Sequence-specific Polymerase Chain-reaction Markers Derived from Randomly Amplified Polymorphic DNA Markers for Fingerprinting Grape (Vitis) Rootstocks

Abstract

Additional index words. sequence characterized amplified region, SCAR Abstract. Randomly amplified polymorphic DNA (RAPD) markers were generated for identifying grape (Vitis) rootstocks. Seventy-seven primers (10 bases long) were screened using CsCl-purified leaf DNA derived from several field samples of nine rootstocks sampled in successive years. Nine RAPD markers were detected from six primers and, in combination, distinguished all nine rootstocks tested. Because inconsistencies were encountered in performing the RAPD assay, sequence-specific primers were derived from cloned RAPD bands for use under more stringent amplification conditions. Southern hybridization analysis of the RAPD gels with cloned RAPD bands as probes revealed deficiencies of scoring RAPD bands based solely on ethidium bromide staining. In some cases, bands of the same size generated by the same primer in different rootstocks-normally scored as the same marker-failed to cross-hybridize, implying lack of homology between the bands. More commonly, bands scored as absent based on ethidium bromide staining were detected by hybridization. Six of the nine cloned RAPD bands were partially sequenced, and sequence-specific primer pairs were synthesized. Two primer pairs amplified a product the same size as the original RAPD band in all rootstocks, resulting in loss of polymorphism. Two other pairs of sequence-specific primers derived from the same marker failed to amplify the expected band consistently. Three of the most useful primer pairs amplified apparent length variants in some accessions and will have value as polymerase chain-reaction markers for fingerprinting.

Detection of genomic regions differentiating two closely related oak species Quercus petraea (Matt.) Liebl. and Quercus robur L.

“…6). Similar results have already been observed for A ilium (Wilkie et a!., 1993), Brassica (Thormann et a!., 1994), Gliricidia (Chalmers et a!., 1992), Petunia (Peltier et a!., 1994) and Vitis (Xu et a!., 1995).…”

Identification of Grape (Vitis) Rootstocks Using Sequence Characterized Amplified Region DNA Markers

“…The need for practical and objective means for identifying grape cultivars has encouraged researchers to investigate DNA-based techniques (Botta et al, 1995;Bourquin et al, 1993Bourquin et al, , 1995Bowers et al, 1993;Guerra and Meredith, 1995;Striem et al, 1990;Thomas et al, 1994). We recently generated six polymer chain reaction (PCR) markers from cloned random amplified polymorphic DNA (RAPD) fragments for identifying winegrape rootstocks (Xu et al, 1995). The markers were designed as sequence characterized amplified region (SCAR) DNA markers (Paran and Michelmore, 1993), and three of the six were found to be informative.…”

“…five markers, UBC204 1140 , UBC231 1840 , UBC231 1660 , UBC251 1440 , and OPG05 500 , are the same as in Xu et al (1995), except that the latter is designated OPG05 550 in the earlier publication, and all were evaluated in the present study at higher than the original annealing temperatures. Markers OPG06 1440 and OPG02 1320 (Xu et al, 1995) were evaluated using newly designed internal primers. Marker OPG02 430 is half the size of the original marker, OPG02 860 , (Xu et al, 1995) because the new internal primers generate a significantly smaller fragment.…”

“…Markers OPG06 1440 and OPG02 1320 (Xu et al, 1995) were evaluated using newly designed internal primers. Marker OPG02 430 is half the size of the original marker, OPG02 860 , (Xu et al, 1995) because the new internal primers generate a significantly smaller fragment. Marker OPG05 1420 has not been described before.…”

Development of SCAR Markers Linked to a Phytophthora fragariae Resistance Gene and Their Assessment in European and North American Strawberry Genotypes

“…To overcome the disadvantages associ- ated with RAPD markers, such as irreproducibility among laboratories, RAPD markers have been converted into highly specific SCAR markers (Paran and Michelmore, 1993). Such SCAR markers have proven useful for fingerprinting, marker-assisted selection, and high-resolution mapping (Kaplan et al, 1996;Paran and Michelmore, 1993;Xu et al, 1995). In this study, a SCAR marker was constructed for the strawberry gene Rpf1, which represents one of the major sources of genetic resistance to P. fragariae.…”