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Karen R. Harris, Brian M. Schwartz, Andrew H. Paterson, and Jeff A. Brady

Thirty-one partial bermudagrass (Cynodon spp.) disease-resistance gene analogs (BRGA) were cloned and sequenced from diploid, triploid, tetraploid, and hexaploid bermudagrass using degenerate primers to target the nucleotide binding site (NBS) of the NBS–leucine-rich repeat (LRR) resistance gene family. Alignment of deduced amino acid sequences revealed that the conserved motifs of the NBS are present and all sequences have non-Drosophila melanogaster Toll and mammalian interleukin-1 receptor (TIR) motifs. Using a neighbor-joining algorithm, a dendrogram was created and nine groups of deduced amino acid sequences from bermudagrass could be identified from those sequences that span the NBS. Four BRGA markers and 15 bermudagrass expressed sequence tags (ESTs) with similarity to resistance genes or resistance gene analogs were placed on a bermudagrass genetic map. Multiple BRGA and EST markers mapped on T89 linkage groups 1a and 5a and clusters were seen on T89 19 and two linkage groups previously unidentified. In addition, three primers made from BRGA groups and ESTs with similarity to NBS-LRR resistance genes amplify NBS-LRR analogs in zoysiagrass (Zoysia japonica or Z. matrella) or seashore paspalum (Paspalum vaginatum). This gives evidence of conservation of NBS-LRR analogs among the subfamilies Chloridoideae and Panicoideae. Once disease resistance genes are identified, these BRGA and EST markers may be useful in marker-assisted selection for the improvement of disease resistance in bermudagrass.

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I.K. Kang, D.A. Starrett, S.G. Suh, J.K. Byun, and K.C. Gross

We are studying β-galactosidase (EC 3.2.1.23) in softening persimmon fruit (Diospyros kaki L.f. cv Fuyu) and hope to decrease the rate of softening by inserting an antisense construct of the β-galactosidase gene. The N-terminal amino acid sequence of persimmon fruit β-galactosidase was recently reported. Here we report the cloning of a putative β-galactosidase gene from persimmons. Degenerate oligonucleotide primers were synthesized based on the amino acid sequence. 5′-RACE (rapid amplification of cDNA ends) was done using persimmon Poly A+ mRNA extracted using a phenol: chloroform/LiCl method. Purification was done on an oligo dT-cellulose column. A fragment of roughly 150 base pairs was purified by agarose gel electrophoresis and subcloned into the pCR-Script cloning vector from Stratagene. After sequencing and verifying the insert's identity, it will be isolated and used to screen a persimmon fruit cDNA library currently being constructed. Ultimately this cDNA clone will be used to make an antisense β-galactosidase construct that will be transformed into persimmon.

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Jiahua Xie, Todd C. Wehner, and Mark A. Conkling

Combining the use of PCR and single-strand conformation polymorphisms (SSCP), nine sequences from the cucumber genome were successfully identified and cloned that encoded two well-conserved asparagine-proline-alanine (NPA) domain homologues to aquaporin genes. The sensitivity and detection efficiency of SSCP and restriction enzyme analysis for detecting DNA sequence variation were evaluated using similar-sized DNA fragments. The SSCP analysis was more sensitive and efficient for discriminating different clones than restriction enzyme analysis, although some sequence variation inside similar-sized DNA fragments could be identified by restriction analysis. Consideration of the results of SSCP analysis with DNA sequence information indicated that one or two base pair changes in the amplified regions could be detected. Moreover, the SSCP analysis results of genomic DNA PCR products that were amplified by degenerate primers can provide rough information about the number of member genes. If the SSCP bands of a cloned fragment (such as CRB7) did not have the corresponding bands from genomic DNA PCR products, that fragment might be a misamplified product. The PCR-based SSCP method with degenerate oligonucleotide primers should facilitate the cloning of member genes.

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H.P.V. Rupasinghe, K.C. Almquist, G. Paliyath, and D.P. Murr

We tested the hypothesis that conversion of 3-hydroxy-3-methylglutaryl co-enzyme A (HMG CoA) to mevalonate (MVA) catalyzed by HMG CoA reductase (HMGR) is the rate limiting step for α-farnesene biosynthesis of apples. In higher plants, isopentenyl pyrophosphate (IPP) is derived via two pathways: 1) the classical mevalonate pathway, and 2) the novel glyceraldehyde-3-phosphate (GAP)/pyruvate pathway independent of HMGR action. When apple skin discs were incubated with MVA, or GAP and pyruvate, MVA increased α-farnesene levels in the skin but not GAP and pyruvate. Treating apple fruits with Lovastatin (1000 ppm), a competitive inhibitor of HMGR, inhibited α-farnesene accumulation in the skin by 20% to 50% during storage. Content of α-farnesene in the skin increased during the first 2 to 4 months in storage, and then decreased. In contrast, HMGR activity, as determined by the conversion of [4-3H]HMG CoA to MVA in the total membrane and soluble fraction, was the highest at the time of harvest and gradually decreased during 5 months of storage in air at 0 °C. The potent ethylene action inhibitor 1-MCP inhibited ethylene production and α-farnesene evolution by 99% and 97%, respectively. The effect of 1-MCP on in vitro activity of HMGR was marginal (≈30% inhibition). 1-MCP inhibited respiratory CO2 evolution by 50%, which suggests also that inhibition by 1-MCP of α-farnesene synthesis in apple could be regulated by the acetyl CoA pool. In plants, HMGR is encoded by a small gene family and differentially expressed. As the first step of studying the molecular mechanism of HMGR regulation, we have isolated a 444-bp fragment of apple hmgr gene using apple skin mRNA and degenerate oligonucleotides designed against conserved regions of plant hmgr genes.

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Akihiro Itai, Takaaki Igori, Naoko Fujita, Mayumi Egusa, Motoichiro Kodama, and Hideki Murayama

of total RNA from O-276-inoculated ‘Nijisseiki’ leaves using M-MLV reverse transcriptase (ReverTra Ace; Toyobo, Tokyo, Japan). The ACC synthase gene was amplified from cDNA by polymerase chain reaction (PCR) using the following degenerate

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Nobutaka Shiraiwa, Kaori Kikuchi, Ichiro Honda, Masayoshi Shigyo, Hiroko Yamazaki, Daisuke Tanaka, Kenji Tanabe, and Akihiro Itai

reaction (PCR) using two degenerate primers; sense: 5′-ATGTGGYMNGARGGNTTYAC-3′ and antisense: 5′-GTRTGNGCNGCNAGNCCCAT-3′ (Y = C/T, M = A/C, N = A/C/G/T, R = A/G). Degenerate oligonucleotide primers were designed from conserved regions of A. thaliana GA4

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Matthew A. Escobar, Andrew Shilling, Pine Higgins, Sandra L. Uratsu, and Abhaya M. Dandekar

., 1983 ). To isolate a fragment of a PPO-encoding gene from the DNA, degenerate oligonucleotides were designed based on the highly conserved CuA and CuB copper-binding domains in plant PPOs. The primers Walt 2 (5′-AAR GAR GCI GAY MGI CCI GCI GGI GCI YTI

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Kelly J. Vining, Q Zhang, C.A. Smith, and T.M. Davis

Shen, K.A. Meyers, B.C. Nurul Islam-Faridi, M. Chin, D.B. Stelly, D.M. Michelmore, R.W. 1998 Resistance gene candidates identified by PCR with degenerate oligonucleotide primers map to clusters of