ITS and LSU sequences, phylogenetic trees

Russula kashmiriana sp. nov (subg. Tenellula, sect. Laricinae) has been collected and described from Himalayan coniferous forest of Azad Jammu and Kashmir (AJ&K), Pakistan. The taxon is characterized by bright reddish orange pileus with cracked margins and obvious reddish orange circle on the pileus, yellow context and light olive to yellowish amyloid basidiospores. Micro and macroscopic features along with molecular phylogenetic analysis based on large subunit (LSU) and internal transcribed spacer (ITS) sequences data confirmed the status of Russula kashmiriana as a distinct species. AJ&K, LSU, Phylogeny, Russulaceae.

Methods
DNA was extracted from gills of dried fruiting bodies by modified CTAB method and the rDNA ITS region was amplified using universal primer pair ITS1F and ITS 4 (Gardes and Bruns, 1993; Bruns, 1995). The LSU regions were amplified by using LR0R and LR5 primers (Ge et al., 2014). The PCR products was sequenced using the same primers (Macrogen, Korea).

To generate consensus sequences of both LSU and ITS regions BioEdit version 7.2.5 (Hall, 1999) was used. Nucleotide sequence comparisons were performed with Basic Local Alignment Search Tool (BLAST) network services using National Center for Biotechnology Information (NCBI), USA database (http://www.ncbi.nlm.nih.gov/). For phylogenetic tree construction and alignment of sequences, closely related sequences were retrieved from GenBank. Published sequences and data from the literature were also added in final data set. An online software MUSCLE was used for alignment purpose (http://www.ebi.ac.uk/Tools/msa/muscle/). Phylogenetic analyses were performed in MEGA6.