Genetic, Genomic, and Transgenic Approaches to Understand Internal Heat Necrosis in Potato.

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Title: Genetic, Genomic, and Transgenic Approaches to Understand Internal Heat Necrosis in Potato.
Author: McCord, Per Hilding
Advisors: Craig Yencho, Committee Co-Chair
Abstract: 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.
Date: 2009-08-12
Degree: PhD
Discipline: Horticultural Science

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