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Yuanfu Ji and John W. Scott

Resistance to begomoviruses tomato mottle virus (ToMoV) and tomato yellow leaf curl virus (TYLCV) has been introgressed to tomato (Lycopersicon esculentum) from L. chilense accessions LA 1932, LA 2779, and LA 1938. Resistance genes have been mapped to three regions on chromosome 6 using randomly amplified polymorphic DNA (RAPD) markers. We call these regions 1, 2, and 3. To facilitate breeding by marker assisted selection, advanced breeding lines with resistance from the above sources were assayed for the presence of RAPD markers to determine which were most tightly linked to begomovirus resistance. The best RAPD markers were then converted to sequence characterized amplified region (SCAR) markers or cleaved amplified polymorphic sequence (CAPS) markers. In addition, selected restriction fragment length polymorphism (RFLP) markers near the three regions were converted into CAPS markers, which were tested for association with the advanced breeding lines. Only LA 2779 derivatives have the L. chilense introgression in region 1, which is near the location of the Ty-1 gene and spans across CAPS markers 32.5Cla and TG118. Two region 1 RAPD markers UBC197 and UBC621 were converted co-dominant SCAR or CAPS markers, which were present in all 16 resistant breeding lines tested. Derivatives from all three accessions have introgressions in region 2. Further assays with more markers in this region are under way to determine the lengths and locations of the introgressions. No tightly linked RAPD markers have been found for the resistance gene from LA 1932 in region 3. RFLP and CAPS markers are being used to more precisely locate the region 3 gene.

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Anne M. Gillen and Fred A. Bliss

Tagging Genes for Nematode Resistance and Tree Growth in Peach.” Provision of the F 2 population HB × Oki by Craig Ledbetter, USDA-ARS, is gratefully acknowledged. Information regarding microsatellite pchgms1 and the L×N CAPs marker was provided by Albert

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Taifeng Zhang, Jiajun Liu, Shi Liu, Zhuo Ding, Feishi Luan and Peng Gao

represent the depth of the bb population derived from X and M, respectively. The stronger the correlations between SNPs and dwarf traits, the closer Δ(SNP index) is to one. Δ(SNP index) = 0 represents no association of SNP with dwarf trait. CAPS marker

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Haejeen Bang, Angela R. Davis, Sunggil Kim, Daniel I. Leskovar and Stephen R. King

yellow and a cleaved amplified polymorphic sequence (CAPS) marker was developed based on a single-nucleotide polymorphism (SNP) within the LCYB gene; the resultant CAPS marker has been used to reliably distinguish canary yellow from red flesh in numerous

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Anne M. Gillen and Fredrick A. Bliss

Peach rootstock breeding may be accelerated by utilization of molecular markers linked to the root-knot nematode resistance locus (Mi) to screen segregating populations. A genetic linkage map was constructed using RFLP markers in an F2 population (PMP2) that is segregating for this locus. PMP2 is derived from a controlled cross of the relatively diverse peach rootstocks Harrow Blood (susceptible) and Okinawa (homozygous resistant). Bulked Segregant Analysis was applied using RAPD markers. A single small (227 base pairs) RAPD marker was found to be linked to the dominant resistant allele of Mi at a distance of 10 cM. This new marker joined the Mi locus to the RFLP linkage map and showed that two dominant RFLP markers are located between the RAPD marker and Mi. RFLPS are expensive, time-consuming and RAPD markers are unreliable, and therefore both are unsuitable for screening breeding populations. We attempted to convert the RAPD marker to a more breeder-friendly CAPS marker. The converted CAP marker was dominant. Attempts to convert the CAP marker to a co-dominant marker were not successful. The utility of the CAP marker was tested in an open pollinated F2 population derived from the F1 parent of PMP2 and in several rootstocks. The genetic linkage map was compared to other Prunus maps. The PMP2 linkage group containing the Mi locus can be related to the peach × almond linkage group which contains the phosphoglucomutase Pgm-1 locus.

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Jeremy A. Pattison*, Suren K. Samuelian and Courtney A. Weber

RAPD and AFLP markers were first used to construct a molecular map in a BC1 red raspberry population consisting of 70 individuals that segregated for Phytophthora root rot resistance. RAPD markers linked to root rot resistance were identified by bulk segregant analysis and through QTL anlaysis. Two common genomic regions were identified by both analyses and were estimated to explain ≈50% of the phenotypic variation. RAPD markers flanking the QTL were cloned and made into sequence specific markers for potential use in marker assisted selection. In addition to the linked markers, RAPDs spread throughout the linkage map were also sequenced and developed into either SCARs, CAPs, or codominant SSRs. Attempts were made to locate red raspberry resistance gene analogs using degenerate primers designed on conserved regions encoding known resistance genes. Results on the type and map position of identified RGA's and selection efficiency of linked markers analyzed in red raspberry cultivars of characterized root rot resistance will be discussed.

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Courtney A. Weber, Gloria A. Moore, Z. Deng, F. Gmitter and Courtney A. Weber

Specific primers were designed for 61 cloned RAPD fragments and from 10 Citrus EST sequences for the production of SCAR, CAPS, and STS markers for a Citrus grandis `DPI 6-4' × Poncirus trifoliata `Rubideaux' F1 pseudo-testcross population. Fifteen SCAR, three CAPS, and one EST/STS markers were developed. An additional 17 SCAR and CAPS primer pairs developed at the Citrus Research and Education Center for a Citrus grandis `Thong Dee' × (Citrus grandis `Thong Dee' × Poncirus trifoliata `Pomeroy') BC1 population were screened in the pseudo-testcross population. A total of 27 markers were identified and scored in the pseudo-testcross population in which 24 were mapped; 13 in the Citrus parental linkage map on seven linkage groups and 11 in the Poncirus parental map on five linkage groups. In the BC1 population, 20 of 27 markers tested were found to be polymorphic and 13 mapped to seven of nine linkage groups. Of these, 11 were mapped in both populations and could be used for aligning presumed homologous regions on the three linkage maps.

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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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M.A. Dalbó, G.N. Ye, N.F. Weeden, W.F. Wilcox and B.I. Reisch

The efficiency of marker-assisted selection for powdery mildew (Uncinula necator (Schw.) Burr) resistance in grapes (Vitis L. sp.) was studied using molecular markers associated with a major QTL (quantitative trait loci) for this trait. Initially, genetic maps were constructed from a segregating population of the cross `Horizon' × Illinois 547-1 (a hybrid between V. rupestris Scheele and V. cinerea Engelm.). A major QTL from Ill. 547-1, the resistant parent, explained 41% of the variation. One RAPD (randomly amplified polymorphic DNA) marker and one AFLP (amplified fragment length polymorphism) marker, obtained by bulked segregant analysis, showed the highest association with powdery mildew resistance in the mapping population. Segregation of the QTL was followed in different crosses by CAPS (cleaved amplified polymorphic sequence) markers developed from these two markers. An allele-specific amplified polymorphism that segregates as present/absent was also developed from the CS25b locus. Powdery mildew resistance was evaluated visually on a 1 to 5 scale in four different seedling populations. Two populations originated from crosses using Ill. 547-1 as the resistant parent. Two other populations were from crosses with NY88.0514.03, a resistant seedling from the original `Horizon' × Ill. 547-1 mapping population. Segregation ratio distortions were observed in some crosses. In these cases, the allele associated with the QTL for powdery mildew resistance was less frequent than the alternate allele. In all crosses, the markers were closely associated with resistance. If selection were based on markers, the percentage of susceptible individuals (classes 4 and 5) would decrease from 24% to 52% to 2% to 18%. Selection efficiency was greatest in crosses where segregation distortion was most intense.

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James M. Bradeen, S. Kristine Ness, Geraldine T. Haberlach, Susan M. Wielgus and John P. Helgeson

Late blight of potato, caused by the fungal pathogen Phytophthora infestans, is of great economic significance and no important U.S. potato cultivars are reliably resistant. The diploid species Solanum bulbocastanum is highly resistant to late blight, even under extreme conditions, but is sexually incompatible with potato. We have generated potato + S. bulbocastanum somatic hybrids. These hexaploid hybrids are highly resistant to late blight and progeny from two successive backcrosses to cultivated potato are either fully resistant or susceptible. The advanced generations are morphologically similar to potato. We have initiated mapping efforts to identify bulbocastanum chromosomal regions responsible for late blight resistance. Using RFLPs, RAPDs, and AFLPs, we identified a single chromosomal region (i.e., single gene or group of linked genes) on chromosome 8 that accounts for 62.2% of the observed resistance. The tomato cDNA clone CT88 cosegregates with late blight resistance in our material and was used to isolate a homeologous BAC clone from a S. bulbocastanum library. Our current efforts include mapping resistance in both fusion-derived and diploid S. bulbocastanum materials via map merging. As markers linked to late blight resistance are identified, we will attempt to convert them to marker forms useful for large-scale breeding efforts. To date, we have successfully converted RAPD and RFLP markers to SCAR and CAPS marker forms. Finally, continued fine mapping and BAC clone characterization will enable future map-based cloning efforts.