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Santiago Vilanova, Carlos Romero, Gerardo Llácer, María Luisa Badenes and Lorenzo Burgos

This report shows the PCR-based identification of the eight known self-(in)compatibility alleles (S 1 to S 7 and S c) of apricot (Prunus armeniaca L.). Two sets of consensus primers, designed from P. armeniaca S-RNase genomic sequences and sweet cherry (P. avium L.) S-RNase-cDNAs, were used to amplify fragments containing the first and the second S-RNase intron, respectively. When the results obtained from the two PCRs were combined, all S-alleles could be distinguished. The identity of the amplified S-alleles was verified by sequencing the first intron and 135 base pairs (bp) of the second exon. The deduced amino acid sequences of these fragments showed the presence of the C1 and C2 Prunus L. S-RNase conserved regions. These results allowed us to confirm S-genotypes previously assigned by stylar ribonuclease analyses and to propose one self-(in)compatibility group (I) and one universal donor group (O) containing unique S-genotypes and self-compatible cultivars (SC). This PCR-based typing system also facilitates the identification of the S c-allele and might be a very useful tool for predicting self-compatibility in apricot breeding progenies.

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Kentaro Kitahara and Shogo Matsumoto

An S-allele cDNA was cloned from pistils of 'McIntosh' apple (Malus ×domestica Borkh.). The allele, designated Si in Japan and S10 in Europe, is an S-RNase that is very similar (94%) to the S3-RNase at the deduced amino acid sequence level. This allele can be detected by amplification using the polymerase chain reaction (PCR) and specific primers, followed by digestion with restriction enzyme EheI. The S10 allele was discovered in 'Empire', 'Maypole', 'Shinano Red', 'Spencer', and 'Vista Bella'. The S-allele cDNAs sequenced to date are listed with their Japanese and European designations.

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Ping Li, Dong Liu, Min Guo, Yuemin Pan, Fangxin Chen, Huajian Zhang and Zhimou Gao

Biotechnology Co., Shanghai, China) on isolates of different mating types and pathogenicity. Twenty-three isolates were selected ( Table 1 ). A 25-µL reaction mixture consisting of 2.5 µL 10X PCR buffer, 200 µM dNTPs, 0.2 µM primer, 0.5 µL template DNA, and 1

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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 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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Chu-Hui Chiang, Tsong-Ann Yu, Shu-Fang Lo, Chao-Lin Kuo, Wen-Huang Peng and Hsin-Sheng Tsay

the ITS sequences ( Baldwin et al., 1995 ; Hillis et al., 1991 ). Based on the ITS sequences, the multiplex PCR method can be used to amplify target-containing samples by mixing specific primers in a single PCR. This approach has been widely used to

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Yonghong Guo, Zong-Ming Cheng and James A. Walla

Five simplified DNA preparation procedures for polymerase chain reaction (PCR) amplification were tested for detection of phytoplasmas from infected herbaceous and woody plants. Thin freehand cross-sections made from infected plant tissues and stored in acetone were used as sources for DNA preparation. The tissue sections were treated by: 1) grinding in sodium hydroxide; 2) sonicating in water; 3) microwaving in water; 4) boiling in sodium hydroxide; or 5) placing directly in PCR tube. PCR amplification was performed with a universal phytoplasma-specific primer pair in a reaction buffer containing 0.5% (v/v) Triton X-100, 1.5 mm magnesium chloride, and 10 mm Tris-HCl. All five procedures provided phytoplasmal template DNA for successful PCR amplification from infected herbaceous plants {periwinkle [Catharanthus roseus (L.) G. Don (periwinkle)], carrot (Daucus carota L.), maize (Zea mays L.)}, while the grinding, microwaving, and boiling procedures also allowed positive amplification from a woody plant [green ash (Fraxinus pennsylvanica Marsh.)]. The quality of the resulting DNA was adequate for subsequent identification of the aster yellows and ash yellows phytoplasmas through nested-PCR using phytoplasma group-specific primer pairs. These methods provide remarkable savings in labor and materials, making disease testing and indexing of plant materials much more attractive.

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Javier Sanzol and Timothy P. Robbins

determined by crossing and S-RNase genotypes as detected by PCR and sequence analysis ( Sanzol et al., 2006 ). Since the first report characterizing S-RNases in european pear ( Zuccherelli et al., 2002 ), sequences for 18 different alleles have been

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Gennaro Fazio, Jack E. Staub and Sang Min Chung

Highly polymorphic microsatellites or simple sequence repeat (SSR), along with sequence characterized amplified region (SCAR) and single nucleotide polymorphisms (SNP), markers are reliable, cost-effective, and amenable for large scale analyses. Molecular polymorhisms are relatively rare in cucumber (Cucumis sativus L.) (3% to 8%). Therefore, experiments were designed to develop SSR, SCAR and SNP markers, and optimize reaction conditions for PCR. A set of 110 SSR markers was constructed using a unique, strategically applied methodology that included the GeneTrapper (Life Technologies, Gaithersburg, Md.) kit to select plasmids harboring microsatellites. Of these markers, 58 (52%) contained dinucleotide repeats (CT, CA, TA), 21 (19%) possessed trinucleotide repeats (CTT, ATT, ACC, GCA), 3 (2.7%) contained tetranucleotide repeats (TGCG, TTAA, TAAA), 4 (3.6%) enclosed pentanucleotide repeat (ATTTT, GTTTT, GGGTC, AGCCC), 3 (2.7%) contained hexanucleotide repeats (CCCAAA, TAAAAA, GCTGGC) and 21 possessed composite repeats. Four SCARs (L18-3 SCAR, AT1-2 SCAR, N6-A SCAR, and N6-B SCAR) and two PCR markers based on SNPs (L18-2H19 A and B) that are tightly linked to multiple lateral branching (i.e., a yield component) were also developed. The SNP markers were developed from otherwise monomorphic SCAR markers, producing genetically variable amplicons. The markers L18-3 SCAR and AT1-2 SCAR were codominant. A three-primer strategy was devised to develop a codominant SCAR from a sequence containing a transposable element, and a new codominant SCAR product was detected by annealing temperature gradient (ATG) PCR. The use of a marker among laboratories can be enhanced by methodological optimization of the PCR. The utility of the primers developed was optimized by ATG-PCR to increase reliability and facilitate technology transfer. This array of markers substantially increases the pool of genetic markers available for genetic investigation in Cucumis.

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Daniel J. Bell, Lisa J. Rowland, James J. Polashock and Frank A. Drummond

among participants in controlled field crosses. Rowland et al. (2003b) developed express sequence tag-polymerase chain reaction (EST-PCR) markers from EST libraries derived from floral buds of cold acclimated and nonacclimated highbush blueberry

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Anna L. Hale, Mark W. Farnham and Monica A. Menz

Breeders of cole crops (Brassica oleracea L.) have an interest in utilizing current and emerging PCR-based marker systems to differentiate elite germplasm. However, until efficiency and cost-effectiveness are determined, most breeders are hesitant to change methods. In this study, our goal was to compare simple sequence repeat (SSR), amplified fragment-length polymorphism (AFLP), and sequence-related amplified polymorphism (SRAP) marker systems for their effectiveness in differentiating a diverse population of 24 elite broccoli (B. oleracea Italica Group) inbreds. Published SSR primer sequences for Brassica L. species were used along with AFLP and SRAP primer combinations. Several SSR primers failed to amplify DNA in the broccoli population, but all AFLP and SRAP primer combinations produced multiple bands. Twenty-nine percent of the SSR primers were monomorphic, while most of the remaining primers detected only one or two differences among inbreds. AFLP and SRAP methods produced multiple differences per primer in almost every case. Phenetic analysis revealed that the type of marker affected the classification of the genotypes. All three marker systems were able to successfully differentiate between the 24 elite inbreds, however, AFLPs and SRAPs were more efficient, making them better alternatives than SSRs over other established methods for fingerprinting B. oleracea inbreds.