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L.J. Grauke, Muhammad J. Iqbal, Avutu S. Reddy, and Tommy E. Thompson

A microsatellite-enriched library was developed from `Halbert', a native pecan [Carya illinoinensis (Wangenh.) K. Koch] selection from Coleman County, Texas. A genomic library enriched for simple sequence repeats (SSR) containing 6144 clones was archived in 384 well plates for screening. In total, 439 clones were identified after Southern hybridization using di- and tri-nucleotide repeats as probes. In total, 125 positive clones were sequenced and primers were designed for 24 repeats. The SSR markers chosen for analysis include di-(CT and GA) and tri-nucleotide repeats (CTT, GAA and GAT). Of the 24 primer pairs tested, 19 successfully amplified microsatellites from `Halbert'. DNA was isolated from 48 pecan and hickory accessions selected to strategically represent the genetic diversity of the National Clonal Germplasm Repository (NCGR) Carya collections. The 19 SSR primers that produced good amplification products in `Halbert' were used to evaluate the collection, with 11 revealing polymorphism. The number of fragments amplified with different primer combinations ranged from 4 to 32 in the 48 genotypes tested. Evaluation of the data confirms the utility of the microsatellites in delimiting known relationships.

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Antonio Figueira, Jules Janick, and Peter Goldsbrough

The size of the haploid genome of Theobroma cacao L. (cacao), estimated using laser flow cytometry, was 0.43 pg. An improved DNA extraction procedure was developed based on isolation of a crude nuclei preparation from leaf tissue that effectively eliminated contamination of the DNA by polysaccharides and produced DNA that was, on average, longer than 50 kb. DNA yields ranged from 2 to 10 μg·g-1 fresh weight of leaf tissue. DNA blot hybridization experiments with a flax (Linum usitatissimum L.) ribosomal DNA probe revealed restriction fragment length polymorphisms between three cacao genotypes. Differences between the DNA of these genotypes were also detected by polymerase chain reaction amplification of polymorphic DNA fragments, using random oligonucleotide primers.

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Vidyasagar R. Sathuvalli, Shawn A. Mehlenbacher, and David C. Smith

resistance alleles would greatly facilitate the development of new cultivars. Random amplified polymorphic DNA and SSR markers linked to EFB resistance have been identified for three sources: ‘Gasaway’, ‘Ratoli’ from Spain, and OSU 759.010 from the Republic

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Fuad Gasi, Naris Pojskić, Mirsad Kurtovic, Clive Kaiser, Stein Harald Hjeltnes, Milica Fotiric-Aksic, and Mekjell Meland

pollinizer cultivars present in six orchards included in the study. Planting density farm 1–5: 1 × 4 m, farm 6: 3 × 5 m. Molecular and phenology analyses. Tissue samples (leaves) for DNA analyses were collected in the Spring of 2014 from a single tree of the

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Li-Qiang Tan, Xin-Yu Wang, Hui Li, Guan-Qun Liu, Yao Zou, Shen-Xiang Chen, Ping-Wu Li, and Qian Tang

leaf teeth (leaf teeth number/leaf length) were also calculated. DNA extraction and SSR marker genotyping. Young leaves were sampled from each plant and then stored in liquid nitrogen until DNA extraction. Genomic DNA of each sample was extracted using

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F.S. Cheng, S.K. Brown, and N.F. Weeden

A DNA extraction protocol was developed for tissues from woody species. DNA was extracted successfully from 11 species and five different types of tissues and was suitable for RAPD and restriction analysis. Spermine precipitation was used to further purify DNA. The protocol can be used for large-scale analysis and mini-preparations.

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Antar Nasr El-Banna, Mohammed Elsayed El-Mahrouk, Mohammed Eraky El-Denary, Yaser Hassan Dewir, and Yougasphree Naidoo

, without epistatic and pleotropic effects and are interpretable as genes and loci. Molecular markers, which detect variation at the DNA level overcome most of the limitations of morphological and biochemical markers. As demonstrated by their use in various

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Hong Lin and M. Andrew Walker

The DNA extracted from cambium tissues of grape (Vitis spp., Muscadinia rotundifolia Small) rootstocks was found to be suitable for molecular analysis. Its quality was equivalent to that of DNA extracted from leaf tissues, although the yield was higher from leaves. The use of cambium tissue allows DNA extractions during dormancy or from grafted rootstocks where leaves are not available. The DNA extracted was suitable for restriction enzyme digestion and for analysis by restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), and simple sequence repeats.

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L.S. Boiteux, M.E.N. Fonseca, and P.W. Simon

Seven plant genomic DNA purification protocols were evaluated for genetic fingerprinting analysis using six tissues obtained from inbred carrot (Daucus carota L.) lines. Evaluations included 1) DNA yield, 2) DNA purity, 3) DNA cleavage with HindIII, 4) DNA integrity, and 5) DNA suitability for amplification in a random amplified polymorphic DNA (RAPD) system. Significant differences were observed among tissues and purification methods for the total amount of DNA. An extraction method using CTAB buffer + organic solvents gave the best results in DNA yield, purity, and HindIII cleavage when compared with the other six nonorganic extraction methods. Of the tissues examined, flowers yielded the most DNA (average value = 115 ng of DNA/mg of fresh tissue); followed by seeds (54 ng·mg-1), fresh leaves (48 ng·mg-1), lyophilized leaves (40 ng·mg-1), calli (22 ng·mg-1), and tap roots (4 ng·mg-1). For most of the preparations, the DNA showed no traces of degradation. However, DNA preparations were not consistently accessible to HindIII cleavage in all tissue-extraction method combinations. Uncut DNA was observed chiefly in extractions from flowers and fresh leaves suggesting a tissue-specific adverse effect on restriction endonuclease activity. Differences in RAPD band (amplicon) intensity and number were observed across tissues and DNA extraction methods using identical PCR conditions for RAPD. Callus was the best type of tissue for RAPD-based fingerprinting yielding a consistently higher number of more intense amplicons when compared to the other tissues. In flowers and seeds, only DNA obtained with the CTAB extraction method could be amplified. Polymorphisms deviating from genetic expectations were mainly observed in root and fresh leaf DNA, indicating that some RAPD markers may not present satisfactory levels of reproducibility. Judicious and uniform selection of DNA purification method as well as tissue source for DNA extraction are, therefore, important considerations for reliable RAPD-based DNA fingerprinting analysis in carrot. In addition, our studies allowed the identification of a better combination of procedures for use in routine manipulations of carrot DNA such as RFLP-RAPD-based cultivar fingerprinting, molecular mapping, screening of transgenic plants, construction of genomic libraries, and gene cloning.

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Vidyasagar R. Sathuvalli, Shawn A. Mehlenbacher, and David C. Smith

chain reaction (PCR) are one of the least expensive types of DNA markers and are suitable to the high sample throughput required for routine use in applied breeding programs ( Welsh and McClelland, 1990 ; Williams et al., 1990 ). RAPD markers are