria (Cy); Cluster III (III); Cluster IA (IA); Paenibacillus (Pb); Frankia (Fr); Epsilonproteobacteria Containing Cluster (Ep); paralogous sequences in Cluster IV (IV).cPrimer coverage queried against sequences recovered from specific environments (Environ.) as described in methods. Environments include: soils (Soil), microbial mats (Mat), and pelagic marine samples (Sea).dSequences are given in the 5′ to 3′ direction, IUPAC characters are used, and I = Inosine.eAbbreviations indicate the Target Group (Tg.) which the primer was intended to amplify as follows: β-Rhizobia (BR); Frankia (Fr); Alphaproteobacteria (AP); Symbiotic rhizobia (R); reamplification of Cluster I (RA); aerobic and microaerophilic diazotrophs (AM); Cyanobacteria (Cy); Alpha- and Betaproteobacteria (AB); Gammaproteobacteria (GP); alternative nitrogenase cluster (AN); designed to match multiple species of Azospirillum, Burkholderia, Gluconoacetobacter, Azotobacter, Herbaspirillum and Azoarcus (MS); species of the cyanobacterial genus Cylindrospermopsis (Cs); Bradyrhizobium sp. prevalent in truffles (TB).fPosition is relative to A. vinelandii nifH (Genbank ACCN# M20568).gDegeneracy is given as the number of oligonucleotides that comprise the primer.hReferences in which the primers are described.iThis binding position for this primer sequence lies beyond the stop codon of Frankia sp. (Genbank ACCN# M21132) and cannot be represented using the A. vinelandii numbering system.NA: Data not available as described in Methods.