For more than four decades after the introduction of cv. Italia (Vitis vinifera L.) in Brazil, several somatic mutations in the genome of cv. Italia and its somatic mutants gave rise to phenotypes which generated at least five new cultivars of fine table grapes. Since no molecular marker proved to be effective in discriminating cv. Italia (V. vinifera L.) and its coloured mutants (Rubi, Benitaka, Brasil, Black Star), primers for the long terminal repeat (LTR) sequences were developed to analyse Inter Retrotransposon Amplified Polymorphism (IRAP) and Retrotransposon-Microsatellite Amplified Polymorphism (REMAP), and investigate how the coloured cultivars derived from clonal propagations of somatic mutations are genetically structured. Primers for LTR sequences of IRAP and REMAP markers were edited from grape sequence databases available at a GenBank. Twenty-four primers, denominated DKS001–DKS024, were edited. Three hundred and forty-nine DNA segments were amplified by individual DKS primers and DKS/ISSR (Inter Simple Sequence Repeats) primer combinations, at an average of 13.96 amplicons per primer pair. High genetic divergence between the five cultivars was inferred from polymorphism in retrotransposons IRAP and REMAP. The analysis of polymorphism of IRAP and REMAP retrotransposons was crucial to show that clonal propagation of somatic mutations may lead towards the formation of genetically divergent cultivars by the formation of genetically structured vineyards and show the mixture of genomes within each cultivar.

ABSTRACT. Amplified fragment length polymorphism (AFLP) analysis was used to evaluate DNA polymorphism in Pilosocereus gounellei with the aim of differentiating samples grown in different Brazilian semiarid regions. Seven primer pairs were used to amplify 703 AFLP markers, of which 700 (99.21%) markers were polymorphic. The percentage of polymorphic markers ranged from 95.3% for the primer combination E-AAG/M-CTT to 100% for

Artificial selection related with important agronomic characteristics of Stevia rebaudiana may cause genetic divergence and formation of genetically structured populations with genetic uniformity or diversity within cultivars. Current study employed inter simple sequence repeats of DNA (ISSR markers) to assess genetic diversity within and among a single cultivated population maintained through sexual propagation (SR1) and four cultivated populations generated by artificial selection and maintained by vegetative propagation (SR2–SR5). Highest polymorphism rate was reported in SR1 (89.24%), whilst the lowest rate of polymorphism occurred in SR2 (60.13%). ISSR markers revealed that selection of plants with traits of vegetative-propagated interest may lead towards the generation of genetically more uniform DNA-level populations, while plants maintained by sexual propagation have high genetic variability. High estimated genetic divergence level indicated that the five areas of stevia form genetically structured populations. SR2 and SR4 are constituted by plants more homogeneous at DNA level for the selected characteristics than plants of SR3 and SR5 populations. Predominant and homogeneous genotypes selected at SR2 and SR4 areas could be valuable for tracing strategies to obtain stevia plants with the desirable agronomic characteristics through crosses between contrasting individuals in future breeding programs.

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RESUMONo presente estudo o objetivo foi selecionar primers ISSR (Inter-Simple Sequence Repeats), adequados para futuros estudos de variabilidade genética em plantas da cultivar 'Itália' (Vitis vinifera L.). Além disso, padronizar o método de quantificação de ADN (Ácido Desoxirribonucleico) e a reação de PCR para os primers selecionados. Para extração do ADN foram utilizadas amostras de folhas jovens de videira coletadas na região de Marialva, PR, e o método de quantificação do ADN escolhido foi o espectrofotômetro Picodrop®. Os primers selecionados por apresentarem um número satisfatório de bandas nítidas (106 no total) em gel de agarose 2% quando submetidos à eletroforese, foram: ISSR-1, ISSR-2, ISSR-5, ISSR-6, ISSR-7, ISSR-8, ISSR-9, ISSR-11, ISSR-12, ISSR-13, ISSR-14 e ISSR-15, usando uma temperatura para ligação dos primers de 50 °C na PCR. O número médio de bandas por primer foi de 8,84, sendo o ISSR-5 o que gerou uma maior quantidade (13) de regiões ISSR amplificadas. SUMMARYThe objective of present study was to select ISSR (Inter-Simple Sequence Repeats) primers suitable for future genetic variability studies of the grape cultivar 'Itália' (Vitis vinifera L.) plants. Besides, it is aimed to standardize the method for quantifying DNA and PCR reaction for the selected primers. For DNA extraction, samples from young vine leaves collected in the region of Marialva-PR were used, and the chosen DNA quantification method was Picodrop® spectrophotometer. The primers selected for presenting a satisfactory number of sharp bands (106 in total) in 2% agarose gel when subjected to electrophoresis were: ISSR-1, ISSR-2, ISSR-5, ISSR-6, ISSR-7, ISSR-8, ISSR-9, ISSR-11, ISSR-12, ISSR-13, ISSR-14 and ISSR-15 using an annealing temperature of 50°C at the PCR. The average number of bands per primer was 8.84, whereas ISSR-5 primer produced the highest number (13) of amplified ISSR regions.Palavras-chave: eletroforese, biologia vegetal, ISSR, uva.