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Shinya Kanzaki, Keizo Yonemori, Akira Sugiura, Akihiko Sato, and Masahiko Yamada

Japanese persimmon (Diospyros kaki Thunb.) cultivars are classified into four types depending upon the nature of astringency loss of the fruit. Among them, the pollination-constant and nonastringent (PCNA) type is the most desirable for fresh fruit consumption due to the trait of stable loss of astringency on the tree with fruit development. Lack of tannin accumulation is the main cause of natural astringency loss in PCNA-type fruit, and is qualitatively inherited. The PCNA trait is recessive to the non-PCNA trait. In this study, we investigated amplified fragment length polymorphism (AFLP) markers for the trait of natural astringency loss of PCNA-type fruit using bulked segregant analysis (BSA) for efficient selection of PCNA type plants in a breeding population. A total of 128 primer combinations were tested and one AFLP marker was found to be linked to the dominant allele controlling the trait for astringency. This marker, EACC/MCTA-400, was absent in all of the PCNA-type plants tested, whereas it was present in about half of the non-PCNA-type plants tested. However, RFLP analysis using this marker enabled the detection of the other dominant allele, and all PCNA-type plants could be distinguished from the non-PCNA-type plants. Application of this marker system will be useful for the selection of PCNA-type plants in persimmon breeding.

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Geunhwa Jung, Dermot P. Coyne, Paul W. Skroch, James Nienhuis, E. Arnaud-Santana, James Bokosi, H.M. Ariyarathne, James R. Steadman, James S. Beaver, and Shawn M. Kaeppler

Random amplified polymorphic DNA (RAPD) markers were used to construct a partial linkage map in a recombinant inbred population derived from the common bean (Phaseolus vulgaris L.) cross BAC 6 × HT 7719 for studying the genetics of disease resistance in common bean. The linkage map spanned 545 cM and included 75 of 84 markers used in this study. The population of 128 recombinant inbred lines was evaluated for resistance to common bacterial blight, foliar resistance to web blight [WB; Thanatephorus cucumeris (Frank) Donk], and resistance to rust [Uromyces appendiculatus var. appendiculatus (Pers.:Pers) Unger]. Common bacterial blight [CBB; Xanthomonas campestris pv. phaseoli (Smith) Dye] resistance was evaluated for CBB strain Epif-IV in later-developed trifoliolate leaves and for CBB strain EK-11 in seeds, first trifoliolate leaves, later-developed trifoliolate leaves, and pods. In addition, lines were rated for plant uprightness and branch density. Two to six markers accounted for 14% to 34% of the phenotypic variation for each trait. Significant marker locustrait associations were found for 14 mapped loci and 7 of the 9 unmapped markers. The distribution of detected QTL appeared to be nonrandom with most significant markers associated with more than one trait or closely linked to markers significantly associated with variation for a different trait. One marker, BC4091250, was significantly associated with WB resistance, resistance for CBB strain Epif-IV in later-developed trifoliolate leaves, and resistance for CBB strain EK-11 in first trifoliolate leaves, later-developed trifoliolate leaves, and pods. A rust resistance gene was mapped in an interval 14.6 cM from RAPD marker H191050 and 12.5 cM from marker AJ16250.

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M.R. Stevens, P.D. Griffiths, J.W. Scott, D.K. Heiny, and D.D Rhoads

Sw-5 is a locus introgressed from Lycopersicon peruvianum to some L. esculentum lines conferring dominant resistance to TSWV. Restriction fragment length polymorphism (RFLP) analyses positions Sw-5 to the long arm of chromosome 9 in the sub-telomeric region between CT71 and CT220. RFLP analyses suggest the introgressed region begins distal to CT71, includes CT220, and may extend to the telomere. Randomly amplified polymorphic DNA (RAPD) analyses with >700 random 10-mer primers identified a single 2.2-kbp band with one primer (primer #72 GAGCACGGGA) that is tightly linked to Sw-5. However, we have also produced an equivalent 2.2-kbp band in analysis of other TSWV-susceptible tomato breeding lines. Thus, this band likely derives from L. esculentum DNA very near to Sw-5 and the introgressed region. Additional analyses have recently detected a potential co-dominant RAPD polymorphism linked to Sw-5.

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Lisa J. Rowland, Smriti Mehra, Anik L. Dhanaraj, Elizabeth L. Ogden, Janet P. Slovin, and Mark K. Ehlenfeldt

Because randomly amplified polymorphic DNA (RAPD) is the only type of molecular marker that has been used extensively in blueberry (Vaccinium spp.) for mapping and DNA fingerprinting of cultivars, there is a need to develop a new, robust marker system. Expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants, were used to develop EST-PCR markers for blueberry. Thirty clones, picked at random from the cDNA library, were single-pass sequenced from the 5' and 3' ends. Thirty PCR primer pairs were designed from the ends of the best quality sequences that were generated and were tested in amplification reactions with genomic DNA from 19 blueberry genotypes, including two wild selections (the original parents of a mapping population), and 17 cultivars. Fifteen of the 30 primer pairs resulted in amplification of polymorphic fragments that were detectable directly after ethidium bromide staining of agarose gels. Several of the monomorphic amplification products were digested with the restriction enzyme AluI and approximately half resulted in polymorphic-sized fragments (cleaved amplified polymorphic sequences or CAPS markers). The polymorphic EST-PCR and CAPS markers developed in this study distinguished all the genotypes indicating that these markers should have general utility for DNA fingerprinting and examination of genetic relationships in blueberry. Similarity values were calculated based on the molecular marker data, and a dendrogram was constructed based on the similarity matrix. Coefficients of coancestry were calculated for each pair of genotypes from complete pedigree information. A fair correlation between similarity coefficients calculated from marker data and coefficients of coancestry was found.

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Darush Struss, Riaz Ahmad, Stephen M. Southwick, and Manuela Boritzki

Simple sequence repeats (SSRs) and amplified fragment-length polymorphisms (AFLPs) were used to evaluate sweet cherry (Prunus avium L.) cultivars using quality DNA extracted from fruit flesh and leaves. SSR markers were developed from a phage library using genomic DNA of the sweet cherry cultivar Valerij Tschkalov. Microsatellite containing clones were sequenced and 15 specific PCR primers were selected for identification of cultivars in sweet cherry and for cross-species amplification in Prunus. In total, 48 alleles were detected by 15 SSR primer pairs, with an average of 3.2 putative alleles per primer combination. The number of putative alleles ranged from one to five in the tested cherry cultivars. Forty polymorphic fragments were scored in the tested cherry cultivars by 15 SSRs. All sweet cherry cultivars were identified by SSRs from their unique fingerprints. We also demonstrated that the technique of using DNA from fruit flesh for analysis can be used to maintain product purity in the market place by comparing DNA fingerprints from 12 samples of `Bing' fruit collected from different grocery stores in the United States to that of a standard `Bing' cultivar. Results indicated that, with one exception, all `Bing'samples were similar to the standard. Amplification of more than 80% of the sweet cherry primer pairs in plum (P. salicina), apricot (P. armeniaca) and peach (P. persica L.) showed a congeneric relationship within Prunus species. A total of 63 (21%) polymorphic fragments were recorded in 15 sweet cherry cultivars using four EcoRI-MseI AFLP primer combinations. AFLP markers generated unique fingerprints for all sweet cherry cultivars. SSRs and AFLP polymorphic fragments were used to calculate a similarity matrix and to perform UPGMA cluster analysis. Most of the cultivars were grouped according to their pedigree. The SSR and AFLP molecular markers can be used for the grouping and identification of sweet cherry cultivars as a complement to pomological studies. The new SSRs developed here could be used in cherry as well as in other Prunus species for linkage mapping, evolutionary and taxonomic study.

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Paul Skroth, Jim Nienhuis, Geunhwa Jung, and Dermont Coyne

30 POSTER SESSION 4 (Abstr. 460-484) Breeding/Genetics/Molecular Markers

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Charles J. Simon and Richard M. Hannan

30 POSTER SESSION 4 (Abstr. 460-484) Breeding/Genetics/Molecular Markers

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S.O. Park, A. Dursun, D.P. Coyne, and G. Jung

106 POSTER SESSION (Abstr. 335–343) Breeding and Genetics–Vegetables II (Molecular Markers and Physiological Genetics)

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M.I. Buteler, D.R. LaBonte, R.L. Jarret, and R.E. Macchiavelli

Using codominant molecular markers (microsatellites) for paternity identification was investigated in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.]. Two experimental populations (CIP and LAES), each consisting of progeny of known parentage, were scored for the presence or absence of alleles segregating at IB-316 and IB-318 microsatellite loci. Paternity was assessed using paternity exclusion and the most-likely parent methods. In the former, paternity is assigned based on the identification of incompatible parent-progeny marker data. In contrast, the latter method incorporates paternity exclusion and a log-likelihood or LOD score that weighs progeny allelic patterns as to the likelihood that they could have come from a given paternal parent. The number of correctly allocated progeny differed for the methods. Paternity exclusion correctly allocated 7% and 25% of the progeny in the LAES and CIP populations, respectively. The most-likely parent method correctly allocated 23% and 88% of the progeny in the LAES and CIP populations, respectively. The greater misassignments in the LAES population were attributed to low allelic diversity at the LAES IB-318 locus and a larger parental population. This study demonstrates the feasibility of identifying paternity in sweetpotato using a minimal number of loci.

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Claudio Cantini, Antonio Cimato, Antonella Autino, Alessandro Redi, and Mauro Cresti

Council of Italy, which has never been previously characterized by molecular markers. We investigated whether the polymorphism displayed by DNA amplification with 12 different primer pairs would be sufficient to distinguish among all the genotypes