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A. Levi, C. E. Thomas, J. Thies, A. Simmons, Y. Xu, X. Zhang, O.U.K. Reddy, A. Davis, S. King, and T. Wehner

Genetic linkage map is being constructed for watermelon based on a testcross population and an F2 population. About 51.0% and 31.8% of the markers in the testcross and F2 populations are skewed form the expected segregation ratios. AFLP markers appeared to be clustered on linkage regions, while ISSR and RAPD markers are randomly dispersed on the genome. AFLP markers also have greater genetic distances as compared with ISSR and RAPD markers, resulting in significant increase of map distance. An initial genetic map (based on the testcross population) that contains 27 ISSR and 141 RAPD markers has a total linkage distance of 1,166.2 cM. The addition of 2 ISSR, 8 RAPD and 77 AFLP markers increased the genetic distance of the map to 2,509.9 cM. Similar results with AFLP markers were also shown in mapping experiments with an F2S7 recombinant inbred line (RIL) population that was recently constructed for watermelon. Although the skewed segregation, marker order appeared to be consistent in linkage groups of the testcross and the F2 population. Experiments with SSR, and EST markers are being conducted to saturate the linkage map of watermelon genome.

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Graham J. King

8 COLLOQUIUM 1 (Abstr. 001–005) Genome Mapping of Horticultural Crops

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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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H.A. Agrama and J.W. Scott

The genetic basis of resistance to tomato yellow leaf curl virus (TYLCV) and tomato mottle virus (ToMoV) was studied in three different mapping populations of tomato (Lycopersicon esculentum Mill.). Bulked segregant analysis (BSA) was used to identify random amplification of polymorphic DNA (RAPD) markers linked to TYLCV and ToMoV resistance. Segregated RAPD markers associated with resistance were linked to morphological markers self-pruning (sp) and potato leaf (c) on chromosome 6. RAPD genetic linkage maps of chromosome 6 were constructed for each of the three populations. Common mapped markers revealed straightforward homologies between the chromosome 6 linkage group of the three populations. Multiple-QTL mapping (MQM) was used to identify quantitative trait loci (QTL) for resistance linked to chromosome 6. These revealed that the resistance against TYLCV and ToMoV was mainly explained by two QTL in two populations and one QTL in another. For all of the resistance QTL detected, the favorable allele was provided by the resistant parents. The presence of three different sources of TYLCV and ToMoV resistance, and the markers in tight linkage with them, provide a means of systemically combining multiple resistance genes. Successful cloning of the R gene from tomatoes would lead to deeper understanding of the molecular basis of resistance to TYLCV and ToMoV, and might also shed light on the evolution of resistance genes in plants in general.

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Amnon Levi, Elizabeth Ogden, and Lisa J. Rowland

Efforts are underway to develop genetic linkage maps for two interspecific blueberry populations (Vaccinium darrowi × V. elliottii and V. caesariense-derived populations). To date, 72 RAPD markers have been mapped, and another 200 markers have been identified as suitable for mapping in the V. darrowi × V. elliottii-derived population. Inheritance of 40 RAPD markers has been followed, and additional 40 RAPD markers have been identified as suitable for mapping in the V. darrowi × V. caesariense population. These two populations are comprised of individual plants that should have a wide range of chilling requirements. At a later date, plants will be classified according to their chilling requirements to identify RAPD markers that cosegregate with chilling requirements. Presently, a bulked-segregant analysis is being performed on a tetraploid breeding population (primarily V. corymbosum) to identify RAPD markers linked to chilling requirement genes.

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Cholani K. Weebadde* and James F. Hancock

While it is important for strawberry breeders to know the genetics of day-neutrality, evidence for inheritance of the trait is still contradictory. It is not known how many genes govern the trait, to what extent each gene affects phenotype and how the environment influences gene expression. Several recent studies point toward a polygenic threshold model and a rejection of the single gene model. A linkage mapping approach is being used to determine if day neutrality can be mapped to several different quantitative trait loci (QTL) that may represent different genes. To confirm that a linkage mapping approach is the method of choice for QTL detection, a small population of the cross `Honeoye' x `Tribute' consisting of 57 progeny segregating for the trait was genotyped with single dose restriction fragment (SDRF) markers and a preliminary genetic map was created using Join Map 3.0. Results separated the molecular markers into at least 24 linkage groups and several putative QTL for day neutrality were identified indicating that the technique will be successful. However, due to the complexity of the octoploid genome of strawberry, over 200 progeny need to be genotyped to build a complete map that includes the 56 linkage groups of the genome. Furthermore, for determining QTL, an accurate phenotypic evaluation is critical. Individuals of the population above were phenotyped under field conditions (East Lansing, Mich.) in 2002 and 2003, and are now being analyzed under controlled temperature and photoperiod conditions for confirmation of the QTL detected for the trait. A larger population of the same cross with over 200 progeny has also been generated and will be mapped using molecular markers after determining their phenotype under the same environmental conditions.

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Thomas M. Davis, Laura M. DiMeglio, Ronghui Yang, Sarah M.N. Styan, and Kim S. Lewers

The cultivated strawberry, Fragaria ×ananassa Duchesne ex Rozier, originated via hybridization between octoploids F. chiloensis (L.) Mill. and F. virginiana Mill. These three octoploid species are thought to share a putative genome composition of AAA`A'BBB`B'. Diploid F. vesca L., is considered to have donated the A genome. Current attention to the development of a diploid model system for strawberry genomics warrants the assessment of simple sequence repeat (SSR) marker transferability between the octoploid and diploid species in Fragaria L. In the present study, 23 SSR primer pairs derived from F. ×ananassa `Earliglow' by genomic library screening were evaluated for their utility in six diploid Fragaria species, including eight representatives of F. vesca, four of F. viridis Weston, and one each of F. nubicola (Hook. f.) Lindl. ex Lacaita, F. mandshurica Staudt, F. iinumae Makino, and F. nilgerrensis Schltdl. ex J. Gay. SSR primer pair functionality, as measured by amplification success rate (= 100% - failure rate) in each species, was ranked (from highest to lowest) as follows: F. vesca (98.4%) > F. iinumae (93.8%) = F. nubicola (93.8%) > F. mandshurica (87.5%) > F. nilgerrensis (75%) > F. viridis (73.4%). The extent to which these octoploid-derived SSR primer pairs generated markers that could be added to the F. vesca linkage map also was assessed. Of the 13 F. ×ananassa SSR markers that segregated codominantly in the F. vesca mapping population, 11 were assigned to linkage groups based upon close linkages to previously mapped loci. These markers were distributed over six of the seven F. vesca linkage groups, and can serve as anchor loci defining these six groups for purposes of comparative mapping between F. vesca and F. ×ananassa.

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Minou Hemmat, Norman F. Weeden, and Susan K. Brown

We mapped DNA polymorphisms generated by 41 sets of Simple Sequence Repeat (SSR) primers, developed independently in four laboratories. All primer sets gave polymorphisms that could be located on our `White Angel' x `Rome Beauty' map for apple [Malus sylvestris (L.) Mill. Var. domestica (Borkh.) Mansf.]. The SSR primers were used to identify homologous linkage groups in `Wijcik McIntosh', NY 75441-58, `Golden Delicious', and `Liberty' cultivars for which relatively complete linkage maps have been constructed from isozyme and Random Amplified Polymorphic DNA (RAPD) markers. In several instances, two or more SSRs were syntenic, and except for an apparent translocation involving linkage group (LG) 6, these linkages were conserved throughout the six maps. Twenty-four SSR primers were consistently polymorphic, and these are recommended as standard anchor markers for apple maps. Experiments on a pear (Pyrus communis L.) population indicated that many of the apple SSRs would be useful for mapping in pear. However some of the primers produced fragments in pear significantly different in size than those in apple.

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Kittipat Ukoskit and Paul G. Thompson

Low-density randomly amplified polymorphic DNA (RAPD) markers of sweetpotato [Ipomoea batatus (L.) Lam.; 2n = 6x = 90] were constructed from 76 pseudotestcross progenies obtained from `Vardaman' × `Regal'. Of 460 primers, 84 generating 196 well-resolved repeatable markers were selected for genetic analysis. `Vardaman' and `Regal' testcross progenies were analyzed for segregation and linkages of RAPD markers. Type of polyploidy, autopolyploidy, or allopolyploidy is uncertain in sweetpotato and was examined in this study using the ratio of nonsimplex to simplex RAPD markers and the ratio of simplex RAPD marker pairs linked in repulsion to coupling. Both measures indicated autopolyploidy. Low-density RAPD linkage maps of `Vardaman' and `Regal' were constructed from simplex RAPD marker linkage analysis. Duplex and triplex markers were then mapped manually into the simplex marker map. Homologous linkage groups were identified using nonsimplex RAPD markers and three homologous groups were found in each of the parent maps. Use of nonsimplex markers increased mapping efficiency. The `Vardaman' map had a predicted coverage of 10.5% at a 25-cM interval of the genome size of 5024 cM. In `Regal', genome coverage was estimated to be 5.6% at a 25-cM interval of the genome size of 6560 cM. Therefore, average chromosome length was ≈56 to 73 cM.

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L.H. Zhang, D.H. Byrne, R.E. Ballard, and S. Rajapakse

Microsatellite or simple sequence repeat (SSR) markers were developed from Rosa wichurana Crépin to combine two previously constructed tetraploid rose (Rosa hybrida L.) genetic maps. To isolate SSR-containing sequences from rose a small-insert genomic library was constructed from diploid Rosa wichurana and screened with several SSR probes. Specific primers were designed for 43 unique SSR regions, of which 30 primer pairs gave rise to clear PCR products. Seventeen SSR primer pairs (57%) produced polymorphism in the tetraploid rose 90-69 mapping family. These markers were incorporated into existing maps of the parents 86-7 and 82-1134, which were constructed primarily with AFLP markers. The current map of the male parent, amphidiploid 86-7, consists of 286 markers assigned to 14 linkage groups and covering 770 cm. The map of the female tetraploid parent, 82-1134, consists of 256 markers assigned to 20 linkage groups and covering 920 cm. Nineteen rose SSR loci were mapped on the 86-7 map and 11 on the 82-1134 map. Several homeologous linkage groups within maps were identified based on SSR markers. In addition, some of the SSR markers provided anchoring points between the two parental maps. SSR markers were also useful for joining small linkage groups. Based on shared SSR markers, consensus orders for four rose linkage groups between parental maps were generated. Microsatellite markers developed in this study will provide valuable tools for many aspects of rose research including future consolidation of diploid and tetraploid rose genetic linkage maps, genetic, phylogenetic and population analyses, cultivar identification, and marker-assisted selection.