PCR primers to amplify and sequence rpb2 (RNA polymerase II second largest subunit)

in the Basidiomycota (Fungi)

P. Brandon Matheny, Biology Department, Clark University

Hibbett Lab


27 December 2006

We perform three separate PCR amplifications to obtain rpb2 3-11 products (ca. 2800 bp) or two PCR amplifications to get rpb2 5-11 products (ca. 2100 bp) in our studies of basidiomycete phylogeny (Matheny et al. 2007a; 2007b). For the study of closely related species, sequences of the most variable region of rpb2 between domains 6 and 7 have proven sufficient (Frøslev et al. 2005; Matheny 2005). In our study on the Agaricales, we utiltized 1100 bp between domains 5 and 7 (Matheny et al. 2006).

The following PCR primer pairs are recommended. Primer sequences are provided at the end of this document.

rpb2 3-11 products

1. b3.1F and b6R2

2. f5F and b7.1R or b7R2 or b7R

3. b6.9F and b11R1 or g11bR

rpb2 5-11 products

1. f5F and b7.1R or b7R2 or b7R

2. b6.9F and b11R1 or g11bR

Primer nomenclature is as follows: b and f refer to "basidiomycete" and "fungal" specific, respectively. The g refers to a "general" primer and is not fungal specific. A number, for example, 5, refers to the conserved domain in which the primer sequence is located. Numbers of domains increase consecutively going downstream in the 3¢ direction. Uppercase F and R refer to "forward" and "reverse".

Below is a general schematic that maps rpb2 pimer positions of primers we most frequently use. Thick bars represent exons and thin lines represent introns.

Presence or absence of introns varies depending on the group being studied but is generally conserved across the Basidiomycota. Several exceptions are noteworthy, however. For example, the Agaricales, Boletales, Ustilago maydis (corn smut), and Cryptococcus neoformans do no possess intron 2. Ustilago, Malassezia (a yeast relative of Ustilago), and Cryptoccocus also lack intron 3. Exemplars of the Trichosporonales share a unique intron with varying phase insertions in domain 6, which dispruts the b6F primer binding site. In addition, Ustilago, Rhodotorula hordea (a yeast relative of rusts), Coniophora arida (Boletales), and Megacollybia platyphylla (Agaricales) lack intron 4. Furthermore, Ustilago and additional smut fungi, as well as the unrelated Rhodotorula hordea possess uniquely placed introns downstream of the g7F primer site. Our data stem primarily from the 5 to 11 region of rpb2, so distribution of introns 1 and 2 across the Basidiomycota is still not well known. The rust Phragmidium however, shares a similar rpb2 gene structure with several major homobasidiomycete lineages (Cantharellales, Phallomycetidae, Hymenochaetales, Russulales, and Polyporales, for example). See the study by Matheny et al. (2007b) for additional details on intron distribution and intron evolution in the Basidiomycota.

Successful PCR programs of varying stringency used to amplify rpb2 fragments:

(1) 95.0 C for 4 min

(2) 95.0 C for 1 min

(3) 50.0 C for 1 min

(4) ramp 0.3 C per sec to 72 C

(5) 72.0 C for 1 min

(6) 34 times to step 2

(7) 72 for 10 min

(8) 15.0 C for 0:00

(9) END

(1) 95.0 for 1 min

(2) 95.0 C for 1 min

(3) 55.0 C for 1 min

(4) 1 min 72.0 C + 1 sec/cycle

(5) 34 times to step 2

(6) 72.0 C for 10 min

(7) 15.0 C for 0:00

(8) END

This overview of an rpb2 5-11 project file for Ampulloclitocybe clavipes (Agaricales) shows the degree of PCR and internal sequencing primer overlap. Blue boxes represent introns 3 and 4.


rpb2 PCR and sequencing primers

Primer Name

Sequence 5¢ — 3¢

Amino acid sequence






Matheny et al. (2007b)




Liu, Whelen, & Hall (1999)




Liu, Whelen, & Hall (1999)





Matheny (2005)




Liu, Whelen, & Hall (1999)




Matheny et al. (2007b)




Liu, Whelen, & Hall (1999)




Matheny et al. (2007b)




Liu, Whelen, & Hall (1999)





Matheny (2005)




Liu, Whelen, & Hall (1999)




Liu, Whelen, & Hall (1999)




Matheny et al. (2007b)





Matheny (2005)




Matheny et al. (2007b)





Matheny et al. (2007b)




Matheny et al. (2007b)




Liu, Whelen, & Hall (1999)




Liu, Whelen, & Hall (1999)

References for rpb2 used in 32 fungal phylogenetic studies to date.


Liu YL, Whelen S, Hall BD (1999). Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799-1808.


Lumbsch HT (2000). Phylogeny of filamentous ascomycetes. Naturwissenschaften 87: 335-342.


Zhang N, Blackwell M (2001). Molecular phylogeny of dogwood anthracnose fungus (Discula destructiva) and the Diaporthales. Mycologia 93: 355-365.


Zhang N, Blackwell M (2002). Molecular phylogeny of Melanospora and similar pyrenomycetous fungi. Mycological Research 106: 148-155.


Chaverri P, Castlebury LA, Overton BE, Samuels GJ (2003). Hypocrea/Trichoderma: species with conidiophore elongation and green conidia. Mycologia 95: 1100-1140.

Chaverri P, Samules GJ (2003). Hypocrea/Trichoderma (Ascomycota, Hyprocreales, Hyprocreaceae): species with green ascospores. Studies in Mycology 48: 1-116.

Matheny PB, Ammirati JF (2003). Inocybe angustispora, I. taedophila, and Cortinarius aureifolius: an unusual inocyboid Cortinarius. Mycotaxon 88: 401-407.


Desjardin DE, Wang Z, Binder M, Hibbett DS (2004). Sparassis cystidiosa sp. nov. from Thailand is described using morphological and molecular data. Mycologia 96: 1010-1014.

Diezmann S, Cox CJ, Schonian G, Vilgalys R, Mitchell TG (2004). Phylogeny and evolution of medical species of Candida and related taxa: a multigenic analysis. Journal of Clinical Microbiology 42: 5624-5635.

Liu YL, Hall BD (2004). Body plan evolution of ascomycetes, as inferred from an RNA polymerase II phylogeny. Proceedings of the National Academy of Sciences, USA 101: 4507-4512.

Lutzoni F, et al. (44 authors) (2004). Assembling the fungal tree of life: progress, classification, and evolution of subcellular traits. American Journal of Botany 91: 1446-1480.

Matheny PB, Watling R (2004). A new and unusual species of Inocybe (Inosperma clade) from tropical Africa. Mycotaxon 89: 497-503.

Miller AN, Huhndorf SM (2004). Using phylogenetic species recognition to delimit species boundaries within Lasiosphaeria. Mycologia 96: 1106-1127.

Reeb V, Lutzoni F, Roux C (2004). Contribution of RPB2 to multilocus phylogenetic studies of the euascomycetes (Pezizomycotina, Fungi) with special emphasis on the lichen-forming Acarosporaceae and evolution of polyspory. Molecular Phylogenetics and Evolution 32: 1036-1060.

Wang Z, Binder M, Dai YC, Hibbett DS (2004). Phylogenetic relationships of Sparassis inferred from nuclear and mitochondrial ribosomal DNA and RNA polymerase sequences. Mycologia 96: 1015-1029.

Zhong Z, Pfister DH (2004). Phylogenetic relationships among species of Leotia (Leotiales) based on ITS and RPB2 sequences. Mycological Progress 3: 237-246.


Frøslev TG, Matheny PB, Hibbett DS (2005). Lower level relationships in the mushroom genus Cortinarius (Basidiomycota, Agaricales): a comparison of RPB1, RPB2, and ITS phylogenies. Molecular Phylogenetics and Evolution 37: 602-618.

Hansen K, LoBuglio KF, Pfister DH (2005). Evolutionary relationships of the cup-fungus genus Peziza and Pezizaceae inferred from multiple nuclear genes: RPB2, b -tubulin, and LSU rDNA. Molecular Phylogenetics and Evolution 36: 1-23.

Matheny PB (2005). Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences (Inocybe, Agaricales). Molecular Phylogenetics and Evolution 35: 1-20.

Miller AN, Huhndorf SM (2005). Multi-gene phylogenies indicate ascomal wall morphology is a better predictor of phylogenetic relationships than ascospore morphology in the Sordariales (Ascomycota, Fungi). Molecular Phylogenetics and Evolution 35: 60-75.

Staats M, van Baarlen P, va Kan JAL (2005). Molecular phylogeny of the plant pathogenic genus Botrytis and the evolution of host specificity. Molecular Biology and Evolution 22: 333-346.


De Fine Licht HH, Boomsma JJ, Aanen DK (2006). Presumptive horizontal symbiont transmission in the fungus-growing termite Macrotermes natalensis. Molecular Ecology 15: 3131-3138.

James TY, et al. (70 authors) (2006). Reconstructing the early evolution of the fungi using a six gene phylogeny. Nature 443: 818-822.

Liu YL, Hodson MC, Hall BD (2006). Loss of the flagellum happened only once in the fungal lineage: phylogenetic structure of kingdom Fungi inferred from RNA polymerase II subunit genes. BMC Evolutionary Biology 6: 74.

Malkus A, Linda Chang PF, Zuzga SM, Chung K, Shao J, Cunfer BM, Arseniuk E, Ueng PP (2006). RNA polymerase II gene (RPB2) encoding the second largest protein subunit in Phaeosphaeria nodorum and P. avenaria. Mycological Research 110: 1152-1164.

Matheny PB, Bougher NL (2006). The new genus Auritella from Africa and Australia (Inocybaceae, Agaricales): molecular systematics, taxonomy and historical biogeography. Mycological Progress 5: 2-17.

Matheny PB, et al. (25 authors) (2006). Major clades of Agaricales: a multi-locus phylogenetic overview. Mycologia 98: in press.

Moncalvo JM, et al. (16 authors) (2006). The cantharelloid clade: dealing with incongruent gene trees and phylogenetic reconstruction methods. Mycologia 98: in press.

Tang AM, Jeewon R, Hyde KD (2006). Phylogenetic utility of protein (RPB2, beta-tubulin) and ribosomal (LSU, SSU) gene sequences in the systematics of Sordariomycetes (Ascomycota, Fungi). Antonie Van Leeuwenhoek: in press.


Hofstetter V, Miadlikowska J, Kauff F, Lutzoni F (2007). Phylogenetic comparison of protein-coding versus ribosomal RNA-coding sequence data: a case study of the Lecanoromycetes (Ascomycota). Molecular Phylogenetics and Evolution: in press.

Matheny PB, Gossmann JA, Zalar P, Arun Kumar TK, Hibbett DS (2007a). Resolving the phylogenetic position of the Wallemiomycetes: an enigmatic major lineage of Basidiomycota. Canadian Journal of Botany: in press.

Matheny PB, et al. (24 authors) (2007b). Contributions of rpb2 and tef1 to the phylogeny of mushrooms and allies (Basidiomycota, Fungi). Molecular Phylogenetics and Evolution: in press.