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Timothy Coolong, William Randle, and Ronald Walcott

Onion (Allium cepa L.) is an economically important vegetable in the United States. Though considered a minor crop in terms of total acreage, onions have high value when compared to other crops and, nationally, their value approaches $800 million. Because harvested onions are routinely stored for long periods, disease can be a major obstacle to the industry. The primary disease reported in stored onions is botrytis neck rot caused by the fungus Botrytis allii (syn. B. aclada). Losses from neck rot can approach 35% of the stored crop. In order to accurately quantify the level of B. allii inoculum in bulbs at harvest to be able to predict potential botrytis neck rot in storage, a quantitative real-time PCR test to quantify levels of B. allii DNA present in onion bulb tissue has been developed. We have employed the TaqMan real time PCR assay and report log-linear (R 2= 0.9915) relationship between B. allii DNA concentration and cycle threshold (Ct) value with a detection limit of 5 pico gram/microliter DNA. In addition, a log-linear standard curve plotting mycelial dry weight against Ct value has been developed to allow prediction of mycelial weight in onion tissue at harvest. Currently, the ability of this test to predict botrytis neck rot during storage is being tested.

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Paola S. Cotroneo, Maria P. Russo, Manuela Ciuni, Giuseppe Reforgiato Recupero, and Angela R. Lo Piero

Genes encoding chalcone synthase (CHS), anthocyanidin synthase (ANS), and UDP-glucose-flavonoid 3-O-glucosyltransferase (UFGT), some of the enzymes of anthocyanin biosynthetic pathway, were assayed in two different experiments using quantitative real-time reverse transcriptase (RT)-PCR, in order to test their transcription levels in the flesh of blood and common orange [Citrus sinensis (L.) Osbeck] fruit, and to investigate their role in anthocyanin accumulation in the same tissue. The first experiment compared a blood orange and a common orange cultivar during seven different fruit maturation stages. This was followed by the test of 11 different genotypes at the end of the winter season. Data collected from the first experiment, over the blood orange cultivar, were statistically analyzed using the Pearson correlation coefficient. Results show that CHS, ANS, and UFGT mRNA transcripts are up- and co-regulated in the blood orange cultivar, whereas they are down-regulated in the common orange cultivar. There is evidence of correspondence between the target genes expression level and the content of the pigment assessed. The second test confirms this correlation and shows that enzyme synthesis levels and pigment accumulation, in plants grown under the same environmental conditions, are dependent on the differences occurring among the genotypes tested. These results suggest that the absence of pigment in the common orange cultivars may be caused by the lack of induction on the structural genes expression. This is the first report on the characterization of the relationships between biosynthetic genes expression and fruit flesh anthocyanin content in blood oranges.

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Takashi Akagi, Yumi Takeda, Keizo Yonemori, Ayako Ikegami, Atsushi Kono, Masahiko Yamada, and Shinya Kanzaki

(arrows indicate the primers). Primers with an asterisk were used in quantitative real-time PCR analysis for M ast ( Table 3 ). Materials and Methods Identification of an ast-linked marker allele conserved among cultivars. To confirm

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K.S. Ling, C.A. Clark, C. Kokkinos, J. R. Bohac, S.S. Hurtt, R. L. Jarret, and A. G. Gillaspie

Sweet potato virus disease (SPVD) is the most devastating virus disease on sweetpotato [Ipomoea batatas (L.) Lam] world wide, especially in East Africa. However, weather it is present in the U.S. is unknown. SPVD is caused by co-infection of sweetpotato feathery mottle virus (SPFMV) and sweetpotato chlorotic stunt virus (SPCSV). Presence of two other potyviruses, sweetpotato virus G (SPVG) and Ipomoea vein mosaic virus (IVMV) has also been confirmed in the U.S. Sweet potato leaf curl virus (SPLCV), a whitefly (Bemisia tabaci) transmitted Begomovirus, also has the potential to spread to commercial sweetpotato fields and poses a great threat to the sweetpotato industry. The U.S. collection of sweetpotato germplasm contains about 700 genotypes or breeding lines introduced from over 20 different countries. Newly introduced sweetpotato germplasm from foreign sources are routinely screened for major viruses with serology and graft-transmission onto indicator plants (Ipomoea setosa). However, a large portion of this collection including heirloom cultivars or old breeding materials has not been systemically screened for these major sweetpotato viruses. In this study, a total of 69 so-called heirloom sweetpotato PI accessions were evaluated for their virus status. We used Real-time PCR to detect five sweetpotato viruses, including four RNA viruses (SPCSV, SPFMV, SPVG, and IVMV) and one DNA virus (SPLCV). A multiplex Real-time RT-PCR system was developed to detect three RNA viruses (SPFMV, SPVG, and IVMV). Preliminary data indicated that about 15% of these heirloom sweetpotato germplasm carried at least one of these viruses tested. Details on virus infection status will be presented.

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Timothy W. Coolong, Ronald R. Walcott, and William M. Randle

could allow for the prediction of storage rot based on inoculum levels at harvest. Conventional diagnostic assays do not have the capacity to reliably quantify mycelial mass in onion tissue; however, quantitative real-time PCR represents one technique by

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Christian A. Wyenandt, Lisa R. Maimone, Kathryn Homa, Angela M. Madeiras, Robert L. Wick, and James E. Simon

cultivars or breeding lines for testing. Table 1. Downy mildew sporulation on leaves of basil cultivars at time of seed collection and detection of Peronospora belbahrii using real-time PCR assay on seed collected following an outbreak of downy mildew at

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Tao Wang, Ruijie Hao, Huitang Pan, Tangren Cheng, and Qixiang Zhang

tested samples as the template performed for each candidate reference gene in triplicate. Reverse transcription quantitative real-time PCR. Quantitative RT-PCR reactions were carried out in 96-well blocks with a PikoReal Real-time PCR System (Thermo

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Douglas C. Whitaker, Mihai C. Giurcanu, Linda J. Young, Pedro Gonzalez, Ed Etxeberria, Pamela Roberts, Katherine Hendricks, and Felix Roman

pathogen (SWFREC). Real-time PCR reactions were performed using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA) using TaqMan ® Fast Advance PCR Master Mix (Applied Biosystems) in a 20 μL reaction. The standard amplification

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Cai-Hong Jia, Ju-Hua Liu, Zhi-Qiang Jin, Qiu-Ju Deng, Jian-Bin Zhang, and Bi-Yu Xu

analyzed by BLAST (< http://ncbi.nlm.nih.gov/blast >). Quantitative real-time RT-PCR analysis of MaMDH expression. For real-time quantitative reverse transcription (RT)–PCR, total RNA was extracted from the roots, rhizomes, leaves, flowers, and fruit

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Zhengke Zhang, Runshan Fu, Donald J. Huber, Jingping Rao, Xiaoxiao Chang, Meijiao Hu, Yu Zhang, and Nina Jiang

-length expansin gene including the complete coding region. The clone was designated as CDK-Exp3 (accession number FJ455264). Real-time polymerase chain reaction analysis. Two oligonucleotide primer pairs used for real-time PCR analysis were designed according to