Ann Microbiol DOI 10.1007/s13213-015-1121-2
ORIGINAL ARTICLE
Restriction fragment length polymorphism and sequence analysis of rRNA-ITS region of somatic hybrids produced between Pleurotus florida and Lentinula edodes Pijush Mallick 1 & Samir Ranjan Sikdar 1
Received: 18 July 2014 / Accepted: 12 June 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015
Abstract Nine pfle somatic hybrids and their parental strains Pleurotus florida and Lentinula edodes were characterized by restriction fragment length polymorphism (RFLP) and sequence analysis of ribosomal RNA internal transcribed spacer (rRNA-ITS) regions. The ITS1-5.8S-ITS2 region was amplified by primer pair ITS1 and ITS4. Restriction digestion of the amplified ITS1-5.8S-ITS2 region with AluI, HinfI, HpaII and HaeIII generated a total of 81 fragments in which HaeIII generated the maximum of 26 restriction fragments and HpaII generated the minimum of 14 fragments for all 11 strains studied. All the pfle hybrids and parents were highly polymorphic in fragment sizes as revealed from the RFLP profiles. The pfle hybrids and parental ITS1-5.8S-ITS2 sequences showed length variations (629–742 bp). Maximum sequence similarity was found in the 5.8S rRNA region followed by ITS1, while maximum polymorphism was identified in the ITS2 region. The nucleotide sequences of the highly conserved 5.8S region of all the pfle hybrids were very similar with minor variations while this variation was greater between the parental strains. The dendrogram generated based on rRNAITS sequences of the pfle hybrids and parents by UPGMA method of clustering revealed five distinct groups, where parent P. florida did not group directly with any of the hybrid Electronic supplementary material The online version of this article (doi:10.1007/s13213-015-1121-2) contains supplementary material, which is available to authorized users. * Samir Ranjan Sikdar
[email protected] Pijush Mallick
[email protected] 1
Division of Plant Biology, Bose Institute, P-1/12, C.I.T. Scheme VII M, Kolkata 700 054, India
strains. The cluster of pfle 6w and pfle 6z remained as separate group in the dendrogram, having no intra cluster linkage. Keywords Lentinula edodes . Pleurotus florida . Polymorphism . RFLP . rRNA-ITS . Somatic hybrid
Introduction Development of efficient selection strategies for recovery of fungal somatic hybrids and methods for their characterization would improve the chances of obtaining desirable hybrid progenies. Protoplast fusion between edible mushroom strains was developed successfully into somatic hybrids, and hybridity was proved by various molecular markers like isozyme, random amplified polymorphic DNA (RAPD) (Chakraborty and Sikdar 2008, 2010), inter simple sequence repeat (ISSR) and genome-specific inter simple sequence repeat (gs-ISSR) (Mallick and Sikdar 2014). Sequencing of the internal transcribed spacer (ITS) of ribosomal RNA (rRNA) can be a useful technique in determining the interspecies (Jorgenson and Cluster 1988; Cullings et al. 1996; Vogler and Bruns 1998) and sometimes intraspecies (Baura et al. 1992) relationships. Nuclear ribosomal DNA represents a high proportion of DNA repeats in whole genomes and has been used for characterization of somatic hybrids (Babiychuk et al. 1992; Lelivelt et al. 1993; Sakata and Monma 1993; Saito et al. 1994; Zanke et al. 1995). Restriction digestion of the rRNA gene has been used in the past to differentiate Saccharomyces cultures (Valente et al. 1996). ITS regions exhibit greater sequence divergence than their flanking coding regions (Hillis and Dixon 1991) and are often used to distinguish related fungal species (Mitchell and Bresinsky 1999; Calvo-Bado et al. 2000; Manian et al. 2001) and to infer phylogenetic relationships from population
Ann Microbiol
to families (O’Donnell 1992; Challen et al. 2003; Callac et al. 2003). Restriction fragment length polymorphism (RFLP) analysis by the polymerase chain reaction (PCR) technique couples two procedures; the first detects polymorphism in DNA sequences that have been amplified by specific oligonucleotide primers, and the second produces different restriction patterns by endonucleases that have been successfully used to analyze the rRNA region of ectomycorrhizal fungi (Gardes et al. 1991; Erland et al. 1994; Henrion et al. 1994; Farmer and Sylvia 1998; Gomes et al. 1999; Glen et al. 2001). Strain identification of the edible mushroom Lentinula edodes revealed nucleotide sequence polymorphism in the 5.8S rRNA gene (Kwan et al. 1991). In the past, RFLP of the rRNA gene was used successfully for species classification or to distinguish among various fungal strains (Gardes and Bruns 1996; Korabecna et al. 2003). This method was also used successfully to characterize plant somatic hybrids in Cucumis (Bordas et al. 1998). Although the rRNA gene is a highly conserved sequence, variation often occurs at different species levels during taxonomic studies (Bruns et al. 1991; Hillis and Dixon 1991). In the present study, we characterized mushroom somatic hybrids for the first time using both RFLP and sequence analysis of the nuclear rRNA-ITS region.
and dTTP) and 5 U/L Taq DNA polymerase (Fermentas, St.Leon-Rot, Germany). A negative control (without template DNA) was included in every reaction run. The amplifications were conducted in a DNA thermal cycler (Applied Biosystems 2027, Foster City, CA) by preliminary denaturation of DNA at 95 °C for 4 min followed by 40 cycles of DNA template denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min, initial extension at 72 °C for 1 min 30 s, followed by a final extension at 72 °C for 8 min. Restriction digestion and gel electrophoresis PCR products were purified by sodium acetate precipitation. Each 10 μL purified PCR product was used for restriction digestion according to the supplier’s specification with each of the four restriction enzymes used for the study viz. AluI, HinfI, HpaII and HaeIII (Fermentas). The digested DNA fragments were size-fractionated in 1.5 % agarose (w/v) gel (run in 1× TAE buffer at 80 V for 3 h) pre-stained with ethidium bromide. The gel profiles were visualized under UV transilluminator and data were recorded in a Molecular Analyst Gel Documentation System (Bio-Rad, Hercules, CA). The sizes of the digested fragments were determined by gene ruler 100 bp plus DNA ladder (MBI, Fermentas) used as a standard molecular weight marker. Sequence analysis
Materials and methods Strains and culture conditions Nine pfle somatic hybrids between Pleurotus florida and Lentinula edodes were developed by protoplast fusion in this laboratory (Mallick and Sikdar 2014). The parental strains were obtained from the National Research Centre for Mushroom, Solan, Himachal Pradesh, India. All 11 strains (9 hybrids and 2 parents) were maintained routinely on PDA medium (potato dextrose agar, pH 6.2) at 24 °C. For DNA isolation all the strains were grown in liquid MYG medium (10 g/L malt, 4 g/L yeast extract and 10 g/L glucose, pH 6.2) at 24 °C.
The purified PCR products were sequenced in our institutional core facility using the Big Dye Terminator 3.1 method and nucleotide bases were read by an automated sequencer (Applied Biosystems). All the sequenced data were submitted to the NCBI GenBank database and aligned in CLUSTALW software. Nucleotide polymorphisms were marked by the box-shaded method. Based on ITS1-5.8S-ITS2 sequences, a dendrogram was generated by the unweighted pair-group with mathematic averages (UPGMA) method of clustering and the genetic distances between hybrids and parents were studied.
Results DNA extraction and PCR conditions RFLP analysis Actively growing mycelial tissues of pfle hybrids and parents were used for DNA extraction by the CTAB method (Dellaporta et al. 1983). The primer pair ITS1 (5′ TCCGTA GGTGAACCTGCGG 3′) and ITS4 (5′ TCCTCCGCTTAT TGATATGC 3′) was used for amplification of the ITS15.8S-ITS2 region (expected fragment ~630–750 bp in size; White et al. 1990). The PCR reaction was performed in a total volume of 25 μL reaction mixture, containing 10 ng template DNA, 20 μM of each primer, 10× Taq buffer (with KCl), 25 mM MgCl2, 2 mM dNTPs (all four dGTP, dCTP, dATP
Primer pair ITS1 and ITS4 amplified the ITS1-5.8S rRNAITS2 region of nine pfle hybrid lines and two parents. The amplified fragments ranged from ~630 bp to ~750 bp in the agarose gel (Fig. 1). Poor PCR amplification was noted in hybrids pfle 1q, pfle 1o and pfle 1r with respect to other hybrids and parents. This might be due to a lower pre-existing copy number of rRNA repeat units present in these hybrid genomes and/or to a partial loss of parental genetic material in the cellular environment, resulting in the unavailability of
Ann Microbiol Fig. 1 Region of ribosomal RNA-internal transcribed spacer (rRNA-ITS) amplified by primers ITS1 and ITS4 from the pfle hybrids and parents. Lanes: M Marker (100 bp DNA ruler plus), C control (without DNA), 1 P. florida, 2 pfle 6w, 3 pfle 1p, 4 pfle 1q, 5 pfle 1v, 6 pfle 1 s, 7 pfle 6 s, 8 pfle 6z, 9 pfle 1o, 10 pfle 1r, 11 L. edodes
complementary sequences of the target region with respect to the primers. The double bands in the gel for hybrid pfle 1o can be seen in Fig. 1; this is probably due to this hybrid containing a corresponding pseudogene somewhere in the pfle 1o genome although the amplification is a CDS (coding DNA sequence) of an rRNA gene. The digested PCR products were polymorphic in all the pfle hybrids and parents (Fig. 2, Supplementary Figs. 1, 2). The approximate size of the DNA fragments observed after restriction digestion for all the strains is summarized in Table 1. Four restriction enzymes viz., AluI, HinfI, HpaII
Fig. 2a,b Restriction digestion of nuclear rRNA-ITS region of pfle hybrids and parents. Digestion by restriction enzymes AluI (a) and HinfI (b). Lanes: M Marker (100 bp DNA ruler plus), C control (without DNA), 1 P. florida, 2 pfle 6w, 3 pfle 1p, 4 pfle 1q, 5 pfle 1v, 6 pfle 1 s, 7 pfle 6 s, 8 pfle 6z, 9 pfle 1o, 10 pfle 1r, 11 L. edodes
and HaeIII generated a total of 81 fragments, of which HaeIII showed the maximum (26) and HpaII the minimum (14) number of restriction fragments in all 11 strains studied. The digested fragment size ranged from ~680 bp to ~120 bp. The AluI fragment of
