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S. Brauner, R.L. Murphy, J.G. Walling, J. Przyborowski, and N.F. Weeden

DNA primers for 37 genes have been developed in pea (Pisum sativum L.). Two-thirds of these primers also amplify orthologous sequences in lentil (Lens culinaris). The primers were designed to be complementary to highly conserved sequences in exons of known genes. In addition, most of the priming sequences were selected to be 1000 to 3000 bp distant on the genomic DNA and to amplify a fragment that contained at least one intron. Segregating sequence polymorphism in mapping populations of recombinant inbred lines (RILs) derived from wide crosses in Pisum was observed by restriction of the amplified fragment with endonucleases recognizing four-base restriction sites. Successful mapping of 36 of these genes in pea demonstrated the utility of these primers for mapping, and it appears likely that the primers should have general utility for comparative mapping in legumes.

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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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Lisa J. Rowland, Anik L. Dhanaraj, James J. Polashock, and Rajeev Arora

Expressed sequence tag-polymerase chain reaction (EST-PCR) markers for DNA fingerprinting and mapping in blueberry (Vaccinium sp.) had previously been developed from expressed sequence tags (ESTs) produced from a cDNA library, derived from RNA from floral buds of cold acclimated plants. Because EST-PCR markers are derived from gene coding regions, they are more likely to be conserved across populations and species than markers derived from random regions of DNA, such as randomly amplified polymorphic DNA (RAPD) or amplified fragment length polymorphism (AFLP) markers. In this study, we tested whether many of the EST-PCR primer pairs developed for blueberry are capable of amplifying DNA fragments in other members of the family Ericaceae. In addition, we cloned and sequenced a selection of 13 EST-PCR fragments to determine if they showed homology to the original blueberry cDNA clones from which the EST-PCR primer pairs were derived. Closely related cranberry genotypes (two wild selections of V. oxycoccus L. and two cultivars of V. macrocarpon Aiton, `Early Black' and `Stevens') and more distantly related rhododendron genotypes (one wild selection each of Rhododendron arboreum Marsh, R. maximum L., and R. ponticum L. and three complex species hybrids, `Sonata', `Grumpy Yellow', and `Roseum elegans') were used. Of 26 primer pairs tested in cranberry, 23 (89%) resulted in successful amplification and eight of those (35%) amplified polymorphic fragments among the cranberry genotypes. Of 39 primer pairs tested in rhododendron, 29 (74%) resulted in successful amplification and 21 of those (72%) amplified polymorphic fragments among the rhododendron genotypes. Approximately 50% of the 13 sequenced EST-PCR fragments were found to be homologous to the original blueberry cDNA clones. These markers should be useful for DNA fingerprinting, mapping, and assessing genetic diversity within cranberry and rhododendron species. The markers which are shown to be homologous to the blueberry cDNA clones by DNA sequencing should also be useful for comparative mapping and genetic diversity studies between some genera of the family Ericaceae.

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Karen R. Harris, W. Patrick Wechter, and Amnon Levi

). Table 3. Similarity of watermelon resistance gene analogs (WRGA) to accessions within GenBank using BLASTX. Development of watermelon resistance gene analog-sequence-tagged site markers. For each WRGA group, WRGA-STS markers were

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Genet Teshome Mekuria, Margaret Sedgley, Graham Collins, and Shimon Lavee

A sequence-tagged site (STS) was developed to identify a genetic marker linked to resistance to olive leaf spot caused by the pathogen, Spilocea oleaginea (Cast) (syn. Cycloconium oleaginum Cast.). The STS was based on a randomly amplified polymorphic DNA (RAPD) marker of about 780 base pairs (bp) linked to olive leaf spot resistance. Several primer pairs were developed to flank the sequence, and one pair produced the expected polymorphism between resistant and susceptible individuals tested, and was used as an STS marker. This primer pair was tested against parents and 34 individuals from a population segregating for resistance to olive leaf spot, and 12 commercial olive (Olea europaea L.) cultivars showing various levels of resistance to the disease. The STS marker was present in 71.4% of the parents and progeny that were designated as resistant, and was absent in 87% of the parents and progeny showing susceptibility. These primers were also able to distinguish cultivars such as `Koroneiki' and `Leccino', that are reported to show resistance to olive leaf spot, from `Barouni' and `Mission', that are reported to be susceptible. This is the first report of a STS marker for olive, and its use will assist greatly in screening olive progeny for resistance to leaf spot in breeding programs.

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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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Minou Hemmat, Norman F. Weeden, Frank S. Cheng, and S.K. Brown

The positions of over 50 SSR loci and other sequence tagged sites (STSs) have been located on the linkage maps of five apple cultivars (Rome Beauty, White Angel, Golden Delicious, Liberty, McIntosh) and two New York accessions. In most cases, the primers used produced single amplification products, permitting identification of homologous loci in the different cultivars and the precise alignment of the linkage maps generated for each. Based on this information, we present a general linkage map for apple with STS markers on each linkage group. The map consists of 17 linkage groups (equal to the haploid chromosome number for the species) with over 500 markers. The positions of several resistant gene analogues have been located on this linkage map. None of these sequences map near genes conferring resistance to scab or powdery mildew. SSR loci exhibited a tendency to cluster in certain regions of the linkage map. This clustering slightly reduces their effectiveness as genome markers for comparative mapping or germplasm diversity. However, the SSR markers definitely displayed a high level of polymorphism, making them particularly useful for genetic studies.

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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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Ke Cao, Lirong Wang, Gengrui Zhu, Weichao Fang, Chenwen Chen, and Pei Zhao

. kansuensis ‘Honggengansutao’. A genetic linkage map was constructed by using SSR, SRAP, and RGA-sequence-tagged site (STS) markers for mapping RKN resistance. This map is a valuable tool for locating the regions involved in RKN resistance and for the

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Patrick J. Conner, Gunawati Gunawan, and John R. Clark

grapevines, whereas reduced expression was suggested as the cause of stenospermocarpic seedlessness in ‘Sultanina’ and derived cultivars ( Malabarba et al., 2017 ; Mejía et al., 2011 ). An STS marker, p3_VvAGL11, was designed to perform genetic analysis of