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R.J. Griesbach, R.M. Beck, and J.R. Stehmann

A method was developed to characterize the genetic heterogeneity of the chalcone synthase gene intron within the Petunia integrifolia (Hook.) Schinz & Thell. species complex. The DNA from wild species collected from known locations was used to amplify the chalcone synthase gene intron through the polymerase chain reaction (PCR). The resulting PCR product was then characterized by Rsa 1 restriction, revealing a degree of heterogeneity that could be used to characterize the species genetically. Of the four different species that were characterized, two could be placed in the same genetic grouping. This study shows that the variation in the intron of the Chs A gene may be species-specific.

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Robert Griesbach* and Ronald Beck

The genetic distance for three Petunia species was determined based upon an intron in the chalcone synthase gene. The sequence of the intron was obtained for P. integriflolia ssp. integriflolia var. depauperata, P. integriflolia ssp. integriflolia Torres ecotype, P. altiplana and P. littoralis. These species are very closely related and believed by some taxonomists to be part of a large single species complex. In all the taxa, the intron contained multiple repeated and inverted sequences. The P. integriflolia ssp. integriflolia Torres ecotype intron differed from the P. integriflolia subsp. integriflolia var. depauperata intron in 3 of 930 nucleotides. While, the P. littoralis intron differed from the P. integrifolia subsp. integrifolia var. depauperata intron in 15 of 930 nucleotides. As compared to the P. integrifolia subsp. integriflolia var. depauperata intron, the intron in P. altiplana intron was longer (1125 bp), had a section of 338 nucleotides with a completely different sequence, and differed by 27 of 787 nucleotides in the common sequence.

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Diane Luth and Gloria A. Moore

Many Citrus species accumulate large amounts of flavonoids, specifically flavanone glycosides, that impart an intensely bitter flavor to the fruit. In grapefruit, this bitterness decreases the acceptability of fresh fruit and juice; in other species, these compounds entirely prevent fruit consumption. No physiological purpose for the accumulation of these compounds has been determined; they do not function in color production or, as far as is known, in defense responses. As has been found in other plants, the accumulation of specific flavonoids in citrus appears to be under genetic control, but no definitive genetic analyses have been done. The long-term objective of this research is to determine whether the production of bitter-tasting flavanone glycosides (neohesperidosides) in citrus can be manipulated using molecular genetic techniques. As a first step, cDNAs for chalcone synthase and chalcone isomerase, the first two biosynthetic enzymes specific to the flavonoid pathway, were isolated from a grapefruit leaf cDNA library using heterologous probes. Southern analyses showed that both genes appear to be part of multigene families, as expected. Northern analyses are underway to determine steady state mRNA levels in various grapefruit tissues, and Western blots to characterize protein expression are also being attempted.

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R.J. Griesbach and R.M. Beck

The sequence of the intron within the chalcone synthase A gene (ChsA) was used to characterize Petunia integrifolia subsp. integrifolia var. depauperata (Fries) Smith et Downs, P. altiplana Ando et Hashimoto, P. littoralis Smith et Downs, and an unknown taxon from the town of Torres in Brazil. Based upon the intron, the Torres taxon most closely resembled P. integrifolia. The unrooted phylogenetic tree suggested that P. integrifolia was more closely related to P. littoralis than P. altiplana.

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R.J. Griesbach and R. Beck

A new method was developed to analyze genetic diversity among Petunia species. The first intron of the chalcone synthase A gene was cloned through the polymerase chain reaction (PCR) and partially sequenced. This sequence was used to dissect the intron into two halves (3' and 5' halves). The PCR primers for the 5' half amplified a single fragment that was the same length for all of the species that were studied. Restriction fragment length polymorphism (RFLP) analysis of the 5' half resulted in the same number and length of fragments for all the species that were evaluated. The PCR primers for the 3' half amplified a number of fragments that were characteristic for each species. This research provides a new tool for measuring genetic diversity. Genetic diversity measured with this tool should be closely related to evolutionary distance.

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E. Firoozabady, C. Lemieux, J. Nicholas, A. Otten, M. Akerboom, K. Robinson, and N. Courtney-Gutterson

We developed an Agrobacterium-mediated leaf disk transformation method for chrysanthemum. We introduced a chimeric chalcone synthase (CHS) gene isolated from chrysanthemum into cv. Moneymaker (pink type) to produce white-flowered plants. The CHS coding sequence was in antisense or sense orientation relative to the CaMV 35S promoter. 3.6% (3/83) antisense-transformed plants and 1.5% (2/133) sense-transformed plants produced completely white flowers. Pigment analysis revealed that this was due to a block at CHS. To study stability of color change of the white Moneymaker plants. Moneymaker, a Moneymaker regenerant, an antisense white (2706),and a sense white (31435) were compared. There was no difference between Moneymaker and the regenerant. Both 2706 and 31435 were vegetatively propagated with good stability; all plants produced white or very pale pink flowers. 2706 flowered 7 days late and 31435 10-12 days late. Flower number was similar for all four lines tested.

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Zhiguo Ju

Delicious apples were harvested and analyzed every 7 days from the start of fruit coloration to commercial harvest. Chalcone synthase (CS) activity increased from 521 to 4120 fkat/g protein during 30 days, while UDPG glucosyltransferase (UDPGTF) activity increased from 0 to 6570 fkat/g protein. These changes significantly correlated with anthocyanin synthesis in fruit skin. Ethephon enhanced activities of both enzymes for 25 days after application, but not beyond. Cycloheximide inhibited CS and UDPGTF activities by 57% and 72%, respectively, and this could not be overcome by ethephon treatment. Bagging prevented fruit from coloring, and removing bags before maturation promoted it. Activity of both enzymes was nil in bagged fruit, and increased dramatically after bag removal. Cycloheximide applied at bag removal reduced CS and UDPGTF activities 74% and 91%, respectively, and decreased anthocyanin synthesis by 82%. The results showed that both CS and UDPGTF were positively correlated with anthocyanin formation and both required de novo synthesis during fruit coloring, although CS had existed before that.

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Rasika G. Mudalige-Jayawickrama, Michele M. Champagne, A. David Hieber, and Adelheid R. Kuehnle

kind support and providing facilities for radioactive work. Two new gene sequences were submitted to GenBank. Accession numbers are AY741318 and AY741319 for dihydroflavonol 4-reductase gene and chalcone synthase gene, respectively.

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Huiling Wang, Wei Wang, Weidong Huang, and Haiying Xu

= chalcone synthase; CHI = chalcone isomerase; F3H = flavanone-3-hydroxylase; FLS = flavonol synthase; DFR = dihydroflavonol reductase; ANS = anthocyanidin synthase; ANR = anthocyanidin reductase; UFGT = UDP-Glc:flavonoid 3-O-glucosyltransferase. Salicylic

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Qianqian Shi, Long Li, Lin Zhou, and Yan Wang

. delavayi ; Pd, purple–red flower of P. delavayi. The TUB gene was used as an internal control, and relative transcript levels are presented as the means with sd s of three technical and three biological replications. CHS , chalcone synthase gene; CHI