Characterization of RAPD markers in Vitis
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A study was initiated to investigate the possibility of using RAPD markers in related populations of Vitis. We also sought to design primers that could amplify translation initiation sites (Kozak sequence) as a mean to maximize the production of RAPD markers from single copy DNA sequences in the genome. RAPD bands were labeled and used as probes on blots with either genomic DNA or RAPD products from cvs Aurore, Cayuga White, Horizon and Illinois 547-1. Reamplification of excised RAPD products produced either several bands of smaller size, a single band of smaller size or a single band of the same size as the original band. Among 16 probes hybridized to genomic DNA blots, three probes, including one from the Kozak primer amplification, hybridized to 1-2 bands, 5 probes hybridized to 3-8 bands and 8, including two from a Kozak primer reaction, to more than 10 bands on the genomic DNA blots. Twelve RAPD bands were also probed on RAPD blots derived from the RAPD reaction that produced each probe. Three of those probes hybridized to 1-2 bands, 8 hybridized to 3-8 and one hybridized to more than 10 bands indicating the presence of probe sequences in more than one RAPD band as amplified with the same primers. This result and the observations on reamplification of RAPD bands support the hypothesis that some of the longer RAPD fragments harbor internal priming sites that are either not amplified unless the reaction mixture is saturated with longer other primers indicating amplification from the same sequence but different sized repetitive DNA. RAPD reactions were also run with 16 primers on parental DNA of 2 crosses used in genetic mapping (Cayuga White x Aurore and Horizon x Illinois 547-1). These reactions rated 140 bands; 100 bands were shared by both populations, including 47 polymorphic bands. Ten polymorphic bands in Cayuga White x Aurore and 22 in Horizon x Illinois 547-1 were population specific. The RAPD analysis as well as hybridization of RAPD markers to the genomic blots suggest that linkage analysis could be used in related segregating populations with carefully chosen markers. Tagging single copy regions with Kozak-sequence-derived primers may be possible, but the low number of probes tested and lack of DNA sequence information prevents any definite conclusions.Keywords:
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Molecular genetic maps are commonly constructed by analyzing the segregation of restriction fragment length polymorphisms (RFLPs) among the progeny of a sexual cross. Here we describe a new DNA polymorphism assay based on the amplification of random DNA segments with single primers of arbitrary nucleotide sequence. These polymorphisms, simply detected as DNA segments which amplify from one parent but not the other, are inherited in a Mendellan fashion and can be used to construct genetic maps in a variety of species. We suggest that these polymorphisms be called RAPD markers, after Random Amplified Polymorphic DNA.
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Random Amplified Polymorphic DNA (RAPD) generated using arbitrary primers of 9, 10, 16 and 20 nucleotide lengths by Polymerase Chain Reaction (PCR) was investigated in 12 species of fishes. We found that the amplification products were best resolved by Urea-SDS-PAGE and detected by silver staining. The amplification products ranged from 25 to 75 depending on the primer and template combination. The random primers generated unique fingerprints for each species of fish in terms of number and position of RAPDs. Our results showed that the fish species can be distinguished from each other by RAPDs. The complexity of the RAPDs in the fingerprints may be manipulated to suit the requirement of the study. The use of RAPD in taxonomy, fishery management and fish culture is discussed.
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Abstract Random amplified polymorphic DNA (RAPD) analysis was assessed as a method to identify polymorphic markers in dogs for potential use in linkage analysis of traits segregating in specific pedigrees. Three sets of primers were evaluated: 294 ten‐nucleotide long arbitrary primers from sets commonly used in RAPD analysis; 40 primers containing simple repeat elements; and 10 long random primers (19‐24 nucleotides). Of the 294 10‐mer RAPD primers, 220 (75%) amplified DNA segments from template DNA yielding primarily of 4‐5 DNA bands. Furthermore, only a few of the primers containing simple repeat elements produced discrete DNA fragments on amplification. On the other hand, all ten of the longer primers yielded amplification products with an average of 10 bands. Polymorphic bands were identified among the test DNA samples in 30% of reactions using 10‐mer RAPD primers, and in 50% of cases utilizing the long primers. RAPD primers with higher GC content amplified more polymorphic DNA fragments. Characteristic differences in amplification patterns were noted among breeds with several of these primers. We conclude from this study that by selecting primers with high GC content RAPD analysis can be successfully applied to identify polymorphic markers even in relatively inbred dog populations, and offers a promising method to search for markers linked to genetic traits of interest.
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Random amplified polymorphic DNA (RAPD) markers have been used in genetic studies of several plant and animal species. However, concerns exist about the reproducibility of RAPD-PCR reactions. Therefore, the use of specific 24mer primer pairs in standard PCR reactions has been suggested for reliable amplification of characterized polymorphic RAPD sequences. The purpose of this article is describe the application of the RAPD-PCR assay to genetic studies of dogs and to investigate the amplification of RAPD sequences with specific primer pairs. Of 240 decanucleotide primers tested by PCR, 34.6% resulted in amplification of at least one polymorphic fragment with samples of a Labrador retriever pedigree. We cloned and sequenced five of these RAPD fragments and synthesized specific 24mer primer pairs for each. Two primer pairs amplified a sequence exclusively from samples that were positive for the RAPD fragment, while three others amplified the respective sequence from all DNA samples. A new polymorphism was observed in the restriction digest products with MseI of one of the amplification products. None of the cloned sequences contains an open reading frame longer than 213 bases. Two sequences hybridized only to specific fragments of genomic DNA from samples that amplified the RAPD, the remaining three sequences hybridized to multiple sequences in all canine samples tested by Southern analysis. None of the five fragments hybridized to human or murine genomic DNA. Data suggested that RAPD sequences can be used as molecular markers in genetic studies of diseases in dogs. However, the use of specific primer pairs leads to loss of the RAPD polymorphism in three of five sequences tested.
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Four new simple sequence repeat (SSR) loci (designated VVMD5, VVMD6, VVMD7, and VVMD8) were characterized in grape and analyzed by silver staining in 77 cultivars of Vitis vinifera. Amplification products ranged in size from 141 to 263 base pairs (bp). The number of alleles observed per locus ranged from 5 to 11 and the number of diploid genotypes per locus ranged from 13 to 27. At each locus at least 75% of the cultivars were heterozygous. Alleles differing in length by only 1 bp could be distinguished by silver staining, and size estimates were within 1 or 2 bp, depending on the locus, of those obtained by fluorescence detection at previously reported loci. Allele frequencies were generally similar in wine grapes and table grapes, with some exceptions. Some alleles were found only in one of the two groups of cultivars. All 77 cultivars were distinguished by the four loci with the exception of four wine grapes considered to be somatic variants of the same cultivar, 'Pinot noir', 'Pinot gris', 'Pinot blanc', and 'Meunier'; two table grapes that are known to be synonymous, 'Keshmesh' and 'Thompson Seedless'; and three table grapes, 'Dattier', 'Rhazaki Arhanon', and 'Markandi', the first two of which have been suggested to be synonymous. Although the high polymorphism at grape SSR loci suggests that very few loci would theoretically be needed to separate all cultivars, the economic and legal significance of grape variety identification requires the increased resolution that can be provided by a larger number of loci. The ease with which SSR markers and data can be shared internationally should encourage their broad use, which will in turn increase the power of these markers for both identification and genetic analysis of grape. Key words : grape, Vitis, microsatellite, simple sequence repeat, DNA typing, identification.
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