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Yiping Zhang and John R. Stommel

The carotenoids have an important influence on tomato fruit quality and enhance the fruit contribution to human nutrition. Expression of the high pigment (hp) locus in tomato results in increased total carotenoids and increased efficiency of utilization of the polyenes. A similar mutant, dark green (dg), contains higher level of chlorophyll in immature fruit and results in darker red pigmentation, both externally and internally in ripe fruit. Random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) analyses were performed using two pairs of near isogenic lines (NILs) designed to be isogenic at the hp and dg loci. Sixty-four AFLP primer pairs and more than 1000 RAPD 10-mer primers were screened for polymorphism between each pair of the NILs. One RAPD marker was identified to be linked to the hp gene, and two AFLP primer pairs showed polymorphic fragments which distinguished the dg NILs. The markers identified in this study will be converted to allele specific SCAR (sequence characterized amplified region) markers, which are more useful in marker-assisted selection breeding programs.

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Jack E. Staub, Fenny Dane, Kathleen Reitsma, Gennaro Fazio, and Anabel López-Sesé

Genetic relationships among 970 cucumber (Cucumis sativus L.) plant introductions (PIs) in the U.S. National Plant Germplasm System (NPGS) were assessed by observing variation at 15 isozyme loci. Allozyme frequency data for these PIs were compared to allozyme variation in heirloom and modern (H&M) cultivars released from 1846-1985 (H&M cultivars; 178 accessions), and experimental commercial (EC) germplasm (EC germplasm; 82 accessions) in use after 1985. Multivariate analysis defined four distinct groups of accessions (Groups A-D), where Group A consisted of PIs received by the NPGS before 1992, Group B contained PIs from India and China obtained by NPGS after 1992, Group C consisted of EC germplasm, and Group D contained H&M cultivars. Morphological, abiotic stress (water and heat stress tolerance) and disease resistance evaluation data from the Germplasm Resources Information Network (GRIN) for the PIs examined were used in conjunction with estimates of population variation and genetic distance estimates to construct test arrays and a core collection for cucumber. Disease resistance data included the evaluation of angular leafspot [Pseudomonas lachrymans (E.F. Smith) Holland], anthracnose [Colletotrichum lagenarium (Ross.) Ellis & Halst], downy mildew [Pseudoperonospora cubensis (Berk. & Curt) Rostow], rhizoctonia fruit rot (Rhizoctonia solani Kuhn), and target leafspot [Corynespora cassiicola (Berk. & Curt) Wei] pathogenicity. The test arrays for resistance-tolerance to angular leafspot, anthracnose, downy mildew, rhizoctonia fruit rot, target leafspot, and water and heat stress consisted of 17, 16, 17, 16, 17, 16, and 16 accessions, respectively. The core collection consisted of accessions in these test arrays (115) and additional 32 accessions that helped circumscribe the genetic diversity of the NPGS collection. The core collection of 147 accessions (115 + 32) represents ≈11% of the total collection's size (1352). Given estimates of genetic diversity and theoretical retention of diversity after sampling, this core collection could increase curatorial effectiveness and the efficiency of end-users as they attempt to identify potentially useful germplasm.

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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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Soon O. Park, Dermot P. Coyne, Nedim Mutlu, James R. Steadman, and Geunhwa Jung

Common bacterial blight, incited by Xanthomonas campestris pv. phaseoli (Xcp), is a serious disease of common bean (Phaseolus vulgaris). RAPD markers and flower color (V gene) previously had been reported to be associated with six QTL affecting leaf and pod resistance to Xcp. However, the markers for the QTL were not confirmed in different populations and environments to indicate their merit in breeding. Our objective was to determine if the associations of RAPD markers and the V gene with QTL for leaf and pod resistance to Xcp in a RI backcross population from the cross BC2F6 `PC-50' × XAN-159 and for leaf resistance to Xcp in a F2 population from a different cross Pinto `Chase' × XAN-159 could be confirmed. Among six QTL previously detected, five in the RI backcross population and three in the F2 population were confirmed to be associated with resistance to Xcp. The V gene and RAPD marker BC437.1050 on linkage group 5 were most consistently associated with leaf and pod resistance to two to five XCP strains in the RI backcross population and with leaf resistance to two Xcp strains in the F2 population. The confirmed marker BC437.1050 and V gene on linkage group 5, along with other resistance genes from other germplasm, could be used to pyramid the different genes into a bean cultivar to enhance the resistance to Xcp.

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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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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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Courtney A. Weber, Gloria A. Moore, Zhanao Deng, and Fred G. Gmitter Jr.

Mapping quantitative trait loci (QTL) associated with freeze tolerance was accomplished using a Citrus grandis (L.) Osb. × Poncirus trifoliata (L.) Raf. F1 pseudo-testcross population. A progeny population of 442 plants was acclimated and exposed to temperatures of -9 °C and -15 °C in two separate freeze tests. A subpopulation of 99 progeny was genotyped for random amplified polymorphic DNA (RAPD), cleaved amplified polymorphic sequence (CAPS), sequence characterized amplified region (SCAR), and sequence tagged site (STS) markers to produce a linkage map for each parent. Potential QTL were identified by interval mapping, and their validity was corroborated with results from means comparison (t test), one-way analysis of variance (F test), and bulked segregant analysis (BSA). Multiple analytical methods provided evidence supporting putative QTL and decreased the probability of missing significant QTL associated with freeze tolerance. QTL with a large effect on freeze tolerance were located on both the Citrus and Poncirus linkage maps. In addition, clusters of markers with significantly different means between marker present and absent classes indicating minor QTL that contribute smaller effects on the level of tolerance were found on the linkage maps of both species.

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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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Frederick J. Ryan and David W. Ramming

The development of grapevines with berries with small seed traces, so-called seedless grapes, is a costly process. Marker assisted selection would save time and money. Adam-Blondin et al. (Vitis 40:147. 2001) demonstrated that a sequence characterized amplified region, SCC8, could identify seedless grapevine cultivars in European accessions of Vitisvinifera L. We have applied this marker to two populations of grapevines in a breeding program in California. One population consisted of 100 individuals while the second had 109. The two crosses had a common female parent, derived from `Flame Seedless'. Fruit were evaluated over several seasons for parameters including total weight of seeds or traces. DNA was isolated from leaves during the spring. Amplification was carried out with SCC8 primers, followed by digestion with Bgl II, and agarose gel electrophoresis. Individuals were scored as homozygous SCC8+ (small seeded), heterozygous SCC8+/scc8-(intermediate sized seeds), or homozygous scc8-(large seeded) and mean total seed weight per berry was calculated for each genetic class. In the first population, the number of individuals in the inferred genotypes fit an expected 1:2:1 distribution (χ2 = 0.480, P> 0.787) and seed weights for each genetic class were reasonable. For the second population, it was necessary to postulate a null allele in one parent, with a 1:1:1:1 expected distribution for genotypes SCC8+/SCC8+, SCC8+/null, SCC8+/scc8-, and scc8-/null. The actual distribution was in agreement with this model (χ2 = 4.379, P> 0.223). The genotype SCC8+/null had the SCC8+ marker and total seed weight >10 mg per berry. Large seeded individuals and heterozygotes could be reliably identified with this marker.

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T. Casey Garvey and John D. Hewitt

An interspecific hybrid was made between an accession of Lycopersicon cheesmanii f. minor Riley (LA 1508) from the Galapagos Islands, Ecuador, and L. pennellii (Corr.) D'Arcy (LA 716). LA 1508 was used because of its high soluble solids content (SSC). It was crossed with LA 716 to test for linkage between isozymes and morphological markers and loci conditioning high SSC. For both accessions, chromosome numbers are equal and there are large differences between SSC and no barriers to crossing. Modified BC1 populations derived from the hybridization were assayed for isozyme markers using starch gel electrophoresis. Associations between marker loci and quantitative-trait loci (QTL) conditioning high SSC were determined using analysis of variance. Six isozymes located on five chromosomes and one morphological marker had significant associations with SSC, indicating linkage to QTL. Digenic epistatic interactions between pairs of independent markers did not appear to play an important role in the interactions between QTL that condition SSC.