β-tubulin paralogue <I>tubC</I> is frequently misidentified as the <I>benA</I> gene in <I>Aspergillus</I> section <I>Nigri</I> taxonomy: primer specificity testing and taxonomic consequences

Abstract

β-tubulin (benA, tub-2) and calmodulin (caM) are crucial genes in the taxonomy of Aspergillus section Nigri. Widely used β-tubulin primers are not specific for the benA gene for some taxa and preferentially amplify the tubC paralogue. Sequences of the tubC paralogue are widely combined with benA sequences in recent taxonomical works as well as other works, resulting in incongruent trees. In this study we newly provide benA sequences for several ex-type strains, which were characterised using the tubC gene only. We designed a highly specific forward primer to benA designated Ben2f for use in Aspergillus section Nigri, and tested specificity of numerous primer combinations to β-tubulin paralogs. The primer pairs with the highest specificity to the benA gene and functional across species in section Nigri includes Ben2f/Bt2b, Ben2f/T22 and T10/T22. We also provide tools based on codon usage bias analysis that reliably distinguish both paralogues. Exon/intron arrangement is the next distinctive characteristic, although this tool is not valid outside section Nigri. The species identity of taxa from the A. aculeatus clade used in previous molecular studies was revised using combined molecular data (ITS, benA, caM). These data together with two different PCR-fingerprinting methods indicated that A. japonicus should be treated as a synonym of A. violaceofuscus. Similarly, A. fijiensis is reduced to synonymy with A. brunneoviolaceus.

Two novel species of Aspergillus section Nigri from indoor air

“…A. fijiensis ) was previously isolated from soil, Fiji (CBS 313.89), Lactuca sativa , Indonesia (CBS 119.49) (Varga et al 2011), guano, Peru (IHEM 18675), corneal scraping keratitis, India (IHEM 22812), droppings of Coenobita sp., Bahamas (IHEM 4062) (Hendricks at al . 2011), and industrial material, China (CCF 108) (Hubka & Kolarik 2012). This is the first report of A. brunneoviolaceus isolated from the indoor air environment and the first reported isolation in the United States.…”

Talaromyces columbinus sp. nov., and Genealogical Concordance Analysis in Talaromyces Clade 2a

“…Initial results indicated that the BT2 primers (BT2a, BT2b) were amplifying paralogous genes. The primers (BT2f and T22) and procedures of Hubka and Kolarik [22], developed to resolve this problem in Aspergillus japonicus, were used to conduct repeated amplification and sequencing of the suspect products. This procedure also produced the paralogous gene products.…”

Notes on some Phytopythium and Pythium species occurring in oak forests in southern Poland

“…Nuclear ITS rDNA region internal transcribed spacers (ITS1 and ITS2) and 5.8S subunits were amplified with primer set ITS5/ITS4 [18]. The reaction mixtures and conventional PCR protocols were the same as in Hubka and Kolarik [19]. Custom purification of PCR amplicons and sequencing was conducted at Macrogen Inc. (Seoul, South Korea) using the same primers.…”