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. Considerable diversity exists in plant tissues for the presence and intensity of water soluble anthocyanin pigments. Whereas the genes responsible for the biosynthesis of these compounds are well characterized, a major gap in our knowledge exists concerning regulatory mechanisms which control differential accumulation of anthocyanins in different plant tissues. Our prior research demonstrated differential expression of anthocyanin structural and regulatory genes for pigment accumulation in fruits and flowers. In contrast, elevated expression of regulatory genes was not required for structural gene expression and anthocyanin accumulation in foliar tissue. Currently, we have isolated and sequenced miRNA from pepper in order to identify regulatory elements that interact with anthocyanin structural and regulatory gene products. Our research has demonstrated that different genetic mechanisms account for pigmentation in reproductive and vegetative tissues and is important for elucidating the genetic control of pigment biosynthesis and development of new plants with enhanced pigmentation. This research is cooperative with Virginia Polytechnic Institute and State University, Blacksburg, VA. The project was monitored through site visits, e-mail, and conference calls.