Variedades de cultivo de líquido amniótico para el diagnóstico in vitro de poliploidías / Amniotic fluid culture varietys for poliploydies in vitro diagnosis

RESUMEN Fundamento: El cultivo celular permite el análisis directo de las células vivas mediante un microscopio. El estudio de las células contenidas en el líquido amniótico, mediante técnicas de cultivo, detecta anomalías en número y morfología de los cromosomas, que pueden relacionarse con enfermedades genéticas. Objetivo: Caracterizar las variedades de cultivo de líquido amniótico para el diagnóstico in vitro de poliploidías. Metodología: Se realizó un estudio descriptivo transversal, en el Centro Provincial de Genética Médica de Camagüey, en el periodo de noviembre de 2016 a abril de 2018.La población de estudio estuvo constituida por 1571 muestras útiles de líquido amniótico obtenidas por amniocentesis, en gestantes en el segundo trimestre, evaluadas en consulta multidisciplinaria con criterios clínicos de estudios cromosómicos según lo establecido en el diagnóstico prenatal citogenético, previo consentimiento informado. Se utilizaron 20 mL de líquido amniótico para la siembra de células fetales, y se aplicaron tres variantes de cultivo abierto (directo, centrifugado y expandido). Se determinó el complemento cromosómico en cada variedad. Resultados: Predominó el complemento cromosómico normal. Las tetraploidías prevalecieron en el cultivo expandido. El índice mitótico fue similar en las tres variedades de cultivo y el cultivo directo tuvo el más bajo índice de poliploidías. Conclusiones: El cariotipo normal fue predominante. Las tetrapolidías fueron las alteraciones más frecuentes y prevalecieron en el cultivo expandido. En el cultivo directo se presentó el más bajo índice de errores inducidos in vitro.

ABSTRACT Background: Cell culture allows direct analysis of live cells under a microscope. The cell study contained in amniotic fluid, by culture techniques, detects abnormalities in chromosome number and morphology, which can be related to genetic diseases. Objective: To describe amniotic fluid culture strains for the in vitro diagnosis of polyploidy. Methodology: A cross-sectional descriptive study was conducted at the Camagüey Provincial Center of Medical Genetics, from November 2016 to April 2018.The study population consisted of 1571 useful amniotic fluid samples obtained by amniocentesis, in pregnant women in the second trimester, evaluated by multidisciplinary discussion with clinical criteria for chromosomal studies as established in the cytogenetic prenatal diagnosis, prior informed consent. 20 mL of amniotic fluid were used for fetal cell seeding, and three open culture strains (direct, centrifuged and expanded) were applied. Chromosomal complement was determined in each variety. Results: Normal chromosome complement was predominant. Tetraploidy prevailed in the expanded culture. The mitotic index was similar in the three culture strains and the direct culture had the lowest polyploidy index. Conclusions: Normal karyotype was predominant. Tetraploidy were the most frequent modifications and prevailed in the expanded culture. Direct culture had the lowest rate of the in vitro induced errors.

Screening and analysis on the differentially expression genes between diploid and autotetraploid watermelon by using of digital gene expression profile / Triagem e análise da expressão diferencial dos genes entre melancia diplóide e autotetraplóide através do perfil de expressão gênica digital

Abstract Synthetic polyploids are key breeding materials for watermelon. Compared with diploid watermelon, the tetraploid watermelon often exhibit wide phenotypic differences and differential gene expression. Digital gene expression (DGE) profile technique was performed in this study to present gene expression patterns in an autotetraploid and its progenitor diploid watermelon, and deferentially expressed genes (DEGs) related to the abiotic and biotic stress were also addressed. Altogether, 4,985 DEGs were obtained in the autotetraploid against its progenitor diploid, and 66.02% DEGs is up-regulated. GO analysis shows that these DEGs mainly distributed in 'metabolic process', 'cell' and 'catalytic activity'. KEGG analysis revealed that these DEGs mainly cover 'metabolic pathways', 'secondary metabolites' and 'ribosome'. Moreover, 134 tolerance related DEGs were identified which cover osmotic adjustment substance, protective enzymes/protein, signaling proteins and pathogenesis-related proteins. This study present the differential expression of stress related genes and global gene expression patterns at background level in autotetraploid watermelons. These new evidences could supplement the molecular theoretical basis for the better resistance after the genome doubling in the gourd family.

Resumo Poliploides sintéticos são materias fundamentais para melhoramento genético da melancia. Comparativamente ao seu homólogo diploide, a melancia tetraploide apresenta amplas diferenças genotípica e fenotípica e diferença de expressão gênica. A expressão gênica digital ou DGE (digital gene expression) foi utilizada neste estudo para representar o perfil de expressão gênica da melancia autotetraploide e seu progenitor diploide e a expressão diferencial de genes relacionados ao estresse biótico e abiótico. Os resultados mostraram que 4.985 DEGs foram observados no organismo autotetraploide, sendo que, deste total, 66.02%foram supra-regulados. A análise de ontologia gênica (GO) mostrou que estes DEGs estão relacionados principalmente com processos metabólicas, célula e atividade catalítica, abrangendo de acordo com a análise de genes e genoma (KEGG) rotas metabólicas, metabolismo secundário e ribossomos. Além disso, 134 genes de defesa foram identificados, abrangendo substâncias de ajuste osmótico, enzimas/proteínas de proteção, proteínas sinalizadoras e proteínas relacionadas à patogênese. Este estudo mostrou a expressão diferencial de genes relacionados ao estresse e o perfil global de expressão gênica de melancia autotetraploide, estes resultados podem complementar, a nível molecular, o entendimento do fator resistência após a duplicação do genoma em cucurbitáceas.

Morphological and stomatal characterization of Heliconia chartacea Var. sexy pink induced polyploidy / Caracterização morfológica e estomática de heliconia chartacea var. sexy pink induzidas a poliploidia

The growth of the tropical flower market has demanded a consistent search for new varieties, primarily those endowed with an exotic profile, but that are also beautiful and durable. The genusHeliconia, naturally found in the Amazon region, is among the most prominent of tropical flowers. Looking to augment the genetic variability available in Heliconia chartacea var. Sexy Pink, biotechnological research was conducted with the application of colchicine to induce polyploidy in plants from this species. With that in mind, this study was undertaken to evaluate the establishment of plants in the field drawn from in vitro polyploidy induction assay and to determine the morphological and physiological characteristics of 38 H. chartacea var. Sexy Pink clones. The characterization analyzes were performed through 49 morphological descriptors and a stomatal density evaluation using microscopy. The genotype 35 exhibited the greatest morphological variations, with alterations in the position and coloring of the inflorescence, in addition to having the edges of the entire limbus. Genotype 18 featured the lowest amounts for plant height and inflorescence size, showing promise for research geared towards use in reduced environments. Some genotypes did not have any flowering and arerecommended exclusively for landscape composition such as foliage, since their exotic characteristics allow for this. The genotypes that were evaluated displayed stomata with tetracytic morphology and guard cells that had no significant changes. However, genotypes with greater equatorial diameter and stomatal density were obtained in relation to the mother-plant. Overall, the induction of polyploidy allowed for clones to be obtained with a high variability for the characteristics of the leaf, pseudostem and inflorescence, with various attributes that confer a more efficient post-harvest management to some genotypes, in addition to favorable aspects for commercialized purposes as a cut flower.

A expansão do mercado de flores tropicais tem demandado uma constante procura por novas variedades, principalmente aquelas dotadas de perfil exótico, mas ainda apresentando beleza e durabilidade. Dentre as flores tropicais de maior destaque, se encontram as do gênero Helicônia, sendo estas naturalmente encontradas na região Amazônica. Visando aumentar a variabilidade genética disponível em Heliconia chartacea var. Sexy Pink, pesquisas biotecnológicas foram realizadas com a aplicação de colchicina para indução a poliploidia em plantas da espécie. Deste modo, o objetivo do presente trabalho foi avaliar as plantas estabelecidas em campo, provenientes dos ensaios de indução à poliploidia in vitro para determinar as características morfológicas e fisiológicas de 38 clones de H. chartacea var. Sexy Pink. As análises de caracterização foram realizadas por meio de 49 descritores morfológicos e avaliação da densidade estomática por microscopia. O genótipo 35 foi o que apresentou as maiores variações morfológicas, com alterações na posição e coloração da inflorescência, além de possuir as bordas do limbo foliar inteiras. O genótipo 18 apresentou os menores valores para altura da planta e tamanho das inflorescências, mostrando-se promissor para pesquisas voltadas ao uso em ambientes reduzidos. Alguns genótipos não tiveram floração, sendo recomendada a sua utilização exclusivamente para composição paisagística como folhagens, já que sua exoticidade permite esta finalidade. Os genótipos avaliados apresentaram estômatos com morfologia tetracítica e células-guarda sem alterações significativas, porém, foram obtidos genótipos com maior diâmetro equatorial e densidade estomática em relação a planta matriz. De modo geral, a indução a poliploidia permitiu a obtenção de clones com alta variabilidade para características da folha, pseudocaule e inflorescência, sendo vários os atributos que conferiram a alguns genótipos um manejo pós-colheita mais eficiente, além de aspectos favoráveis para comercialização como flor de corte.

Polyploidy as a chromosomal component of stochastic noise: variable scalar multiples of the diploid chromosome complement in the invertebrate species Girardia schubarti from Brazil / Poliploidia como componente cromossômico de ruído estocástico: variações escalares múltiplas do componente cromossômico diploide do invertebrado Girardia schubarti ocorrente no Brasil

Abstract Chromosome stoichiometry, a form of genetic plasticity, specifically refers to variation in the standard diploid genomic composition of an individual or species. In the present work, freshwater planarians (Girardia schubarti) were analyzed to recognize variations in chromosomal stoichiometry especially of complete ploidal change between specimens, within specimens and between cells within specimens and any relations they might have with selected components of phenotypic plasticity. Homoploid polyploids for the group reached rational scalar multiples (e.g. tetraploids) or irrational scalar multiples (e.g. triploids). Karyotypic mosaics emerged where individual cells presented polyploid multiples in arithmetic and geometric progressions. Ploidal multiplicity, a chromosomal component of stochastic noise, had positive phenotypic effects (increased dimensions) on morphologic criteria of body length, body width and dorsal surface reflecting a significant genotypic plasticity (GP) and robust phenotypic plasticity (PP). Variable but significant association of genotypic plasticity with robust phenotypic variance suggests kinetics of phenotypic homeostasis that is species-specific permitting phenotypic adaptability to environmental variables by means of GP. That association is diminished, deactivated or lost in more advanced and more complex organisms.

Resumo A estequiometria cromossômica, uma forma de plasticidade genotípica, representa variações na composição genômica diploide de um indivíduo ou espécie. Planárias límnicas (Girardia schubarti) foram analisadas para verificar a estequiometria cromossômica, especialmente alterações na ploidia entre espécimes, em cada espécime e entre células do mesmo espécime, além de relações dessas alterações com a plasticidade fenotípica. Espécimes poliploides homoploides apresentaram múltiplos escalares racionais ou irracionais, tais como triploides. Mosaicos cariotípicos ocorreram quando células apresentaram poliploides múltiplos em progressões aritméticas e geométricas. Nas planárias estudadas, a multiplicidade ploidal, um componente cromossômico de ruído estocástico, apresentou efeitos fenotípicos positivos, causando aumento das dimensões dos indivíduos, tais como comprimento corporal, largura do corpo e superfície dorsal, indicando plasticidade genotípica (GP) significativa e plasticidade fenotípica (PP) robusta. Associações significativas da plasticidade genotípica com variâncias fenotípicas robustas, embora variáveis, sugerem que a homeostase fenotípica, a qual é espécie-específica, possibilita adaptações a variáveis ambientais através da GP. Tal associação apresenta-se reduzida, desativada ou perdida em organismos mais complexos.

Lavandula L. - aplicação da cultura in vitro à produção de óleos essenciais e seu potencial económico em Portugal / Lavandula L. - application of in vitro cultivation for the essential oils production and their economic potential in Portugal

RESUMO Os óleos essenciais são metabolitos secundários que possuem diversas propriedades com elevado interesse, nomeadamente as biológicas. Estas propriedades englobam todas as atividades que esta mistura de compostos voláteis (principalmente monoterpenos, sesquiterpenos e fenilpropanóides) exerce sobre os seres humanos, animais e outras plantas. Os óleos essenciais apresentam grande valor económico, sendo os do género Lavandula dos mais comercializados e estudados devido à sua aplicabilidade industrial e propriedades terapêuticas As lavandulas são colhidas na natureza ou propagadas por técnicas convencionais, nomeadamente por estacaria. Mais recentemente, protocolos de micropropagação foram desenvolvidos para algumas espécies, permitindo a produção de plantas em larga escala disponíveis em qualquer período do ano e sem comprometer a biodiversidade das espécies. O desenvolvimento de plantas tetraplóides capazes de aumentar a produção de óleo essencial nas suas flores é outro meio eficaz para aumentar potencialmente o valor das espécies de Lavandula. Em Portugal existem 5 espécies nativas do género Lavandula, amplamente distribuídas pelo país. Contudo, o seu potencial industrial permanece praticamente inexplorado e em termos de mercado o seu reconhecimento é muito reduzido. Trabalhos recentes, baseados na avaliação das propriedades biológicas dos óleos essenciais e a forma como eles podem exercer os seus efeitos contribuíram para a valorização do potencial económico das lavandulas em Portugal. Esta revisão tenta dar uma visão geral de que forma a aplicação das culturas in vitro pode levar a uma maior produção de óleos essenciais em Lavandula spp., dando especial ênfase às lavandulas nativas de Portugal.

ABSTRACT Essential oils are secondary metabolites plants and, among other features, they own several biological properties. The term “biological” includes all activities that these mixtures of volatile compounds (mainly monoterpenes, sesquiterpenes, and phenylpropanoids) maintain on humans, animals and other plants. Essential oils have great economic value, being the genus Lavandula one of the most commercialized and studied type due to its dits industrial applicability and therapeutic properties. Lavenders are harvested in the nature or propagated using traditional methodologies. More recently micropropagation protocols have been developed for several Lavandula species allowing a production of a high amount of plants available at any time of the year without compromising the biodiversity of the species. Another efficient way to potentially increase the value of Lavandula species is through the development of tetraploid plants, capable of raising the production of essential oils in flowers. In Portugal, there are 5 native species of the genus Lavandula, widely distributed throughout the country. However, native Portuguese lavenders remain mainly unexplored and have received poor recognition in markets. A recent study was carried out in order to evaluate the biological properties of the oils and also to understand how they may exercise their effects, contributing to enhance the economic potential of lavandulas in Portugal. This review attempts to provide an overview about the application of in vitrocultivations for the production of essential oils in Lavandulaspp., with special emphasis on native lavandulas of Portugal.

Caracterização citogenética para identificação dos níveis de ploidia em cinco espécies do gênero Mentha L / Citogenetic characterization to determination of ploidy level on five Mentha L. species

Neste trabalho foi feita a caracterização citogenética da: microsporogênese, tétrades, estimativa da viabilidade do pólen pelo método de coloração e contagem do número máximo de nucléolos por célula interfásica, para identificação dos níveis de ploidia, em cinco espécies do gênero Mentha L. Foram coletadas inflorescências em 30 plantas de cada espécie, em duas florações sucessivas, nos anos 2006 e 2007. As inflorescências foram tratadas em etanol-ácido acético (3:1), em temperatura ambiente durante seis horas, transferidas para álcool 70% (v/v) e conservadas em geladeira até análise. Nas análises da microsporogênese, tétrades e pólen o corante usado foi carmin propiônico 2% e na identificação dos nucléolos nitrato de prata (AgNO3). Os resultados demonstraram que as cinco espécies são poliplóides. M. crispa heptaplóide (2n=7x=84) com 11 nucléolos, M. spicata tetraplóide (2n=4x=48) com 8 nucléolos, M.x gentilis pentaplóide (2n=5x=60) com 12 nucleólos, M. piperita e M.x piperita ambas hexaplóides (2n=6x=72) com 8 e 9 nucléolos respectivamente. M. spicata e M. crispa mantiveram as mais altas porcentagens de células normais na microsporogênese, na formação de tétrades e na estimativa da viabilidade do pólen por coloração, sugerindo maior estabilidade meiótica quando comparados aos demais poliplóides estudados.

The cytogenetic characterization of five species of Mentha L. genus, including the data: regularity of microsporogenesis and tetrads, and polen viability, using the coloration method and the counting of the maximum number of nucleolus by interphasic cell were carried out in this study to identify the ploid levels. These analyses were performed from inflorescences collected in 30 plants of each species, during two successive florations in 2006 and 2007. Inflorescences were treated in 3:1 ethanol:acetic acid mixture at room temperature during six hours, then transferred to 70%(v/v) ethanol solution and refrigerated until the analysis. For microsporogenis, tetrad and pollen analysis, we used carmine propionic 2% (m/v) and for nucleolus identification, we used AgNO3 solution. It was possible to observe that all five species were polyploids. M. crispa heptaploid (2n=7x=84) with 11 nucleolus, M. spicata tetraploid (2n=4x=48) with 8 nucleolus, M. x gentilis pentaploid (2n=5x=60) with 12 nucleolus, M. piperita and M. x piperita both hexaploid (2n=6x=72) with 8 and 9 nucleolus respectively. M. spicata and M. crispa kept the highest percentual values of normal cells in microsporogenesis as well as in tetrads formation and pollen viability, suggesting a higher meiotic stability when compared to the other polyploids studied.

A polymorphic microsatellite from the Squalius alburnoides complex (Osteichthyes, Cyprinidae) cloned by serendipity can be useful in genetic analysis of polyploids

A new microsatellite locus (SAS1) for Squalius alburnoides was obtained through cloning by serendipity. The possible usefulness of this new species-specific microsatellite in genetic studies of this hybrid-species complex, was explored. The polymorphism exhibited by SAS1 microsatellite is an important addition to the set of microsatellites previously used in genetic studies in S. alburnoides complex, that mostly relied in markers described for other species. Moreover, the SAS1 microsatellite could be used to identify the parental genomes of the complex, complementing other methods recently described for the same purpose.

Chromosome number and cytogenetics of Euphorbia heterophylla L

Euphorbia heterophylla L. (Euphorbiaceae) is a herbaceous species of great economic importance due to its invasive potential and consequent damage to agriculture and pasture land. For the first time, we provide information on its chromosome number, morphology, and behavior of mitotic chromosomes. Seeds were germinated and submitted to four treatments to obtain metaphases: 0.5% colchicine for 2 to 5 h, at ambient temperature; 0.5% colchicine for 16 to 24 h; 0.0029 M 8-hydroxyquinoline (8-HQ) for 2 to 5 h at ambient temperature, and 0.0029 M 8-HQ for 16 to 24 h at 4°C. The material was then fixed in methanol:acetic acid (3:1) and kept at -20°C for 24 h. Roots were macerated in the enzyme solution of Flaxzyme™ (NOVO FERMENT™)-distilled water (1:40) at 34°C for 2 h and later fixed again. Chromosome preparations were obtained by the dissociation of the apical meristems. The best chromosome preparations were obtained with the use of 8-HQ for 21 h 30 min at 4°C. E. heterophylla showed 2n = 28 chromosomes. The short arm of the largest pair of chromosomes of the complement (pair number 1) displayed a secondary constriction while the nucleolus was observed in the interphasic cell. Structural rearrangements were also observed in the E. heterophylla L. genome. The genomic instability associated with polyploidy may be the result of selection shaped by environmental adaptations and/or human-induced manipulation through agricultural practices.

Unusual chromosome numbers in Paspalum L. (Poaceae: Paniceae) from Brazil

Somatic chromosome numbers were determined for 20 new germplasm accessions of Paspalum, belonging to 17 species collected in Brazil. Chromosome number is reported for the first time for P. reduncum (2n = 18), P. cinerascens (2n = 20), P. cordatum (2n = 20), P. filgueirasii (2n = 24), P. ammodes (2n = 36), P. bicilium (2n = 40), P. heterotrichon (2n = 40), and P. burmanii (2n = 48). New cytotypes were confirmed for two germplasm accessions of P. carinatum (2n = 30) and P. trachycoleon (2n = 36), one of P. clavuliferum (2n = 40) and one of P. lanciflorum (2n = 40), indicating variability in these species. The remaining chromosome numbers reported here confirm previous counts. The unexpected chromosome numbers 2n = 18, 24, 36, and 48 in Paspalum species, which are usually shown to be multiples of 10, suggest that much more collection and cytogenetic characterization are necessary to assess the whole chromosomal and genomic multiplicity present in the genus, which seems to be much more diverse than currently thought to be.

Anatomical alterations due to polyploidy in cassava, Manihot esculenta Crantz

Information on anatomical structure is needed by breeders working on improvement for drought tolerance. For studying the effect of polyploidy on cassava anatomy and its significance to tolerance to drought, we induced a polyploidy type of a selected clone (UnB 530) by applying an aqueous solution of 0.2% colchicine on lateral buds for a period of 12 h. The stem identified as tetraploid was propagated to produce the whole plant. Free-hand cross-sections of the median portion between stem internodes were made. They were clarified using 50% sodium hypochlorite solution, stained with 1% safranin-alcian blue, passed through an ethanol series and butyl acetate and mounted in synthetic resin. The tetraploid type showed more prismatic and druse crystals in the cortical parenchyma, and its pericycle fibers had thicker walls. The secondary xylem of tetraploid types was wider than diploid ones, having thinner walls and less starch.

Cassava genetic resources and their utilization for breeding of the crop

Wild cassava relatives are perennials and vary in growth pattern from nearly acaulescent subshrubs to small trees. They have been used as a source of useful characters such as high protein content, apomixis, resistance to mealybug and mosaic disease, and tolerance to drought. Indigenous clones are a potential source of beta-carotene and lycopene. Apomixis genes have been transferred to the crop successfully through interspecific hybridization, and apomictic clones arising from these hybrids are now being grown at the Universidade de Brasília. Interspecific hybrids produced earlier were polyploidized and had their fertility restored. Different useful types of chimera were also produced.

Microsporogenesis in sexual Brachiaria hybrids (Poaceae)

Three sexual interspecific hybrids of Brachiaria (HBGC076, HBGC009, and HBGC014) resulting from crosses between B. ruziziensis (female genitor) and B. decumbens and B. brizantha (male genitors) produced by Embrapa Beef Cattle in the 1980s were cytologically analyzed by conventional methods for meiotic studies. The cytogenetic analysis showed the occurrence of common meiotic abnormalities among them. The most frequent abnormalities were those related to irregular chromosome segregation due to polyploidy. Other abnormalities, such as chromosome stickiness, absence of cytokinesis, irregular cytokinesis, abnormal spindle orientation, and abnormal nucleolus disintegration, were found in the three hybrids, while, chromosome disintegration was detected only in HBGC014. All the abnormalities, except for abnormal nucleolus disintegration, can cause unbalanced gamete formation, leading to pollen sterility. Multivalent chromosome association at diakinesis revealed genome affinity between the two parental species in the hybrids, suggesting some possibility for gene introgression. Presently, the Brachiaria breeding program has the objective of releasing, primarily, apomictic hybrids as new cultivars since they do not segregate but preserve the genetic makeup indefinitely. Besides, they result in homogeneous pastures which are easier to manage. The sexual hybrids, however, are paramount in the breeding program: they work as ‘bridges’ to introgress traits of interest into the apomictic genotypes. The cytogenetic analyses of these three hybrids substantiate their maintenance in the breeding program due to low frequency of meiotic abnormalities, complemented by interesting agronomic traits. They may be used in crosses to generate new cultivars in the future.

Differentiation of Nijmegen breakage syndrome from Fanconi anemia

Nijmegen breakage syndrome (NBS) is a rare auto-somal recessive condition with chromosomal instability. Clinical and biological overlap between Fanconi anemia and ataxia telangiectasia has been reported. We report two cases of NBS born to consanguineous parents. Case one had NBS and Falconi anemia clinical features but relatively little chromosome breakage. The second case had mild NBS features, while cytogenetic evaluation with mitomycin C induction showed chromosome damage. Chromosomal analysis of bone marrow cells revealed tetraploidy, which indicates progression towards leukemia. On the basis of clinical and cytogenetic evaluation, these two cases were confirmed as NBS. However, detailed molecular studies are essential for accurate diagnosis and management of this disease.

The synthesis of a new cassava-derived species, Manihot vieiri Nassar

A new species was synthesized artificially by chromosome doubling in a hybrid. The ensuing polyploid type exhibits an apomictic nature and maintains its morphological characteristics in the progeny. It showed a frequency of multiembryonic sacs of 29% in the ovules examined, whereas sacs were absent in the diploid type.

Microsporogenesis in Brachiaria dictyoneura (Fig. & De Not.) Stapf (Poaceae: Paniceae)

icrosporogenesis was analyzed in five accessions of Brachiaria dictyoneura presenting x = 6 as the basic chromosome number. All accessions were tetraploid (2n = 4x = 24) with chromosome pairing in bi-, tri-, and quadrivalents. The recorded meiotic abnormalities were those typical of polyploids, including precocious chromosome migration to the poles, laggard chromosomes, and micronucleus formation. The frequency of these abnormalities, however, was lower than those reported for other polyploid accessions previously analyzed for other Brachiaria species. Cell fusion and absence of cytokinesis were also recorded in some accessions, leading to restitutional nucleus formation in some cells. Genetically unbalanced microspores, binucleate, and 2n microspores were found among normal meiotic products as results from these abnormalities. The limitation in using these accessions as pollen donor in interspecific crosses with sexual species with x = 7 or x = 9 in breeding programs is discussed.

Evidence of allopolyploidy in Brachiaria brizantha (Poaceae: Paniceae) through chromosome arrangement at metaphase plate during microsporogenesis

In the hexaploid (2n = 6x = 54) accession B176 of Brachiaria brizantha, one cytological characteristic differentiated it from the other accessions previously analyzed with the same ploidy level. Nearly 40% of meiocytes displayed the chromosome set arranged at two metaphase plates at the poles of the cell, close to the membrane. In these cells, both metaphase plates were arranged in an angle to form a typical tripolar spindle. Therefore, cells did not show normal chromosome segregation at anaphase I. Only nine univalent chromosomes migrated from each plate to the opposite pole with the remainder staying immobile on the plate. As a result of such spindle orientation and chromosome behavior, trinucleate telophases I were recorded. After telophase, cytokinesis eliminated the small nuclei into a microcyte. The second division proceeded normally, with the presence of microcytes in all phases. The origin of such an abnormality was explained on the hexaploid level of the accession which could have resulted by chromosome doubling of a triploid derived from species that did not display the same behavior for spindle organization. The high percentage of meiotic abnormalities recorded in this accession compromises fertility and renders it inadequate for the breeding program.

Microsporogenesis in tetraploid accessions of Brachiaria nigropedata (Ficalho & Hiern) Stapf (Gramineae)

The genus Brachiaria (Trin.) Griseb. has achieved considerable importance to cattle production systems, as a result of the good production and adaptation of a few cultivars to poor and acid soils of the Brazilian savannas. Many of its species and accessions are polyploid and apomictic, which limits direct hybridization. To assist the breeding program, cytogenetic characterization has been undertaken on the accessions of Brachiaria collection at the Embrapa Beef Cattle Research Center. In this study, chromosome number and meiotic behavior are reported for the Brachiaria nigropedata (Ficalho & Hiern) Stapf collection. The 20 available accessions are tetraploid (2n = 4x = 36). Chromosomes paired preferentially as bivalents, but quadrivalents were found in high frequencies in some cells. Meiotic behavior was, in general, irregular, and varied among accessions. Most accessions presented more than 20% of abnormal tetrads. The most common meiotic abnormalities were those related to irregular chromosome segregation due to polyploidy, leading to micronuclei formation in the tetrad stage. A low frequency of other meiotic abnormalities such as the absence of cytokinesis, chromosome stickiness, cell fusion, anaphase bridges, and chromosome transfer among microsporocytes were also recorded in some accessions. Limitations of these accessions for use in hybridization programs are discussed.

Symmetric pollen mitosis I and suppression of pollen mitosis II prevent pollen development in Brachiaria jubata (Gramineae)

Microsporogenesis and pollen development were analyzed in a tetraploid (2n = 4x = 36) accession of the forage grass Brachiaria jubata (BRA 007820) from the Embrapa Beef Cattle Brachiaria collection that showed partial male sterility. Microsporocytes and pollen grains were prepared by squashing and staining with 0.5 percent propionic carmine. The meiotic process was typical of polyploids, with precocious chromosome migration to the poles and laggards in both meiosis I and II, resulting in tetrads with micronuclei in some microspores. After callose dissolution, microspores were released into the anther locule and appeared to be normal. Although each microspore initiated its differentiation into a pollen grain, in 11.1 percent of them nucleus polarization was not observed, i.e., pollen mitosis I was symmetric and the typical hemispherical cell plate was not detected. After a central cytokinesis, two equal-sized cells showing equal chromatin condensation and the same nuclear shape and size were formed. Generative cells and vegetative cells could not be distinguished. These cells did not undergo the second pollen mitosis and after completion of pollen wall synthesis each gave rise to a sterile and uninucleate pollen grain. The frequency of abnormal pollen mitosis varied among flowers and also among inflorescences. All plants were equally affected. The absence of fertile sperm cells in a considerable amount of pollen grains in this accession of B. jubata may compromise its use in breeding and could explain, at least in part, why seed production is low when compared with the amount of flowers per raceme.

Long survival in a 69, XXX triploid infant in Greece

The live birth of a triploidy infant is a very rare event and death usually occurs within the first hours of life. Triploid cases with a survival of more than two months are infrequent. We report on an infant with a 69,XXX chromosome constitution who survived 164 days. Chromosomal analysis demonstrated a 69,XXX karyotype with no evidence of mosaicism. This is the longest survival reported for this condition to date in Greece and the fourth longest worldwide. The infant was admitted to our clinic several times due to respiratory problems, and supplementary oxygen was required. The improved survival of our case was possibly due to better management of respiratory illness and prematurity, and these are essential factors that physicians should consider carefully with such rare cases.

Evolution by polyploidy and gene regulation in Anura

The evolution of the metazoa has been characterized by gene redundancy, generated by polyploidy, tandem duplication and retrotransposition. Polyploidy can be detected by looking for duplicated chromosomes or segments of orthologous chromosomes in post-polyploid animals. It has been proposed that the evolutionary role of polyploidy is to provide extra-copies of genes, whose subsequent alteration leads to new functions, increased biological complexity, and, ultimately, speciation. We review the theory of evolution by genome duplication, basing our arguments on findings from autopolyploid anurans and fish, undergoing post-polyploidy diploidization. We conclude that: 1) the high genetic variability of autotetraploid anurans is a result of tetrasomic expression, based on studies of isozymes and other proteins. 2) Epigenetic mechanisms mediate the reduced expression or silencing of redundant copies of genes in the regulation of gene expression of these tetraploids. This conclusion is based on data concerning ribosomal and hemoglobin gene activity. 3) Duplication of the genome may have occurred more than once in the phylogeny of the anurans, as exemplified by 4n and 8n Leptodactylidae species.