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Thursday, 12 January 2017

rDNA Sequencing

                      PCR-amplification and subsequent sequencing of parts of the ribosomal DNA avoid the main limitations of RFLPs because the whole information of hundreds of nucleotides of the target DNA is used. rDNA sequences may be used for di-agnosis and for phylogenetic analyses (dendrograms) on relationships among fungi. Sequencing is nowadays the most important tool for molecular system-atics and led to taxonomic rearrangements and changes in nomenclature. 

The ITS sequences of a great number of wood fungi are known, e.g., from mycorrhizal fungi like Hebeloma velutipes (Aanen et al. 2001), from para-sites like Armillaria species (Chillali et al. 1998) and Laetiporus sulphureus (Rogers et al. 1999), and from the red streaks producing Trichaptum abietinum (Kauserud and Schumacher 2003). Regarding wood decay fungi, a data set of rDNA-ITS sequences of 18 house-rot fungi is shown in Table 2.8 (Schmidt and Moreth 2002/2003) complemented by the 18S and 28S rDNA sequences of some important species (Moreth and Schmidt 2005). 

Sequences of the ITS region (and the 18S and 28S rDNA) may be used to identify unknown fungal samples through sequence comparison by Basic lo-cal alignment search tool (BLAST) (e.g., www.ncbi.nlm.nih.gov/blast/bast.cgi). BLAST revealed ITS-sequence identity of a "wild" S. lacrymans isolate from the Himalayas with indoor isolates (White et al. 2001), identified misnamed isolates of S. lacrymans (Horisawa et al. 2004), identified Antrodia spp. and Serpula spp. isolations from fruit bodies and wood samples (Hogberg and Land 2004), and confirmed Coniophora puteana isolates (Raberg et al. 2004). Kim et al. (2005) used a part of the 28S rDNA for identification of a number of basidiomycete fungi from playground wood products by BLAST. 
Partial 18S rDNA sequence of Sirococcus conigenus isolated from Norway spruce cankers was used by Lilja et al. (2005) to confirm the identification of the fungus. The whole IGS was sequenced to investigate intraspecific variation of mycorrhizal fungi like Laccaria bicolor (Martin et al. 1999). IGS I sequence analysis was used for Hebeloma cylindrosporum (Guidot et al. 1999) and Xerocomus pru-inatus (Haese and Rothe 2003). 
IGS I analysis suggested that three different morphotypes/genotypes of an ectomycorrhizal fungus present in Kenya repre-sent separate biological species (Martin et al. 1998). The IGS I region grouped isolates of Armillaria mellea s.s. in Asian, western North American, eastern North American and European populations (Coetzee et al. 2000). Sequences are used to construct phylogenetic trees (dendrograms) for phy-logenetic analyses (molecular systematics). 

It is not unusual for those inten-. from the databases. 
Nuclear and mitochondrial genes have different inheritance. Selosse et al. (1998) showed intraspecific polymorphism of the larg_esubu ectomycorrhizal miotorcrhahiozn-i drial rDNA in Lucca ria bicolor.

 A sequence database of several  basidiomycetes based on a portion of the large subunit of mitochondrial rDNA was assembled in view of identification (Bruns et al. 1998). rDNA sequencing yields a comprehensivepool of information, but is tedious and expensive. Further costs emerge if the PCR products are previously cloned. An automatic sequencer is too expensive for small laboratories. However, spe-cialized sequencing services are meanwhile inexpensive,providing a sequence of about 800 by length for about € 10. 

The ITS of some brown-rot and white-rot fungi was sequenced by Jellison et al. (2003). It is normal to deposit sequences in the international electronic databases for everyone's use (European Molecular Biology Laboratory EMBL: www.ebi. ac.uk/embl; American GenBank: www.ncbi.nlm.nih.gov/genbank; DNA Data Bank of Japan DDBJ: www.ddbj.nig.ac.jp). 

isolates of Armillaria s.s. in Asian, western North American,e e weste, "u' vasutTin North American and European populations (Coetzee et al. 2000). Sequences are used to construct phylogenetic trees (dendrograms) for phv-logenetic analyses (molecular systematics). It is not unusual for those inten-tions to complement own data with sequences downloaded from the databases. For closely related fungi, like Armillaria species, IGS sequences were used for phylogenetic analysis (e.g., Terashima et al. 1998b). Also, ITS sequences may be applied to phylogenetic trees. An example of S. lacrymans and S. himantioides is shown in Fig. 2.22. The tree shows that isolates of S. lacrymans collected in nature in Czechoslovakia, India, Pakistan and Russia group in the branch of indoor isolates ("Domesticus group") but differ from wild Californian isolates ("Shastensis group") (Kauserud et al. 2004b; also White et al. 2001; Palfreyman et al. 2003), suggesting a North American link between the anthropogenic isolates and the wild relative S. himantioides. Yao et al. (1999) applied ITS sequences to a phylogenetic study of Tyromyces s.l. For phylogenetic analyses of higher groups, genera, families and orders, often the conserved 18S and 28S rDNA are used. Bresinsky et al. (1999) and Jarosch and Besl (2001) sequenced 900 bases of the 28S rDNA ofS. lacrymans, S. himantioides, Meruliporia incrassata and of Coniophora and Leucogyrophana species. Although it is not necessary to sequence the whole rRNA genes to con-struct trees, complete 18S and 28S rDNA sequences of a number of important wood-decay fungi are already known . 

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