Curr Genet DOI 10.1007/s00294-014-0470-x
TECHNICAL NOTES
Multiple displacement amplification of whole genomic DNA from urediospores of Puccinia striiformis f. sp. tritici R. Zhang · Z. H. Ma · B. M. Wu
Received: 4 September 2014 / Revised: 4 December 2014 / Accepted: 20 December 2014 © Springer-Verlag Berlin Heidelberg 2015
Abstract Biotrophic fungi, such as Puccinia striiformis f. sp. tritici, because they cannot be cultured on nutrient media, to obtain adequate quantity of DNA for molecular genetic analysis, are usually propagated on living hosts, wheat plants in case of P. striiformis f. sp. tritici. The propagation process is time-, space- and labor-consuming and has been a bottleneck to molecular genetic analysis of this pathogen. In this study we evaluated multiple displacement amplification (MDA) of pathogen genomic DNA from urediospores as an alternative approach to traditional propagation of urediospores followed by DNA extraction. The quantities of pathogen genomic DNA in the products were further determined via real-time PCR with a pair of primers specific for the β-tubulin gene of P. striiformis f. sp. tritici. The amplified fragment length polymorphism (AFLP) fingerprints were also compared between the DNA products. The results demonstrated that adequate genomic DNA at fragment size larger than 23 Kb could be amplified from 20 to 30 urediospores via MDA method. The real-time PCR results suggested that although fresh urediospores collected from diseased leaves were the best, spores picked from diseased leaves stored for a prolonged period could also be used for amplification. AFLP fingerprints exhibited no significant differences between amplified DNA and DNA extracted with CTAB method, suggesting amplified DNA can represent the pathogen’s genomic DNA very
Communicated by Z. Zhang. R. Zhang · Z. H. Ma (*) · B. M. Wu (*) Department of Plant Pathology, China Agricultural University, 2 West Yuanmingyuan Rd., Beijing 100193, China e-mail: [email protected] B. M. Wu e-mail: [email protected]
well. Therefore, MDA could be used to obtain genomic DNA from small precious samples (dozens of spores) for molecular genetic analysis of wheat stripe rust pathogen, and other fungi that are difficult to propagate. Keywords Wheat stripe rust · AFLP · Multiple displacement amplification · Real-time PCR · β-Tubulin gene
Introduction For precious biological samples and microorganisms difficult to propagate, whole genome amplification (WGA) techniques could be used for amplification of genomic DNA from limited biological samples to meet the requirements of molecular genetic analyses (Silander and Saarela 2008). In early studies, some PCR-based WGA techniques such as degenerate oligonucleotide primed PCR (Telenius et al. 1992) and primer extension pre-amplification (Zhang et al. 1992) were developed. However, these techniques amplify relatively small DNA fragment of
TECHNICAL NOTES
Multiple displacement amplification of whole genomic DNA from urediospores of Puccinia striiformis f. sp. tritici R. Zhang · Z. H. Ma · B. M. Wu
Received: 4 September 2014 / Revised: 4 December 2014 / Accepted: 20 December 2014 © Springer-Verlag Berlin Heidelberg 2015
Abstract Biotrophic fungi, such as Puccinia striiformis f. sp. tritici, because they cannot be cultured on nutrient media, to obtain adequate quantity of DNA for molecular genetic analysis, are usually propagated on living hosts, wheat plants in case of P. striiformis f. sp. tritici. The propagation process is time-, space- and labor-consuming and has been a bottleneck to molecular genetic analysis of this pathogen. In this study we evaluated multiple displacement amplification (MDA) of pathogen genomic DNA from urediospores as an alternative approach to traditional propagation of urediospores followed by DNA extraction. The quantities of pathogen genomic DNA in the products were further determined via real-time PCR with a pair of primers specific for the β-tubulin gene of P. striiformis f. sp. tritici. The amplified fragment length polymorphism (AFLP) fingerprints were also compared between the DNA products. The results demonstrated that adequate genomic DNA at fragment size larger than 23 Kb could be amplified from 20 to 30 urediospores via MDA method. The real-time PCR results suggested that although fresh urediospores collected from diseased leaves were the best, spores picked from diseased leaves stored for a prolonged period could also be used for amplification. AFLP fingerprints exhibited no significant differences between amplified DNA and DNA extracted with CTAB method, suggesting amplified DNA can represent the pathogen’s genomic DNA very
Communicated by Z. Zhang. R. Zhang · Z. H. Ma (*) · B. M. Wu (*) Department of Plant Pathology, China Agricultural University, 2 West Yuanmingyuan Rd., Beijing 100193, China e-mail: [email protected] B. M. Wu e-mail: [email protected]
well. Therefore, MDA could be used to obtain genomic DNA from small precious samples (dozens of spores) for molecular genetic analysis of wheat stripe rust pathogen, and other fungi that are difficult to propagate. Keywords Wheat stripe rust · AFLP · Multiple displacement amplification · Real-time PCR · β-Tubulin gene
Introduction For precious biological samples and microorganisms difficult to propagate, whole genome amplification (WGA) techniques could be used for amplification of genomic DNA from limited biological samples to meet the requirements of molecular genetic analyses (Silander and Saarela 2008). In early studies, some PCR-based WGA techniques such as degenerate oligonucleotide primed PCR (Telenius et al. 1992) and primer extension pre-amplification (Zhang et al. 1992) were developed. However, these techniques amplify relatively small DNA fragment of
