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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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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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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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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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Jaeho Yoon, Dongcheng Liu, Wonseob Song, Weisheng Liu, Aimin Zhang, and Shaohua Li

The genetic relationships among 96 peach and nectarine [Prunus persica (L.) Batsch.] genotypes and botanical varieties originating from different ecogeographical regions of China, Japan, North America, and South Korea were evaluated with 33 SSR markers screened from 108 published SSR markers developed for peach or sweet cherry (P. avium L.). The 33 SSRs detected polymorphisms among 96 genotypes and revealed a total of 283 alleles with an average of 8.6 alleles per locus. The polymorphism information content (PIC) value ranged from 0.40 (BPPCT041) to 0.98 (BPPCT009) with an average of 0.80. Unweighted pair group method average (UPGMA) cluster analysis based on Nei's genetic distances classified genotypes into six groups, corresponding to their ecogeographical origin. Group I consisted of northern Chinese and northwestern Chinese local cultivars, and was divided into two subgroups, white and yellow peaches. Group II contained mainly southern Chinese local, Japanese, and North American cultivars and can be divided into four subgroups: Japanese white, Chinese flat, North American yellow, and some Chinese local ornamental peach cultivars. Groups III, IV, and V were comprised of Chinese local ancient cultivars, and contained `Xinjiangdatianren' and `Renmiantao', Chinese dwarf cultivars, and `Fenshouxing', respectively. Group VI had only `Baishanbitao', a Chinese ornamental cultivar. Northern and northwestern Chinese local cultivars clustered together with a greater diversity than southern Chinese local cultivars, indicating that the northern and northwestern Chinese local cultivars are similar ecotypes, and southern Chinese local cultivars are a subset of the northern Chinese group. Moreover, the Japanese and North American genotypes had a close phylogenetic relationship with southern Chinese local cultivars. The taxonomic placement of P. ferganensis (Kost. et Kiab) Kov. et Kost. and the phylogenetic relationship of `Baishanbitao' with peaches are discussed.

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Ram K. Birhman, Sylvain R. Rivard, and Mario Cappadocia

Using restriction fragment length polymorphism (RFLP) analysis, the genetic architecture of some anther-culture-derived S. chacoense Bitt. plants was studied, and their origins were elucidated. Our RFLP analyses showed that 1) several plants, even of different ploidy but otherwise genetically identical (clones), can be regenerated from callus originating from a single microspore and, conversely, that 2) some plants regenerated from single callus can have different genetic constitutions and, therefore, must have originated from two different microspore. These findings imply that previous anther culture efficiency estimates might have to be reconsidered.