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Kim S. Lewers*, Eric T. Stafne, John R. Clark, Courtney A. Weber, and Julie Graham

Some raspberry and blackberry breeders are interested in using molecular markers to assist with selection. Simple Sequence Repeat markers (SSRs) have many advantages, and SSRs developed from one species can sometimes be used with related species. Six SSRs derived from the weed R. alceifolius, and 74 SSRs from R. idaeus red raspberry `Glen Moy' were tested on R. idaeus red raspberry selection NY322 from Cornell Univ., R. occidentalis `Jewel' black raspberry, Rubus spp. blackberry `Arapaho', and blackberry selection APF-12 from the Univ. of Arkansas. The two raspberry genotypes are parents of an interspecific mapping population segregating for primocane fruiting and other traits. The two blackberry genotypes are parents of a population segregating for primocane fruiting and thornlessness. Of the six R. alceifolius SSRs, two amplified a product from all genotypes. Of the 74 red raspberry SSRs, 56 (74%) amplified a product from NY322, 39 (53%) amplified a product from `Jewel', and 24 (32%) amplified a product from blackberry. Of the 56 SSRs that amplified a product from NY322, 17 failed to amplify a product from `Jewel' and, therefore, detected polymorphisms between the parents of this mapping population. Twice as many detected polymorphisms of this type between blackberry and red raspberry, since 33 SSRs amplified a product from NY322, but neither of the blackberry genotypes. Differences in PCR product sizes from these genotypes reveal additional polymorphisms. Rubus is among the most diverse genera in the plant kingdom, so it is not surprising that only 19 of the 74 raspberry-derived SSRs amplified a product from all four of the genotypes tested. These SSRs will be useful in interspecific mapping and cultivar development.

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Claudio Cantini, Amy F. Iezzoni, Warren F. Lamboy, Manuela Boritzki, and Darush Struss

The U.S. Department of Agriculture (USDA), Agricultural Research Service (ARS) tetraploid cherry (Prunus L. sp.) collection at Geneva, N.Y., contains ≈75 accessions of sour cherry (P. cerasus L.), ground cherry (P. fruticosa Pall.), and their hybrids. Accurate and unambiguous identification of these accessions is essential for germplasm preservation and use. Simple sequence repeats (SSRs) are currently the markers of choice for germplasm fingerprinting because they characteristically display high levels of polymorphism. Recently SSR primer pairs from sweet cherry (P. avium L.), sour cherry, and peach [(P. persica L. Batsch (Peach Group)] have been reported. Ten SSR primer pairs were tested on 59 tetraploid cherry accessions to determine if they could differentiate among the accessions. Scorable SSR fragments were produced with all primer-accession combinations. The cherry accessions exhibited high levels of polymorphism with 4 to 16 different putative alleles amplified per primer pair. Most of the putative alleles were rare with frequencies <0.05. Heterozygosity values ranged from 0.679 to 1.00, while gene diversity values ranged from 0.655 to 0.906. The primer pairs differentiated all but two of the 59 cherry accessions. Based upon the ability of the SSR data to differentiate the cherry accessions and the high level of gene diversity, we propose that all the tetraploid cherry accessions in the USDA/ARS collection be fingerprinted to provide a mechanism to verify the identity of the individual accessions. The fingerprinting data are available on the World Wide Web (http://www.ars-grin.gov/gen/cherry.html) so that other curators and scientists working with cherry can verify identities and novel types in their collections and contribute to a global database.

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Emmanouil N. Tzitzikas, Antonio J. Monforte, Abdelhak Fatihi, Zacharias Kypriotakis, Tefkros A. Iacovides, Ioannis M. Ioannides, and Panagiotis Kalaitzis

fragment length polymorphism ( Garcia-Mas et al., 2000 ), random amplified polymorphic DNA (RAPD) ( Sensoy et al., 2007 ; Staub et al., 2004 ), and simple sequence repeat (SSR) ( Danin-Poleg et al., 2001 ), using diverse germplasm from different locations

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B. Khadari, A. Oukabli, M. Ater, A. Mamouni, J.P. Roger, and F. Kjellberg

A study was conducted to identify genotypes present in a Moroccan fig germplasm collection and provide the first database for a reference collection in northern Morocco. In total, 75 fig samples were analyzed using 8 intersimple sequence repeat primers and 6 simple sequence repeat loci. From these samples, we identified 72 fig genotypes. In genetically heterogeneous cultivars, genotypes under the same denomination were distinguished by both molecular markers and pomological traits. Molecular analysis was used to classify the germplasm into 46 well-defined cultivars and 6 caprifig trees. The remaining genotypes were not clearly identified due to three cases of mislabeling and four cases of homonymy. No evidence was found for the occurrence of geographically widespread genotypes.

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Dror Sharon, Avital Adato, Samir Mhameed, Uri Lavi, Jossi Hillel, Maria Gomolka, Conny Epplen, and Jorg Thomas Epplen

Plant genomes contain polymorphic repetitive sequences that can be used as DNA markers. Minisatellites (16 to 64 bp per repeat) and simple-sequence repeats (2 to 6 bp per repeat) are the most polymorphic markers found in plant and animal genomes. In this study, the hybridizations between genomic DNA and variable number of tandem repeat probes were examined in Arabidopsis thaliana L. (Heynn), onion (Allium cepa L.), tomato (Lycopersicon esculentum L.), wheat (Triticum aestivum L.), avocado (Persea americana Mill.), litchi (Chinensis Sonn.), mango (Mangifera indica L.), and Carica species. Some of the probes detected polymorphic sequences in all the species, but others were useful only for one or two species. None of the probes gave clear band patterns in either onion or wheat. The in-gel hybridization method was similar to Southern blot hybridization using the simple-sequence repeat probes.

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M.J. Iqbal, L.J. Grauke, A.S. Reddy, and T.E. Thompson

A microsatellite library has been developed from `Halbert', a native pecan selection from Coleman County, Texas, using methods developed at the Texas A&M Univ. Crop Biotechnology Center. A total of 6144 DNA fragment clones were archived in 384 well plates for screening. Four-hundred-thirty-nine clones were positive after Southern hybridization using di- and tri-nucleotide repeats as probes. One-hundred-twenty-five positive clones were sequenced on an ABI 377 automated DNA sequencer. Of these, 24 repeats had enough sequences at the two ends to design primers. Primers were designed using Primer Express software, and were synthesized by Genosys, USA. The simple sequence repeats (SSRs) chosen for primer analysis include di- (CA and GA) and tri-nucleotide repeats (CTT, GAA and GAT). The SSRs were amplified under high stringency conditions with temperatures based on length and GC content. Reproducibility was verified using `Halbert' DNA isolated from different inventories. Of the 24 primer pairs tested, 20 successfully amplified microsatellites from `Halbert'. DNA was isolated from 48 pecan and hickory accessions selected to strategically represent the genetic diversity of the NCGR Carya collections (a core collection). The accessions included parent-progeny combinations, individuals from geographically distant native populations, species, and interspecific hybrids. The 20 SSR primers that produced good amplification products in `Halbert' were used to evaluate the collection, with 11 revealing multiple sizes of the repeat. The number of bands amplified with different primer combinations ranged from 4 to 32 in the 48 genotypes tested. We used RFLPscan software to aid in gel scoring (sizing amplified fragments, and comparing amplification profiles), and NTSYSpc software to evaluate genetic similarities. Evaluation of the data confirms the utility of the primers in delimiting known relationships.

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Minou Hemmat, Norman F. Weeden, Patrick J. Conner, and Susan K. Brown

The columnar mutation `Wijcik McIntosh' has attracted much attention because of its compact growth habit, which is compatible with high-density plantings. Using bulked segregant analysis, we identified several randomly amplified polymorphic DNA (RAPD) markers that displayed a close linkage with the columnar locus (Co). The RAPD marker that displayed the closest linkage was end sequenced to develop a sequence tagged site for rapidly screening segregating populations. A simple sequence repeat (SSR) of (GA)17 was identified within the DNA fragment. Four allelic forms, including an apparent null allele, could be distinguished among the cultivars tested. The null allele displayed close linkage with Co in two progenies, and we used this marker to identify the location of the gene on the apple linkage map.

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Laura L. Benson, Warren F. Lamboy, and Richard H. Zimmerman

Simple sequence repeats (SSRs) are highly polymorphic regions of DNA that can be used for the molecular characterization of apple (Malus) germplasm. SSR markers are sufficiently variable to distinguish between individual plants in wild Malus species. In this study, accessions of Malus hupehensis were screened for fragment length variation in PCR amplified simple sequence repeat regions of DNA. The fragment length phenotype produced by five SSR primer pairs showed no variation between two lineages of M. hupehensis collected in the Changjiang (Yangtse) River valley. One lineage was collected by E.H. Wilson in 1908 near the city of Ichang, Hubei Province. The second lineage was collected by cooperators at China's Southwest Agricultural University (SWAU) in 1997 near the city of Chongqing (Chungking). M. hupehensis Plant Introduction No. 588760 from the National Plant Germplasm System lacks provenance, but displays a fragment length phenotype identical to both the Wilson and SWAU lineages. The spread of a clone may be aided by asexual reproduction through seed, which is not uncommon in polyploid apples. Two seedlings each of 15 maternal trees from the SWAU lineage were assayed for ploidy level by flow cytometry. The DNA content per nucleus for all SWAU progeny fell within the range for triploids, 2.19 to 2.68 pg DNA/nucleus. It appears that plant explorers in China separated by almost 90 years have succeeded in sampling a single clonal lineage of M. hupehensis.

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Laura L. Benson, Warren F. Lamboy, and Richard H. Zimmerman

The U.S. National Plant Germplasm System (NPGS) currently holds 36 separate accessions of the `Yichang' clone of Malus hupehensis (Pamp.) Rehd. The `Yichang' clone originally entered the United States in 1908 as seed collected for the Arnold Arboretum by E.H. Wilson near Yichang, Hubei Province, China. The original description of M. hupehensis omits fruit characters, and botanists frequently augment these omissions with descriptions of the `Yichang' clone. Apomixis occurs in Malus, including M. hupehensis, and is strongly associated with elevated ploidy levels. Simple sequence repeats (SSRs) were used to characterize 65 accessions of M. hupehensis. To check for polyploidy, a set of M. hupehensis accessions was evaluated with flow cytometry. The simple sequence repeat phenotypes and ploidy information revealed the `Yichang' clone under various accession names in arboreta. It was neither known nor suspected that the U.S. National Plant Germplasm System held many duplicate accessions of the `Yichang' clone prior to their molecular characterization. Germplasm conservation decisions for Malus species can benefit from an increased knowledge of the genetic variation or lack thereof in naturalized populations and ex situ collections.

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P. Boccacci, A. Akkak, D. Torello Marinoni, G. Bounous, and R. Botta

The authors thank H. Steinkellner (Zentrum fuer Angewandte Genetik, Universitaet fuer Bodenkultur, Vienna, Austria) for providing unpublished primer sequences of SSR loci, and S.A. Mehlenbacher (Oregon State University, Corvallis, Ore.) for