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MCCORD, PER HILDING. Genetic, Genomic, and Transgenic Approaches to Understand Internal Heat Necrosis in Potato. (Under the direction of G. Craig Yencho and Bryon R. Sosinski.) Internal heat necrosis (IHN) is a physiological disorder of potato tubers. A multifaceted approach was followed to better understand IHN. This approach entailed three major projects, involving genetic, genomic, and transgenic techniques. The genetics approach involved developing linkage maps of tetraploid potato using AFLP and SSR markers, and identifying QTL for IHN. Our mapping population consisted of 160 individuals from a cross between ‘Atlantic’, an IHN-susceptible cultivar, and B1829-5, an IHN-resistant breeding line. Phenotypic data indicated that the distribution of IHN is highly skewed toward resistance. Early foliage maturity was positively correlated with reduced IHN. QTL for resistance to IHN were located in multiple years on chromosomes IV, V, and group VII of ‘Atlantic’, and on group VII of B1829-5. The QTL explained between 6.7 and 40.9 percent of the variation for mean severity, and from 4.9 to 32.5 percent of the variation for percent IHN incidence. Two IHN-linked markers from chromosomes I and V were also associated with IHN in a secondary mapping population. The correlation between maturity and IHN may be partially explained by loosely linked QTL on chromosomes II and V. QTL were also detected in this population for tuber dry matter content, specific gravity, skin texture, yield, and flower color. The genomic approach entailed a microarray-based gene expression analysis of ‘Atlantic’ potatoes grown under normal and high temperatures in a growth chamber. Although overall levels of IHN were not significantly different between treatments, IHN symptoms increased more rapidly under high temperature. Mixed model analysis identified 17 genes whose expression was significantly different between high and normal temperatures at the first and later harvest dates. Expression levels of four of these genes were tested via quantitative RT-PCR (qRT-PCR). Confirmation of expression was obtained for one gene, encoding a heat shock protein, under all experimental conditions. Testing of these genes using field-grown potatoes showed no differences between clean and IHN-affected tubers; this may have been due to cold storage of the tubers prior to RNA extraction. Tuber calcium levels may be involved in the expression of IHN, and the transgenic project was designed to test this association. ‘Atlantic’ was transformed via Agrobacterium tumefaciens with a construct containing a maize calreticulin-derived calcium binding peptide (CBP) shown to elevate calcium levels in Arabidopsis. Plants were grown under mild heat stress conditions in walk-in growth chambers. Transgenic lines generally had higher yields than wild-type plants. Levels of IHN in transgenic tubers were equal to or higher than wild-type tubers. Quantitative RT-PCR of CBP showed no significant differences between transgenic lines, suggesting that differing protein abundance underlies phenotypic differences between transgenic lines. Two native calreticulin genes were assayed by qRT-PCR for evidence of silencing, but none was found. Nutrient analysis of calcium and 11 other nutrients was also performed. Calcium levels in leaves of two transgenic lines were higher than wild-type ‘Atlantic’. Leaf levels of magnesium, manganese, sulfur, and sodium were also higher than ‘Atlantic’ in at least one line. Most of these minerals are involved in photosynthesis; increased amounts in leaves could be responsible for the yield increase. In tubers, mineral levels were not significantly different. It is possible that expression of CBP in tubers is much lower than in leaves, which could explain the failure to detect any mineral differences. The increase in IHN could be due to the interruption of a signal from the leaves, or a subtle effect on tuber minerals that would require larger sample sizes to detect.

Peach (Prunus persica (L.) Batsch) has been proposed as a model organism for structural genomics in the family Rosaceae. Several resources have been or are currently being developed to study the structural genomics of the species. A highly saturated genetic linkage map has been developed from an interspecific 'Texas' almond X 'Earligold' peach cross (T X E), and two peach BAC libraries from the cultivars 'Nemared' and 'Lovell' have been generated for the creation of a physical mapping resource. A framework map has been generated by the hybridization of markers from the T X E and other Prunus maps to the 'Nemared' library. In addition, peach and almond unigenes are being hybridized to the library in order to generate an anchored transcript map, identify contigs for overall map construction, and provide information on genome organization. A subgroup of the Prunus unigene set comprising 942 ESTs derived from peach fruit mesocarp was hybridized to the 44,150-clone 'Nemared' library. Eight hundred ninety-nine of these ESTs successfully hybridized to the library, identifying 3475 BACs, of which 2725 were unique. Seventy-six of these ESTs are now anchored to the reference map by hybridization to genetically anchored BAC clones or contigs. Twenty-six of these ESTs mapped near loci for important agronomic traits, including flesh acidity, fruit skin pubescence, and almond shell hardness. Unanchored ESTs generated contigs of 2 to 44 clones, which are being fingerprinted and integrated into the growing peach physical map in preparation for sequencing of the peach genome. Internal heat necrosis (IHN) is a physiological disorder of potato tubers resulting in the discoloration of parenchymal tissue. Environmental conditions and the large acreage of the IHN-susceptible cultivar 'Atlantic' result in significant losses due to the disorder in the mid-Atlantic states and Florida. A combination of bulked segregant analysis and AFLP marker technology was used to search for molecular markers for IHN in a population of tetraploid 4x-2x S. tuberosum x S. phureja-S. stenotomum (tub x phu-stn) hybrids. These clones are being used to breed an IHN-resistant, high specific-gravity replacement for 'Atlantic'. One marker, potPCR13-HindIII-R, was identified in two small test populations and was strongly associated with resistance to IHN, explaining 69.9% and 64 % of the observed variation for IHN incidence and severity, respectively. This marker showed strong sequence homology to calcium-dependent protein kinases (CDPKs), reinforcing prior evidence of the importance of calcium to the manifestation of IHN. When tested on a different set of clones from a combining ability study for IHN, this marker was no longer significant. However, a second putative marker from the BSA-AFLP screen (potPCR31-A) that was not statistically significant in the small populations, was significantly associated with IHN susceptibility in this second population, and the regression models of potPCR-31-A on IHN severity and incidence explained 17.8% and 18.6% of the phenotypic variation, respectively. This marker showed homology to plastid terminal oxidases (PTOXs), which are involved in desaturation of carotenoids, and may have a role in protecting biomolecules under oxidative stress. The small sample sizes used in marker development and initial testing may have resulted in these markers being significant predictors only in certain populations. It is also possible that, given the quantitative nature of IHN, only a subset of all IHN-associated loci may be necessary for the disorder to be manifest. This research is the first molecular marker study of the genetics of IHN, and has set a foundation for future studies.