Development of allele-specific PCR and RT–PCR assays for clustered resistance genes using a potato late blight resistance transgene as a model

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.

Disease resistance gene transcription in transgenic potato is unaltered by temperature extremes and plant physiological age

“…The fragment, labeled "RB" is an amplicon specific to the RB transgene and is present in all transgenic plants, regardless of growing temperature, and is absent in all nontransgenic plants. The lower fragment, present in all RTPCRs (except water) originates from a template unrelated to RB and serves as an additional internal control (Millett and Bradeen 2007) Until now, no study has reported the impact of growing temperature on the transcription of P. infestans resistance genes. At a phenotypic level, high temperatures decreased potato resistance to P. infestans (Rubio-Covarrubias et al 2006).…”

“…RNA was quantified using a NanoDrop ND-1000 (NanoDrop Technologies, LLC, Wilmington, DE). Qualitative RT-PCRs were performed as described by Millett and Bradeen (2007). Quantitative RT-PCRs were performed as described by Bradeen et al (2009), and standardized against Elongation Factor 1-α, an appropriate standard for transcriptional study of the potato-P. infestans pathosystem (Nicot et al 2005).…”

“…Each encodes an NBS-LRR R protein and each holds significant promise for the transgenic control of late blight disease. Previously, we developed an RT-PCR assay specific for the RB transgene (Millett and Bradeen 2007). Capitalizing upon single nucleotide polymorphisms introduced during the cloning process and unique to the transgene, our assay is so precise as to differentiate between the RB transgene and the native, endogenous S. bulbocastanum gene from which it was cloned.…”

“…A photograph of an agarose gel documenting transgene RB transcription. Our previously published RT-PCR assay(Millett and Bradeen 2007) was employed to test for RB transcription in transgenic plants of line SP2577 grown at 10°C, 20°C, and 30°C. Lanes are (left to right): DNA size standard, RT-PCRs of SP2577 (+) and nontransformed 'Dark Red Norland' (−) grown at each temperature, and RT-PCR technical controls [S-nontransformed 'Superior' (negative control), K+-transgenic 'Katahdin' (positive control), Wwater; no RNA template (negative control)]…”

A Novel Class of Simple PCR Markers with SNP-Level Sensitivity for Mapping and Haplotype Characterization in Solanum Species

“…Similar allele-specific PCR techniques have been employed for the generation of SNP-based markers for mapping in Arabidopsis (Drenkard et al 2000), genotyping in potato (Sattarzadeh et al 2006), and for blast disease resistance genes in rice (Hayashi et al 2006). Previously, we adopted a MAMA approach to develop transgene-specific PCR and RT-PCR assays for the RB transgene (Millett and Bradeen 2007). Here, we demonstrate the utility of MAMA-PCR for mapping applications and analysis of marker association and haplotype frequency near the RB late blight resistance locus in S. bulbocastanum.…”