Limits...
Towards barcode markers in Fungi: an intron map of Ascomycota mitochondria.

Santamaria M, Vicario S, Pappadà G, Scioscia G, Scazzocchio C, Saccone C - BMC Bioinformatics (2009)

Bottom Line: A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality.A new query approach has been developed to retrieve effectively introns information included in these entries.Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNR - Istituto di Tecnologie Biomediche, Sede di Bari, Via Amendola 122/D, Bari, 70126, Italy. monica.santamaria@ba.itb.cnr.it

ABSTRACT

Background: A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality. In particular the potential use of mitochondrial DNA species markers has been taken in account. Unfortunately, a serious difficulty in the PCR and bioinformatic surveys is due to the presence of mobile introns in almost all the fungal mitochondrial genes. The aim of this work is to verify the incidence of this phenomenon in Ascomycota, testing, at the same time, a new bioinformatic tool for extracting and managing sequence databases annotations, in order to identify the mitochondrial gene regions where introns are missing so as to propose them as species markers.

Methods: The general trend towards a large occurrence of introns in the mitochondrial genome of Fungi has been confirmed in Ascomycota by an extensive bioinformatic analysis, performed on all the entries concerning 11 mitochondrial protein coding genes and 2 mitochondrial rRNA (ribosomal RNA) specifying genes, belonging to this phylum, available in public nucleotide sequence databases. A new query approach has been developed to retrieve effectively introns information included in these entries.

Results: After comparing the new query-based approach with a blast-based procedure, with the aim of designing a faithful Ascomycota mitochondrial intron map, the first method appeared clearly the most accurate. Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals.

Conclusion: The development of the new query system described here would answer the pressing requirement to improve drastically the bioinformatics support to the DNA Barcode Initiative. The large scale investigation of Ascomycota mitochondrial introns performed through this tool, allowing to exclude the introns-rich sequences from the barcode candidates exploration, could be the first step towards a mitochondrial barcoding strategy for these organisms, similar to the standard approach employed in metazoans.

Show MeSH
Intron size distribution as estimated by the Query-based protocol. The distribution of size in each gene are depicted as for the blast protocol results. From the distribution of intron sizes one record (AY955840 and its equivalent from full genome collection NC_007935) was removed because with a value too large (14969 bp) to be easily recorded on the graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2697638&req=5

Figure 7: Intron size distribution as estimated by the Query-based protocol. The distribution of size in each gene are depicted as for the blast protocol results. From the distribution of intron sizes one record (AY955840 and its equivalent from full genome collection NC_007935) was removed because with a value too large (14969 bp) to be easily recorded on the graph.

Mentions: The Query-based approach generally confirmed the results obtained with the Blast-based one, but it seems more accurate in recognizing also small exons (almost the totality of intron sizes are lower than 3500 bp). Indeed, as shown in Figure 7, the intron sizes are still included in a range between 500 and 3000 bp, with few exceptions. The majority of them are found in a range between 1000 and 2000 bp and the unrealistic intron sizes revealed by the Blast approach are not found any longer. Figure 8 displays that CO1 and CYTB genes show again a high density of introns. As for the rRNA genes, the situation is not clear: the large amount of introns highlighted through the Blast method disappeared almost completely as a result of the Query-based method. Therefore, further investigation of the ribosomal RNA genes is required. The ND3 gene does not appear to be completely intron-free as found with the Blast-based approach. Finally, even if several Ascomycota mitochondrial genes show a low incidence of introns, such as ND3, ND4 and the terminal 3' of ND5, ND6 appears to be completely intron-free and thus it constitutes the best potential barcode candidate. A summary of a number of records and different species retrieved for each gene is displayed in Figure 9, respectively. See additional file 3 for the original data about the presence and the position of introns in the totality of mitochondrial Ascomycota records present in Genbank used to obtain the graphs reported in Fig. 8.


Towards barcode markers in Fungi: an intron map of Ascomycota mitochondria.

Santamaria M, Vicario S, Pappadà G, Scioscia G, Scazzocchio C, Saccone C - BMC Bioinformatics (2009)

Intron size distribution as estimated by the Query-based protocol. The distribution of size in each gene are depicted as for the blast protocol results. From the distribution of intron sizes one record (AY955840 and its equivalent from full genome collection NC_007935) was removed because with a value too large (14969 bp) to be easily recorded on the graph.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2697638&req=5

Figure 7: Intron size distribution as estimated by the Query-based protocol. The distribution of size in each gene are depicted as for the blast protocol results. From the distribution of intron sizes one record (AY955840 and its equivalent from full genome collection NC_007935) was removed because with a value too large (14969 bp) to be easily recorded on the graph.
Mentions: The Query-based approach generally confirmed the results obtained with the Blast-based one, but it seems more accurate in recognizing also small exons (almost the totality of intron sizes are lower than 3500 bp). Indeed, as shown in Figure 7, the intron sizes are still included in a range between 500 and 3000 bp, with few exceptions. The majority of them are found in a range between 1000 and 2000 bp and the unrealistic intron sizes revealed by the Blast approach are not found any longer. Figure 8 displays that CO1 and CYTB genes show again a high density of introns. As for the rRNA genes, the situation is not clear: the large amount of introns highlighted through the Blast method disappeared almost completely as a result of the Query-based method. Therefore, further investigation of the ribosomal RNA genes is required. The ND3 gene does not appear to be completely intron-free as found with the Blast-based approach. Finally, even if several Ascomycota mitochondrial genes show a low incidence of introns, such as ND3, ND4 and the terminal 3' of ND5, ND6 appears to be completely intron-free and thus it constitutes the best potential barcode candidate. A summary of a number of records and different species retrieved for each gene is displayed in Figure 9, respectively. See additional file 3 for the original data about the presence and the position of introns in the totality of mitochondrial Ascomycota records present in Genbank used to obtain the graphs reported in Fig. 8.

Bottom Line: A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality.A new query approach has been developed to retrieve effectively introns information included in these entries.Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNR - Istituto di Tecnologie Biomediche, Sede di Bari, Via Amendola 122/D, Bari, 70126, Italy. monica.santamaria@ba.itb.cnr.it

ABSTRACT

Background: A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality. In particular the potential use of mitochondrial DNA species markers has been taken in account. Unfortunately, a serious difficulty in the PCR and bioinformatic surveys is due to the presence of mobile introns in almost all the fungal mitochondrial genes. The aim of this work is to verify the incidence of this phenomenon in Ascomycota, testing, at the same time, a new bioinformatic tool for extracting and managing sequence databases annotations, in order to identify the mitochondrial gene regions where introns are missing so as to propose them as species markers.

Methods: The general trend towards a large occurrence of introns in the mitochondrial genome of Fungi has been confirmed in Ascomycota by an extensive bioinformatic analysis, performed on all the entries concerning 11 mitochondrial protein coding genes and 2 mitochondrial rRNA (ribosomal RNA) specifying genes, belonging to this phylum, available in public nucleotide sequence databases. A new query approach has been developed to retrieve effectively introns information included in these entries.

Results: After comparing the new query-based approach with a blast-based procedure, with the aim of designing a faithful Ascomycota mitochondrial intron map, the first method appeared clearly the most accurate. Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals.

Conclusion: The development of the new query system described here would answer the pressing requirement to improve drastically the bioinformatics support to the DNA Barcode Initiative. The large scale investigation of Ascomycota mitochondrial introns performed through this tool, allowing to exclude the introns-rich sequences from the barcode candidates exploration, could be the first step towards a mitochondrial barcoding strategy for these organisms, similar to the standard approach employed in metazoans.

Show MeSH