title: Multiple displacement amplification of whole genomic DNA from urediospores of Puccinia striiformis f. sp. tritici.
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 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.
authors: Department of Plant Pathology, China Agricultural University, 2 West Yuanmingyuan Rd., Beijing, 100193, China.
authors: R
authors: R
authors: Zhang
authors: Z H
authors: ZH
authors: Ma
authors: B M
authors: BM
authors: Wu
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D001487:Basidiomycota / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D004271:DNA, Fungal / Q000235:genetics*; D016681:Genome, Fungal; D023281:Genomics; D021141:Nucleic Acid Amplification Techniques; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D014908:Triticum / Q000382:microbiology
chemical_list: D004271:DNA, Fungal
references: Mol Hum Reprod. 2007 Jun;13(6):431-6
references: 17439955
references: Genomics. 2013 Jan;101(1):24-9
references: 22982297
references: Fungal Genet Biol. 2009 Apr;46(4):299-307
references: 19570502
references: Hum Mutat. 2005 Sep;26(3):262-70
references: 16086324
references: FEMS Microbiol Lett. 2005 Jan 1;242(1):65-71
references: 15621421
references: Hum Mutat. 2006 May;27(5):496-503
references: 16619243
references: J Microbiol Methods. 2005 Nov;63(2):157-64
references: 15936100
references: Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5847-51
references: 1631067
references: Mol Hum Reprod. 2011 Jun;17(6):335-43
references: 21177337
references: Methods Mol Biol. 2008;439:1-18
references: 18370092
references: Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5261-6
references: 11959976
references: Fungal Genet Biol. 2005 May;42(5):367-75
references: 15809002
references: J Microbiol Methods. 2009 May;77(2):229-34
references: 19233233
references: Genomics. 1992 Jul;13(3):718-25
references: 1639399
references: Appl Environ Microbiol. 2005 Jun;71(6):3342-7
references: 15933038
references: Curr Opin Microbiol. 2007 Oct;10(5):510-6
references: 17923430
references: Curr Genet. 2007 Mar;51(3):209-19
references: 17256172
references: Mycol Res. 2007 Feb;111(Pt 2):163-75
references: 17324755
references: Trends Biotechnol. 2003 Dec;21(12):531-5
references: 14624861
references: Gene. 2014 Oct 15;550(1):131-5
references: 25010725
references: Nat Protoc. 2006;1(4):1965-70
references: 17487184
references: Biochem Soc Trans. 2009 Apr;37(Pt 2):450-3
references: 19290880
references: Hum Mutat. 2006 Jul;27(7):603-14
references: 16786504
references: Vet Med Int. 2010 Oct 24;2010:192787
references: 20981298