Positioning of introns in different laccase genes, a relevant tool for solving phylogenetic position ambiguity of Volvariella volvacea laccase genes

Volvariella volvacea (paddy straw mushroom) is a high temperature-loving mushroom with the shortest cropping cycle in the basidiomycete family Pluteaceae. This mushroom is cultivated extensively in many South-East Asian and African countries by employing both the outdoor and indoor cultivation techniques. The objective of this study is to analyze intron positions in different laccase genes of this mushroom. Primer pairs for laccase 1, 2, 3, 4, 5 and 6 were designed by using the mRNA sequences of these genes available in NCBI nucleotide database and the Primer 3 free software. These primers were used to amplify different laccase genes in one heterozygotic strain, Vv-01 and one putative homozygotic strain, BBSR-003 at an annealing temperature range of 53 to 58°C to obtain amplicons of requisite sizes. PCR products were then sequenced and annotated using the BioEdit and Artemis softwares. Out of 6 laccase genes, partial sequences of laccase 1, 2, 3 and 6 in the heterozygotic strain Vv-01 and partial sequence of laccase 3 in homozygotic strain BBSR-003 were obtained. The lcc 3 sequences of two strains, Vv-01 and BBSR-003 have been submitted to NCBI GenBank with accession numbers HQ687205 and JF313903, respectively and they showed differences at 10 different base positions. In lcc 3, there was an intron 8 found earlier in laccase genes from Coprinopsis cinerea, but same has not been spliced in published mRNA sequence of V. volvacea. This intron has to be spliced in strain Vv-01 to give a functional protein and by virtue of which, the phylogenetic position of lcc3 in laccase dendrograms is probably wrong. In lcc 1, 16 different introns have been recorded and the amino acid sequence of Vv-01 strain differs by one amino acid less than the published sequence of lcc1. No evidence of heterozygocity has been recorded in this sequence. The lcc 2 sequence also revealed 16 different introns. Although only a small portion of sequence could be validated but evidence for heterozygocity was found in strain Vv-01. The small sequence of lcc 2 in strain BBSR-003 also differed at some bases from strain Vv-01. At the protein sequence level, 26 amino acids of the published mRNA seemed to be derived from the wrong reading frame. This could be due to errors in sequencing, or that the strain used had a pseudo gene or a very original allele for lcc 2. Based on these observations, the position of laccase 2 in the dendrogram is likely to be changed. The sequence of lcc 6 helped in identifying 5 introns. The positions of introns in different laccase genes (lcc 1, lcc 2 and lcc 3) of V. volvacea grouped them to sub-family A .