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Chandra S. Thammina, David L. Kidwell-Slak, Stefan Lura and Margaret R. Pooler

repeat types with high polymorphism information content developed by Wadl et al. (2012) were selected for gradient-polymerase chain reaction (PCR) tests using C. racemosa NA49084 to determine optimal annealing temperatures. PCR primers were

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Boyang R. Cao and Chih-Cheng T. Chao

Polymorphisms of 21 date cultivars (Phoenix dactylifera L.) in California were determined by amplified fragment length polymorphism (AFLP) analysis with near infrared fluorescence labeled primers. Four primer sets were used to detect polymorphisms. Based on the UPGMA-cluster analysis of 328 polymorphic bands, the majority of the cultivars was separated into two major groups. Cultivars Abada, Amir Hajj, Ashrasi, Bentamoda, Boyer No. 11, Deglet Beida, Horra, Javis No. 1, Khadrawy, and Thoory belonged to group I. Cultivars Badrayah, Dayri, Halawy, Haziz, Khir, Medjool, Sayer, and Zahidi belonged to group II. Cultivars Barhee and Deglet Noor were further separated from groups I and II. `Hayany' was distinct from all other date cultivars tested. These results demonstrated that AFLP markers could efficiently identify individual date cultivars. The information will be useful for future date germplasm collection and facilitated selection of diverse parents for cross hybridization in a breeding program.

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Elizabeth J. Parks and James W. Moyer

Fingerprinting using molecular markers is a highly effective method of cultivar identification that is a powerful aid to traditional methods based on morphology. Amplified fragment length polymorphism (AFLP) is a robust and reliable method for generating molecular markers that has been used to evaluate many crops for a variety of applications. In this study, AFLP was used to develop and validate robust genetic fingerprints for poinsettia (Euphorbia pulcherrima Willd. ex Klotzch) cultivars. Polymorphism selection was completed to facilitate the identification of useful polymorphisms and minimize future fingerprinting costs and time. Poinsettia is a highly variable crop subject to genetic drift and variable cultivars. Validation of polymorphisms to remove those associated with intracultivar variation improved the reliability of the fingerprinting. The result was a poinsettia AFLP database that defines the genetic fingerprints of 104 cultivars. Cluster analysis illustrated differentiation of most poinsettia cultivars tested. Selection of a subset of AFLP polymorphisms resulted in clustering of cultivars according to known origin and breeding program. This method has applications not only for cultivar identification for cultivar protection, and maintenance of cultivar uniformity, but also has the potential application of developing markers for important traits.

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A. Virginia Freire, John E. Preece and David A. Lightfoot

Silver maple has great potential as a biomass feedstock. We selected 21 elite silver maple clones representing 7 provenances located on east to west and north to south transects across the natural area of distribution. In addition five different red maples including one commercial cultivar as well as four interspecific hybrids between red and silver maple were compared to the silver maples. DNA was extracted using a modification of the CTAB technique (Murray and Thompson, 1980). Polymerase chain reaction was used with random primers from the OPF series (1-20) and primers used by Krahl et al. (1993). Polymorphism was detected at high frequency. Greater polymorphism was observed between species than within species. However, we have observed DNA concentration dependent polymorphism. RAPD technology has potential for determination of genetic relationship among silver maple clones.

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J. Fang, Y. Qiao, Z. Zhang and C.T. Chao

We used amplified fragment length polymorphism (AFLP) markers to analyze 14 fruiting mei cultivars from China and Japan. The levels of polymorphism and genetic relationship among cultivars were studied using two types of AFLP primer combinations [EcoR I + Mse I (E+M) and EcoR I + Taq I (E+T)] and the combined data from both types of primer combinations (E+M+T). The polymorphism among the cultivars was 57.92% based on E+M primers and 63.04% based on E+T primers. All three dendrograms generated by the three sets of data showed similar relationships among the fruiting mei cultivars. The corresponding main clusters contained the same cultivars and the subgroups correlated closely with the known geographic origins of the cultivars.

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R. Biffi, F.M. Restivo, F. Tassi, E. Caporali, A. Carboni, G.P. Marziani, A. Spada and A. Falavigna

The use of a sex-linked molecular marker for early sex diagnosis in the dioecious species Asparagus officinalis L. was evaluated. Screening of random genomic probes as a part of a restriction fragment length polymorphism mapping project resulted in the identification of a sex-linked (6.9 cM) marker. The usefulness of this molecular tool was compared to morphological markers for prediction of gender in several genotypes. The level of polymorphism detected by this probe was high, and the level of incorrect sex attribution, as determined by this method, was low (≈7%).

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M. Hubbard, J. Kelly, S. Rajapakse, A. Abbott and R. Ballard

We have identified cloned rose DNA fragments that detect restriction fragment length polymorphisms (RFLP) in rose (Rosa ×hybrida) cultivars. RFLP can be used as genetic markers for identification, certification, and patent protection. By comparing RFLP patterns for each of six probes, we have been able to characterize eight cultivars. These results confirm that RFLP analyses are useful for rose cultivar identification and may provide a means for protecting patent rights to new cultivars.

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Jinggui Fang, Pachanoor S. Devanand and ChihCheng T. Chao

Single-nucleotide-polymorphism (SNP) is the most abundant genetic variation among individuals within a species. SNPs can be used as markers for gene discovery and for assessment of biodiversity. We established a practical strategy for discovering candidate SNPs in fruiting-mei (Prunusmume Sieb. et Zucc.), a non-model tree fruit, from amplified-fragment-length-polymorphism (AFLP) fragments. Eighty-one of the 150 chosen bands from 10 cultivars of fruiting-mei were successfully re-amplified and 67 of these re-amplified PCR products yielded 13 groups of reliable sequences. The sequencing results from both directions of 23 randomly selected PCR products using the corresponding selective primers showed that all the purified fragments from the gels were EcoR I-EcoR I fragments. The sequence alignment of 13 groups of sequence yielded 95 SNPs from a total of 5252 bp, averaging one SNP every 55 bp. Among these SNPs, 73 were heterozygous in the loci of some individual cultivars. The SNPs distribution were: 58% transition, 40% transversion, and 2% InDels. There were also one di-nucleotide polymorphism and one tetra-nucleotide deletion. The procedure of SNP isolation from AFLP fragments can be useful for transferring AFLP markers into sequence-tagged-site markers.

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P. Obara-Okeyo, Kouei Fujii and Shunji Kako

Seventy Cymbidium (Swartz.) cultivars were analyzed for isozyme variability in eight enzyme systems by starch gel electrophoresis. All systems studied [aspartate aminotransferase (AAT), malate dehydrogenase (MDH), alcohol dehydrogenase (ADH), phosphoglucomutase (PGM), glucose phosphate isomerase (GPI), triosephosphate isomerase (TPI), and shikimate dehydrogenase (SKDH)] showed polymorphism. When all enzyme systems were evaluated, 68 of the 70 Cymbidium cultivars could be distinguished. Isozymes could not distinguish betwen the cultivars Golden Star `Kumamoto' and Golden Star `Sunrise'. No cultivar showed a single unique pattern, but the TPI system gave one “diagnostic” pattern. Segregation ratios from controlled crosses suggested that LAP-1 is simply inherited and controlled by at least two alleles.

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Michael J. Havey

Restriction fragment length polymorphisms (RFLPs) in the chloroplast and nuclear genome are useful for estimation of phylogenetic relationships. Fifteen mutations at restriction enzyme sites in the chloroplast DNA were discovered. The wild species A. oschaninii and A. vavilovii were identical to A. cepa for all mutations. These species represent sources of wild germplasm closely related to the bulb onion. Nuclear RFLPs are now being used to estimate the genetic distances between accessions of A. oschaninii A. vavilovii, and open-pollinated populations of the cultivated bulb onion.