Only a few plant ITS sequences were amplified using the fungus-specific primer ITS1-F (ranging from 20 to 24 sequences under different stringency conditions). Assessing these sequences using Blast, 20 out of 24 were revealed to be fungal sequences erroneously deposited as algae from an unpublished study (six Liagora species, two

Caulerpa species, Helminthocladia australis, and Ganonema farinosum). There was a sequence deposited as Chorella matching a fungal sequence. The three others were Chlorarachniophyte species that did not match any known fungal sequence. Some of the other primer combinations, including ITS1-ITS2, amplified a high number of plant sequences from different orders. We also AZD1480 molecular weight confirmed that the assumed basidiomycete-specific primer ITS4-B did not amplify any plant sequences even when allowing 3 mismatches. Table 1 Number of plant and fungi ITS sequences amplified in silico from EMBL fungal and plant databases, using the various primer combinations and allowing none to three mismatches. Primer comb. Fungal ITS sequences Plant ITS sequences S63845 in vitro Number of mismatches * 0 1 2 3 0 1 2 3 ITS5-ITS4 5482 5924 6026 6123 500 514 5667 5996 NS7-ITS2 1067 1291 1313 1320 23 190 231 403 ITS3-LR3 2070 2459 2499 2548 51 168 248 300 ITS1-ITS2 17545 19816 25223 25457 1107

17665 18755 19084 ITS1-F-ITS2 2112 4169 4592 4658 20 21 21 24 ITS5-ITS2 7713 8993 9180 9293 94 703 11123 12100 ITS1-ITS4 10013 10610 12488 12656 5783 6740 7500 7620 ITS3-ITS4 18815 21195 21663 22078 415 7829 8583 8852 ITS3-ITS4-B 1269 1673 1811 1863 0 0 0 0 * The number of mismatches allowed Selleck LY2606368 between the primer and the DNA strand reflects the stringency level of the PCR, i.e. strict PCR conditions such as annealing temperature close to or above the recommended Tm will not allow unspecific sequences (including one or more mismatches) to be amplified. Primer mismatches in sequence subsets The selected ITS primers showed large variation in their ability to amplify fungal sequences from the three subsets when allowing different number of

mismatches (Figure 2). All primer pairs amplified at least 90% of the sequences when allowing two or three mismatches, with the exception of ITS4-B (see below). It is noteworthy that the percentages of sequences were quite similar for two and three mismatches, indicating that Tacrolimus (FK506) rather few sequences included three mismatches. Under strict conditions (i.e. allowing no mismatches), the proportion of amplified sequences varied considerably between primer pairs, ranging from 36% for ITS1-F to 81% for ITS5 (Figure 2). Figure 2 Percentage of sequences amplified from each subset using different primer pairs allowing a maximum of 0, 1, 2, or 3 mismatches. Allowing one mismatch increased the proportion of amplified sequences from 36% to 91.6% for the commonly used primer ITS1-F, implying that more than half of the amplified sequences included one mismatch. ITS5 amplified the highest proportion of the sequences when allowing for a single mismatch (97.