RESUMEN
Assessment of the existing PCR-gel electrophoresis-based methods for detection of Rx1 and Rx2, the genes that independently control extreme resistance (ER) to Potato virus X (PVX), indicated that the 5Rx1F/5Rx1R primer pair led to reliable detection of Rx1, whereas the 106Rx2F/106Rx2R primer pair detected Rx2 despite some nonspecific reactions in potato clones/cultivars without Rx2. However, the methodology is time consuming and does not differentiate the absence of Rx1/Rx2 from a failed PCR reaction. A newly designed primer pair that targets Rx1 and Rx2 as well as rx1 and rx2 produced an amplicon for all alleles. When the primer pair was combined with 5Rx1F/5Rx1R, respective amplicons were produced, although they were not distinguishable by regular agarose gel electrophoresis. When subjected to a high-resolution DNA melting (HRM) assay, two distinct melting profiles for Rx1 and rx1, respectively, were detected. Triplex PCR-gel electrophoresis and -HRM assay for detection of Rx1, Rx2, and rx1/rx2 were also performed. The efficacy of the HRM assays were validated in potato cultivars/clones with known phenotypes, indicating its potential for high-throughput selection of potato clones/cultivars carrying Rx1 or Rx2. Duplex PCR-HRM assays of over 600 progeny from 12 crosses involving various parents correctly detected the presence or absence of Rx1 in each progeny, allowing accurate prediction of the phenotype. Progeny that tested positive for Rx1 by HRM exhibited ER to PVX whereas progeny that tested negative for Rx1 were susceptible to PVX infection. The genotype of each parent and the possible presence of Nx in two Rx1-possessing parents are also discussed.
Asunto(s)
Antibiosis/genética , Desnaturalización de Ácido Nucleico , Enfermedades de las Plantas/genética , Potexvirus/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Selección Genética , Solanum tuberosum/genética , Marcadores Genéticos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Solanum tuberosum/virología , VirulenciaRESUMEN
Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Rysto, a gene responsible for extreme resistance to Potato virus Y (PVY) in potato, was performed in tetraploid potato 'Barbara' (Rrrr) and 'AC Chaleur' (rrrr) as well as their progeny selections. Three and two sequence variants were identified in Barbara resistant (R) selections and AC Chaleur susceptible (S) selections, respectively. Further analysis indicates that the variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harboring the STS markers. Two primer pairs, one targeting the region containing a 20-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in Barbara-R bulk and one visible fragment in AC Chaleur-S bulk; pair two produced one visible fragment in all samples. When subjected to high-resolution melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progeny of Barbara × AC Chaleur revealed 72 and 75 progeny with R and S melting profiles, respectively, which was consistent with YES3-3A and YES3-3B assays and phenotyping analysis, thus demonstrating the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for high-throughput marker-assisted selection for Rysto-conferred resistance to PVY was validated further with three populations involving Barbara as the R parent.
Asunto(s)
Enfermedades de las Plantas/inmunología , Polimorfismo de Nucleótido Simple/genética , Potyvirus/fisiología , Lugares Marcados de Secuencia , Solanum tuberosum/genética , Secuencia de Bases , Cruzamiento , Cartilla de ADN/genética , Marcadores Genéticos/genética , Variación Genética , Desnaturalización de Ácido Nucleico , Enfermedades de las Plantas/virología , Alineación de Secuencia , Análisis de Secuencia de ADN , Solanum tuberosum/inmunología , Solanum tuberosum/virología , Tetraploidía , Temperatura de TransiciónRESUMEN
The purpose of this study was to investigate whether the effects of cooling and reheating on the glycaemic index (GI) of novel potato clones (selections) differed depending on selection and whether cooling altered starch absorption in vivo. We conducted 3 experiments using 4 novel potato clones in healthy subjects. Experiment 1: the GI of 4 selections each prepared in 3 ways (freshly boiled, cooled, or cooled and reheated) was measured in 2 groups of 10 subjects (each group tested 2 selections). Experiment 2 (n=10): two selections from Experiment 1 were re-tested one year later, by a different subject group. Experiment 3 (n=10): two selections from Experiment 1 were tested by subjects from Experiment 2 to assess the rate and extent of starch absorption using the second-meal effect and the breath hydrogen method, respectively. Experiment 1 demonstrated a selection×treatment interaction for GI (p=0.024); cooling reduced the GI of two selections by 40-50% (p<0.05) but reduced GI of the other 2 by only 8-10% (ns). Experiment 2 confirmed the selection×treatment interaction (p=0.018) seen in Experiment 1. Experiment 3: cooling reduced the GI by an average of 37% (p<0.05) but only increased starch malabsorption in vivo from 3% to 5% (p=0.021); there was no significant second-meal effect. It is concluded that the effect of cooling on the GI of potatoes may vary from 0-50% depending on selection. However, the mechanism for the effect is not clear: the 2% increase in starch malabsorption seen upon cooling potatoes was not nearly enough to account for the 37% reduction in GI.