Abstract
Members of the NBS-LRR gene family impart resistance to a wide variety of pathogens and are often found clustered within a plant genome. This clustering of homologous sequences can complicate PCR-based characterizations, especially the study of transgenes. We have developed allele-specific PCR and RT–PCR assays for the potato late blight resistance gene RB. Our assay utilizes two approaches toward primer design, allowing discrimination between the RB transgene and both the endogenous RB gene and numerous RB homeologs. First, a reverse primer was designed to take advantage of an indel present in the RB transgene but absent in rb susceptibility alleles, enhancing specificity for the transgene, though not fully discriminating against RB homeologs. Second, a forward primer was designed according to the principles of mismatch amplification mutation assay (MAMA) PCR, targeting SNPs introduced during the cloning of RB. Together, the indel reverse primer and the MAMA forward primer provide an assay that is highly specific for the RB transgene, being capable of distinguishing the transgene from all RB endogenous gene copies and from all RB paralogs in a diverse collection of wild and cultivated potato genotypes. These primers have been successfully multiplexed with primers of an internal control. The multiplexed assay is useful for both PCR and RT–PCR applications. Double MAMA-PCR, in which both PCR primers target separate transgene-specific SNPs, was also tested and shown to be equally specific for the RB transgene. We propose extending the use of MAMA for the characterization of resistance transgenes.
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Ballvora A, Ercolano MR, Weis J, Meksem K, Bormann CA, Oberhagen P, Salamini F, Gebhardt C (2002) The R1 gene for potato resistance to late blight (Phytophthora infestans) belongs to the leucine zipper/NBS/LRR class of plant resistance genes. Plant J 30:361–371
Bleicher KB, Pippert TR, Glaab WE, Skopek TR, Sina JF, Umbenhauer DR (2001) Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4A. J Biochem Mol Toxicol 15:133–142
Borucki MK, Call DR (2003) Listeria monocytogenes serotype identification by PCR. J Clin Microbiol 41:5537–5540
Burnouf DY, Fuchs RPP (2000) The early detection of frameshift mutations induced by a food-borne carcinogen in rats: a new tool for molecular epidemiology. Mutat Res 462:281–291
Buzard GS, Enomoto T, Hongyo T, Perantoni AO, Diwan BA, Devor DE, Reed CR, Dove LF, Rice JM (1999) neu mutation in schwannomas induced transplacentally in Syrian golden hamsters by N-nitrosoethylurea: high incidence by low allelic representation. J Cancer Res Clin Oncol 125:529–540
Carnegie SF, Colhoun J (1982) Susceptibility of potato leaves to Phytophthora infestans in relation to plant age and leaf position. Phytopath Z 104:157–167
Cebula TA, Payne WL, Feng P (1995) Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J Clin Microbiol 33:248–250
Cha RS, Zarbl H, Keohavong P, Thilly WG (1992) Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl 2:14–20
Colton LM, Groza HI, Wielgus SM, Jiang J (2006) Marker-assisted selection for the broad-spectrum potato late blight resistance conferred by gene RB derived from a wild potato species. Crop Sci 46:589–594
Creighton S, Huang M-M, Cai H, Arnheim N, Goodman MF (1992) Base mispair extension kinetics: binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini. J Biol Chem 267:2633–2639
Drenkard E, Richter BG, Rozen S, Stutius LM, Angell NA, Mindrinos M, Cho RJ, Oefner PJ, Davis RW, Ausubel FM (2000) A simple procedure for the analysis of single nucleotide polymorphisms facilitates map-based cloning in Arabidopsis. Plant Physiol 124:1483–1492
Fry WE, Goodwin SB (1997) Resurgence of the Irish potato famine fungus: after 150 years, the late blight fungus is again menacing farmers. Bioscience 47:363–372
Fulton TM, Chunwongse J, Tanksley SD (1995) Miniprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep 13:207–209
Gale JM, Tafoya GB (2004) Evaulation of 15 polymerases and phosphorothioate primer modification for detection of UV-induced C:G to T:A mutations by allele-specific PCR. Photochem Photobiol 79:461–469
Garelik G (2002) Taking the bite out of potato blight. Science 298:1702–1704
Glaab WE, Skopek TR (1999) A novel assay for allelic discrimination that combines the fluorogenic 5′ nuclease polymerase chain reaction (TaqMan®) and mismatch amplification mutation assay. Mutat Res 430:1–12
Grube RC, Radwanski ER, Jahn M (2000) Comparative genetics of disease resistance within the Solanaceae. Genetics 155:873–887
Hayashi K, Hashimoto N, Daigen M, Ashikawa I (2004) Development of PCR-based SNP markers for rice blast resistance genes at the Piz locus. Theor Appl Genet 108:1212–1220
Hayashi K, Yoshida H, Ashikawa I (2006) Development of PCR-based allele-specific and InDel marker sets for nine rice blast resistance genes. Theor Appl Genet 113:251–260
Huang M-H, Arnheim N, Goodman MF (1992) Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. Nucleic Acids Res 20:4567–4573
Huang S, van der Vossen EAG, Kuang H, Vleeshouwers VGAA, Zhang N, Borm TJA, van Eck HJ, Baker B, Jacobsen E, Visser RGF (2005) Comparative genomics enabled the isolation of the R3a late blight resistance gene in potato. Plant J 42:251–261
Jinneman KC, Hill WE (2001) Listeria monocytogenes lineage group classification by MAMA–PCR of the listeriolysin gene. Curr Microbiol 43:129–133
Kamoun S (2001) Nonhost resistance to Phytophthora: novel prospects for a classical problem. Curr Opin Plant Biol 4:295–300
Leister D, Ballvora A, Salamini F, Gebhardt C (1996) A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plants. Nature Genet 14:421–429
Lozoya-Saldana H, Belmar-Diaz C, Bradeen JM, Helgeson JP (2005) Characterization of Phytophthora infestans isolates infecting transgenic and somatic hybrid potatoes resistant to the pathogen in the Toluca Valley, Mexico. Am J Potato Res 82:79
Mendelman LV, Petruska J, Goodman MF (1990) Base mispair extension kinetics: comparison of DNA polymerase α and reverse transcriptase. J Biol Chem 265:2338–2346
Meyers BC, Dickerman AW, Michelmore RW, Sivaramakrishnan S, Sobral BW, Young ND (1999) Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. Plant J 20:317–332
Meyers BC, Kozik A, Giego A, Kuang H, Michelmore RW (2003) Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15:809–834
Michelmore RW, Meyers BC (1998) Clusters of resistance genes in plants evolve by divergent selection and a birth-and-death proces. Genome Res 8:1113–1130
Nie X, Singh RP (2001) A novel usage of random primers for multiplex RT–PCR detection of virus and viroid in aphids, leaves, and tubers. J Virol Methods 91:37–49
Nimchuk Z, Rohmer L, Chang JH, Dangl JL (2001) Knowing the dancer from the dance: R-gene products and their interactions with other proteins from host and pathogen. Curr Opin Plant Biol 4:288–294
Pan Q, Liu Y-S, Budai-Hadrian O, Sela M, Carmel-Goren L, Zaamir D, Fluhr R (2000a) Comparative genetics of nucleotide binding site-leucine rich repeat resistance gene homologues in the genomes of two dicotyledons: tomato and Arabidopsis. Genetics 155:309–322
Pan Q, Wendel J, Fluhr R (2000b) Divergent evolution of plant NBS-LRR resistance gene homologues in dicot and cereal genomes. J Mol Evol 50:203–213
Park T-H, Gros J, Sikkema A, Vleeshouwers VGAA, Muskens M, Allefs S, Jacobsen E, Visser RGF, van der Vossen EAG (2005) The late blight resistance locus Rpi-blb3 from Solanum bulbocastanum belongs to a major late blight R gene cluster on chromosome 4 of potato. Mol Plant Microbe Interact 18:722–729
Qiang YZ, Qin T, Fu W, Cheng WP, Li YS, Yi G (2002) Use of a rapid mismatch PCR method to detect gyrA and parC mutations in ciproflaxacin-resistant clinical isolates of Escherichia coli. J Antimicrob Chemother 49:549–552
Radonić A, Thulke S, Mackay IA, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun 313:856–862
Richter TE, Ronald PC (2000) The evolution of disease resistance genes. Plant Mol Biol 42:195–204
Rossi M, Goggin FL, Milligan SB, Kaloshian I, Ullman DE, Williamson VM (1998) The nematode resistance gene Mi of tomato confers resistance against the potato aphid. Proc Natl Acad Sci USA 95:9750–9754
Sanchez MJ, Bradeen JM (2006) Towards efficient isolation of R gene orthologs from multiple genotypes: Optimization of long range-PCR. Mol Breed 17:137–148
Song J, Bradeen JM, Naess SK, Raasch JA, Wielgus SM, Haberlach GT, Liu J, Kuang H, Austin-Phillips S, Buell CR, Helgeson JP, Jiang J (2003) Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight. Proc Natl Acad Sci USA 100:9128–9133
Stewart HE (1990) Effect of plant age and inoculum concentration on expression of major gene resistance to Phytophthora infestans in detached potato leaflets. Mycol Res 94:823–826
Stewart HE, Taylor K, Wastie RL (1983) Resistance to late blight in foliage (Phytophthora infestans) of potatoes assessed as true seedlings and as adult plants in the glasshouse. Potato Res 26:363–366
Swaminathan S, Ellis HM, Waters LS, Yu D, Lee E-C, Court DL, Sharan SK (2001) Rapid engineering of bacterial artificial chromosomes using oligonucleotides. Genesis 29:14–21
van der Vossen EAG, Rouppe van der Voort JNAM, Kanyuka K, Bendahmane A, Sandbrink H, Baulcombe DC, Bakker J, Stiekema WJ, Klein Lankhorst RM (2000) Homologues of a single resistance-gene cluster in potato confer resistance to distinct pathogens: a virus and a nematode. Plant J 23:567–576
van der Vossen E, Sikkema A, te Lintel Hekkert B, Gros J, Stevens P, Muskens M, Wouters D, Pereira A, Stiekema W, Allefs S (2003) An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato. Plant J 36:867–882
van der Vossen EAG, Gros J, Sikkema A, Muskens M, Wouters D, Wolters P, Pereira A, Allefs S (2005) The Rpi-blb2 gene from Solanum bulbocastanum is an Mi-1 gene homolog conferring broad-spectrum late blight resistance in potato. Plant J 44:208–222
Vleeshouwers VGAA, Martens A, van Dooijeweert W, Colon LT, Govers F, Kamoun S (2001) Ancient diversification of the Pto kinase family preceded speciation in Solanum. Mol Plant Microbe Interact 14:996–1005
Wagner J, Kamiya H, Fuchs RPP (1997) Leading versus lagging strand mutagenesis induced by 7,8-dihydro-8-oxo-2′-deoxyguanosine in Escherichia coli. J Mol Biol 265:302–309
Yu H, Goodman MF (1992) Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates. J Biol Chem 267:10888–10896
Zirnstein G, Li Y, Swaminathan B, Angulo F (1999) Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis. J Clin Microbiol 37:3276–3280
Zirnstein G, Helsel L, Li Y, Swaminathan B, Besser J (2000) Characterization of gyrA mutations associated with fluoroquinolone resistance in Campylobactyer coli by DNA sequence analysis and MAMA PCR. FEMS Microbiol Lett 190:1–7
Acknowledgments
This research was funded in part by a National Science Foundation Graduate Research Fellowship to B. P. M. The authors wish to thank Drs. John Helgeson, Jiming Jiang, Sandra Austin-Phillips, Bill Campbell, and Shelley Jansky for plant materials. The greenhouse and field assistance of Dimitre Mollov and computer support from the University of Minnesota Supercomputing Institute are gratefully acknowledged.
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Communicated by R. Waugh.
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Millett, B.P., Bradeen, J.M. Development of allele-specific PCR and RT–PCR assays for clustered resistance genes using a potato late blight resistance transgene as a model. Theor Appl Genet 114, 501–513 (2007). https://doi.org/10.1007/s00122-006-0449-1
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DOI: https://doi.org/10.1007/s00122-006-0449-1