1a.Objectives (from AD-416)
Determine the expression and control of structural and regulatory genes for anthocyanin coloration of leaves, flowers, and fruit in pepper. Develop selectable markers useful in breeding for tissue-specific anthocyanin accumulation.

1b.Approach (from AD-416)
Published sequence data of the Petunia x hybrida An1, An2, An4 and An11 regulatory genes, and the various anthocyanin-related structural genes (Chs, Dfr, Dhr, An6, etc.) will be used to obtain cDNA or partial genomic DNA clones of >500bp by PCR, in order to study the expression of Capsicum genes and obtain selectable markers for these genes in pepper. We have already developed Capsicum PCR primers for Chs, Dfr and Ans. Expression of anthocyanin-related genes will be examined in pepper fruit, leaf and flower tissue in plants of varying genotype and pigmentation using viral-induced break and related to viral RNA distribution. HPLC profiling will be used to determine which pigments are reduced or increased in viral-induced color break. Gene expression of the affected steps will be examined, and comparisons made between the plant genes and viral sequences for localized homology that might cause silencing. Northern analysis and/or microarray analysis of mRNA extracted from the various tissues will be performed to determine whether the pattern of viral distribution coincides with changes in gene expression and the increase or decrease in anthocyanin concentrations in color break.

3.Progress Report
Anthocyanin gene regulation. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Virginia Polytechnic Institute and State University. Additional details of the research can be found in the report for the parent project 1275-21000-172-00D “Genetic enhancement of quality constituents and disease resistance in Solanaceous vegetables.” Color attributed to anthocyanins contributes to product sensory quality attributes and potential nutritive value. A Capsicum (pepper) line we have developed, 02C27, accumulates anthocyanin in various tissues and accumulation is markedly sensitive to environmental stress. Real-time PCR analysis of tissues revealed functional but differentially expressed structural and regulatory genes in the anthocyanin biosynthetic pathway coincident with anthocyanin accumulation. Similar results were obtained upon evaluation of gene expression in tissues from genotypes that accumulate anthocyanin in different plant parts. These results enable us to now investigate transcription factor protein interactions to further characterize mechanisms of gene regulation. The results are important for identification of key regulatory elements that influence anthocyanin accumulation and will result in new knowledge to facilitate enhancement of anthocyanin content in pepper and related crops. The project was monitored through site visits and numerous conference calls. This research is under National Program 301, Action Plan Component 2, Problem Area 2c and Component 3, Problem Areas 3b and 3c; ARS Strategic Plan Goal 2, Objective 2.2.