Extreme Resistance to Potato Virus A in Potato Cultivar Barbara is Independently Mediated by Ra and Rysto.

Potato virus A (PVA) and potato virus Y (PVY) are two members of genus Potyvirus infecting potato crops worldwide. Host resistance offers an economical and effective means for the control or management of these viruses. In this study, 20 potato clones were screened for their resistance against PVA and PVY by mechanical or graft inoculation assay, and were explored for the relationship between extreme resistance genes Ra and Ry by the detection of molecular markers linked to Ryadg, Rysto, and Rychc. Six clones, including Barbara, Jizhangshu 8, Longshu 7, Longshu 8, M6, and Solara, were found to be extremely resistant to both PVA and PVY; three clones (AC142, Eshu 3, and Shepody) were deemed to be extremely resistant to PVA but susceptible to PVY. To further reveal the inheritance of the extreme resistance (ER) against PVA, a tetraploid F1 population of Barbara × F58050 (susceptible to both PVY and PVA) and a tetraploid BC1 population of BF145 (a PVA-resistant but PVY-susceptible progeny of Barbara × F58050) × F58050 were obtained. Phenotyping of the F1 and BC1 populations by graft inoculation with PVA showed segregation ratios of 3:1 and 1:1 (resistant:susceptible), respectively. These results suggest that two independent loci control ER against PVA in Barbara: one confers ER to both PVA and PVY and the other confers ER to PVA only. The deduced genotype of Barbara is RyryryryRararara.

High Resolution DNA Melting Assays for Detection of Rx1 and Rx2 for High-Throughput Marker-Assisted Selection for Extreme Resistance to Potato virus X in Tetraploid Potato.

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.

Development and Validation of High-Resolution Melting Markers Derived from Rysto STS Markers for High-Throughput Marker-Assisted Selection of Potato Carrying Rysto.

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.

Quantitative resistance in potato leaves to late blight associated with induced hydroxycinnamic acid amides.

Late blight is a serious economic threat to potato crop, sometimes leading to complete crop loss. The resistance in potato to late blight can be qualitative or quantitative in nature. Qualitative resistance is not durable. Though quantitative resistance is durable, the breeding is challenging due to polygenic inheritance. Several quantitative trait loci (QTLs) have been identified, but the mechanisms of resistance are largely unknown. A nontargeted metabolomics approach was used to identify resistance-related (RR) metabolites in a resistant genotype (F06025), as compared to a susceptible (Shepody) genotype, mock- or pathogen-inoculated. The RR metabolites, which had high fold change in abundance, mainly belonged to phenylpropanoid, flavonoid, fatty acid, and alkaloid chemical groups. The most important phenylpropanoids identified were hydroxycinnamic acid amides, the polyaromatic domain of suberin that is known to be associated with cell wall reinforcement. These metabolites were mapped on to the potato metabolic pathways, and the candidate enzymes and their coding genes were identified. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay revealed a higher upregulation of 4-coumarate: CoA ligase (4-CL), tyrosine decarboxylase (TyDC), and tyramine hydroxycinnamoyl transferase (THT) in the pathogen-inoculated resistant genotype than in susceptible. These genes were sequenced in both resistant and susceptible genotypes, and nonsynonymous single-nucleotide polymorphisms (nsSNPs) were found. The application of these genes in potato resistance improvement, following validation, is discussed.

Decreased defense gene expression in tolerance versus resistance to Verticillium dahliae in potato.

Verticillium dahliae Kleb., a soil-borne fungus that colonizes vascular tissues, induces wilting, chlorosis and early senescence in potato. Difference in senescence timing found in two diploid potato clones, 07506-01 and 12120-03, was studied and genetic variation in response to V. dahliae infection was identified as a causal factor. The clone, 07506-01, was infected with V. dahliae but did not develop symptoms, indicating tolerance to the pathogen. The other diploid clone, 12120-03 had low levels of pathogen with infection and moderate symptoms indicating partial resistance. 07506-01 was found to carry two susceptible alleles of the Ve2 gene and 12120-03 carried one Ve2 resistant and one susceptible allele. Infected leaves of the two clones were compared using gene expression profiling with the Potato Oligonucleotide Chip Initiative (POCI) microrarray. The results provide further evidence for differences in response of the two clones to infection with V. dahliae. Chlorophyll biosynthesis was higher in the tolerant 07506-01 compared to partially resistant 12120-03. On the other hand, expression of fungal defense genes, Ve resistance genes and defense phytohormone biosynthetic enzyme genes was decreased in 07506-01 compared to 12120-03 suggesting defense responses were suppressed in tolerance compared to resistance. Transcription factor gene expression differences pointed to the WRKY family as potential regulators of V. dahliae responses in potato.

Response of Potato Cultivars to Five Isolates Belonging to Four Strains of Potato virus Y.

The responses of 14 potato cultivars to five Potato virus Y (PVY) isolates belonging to four strains (ordinary [PVYO], tobacco veinal necrosis [PVYN], N:O group [PVYN:O], and nonrecombinant potato tuber necrotic [PVYNTN]) were studied in primary and secondary infections. For the primary infection experiments, foliage symptoms were monitored daily after mechanical inoculation with a PVY isolate until harvest; and, for the secondary infection experiments, foliage symptoms were monitored regularly from plant emergence until harvest. Tuber symptoms (namely, tuber necrotic ringspots) were checked at harvest and monthly postharvest for up to 4 months. In both infections, symptoms varied significantly depending on potato cultivar and virus strain or isolate. In primary infections, local lesions occurred on inoculated leaves of 'AC Chaleur', 'Eramosa', 'Goldrush', 'Jemseg', 'Katahdin', 'Ranger Russet', and 'Yukon Gold' after inoculation with PVYO isolates, followed by systemic necrosis on latterly emerged uninoculated leaves. In contrast, plants of 'CalWhite', 'La Rouge', 'Red LaSoda', 'Russet Burbank', 'Russet Norkotah', and 'Superior' did not exhibit any visible symptoms on inoculated leaves but developed mild to severe mosaic on latterly emerged leaves after infection with PVYO isolates. In all cultivars, near-symptomless to mild mosaic was induced by PVYN and mild to severe mosaic by PVYN:O. PVYNTN induced mild to severe mosaic in plants of all cultivars except AC Chaleur, 'Cherokee', and Yukon Gold, which developed visible systemic necrosis. Necrotic ringspots were observed in tubers of PVYNTN-infected plants of AC Chaleur, Cherokee, and Yukon Gold. The tuber symptoms were also incited by PVYN-Jg on Cherokee. In secondary infections, the symptoms were generally more severe than primary infections even though the symptom types did not alter. As in the greenhouse, a clear symptom severity pattern (PVYO-FL > PVYO-RB > PVYNTN-Sl > PVYN:O-Mb58 > PVYN-Jg) was observed in AC Chaleur, Cherokee, Eramosa, Goldrush, Jemseg, Katahdin, Ranger Russet, and Yukon Gold in the field.

Tuber transcriptome profiling of eight potato cultivars with different cold-induced sweetening responses to cold storage.

Tubers are vegetative reproduction organs formed from underground extensions of the plant stem. Potato tubers are harvested and stored for months. Storage under cold temperatures of 2-4 °C is advantageous for supressing sprouting and diseases. However, development of reducing sugars can occur with cold storage through a process called cold-induced sweetening (CIS). CIS is undesirable as it leads to darkened color with fry processing. The purpose of the current study was to find differences in biological responses in eight cultivars with variation in CIS resistance. Transcriptome sequencing was done on tubers before and after cold storage and three approaches were taken for gene expression analysis: 1. Gene expression correlated with end-point glucose after cold storage, 2. Gene expression correlated with increased glucose after cold storage (after-before), and 3. Differential gene expression before and after cold storage. Cultivars with high CIS resistance (low glucose after cold) were found to increase expression of an invertase inhibitor gene and genes involved in DNA replication and repair after cold storage. The cultivars with low CIS resistance (high glucose after cold) showed increased expression of genes involved in abiotic stress response, gene expression, protein turnover and the mitochondria. There was a small number of genes with similar expression patterns for all cultivars including genes involved in cell wall strengthening and phospholipases. It is proposed that the pattern of gene expression is related to chilling-induced DNA damage repair and cold acclimation and that genetic variation in these processes are related to CIS.

Verticillium dahliae Disease Resistance and the Regulatory Pathway for Maturity and Tuberization in Potato.

Kleb. is a pathogenic fungus causing wilting, chlorosis, and early dying in potato ( L.). Genetic mapping of resistance to was done using a diploid population of potato. The major quantitative trait locus (QTL) for resistance was found on chromosome 5. The gene, controlling earliness of maturity and tuberization, was mapped within the interval. Another QTL on chromosome 9 co-localized with the wilt resistance gene marker. Epistasis analysis indicated that the loci on chromosomes 5 and 9 had a highly significant interaction, and that functioned downstream of The alleles were sequenced and found to encode StCDF1.1 and StCDF1.3. Interaction between the resistance allele and the was demonstrated, but not for Genome-wide expression QTL (eQTL) analysis was performed and genes with eQTL at the and loci were both found to have similar functions involving the chloroplast, including photosynthesis, which declines in both maturity and wilt. Among the gene ontology (GO) terms that were specific to genes with eQTL at the , but not the locus, were those associated with fungal defense. These results suggest that controls fungal defense and reduces early dying in wilt through affecting genetic pathway controlling tuberization timing.

Differences between the Bud End and Stem End of Potatoes in Dry Matter Content, Starch Granule Size, and Carbohydrate Metabolic Gene Expression at the Growing and Sprouting Stages.

Potatoes usually have the tuber bud end dominance in growth during tuber bulking and in tuber sprouting, likely using carbohydrates from the tuber stem end. We hypothesized that the tuber bud end and tuber stem end coordination in carbohydrate metabolism gene expression is different between the bulking dominance and sprouting dominance of the tuber bud end. After comparing the growing tubers at harvest from a green vine and the stage that sprouts just started to emerge after storage of tubers at room temperature, we found the following: (1) Dry matter content was higher in the tuber stem end than the tuber bud end at both stages. (2) The starch granule size was larger in the tuber bud end than in the tuber stem end. (3) The tuber bud end had higher gene expression for starch synthesis but a lower gene expression of sucrose transporters than the tuber stem end during tuber growing. (4) The tuber stem end at the sprouting stage showed more active gene expression in both starch degradation and resynthesis, suggesting more active export of carbohydrates, than the tuber bud end. The results indicate that the starch accumulation mechanism in the tuber bud end was different between field growing and post-harvest sprouting tubers and that tubers already increased dry matter and average starch granule sizes in the tuber bud end prior to the rapid growth of sprouts.

Effect of preparation method on the glycaemic index of novel potato clones.

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.