Endosperm genotyping as a strategy to differentiate the allele source in maize heterozygous progeny

The objective of this work was to distinguish the parental source of alleles in heterozygous progeny using semiquantitative polymerase chain reaction (PCR) in maize endosperm. Endosperms derived from direct and reciprocal single-cross hybrids between maize inbred lines L3 and L1113-01 were genotyped by semiquantitative PCR methodology (SQ-PCR) using fluorescent microsatellite primers. The amplification products were evaluated by the ratios of fluorescence intensity (RFI), calculated between the peaks corresponding to the alleles derived from each parental line. Based on the statistically significant contrast between RFI mean values of direct and reciprocal single-cross hybrids, it was possible to distinguish the number of alleles received from each parental line and, ultimately, to determine the origin of the alleles of each cross. Thus, endosperm genotyping using SQ-PCR is a promising strategy to map QTL in maize outbred populations.

Agronomical and molecular characterization of banana germplasm

The objective of the present work was to characterize banana accessions from the Germplasm Bank at Embrapa Mandioca e Fruticultura Tropical (Brazil), using agronomical, physical and physicochemical characteristics of fruit and simple sequence repeats (SSR) markers. Twenty-six accessions were analyzed, in which high genetic variability was found, especially for the agronomical characters number of fruit and weight of bunch. Accessions with high contents of carotenoids (diploid 'Jaran'), polyphenols (triploid 'Caipira' and tetraploid 'Teparod') and vitamin C (diploid 'Tuugia' and an unknown triploid AAA) in the fruit were identified. Thirteen microsatellite primers revealed an average of 7.23 alleles, which showed high variability. A dendrogram was prepared using the Gower algorithm for the distance matrices obtained from the agronomical, physical and physicolchemical analysis of fruit and SSR markers. Adopting the average genetic divergence as the cut-off point, three clusters were found: G1, formed by the diploids 'Jaran', 028003-01 and M-48; G2, by the diploids 'Malbut' and 'Ido 110'; and G3, by 21 tri-and tetraploid accessions, including one diploid, 'Tuugia'. The triploids with the B genome 'Thap Maeo', 'Walha', 'Pacha Nadan' and 'Champa Madras' were grouped in G2. Results from this work can be used for breeding hybrids with good agronomical traits and fruit quality.

Quantification of the diversity among common bean accessions using Ward-MLM strategy

The present work aimed at evaluating the divergence among common bean accessions by their agronomic, morphological and molecular traits, based on the Ward-MLM procedure. A collection of 57 accessions from the gene bank of Universidade Federal do Espírito Santo was used in this study, from which: 31 were landraces belonging to the community Fortaleza, in the municipality of Muqui, ES, Brazil; 20 accessions were provided by Embrapa Trigo; and 6 were commercial cultivars. Five agronomic traits (plant cycle, number of seeds per pod, number of pods per plant, weight of 100 seeds, and grain yield), five morphological traits (growth habit, plant size, seed shape, seed color, and commercial group) and 16 microsatellite primers were evaluated. High genetic variability was detected considering morphological, agronomic and molecular traits in the 57 common bean accessions studied. The Ward-MLM procedure showed that the ideal number of groups was five, according to the pseudo F and pseudo t² criteria. The accessions from Andean origin had heavier seeds than others and formed a cluster. The Ward-MLM statistical procedure is a useful technique to detect genetic divergence and to cluster genotypes by simultaneously using morphological, agronomic and molecular data.

Identificação de marcadores microssatélites relacionados ao escurecimento de grãos em feijão

O objetivo deste trabalho foi identificar marcadores microssatélites ligados ao loco de características quantitativas (QTL) responsável pelo escurecimento tardio do tegumento de feijões do tipo carioca, a fim de reduzir o tempo de avaliação necessário para seleção quanto a essa característica. Foram utilizados dados de avaliação fenotípica de 185 progênies F2:3 derivadas do cruzamento VC-3 x 'BRSMG Majestoso', para o estudo do controle genético do escurecimento dos grãos. Com esses dados, foram confeccionados dois "bulks" segregantes de DNA, empregados para a avaliação de 444 pares de primers SSR. Os aplicativos computacionais GQMOL e Sisvar foram utilizados para avaliar as segregações, confeccionar um grupo de ligação e realizar análises de marca simples e de regressão múltipla pelo método "backward". Oito marcadores apresentaram polimorfismo nos "bulks". Seis desses marcadores foram agrupados em um grupo de ligação de 80,49 cM, e destes, três mostraram-se estreitamente ligados ao QTL responsável pelo escurecimento tardio dos grãos. O marcador PVM02TC116 cossegregou com o QTL em questão, e os marcadores PVESTBR-98 (2,00 cM) e PV176 (12,24 cM) flanqueiam essa região, o que sugere elevada eficiência para possível uso na seleção assistida.

Outcrossing rate between 'Haden' and 'Tommy Atkins' mangoes estimated using microsatellite and AFLP markers

The objective of this work was to estimate outcrossing rates between Haden and Tommy Atkins mango cultivars, using AFLP and microsatellite markers. Progenies of an isolated 'Haden' plant, identified in a 'Tommy Atkins' commercial orchard, in Petrolina, PE, Brazil, were analyzed. Total DNA was isolated from the progeny leaves and used for AFLP and microsatellite reactions. Multilocus outcrossing rates (t m) were estimated by direct count of AFLP or microsatellite markers and by the mLTR software. Outcrossing rates ranged from 0.85 to 0.87 with the analysis based on seven AFLP markers, and from 0.83 to 0.91 based on three microsatellite primers. No unexpected band patterns were observed for 'Haden' and 'Tommy Atkins'. The estimates obtained with the mLTR software were close to those obtained by direct AFLP and microsatellite allele counting, which indicates that the multilocus model was appropriate for this kind of study. The microsatellites mMiCIR005, mMiCIR030, and mMiCIR036 can be used to elucidate the origin of 'Haden' and 'Tommy Atkins' seedlings.

Genetic similarity in a hybrid population of 'Montenegrina' and 'King' mandarins

The objective of this work was to confirm the hybrids obtained in plants originated from the crossing between the mandarins Citrus deliciosa 'Montenegrina' and C. nobilis 'King', and to estimate the genetic similarity among hybrids, and between each hybrid and its parents. Twenty‑three pairs of microsatellite primers were tested. Fourteen of these pairs showed polymorphic bands between parents. Primers CCSM 129 and CCSME 52 were sufficient to identify the 12 nucellar clones observed in the studied population. Genetic similarity analysis of the population (hybrids and parents) showed 0.56 average similarity. Besides the 12 clones of 'Montenegrina' identified, 25 hybrids were found of which D18, C32, D06, C05 and D09 are the more similar to 'Montenegrina'.

Genetic diversity between improved banana diploids using canonical variables and the Ward-MLM method

The objective of this work was to estimate the genetic diversity of improved banana diploids using data from quantitative analysis and from simple sequence repeats (SSR) marker, simultaneously. The experiment was carried out with 33 diploids, in an augmented block design with 30 regular treatments and three common ones. Eighteen agronomic characteristics and 20 SSR primers were used. The agronomic characteristics and the SSR were analyzed simultaneously by the Ward-MLM, cluster, and IML procedures. The Ward clustering method considered the combined matrix obtained by the Gower algorithm. The Ward-MLM procedure identified three ideal groups (G1, G2, and G3) based on pseudo-F and pseudo-t² statistics. The dendrogram showed relative similarity between the G1 genotypes, justified by genealogy. In G2, 'Calcutta 4' appears in 62% of the genealogies. Similar behavior was observed in G3, in which the 028003-01 diploid is the male parent of the 086079-10 and 042079-06 genotypes. The method with canonical variables had greater discriminatory power than Ward-MLM. Although reduced, the genetic variability available is sufficient to be used in the development of new hybrids.

Identification of zygotic and nucellar seedlings in polyembryonic mango cultivars

The objective of this work was to evaluate the occurrence of polyembryony in the mango cultivars Manila and Ataulfo, and to determine whether seedlings cultured in vitro are zygotic or nucelar. Percentage of polyembryony was calculated and the number of embryos in 100 seeds of each cultivar was recorded. 'Manila' exhibited 97% polyembryony with 3.4 embryos per seed, while 'Ataulfo' had 95% polyembryony with 3.2 embryos per seed. Later, 20 seeds of each cultivar were established in vitro, and it was analyzed those in which all embryos germinated (12 seeds from 'Manila' and 7 from 'Ataulfo'). DNA was extracted from seedling leaf tissue, and its origin was identified with 14 RAPD primers. The polymorphic markers recognized the seedlings of sexual origin in seven of nine 'Manila' polyembryonic seeds, and in four of seven 'Ataulfo' ones. Also, in polyembryonic seeds not all zygotic seedlings were produced by small embryos located at the micropyle.

Molecular characterization of soybean cultivars by microsatellite markers with universal tail sequence

The objective of this work was to standardize a semiautomated method for genotyping soybean, based on universal tail sequence primers (UTSP), and to compare it with the conventional genotyping method that uses electrophoresis in polyacrylamide gels. Thirty soybean cultivars were genotypically characterized by both methods, using 13 microsatellite loci. For the UTSP method, the number of alleles (NA) was 50 (2-7 per marker) and the polymorphic information content (PIC) ranged from 0.40 to 0.74. For the conventional method, the NA was 38 (2-5 per marker) and the PIC varied from 0.39 to 0.67. The genetic dissimilarity matrices obtained by the two methods were highly correlated with each other (0.8026), and the formed groups were coherent with the phenotypic data used for varietal registration. The 13 markers allowed the distinction of all analyzed cultivars. The low cost of the UTSP method, associated with its high accuracy, makes it ideal for the characterization of soybean cultivars and for the determination of genetic purity.

DIVERGÊNCIA GENÉTICA ENTRE DOZE GENÓTIPOS DE ABACAXIZEIRO (Ananas comosus L, Merril.) ESTIMADA POR ANÁLISE DE MARCADORES RAPD

Por meio de estudos moleculares, este trabalho determinou a distância genética entre 12 genótipos de A. comosus por marcadores RAPD (Random Amplified Polymorphic DNA), utilizando 11 "primers" decâmeros da OPERON Technologies Inc. Dos 12 genótipos , 1 foi proveniente da Jamaica, 2 do Estado do Acre (Quinari e RBR-1), 2 do Estado do Maranhão (Turiaçu e São Domingos), 3 do Estado do Piauí (Cefas, Floriano-1 e Floriano-2), 2 do Estado da Bahia (Monte Alegre-1 e Monte Alegre-2) e 2 de Minas Gerais (Pérola e Smouth Cayenne). Pela análise de "cluster", utilizando o método de UPGMA, foi constatada uma grande divergência entre os genótipos de A. comosus estudados com a separação destes em dois grupos a uma distância genética de 31,1%.

AVALIAÇÃO DE PLANTAS MATRIZES DE ABACAXIZEIRO CULTIVAR SMOOTH CAYENNE UTILIZANDO MARCADORES RAPD E PADRÕES ISOENZIMÁTICOS

Foram coletadas, em área comercial da fazenda Córrego dos Bois, município de Canápolis -- MG, 20 plantas matrizes de abacaxizeiro cultivar Smooth Cayenne, para avaliação de similaridade e padrões genotípicos através de marcadores moleculares RAPD e padrões isoenzimáticos. As plantas matrizes foram selecionadas mediante as seguintes características: planta sadia, frutos cilíndricos, ausência de fasciação, pedúnculo curto, ausência de espinhos nas folhas, "olho" do fruto chato e peso dos frutos entre 1,6 a 2,2kg. Para análise de marcadores RAPD, foram testados 100 "primers", dos quais, 43 foram eficientes na amplificação das amostras, onde foram observados padrões de bandas diferentes entre as plantas matrizes utilizadas, indicando a existência de variabilidade genética. Nos padrões isoenzimáticos, dos 15 sistemas utilizados para revelação das amostras, 8 apresentaram atividade enzimática, sendo 5 deles com baixa resolução; entretanto, estes sistemas não foram eficientes em diferenciar as amostras devido à ausência de polimorfismo

MARCADORES RAPD PARA MAPEAMENTO GENÉTICO E SELEÇÃO DE HÍBRIDOS DE CITROS

Os marcadores moleculares apresentam várias aplicações no melhoramento de plantas, permitindo uma série de análises genéticas. Este trabalho foi realizado com o objetivo de estabelecer marcadores RAPD para serem utilizados em estudos de mapeamento genético e na seleção de híbridos entre tangerina-'Cravo' (Citrus reticulata Blanco) e laranja-'Pêra' (C. sinensis (L.) Osbeck). Extraiu-se DNA de folhas dos parentais e de seis híbridos F1. As reações de amplificação foram preparadas em 13 uL de solução, constituída por tampão 1x GIBCO BRL; soluções 1,54 mM de MgCl2 e 0,2 mM de cada dNTP; 15 ng de cada 'primer'; 1,5 unidade de 'Taq DNA Polymerase' e 15 ng de DNA genômico. As reações foram realizadas em termocicladores programados para 36 ciclos de 1 min a 92ºC, 1 min a 36ºC, 2 min a 72ºC e 10 min de extensão a 72ºC. Foram testados 'primers' decâmeros arbitrários dos 'kits' A, AB, AT, AV, B, C, D, E, G, H, M, N, P, Q, R e U da Operon, sendo selecionados 113 por apresentarem polimorfismo, com número de marcadores variando de 1 a 6 por 'primer'. Esses 'primers' amplificaram 201 (23,13%) bandas polimórficas, aplicáveis no mapeamento genético e seleção de híbridos. A freqüência de 'primers' com 1; 2; 3; 4; 5 e 6 bandas polimórficas foi de 49,5%, 33,6%, 9,7%, 4,4%, 1,8% e 1,0%, respectivamente.

Uso de marcadores moleculares na análise da variabilidade genética em acerola (Malpighia emarginata D.C.)

A acerola (Malpighia emarginata) é uma frutífera tropical encontrada nativa na América Central e no Norte da América do Sul, sendo de grande importância econômica e social devido ao seu alto conteúdo de vitamina C (ácido ascórbico). Pomares de acerola têm sido preferencialmente estabelecidos por métodos de propagação vegetiva. No entanto, a propagação sexuada por sementes é igualmente utilizada e permite revelar um alto grau de polimorfismo na cultura, possibilitando a identificação de genótipos portadores de características de interesse agronômico. Vinte e quatro acessos de acerola, pertencentes ao Banco Ativo de Germoplasma da Universidade Estadual de Londrina, foram analisados, usando marcadores RAPD (Random amplified Polymorphic DNA) e obtidos com iniciadores (primers) de seqüência simples repetidas (SSRs). Um total de 164 e 73 marcadores foram obtidos com primers de RAPD e SSR, respectivamente. Os marcadores obtidos foram analisados, usando o método de agrupamentos UPGMA. A análise comparativa dos dendrogramas gerados com os primers de RAPD e com os primers SSR mostrou que, enquanto alguns acessos se associaram em grupos diferentes, outros apresentaram a mesma associação. Entretanto, maior polimorfismo entre acessos foi detectado com os primers de RAPD. A análise dos resultados revelou a alta variabilidade contida na coleção, permitindo associar o grau de similaridade genética, obtido por marcadores de DNA, com caracteres morfológicos compartilhados entre os acessos.

Caracterização e identificação de cultivares e seleções de pereiras através de marcadores RAPD

Trinta e seis acessos de pereira representando diversas espécies, híbridos e seleções do banco de germoplasma do Instituto Agronômico (IAC) foram geneticamente caracterizados através de marcadores RAPD. Cada primer originou de 10 a 19 bandas, sendo que 26 deles forneceram 250 bandas polimórficas, de um total de 353. Os primers OPC02, OPC08, OPD02, OPD19, OPD20 e OPE06 revelaram bandas específicas para as peras orientais e OPA01, OPA11, OPC08, OPD04, OPD09 e OPD15 para as ocidentais. O dendograma obtido foi confirmado pela análise de coordenada principal, originando três principais agrupamentos: 1) Todas as pereiras lançadas pelo IAC, como 'Seleta', 'Triunfo', 'Primorosa', 'Tenra', IAC 16-41, 'Centenária', além de 'William's', 'Packham's Triumph', 'D'água', 'Hood', 'M. Sieboldt', 'Kieffer','Branca Francesa' e 'Schimidt'. 2) As pereiras asiáticas, como 'Okusankichi', 'Shinseiki', 'Atago', 'Hakko', 'Hosui', 'Nijiseiki', 'Kosui' e 'Ya-li', além de 'Nodji', 'Limeira' e todas as seleções IAC das séries 193; 293 e 393. 3) Todas as pereiras porta-enxertos da série Taiwan (P. calleryana D.), além de 'Manshu Mamenashi' (P. betulaefolia B.). Evidenciou-se que os cultivares IAC possuem maior proximidade genética com as peras ocidentais (Pyrus communis L.), mesmo sendo descendentes de 'Hood', material suspeito de ser híbrido interespecífico entre P. communis e P. serotina R.. Os resultados ratificaram a importância dos marcadores RAPD para a identificação de cultivares, seleções e híbridos pertencentes aos diferentes grupos botânicos, mostrando ser ferramenta de apoio adequada a programas de melhoramento genético de fruteiras.

Genetic variations among passion fruit species using rapd markers

It has been evaluated the genetic variability through the use of RAPD molecular markers on the following passionflower species: Passiflora amethystina, P. caerulea, P. cincinnata, P. coccinea, P. serrato digitata, P. foetida, P. maliformis, P. alata, P. giberti, P. laurifolia, P. macrocarpa, P. nitida, P. setacea, P. suberosa, P. ligularis, P. capsularis, P. edulis Sims and its botanical variety P. edulis Sims f. flavicarpa Deg. In this research work, the analyses of the random amplified polymorphic DNA products (RAPD) were employed to estimate the genetic diversity and the taxonomic linkage within the species above. The total of 21 primers were used in this study which generated 270 different polymorphic products. It was possible to detect that the Passiflora species had shown a similarity of 17,3%, and between Passiflora edulis Sims and Passiflora edulis Sims f. flavicarpa a similarity of 34,35% has been found. The rate of similarity within edulis specie is low, making it clear that a large variability between the yellow and the purple forms exists.