Escaping introns in COI through cDNA barcoding of mushrooms: Pleurotus as a test case.

Farhat A Avin,Thomas W A Braukmann,Paul D N Hebert,Sabaratnam Vikineswary,Yee‐Shin Tan,Bhassu Subha

doi:10.1002/ece3.3049

Abstract

DNA barcoding involves the use of one or more short, standardized DNA fragments for the rapid identification of species. A 648‐bp segment near the 5′ terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the universal DNA barcode for members of the animal kingdom, but its utility in mushrooms is complicated by the frequent occurrence of large introns. As a consequence, ITS has been adopted as the standard DNA barcode marker for mushrooms despite several shortcomings. This study employed newly designed primers coupled with cDNA analysis to examine COI sequence diversity in six species of Pleurotus and compared these results with those for ITS. The ability of the COI gene to discriminate six species of Pleurotus, the commonly cultivated oyster mushroom, was examined by analysis of cDNA. The amplification success, sequence variation within and among species, and the ability to design effective primers was tested. We compared ITS sequences to their COI cDNA counterparts for all isolates. ITS discriminated between all six species, but some sequence results were uninterpretable, because of length variation among ITS copies. By comparison, a complete COI sequences were recovered from all but three individuals of Pleurotus giganteus where only the 5′ region was obtained. The COI sequences permitted the resolution of all species when partial data was excluded for P. giganteus. Our results suggest that COI can be a useful barcode marker for mushrooms when cDNA analysis is adopted, permitting identifications in cases where ITS cannot be recovered or where it offers higher resolution when fresh tissue is. The suitability of this approach remains to be confirmed for other mushrooms.

Highlights

DNA barcoding employs short, standardized DNA fragments for the rapid identification of species (Gilmore, Graefenhan, Louis Seize, & Seifert, 2009; Hebert, Cywinska, & Ball, 2003; Nguyen & Seifert, 2008; Vialle et al, 2009)
A 648-bp segment near the 5′ terminus of the mitochondrial cytochrome c oxidase subunit I (COI) gene has been adopted as the DNA barcode region for animals because its performance in species discrimination is high and it is usually easy to recover (Hebert et al, 2003)
A multi-locus barcode approach has been adopted to improve resolution across plants and fungi (Hollingsworth et al, 2009; James et al, 2006), and ITS has been adopted as the standard barcode region for fungi (Avin, Bhassu, Shin, & Sabaratnam, 2012; Begerow, Nilsson, Unterseher, & Maier, 2010; Schoch, Seifert, Huhndorf, et al, 2012; Seifert, 2009) studies have shown that this gene region often fails to distinguish closely related fungal species (Schoch, Seifert, Caldeira, et al, 2012)

Summary

| INTRODUCTION

These introns are often long, leading to extreme variation in length of the COI gene from approximately 1,584 bp in species lacking introns to over 22 kb in those with many introns (Férandon et al, 2010; Gonzalez et al, 1998; Haridas & Gantt, 2010; Wang et al, 2008) The presence of these introns impedes sequence recovery by conventional PCR (Seifert, 2009; Seifert et al, 2007), a factor which has supported the adoption of ITS as the sole DNA barcode for mushrooms (Schoch & Seifert, 2012; Vialle et al, 2009).

| MATERIALS AND METHODS

| DISCUSSION

Findings

DATA ACCESSIBILITY