Production of wheat-doubled haploids resistant to eyespot supported by marker-assisted selection

BACKGROUND: Wheat is one of the most important crops cultivated all over the world. New high-yielding cultivars that are more resistant to fungal diseases have been permanently developed. The present study aimed at the possibility of accelerating the process of breeding new cultivars, resistant to eyespot, by using doubled haploids (DH) system supported by marker-assisted selection. RESULTS: Two highly resistant breeding lines (KBP 0916 and KBH 4942/05) carrying Pch1 gene were crossed with the elite wheat genotypes. Hybrid plants of early generations were analyzed using endopeptidase EpD1 and two SSR markers linked to the Pch1 locus. Selected homozygous and heterozygous genotypes for the Pch1-linked EpD1b allele were used to produce haploid plants. Molecular analyses were performed on haploids to identify plants possessing Pch1 gene. Chromosome doubling was performed only on haploid plants with Pch1 gene. Finally, 65 DH lines carrying eyespot resistance gene Pch1 and 30 lines without this gene were chosen for the eyespot resistance phenotyping in a field experiment. CONCLUSIONS: Results of the experiment confirmed higher resistance to eyespot of the genotypes with Pch1 in comparison to those without this gene. This indicates the efficiency of selection at the haploid level.

Pleiotropic effects of the sdw1 locus in barley populations representing different rounds of recombination

Background In the present study populations, representing different rounds of recombination were used for the analysis of phenotypic effects associated with the sdw1/denso locus. Other studies have mostly focused only on one type of population. Many different QTLs mapped at the same position as the sdw1/denso locus may indicate a pleiotropy of this gene or a tight linkage between genes conditioning quantitative traits. To date, results of studies have not unequivocally proven either of these two phenomena. Results Both breeding and molecular mapping experiments were undertaken to examine 200 single seed descent (SSD) and 60 doubled haploid (DH) lines obtained from the Maresi (with a semi-dwarfing gene) and Pomo cross combination. They were evaluated for the type of juvenile growth habit and certain agronomic traits were measured after harvesting. The estimates of mean values, standard errors and significance of effects were analyzed. In terms of the analyzed characteristics, the greatest variability was obtained for genotypes with the prostrate growth habit. Microsatellite markers (SSR) were also used to identify co-segregation with the sdw1/denso locus and Bmag0013, Bmag0877, Bmag0306b markers were linked the closest. A partial linkage map of chromosome 3H with the sdw1/denso semi-dwarfing gene was constructed and QTLs were identified. Conclusions Our experiments confirmed the impact of the semi-dwarfing gene on plant height, heading and flowering date both in SSD and DH populations, which may indicate pleiotropy. Moreover, a partial linkage between sdw1/denso locus and grain weight per spike and 1000-grain weight was found in the SSD population.

Trasplante alogénico haploidéntico de progenitores hematopoyéticos en pediatría en Uruguay / Haploidentical allogeneic hematopoietic transplant of hematopoietic parents in pediatrics in Uruguay

Introducción: el trasplante alogénico de progenitores hematopoyéticos (TPH) es actualmente la única opción de tratamiento curativo disponible para un número de neoplasias hematológicas de alto riesgo, así como para algunas enfermedades no malignas hereditarias o adquiridas. El TPH haploidéntico (HI) es una opción válida para pacientes que no tienen un hermano HLA-idéntico. Objetivo: describir los resultados obtenidos con TPH HI en pediatría. Material y método: en el año 2005 se inició en el Centro Hemato-Oncológico Pediátrico del Centro Hospitalario Pereira Rossell un programa de TPH HI para aquellos pacientes sin donante relacionado HLA-idéntico. Resultados: se trasplantaron 32 pacientes, 24 con neoplasias hematológicas y 8 con enfermedades no malignas. Se utilizaron dos estrategias de prevención de la enfermedad injerto contra huésped (EICH), depleción de linfocitos T (DLT) in vitro (28 pacientes) y DLT alorreactivos in vivo con altas dosis de ciclofosfamida postrasplante (4 pacientes). Veintisiete pacientes (84%) tuvieron un implante con quimerismo total del donante. La incidencia de EICH agudo y crónico fue de 26,9% y 11,8%, respectivamente. La muerte no relacionada a recaída al año del trasplante fue de 21,9%. Con una mediana de seguimiento de 32 meses, la sobrevida global a dos años fue de 52,4%. Conclusiones: el TPH HI ha demostrado ser una opción factible en nuestro medio para aquellos pacientes sin donante HLA-idéntico. Los resultados son comparables a los obtenidos con otros donantes alternativos y con costos más accesibles. Uruguay está hoy día mejor posicionado para ofrecer un TPH a los pacientes que así lo requieran...

Comparison of haploid and doubled haploid sugar beet clones in their ability to micropropagate and regenerate

Background: Haploid plant material is considered as recalcitrant to organogenesis, propagation, and maintenance in vitro. However, sugar beet (Beta vulgaris L.) breeders utilizing doubled haploid (DH) technology in their breeding programs indicate that sugar beet haploids may be cultured in vitro as well as diploids. Thus in this paper the in vitro performance of haploid and the doubled haploid sugar beet of various origin was evaluated. The DHs were derived from haploids by diploidization and twelve such haploid and corresponding DH clone pairs were obtained thus the comparison included haploid and DH clones that had identical allelic composition and differed only in their ploidy level. Results: The genotypes differed in shoot morphology and susceptibility to blackening during culture in vitro, but no significant differences were observed between the haploids and DHs. The micropropagation rate was, on average, higher for the haploids than DHs. Viability of the midrib and petiole explants after a 6-week culture was highly genotype dependent, but not affected by explant ploidy level. However, regeneration efficiency depended on both the genotype and ploidy level. The explants of several haploids regenerated more frequently and developed more adventitious shoots than the corresponding DHs thus overall efficiency was higher for haploids. Conclusions: The results obtained indicate that most of the haploids used in the comparison performed similar to or even better than DHs. This suggests that sugar beet haploid material can be successfully used not only for the production of DHs, but also maintained in vitro and utilized in projects requiring haploid tissues as the source material.

Low productivity of ribonucleotide reductase in Saccharomyces cerevisiae increases sensitivity to stannous chloride

Ribonucleotide reductase (RNR) of the yeast Saccharomyces cerevisiae is a tetrameric protein complex, consisting of two large and two small subunits. The small subunits Y2 and Y4 form a heterodimer and are encoded by yeast genes RNR2 and RNR4, respectively. Loss of Y4 in yeast mutant rnr4delta can be compensated for by up-regulated expression of Y2, and the formation of a small subunit Y2Y2 homodimer that allows for a partially functional RNR. However, rnr4delta mutants exhibit slower growth than wild-type (WT) cells and are sensitive to many mutagens, amongst them UVC and photo-activated mono- and bi-functional psoralens. Cells of the haploid rnr4delta mutant also show a 3- to 4-fold higher sensitivity to the oxidative stress-inducing chemical stannous chloride than those of the isogenic WT. Both strains acquired increased resistance to SnCl2 with age of culture, i.e., 24-h cultures were more sensitive than cells grown for 2, 3, 4, and 5 days in liquid culture. However, the sensitivity factor of three to four (WT/mutant) did not change significantly. Cultures of the rnr4delta mutant in stationary phase of growth always showed higher frequency of budding cells (budding index around 0.5) than those of the corresponding WT (budding index <0.1), pointing to a delay of mitosis/cytokinesis.

Asexual Recombination in a uvsH Mutant of Aspergillus nidulans

Mutations in the gene uvsH of Aspergillus nidulans result in increased spontaneous chromosome instability and increased intragenic and intergenic mitotic recombination in homozygous diploids. The aim of the present work was to obtain a uvs mutant of A. nidulans and to use it for the isolation of asexual recombinants (parameiotic segregants). The mutant uvsH, named B511, showed normal frequency of meiotic recombination in sexual crosses and high frequency of parameiotic segregants in the parasexual crossings with master strains (B511//A757 and B511//A288). Asexual haploid recombinants (parameiotic segregants), diploid and aneuploid segregants were recovered directly from the uvs//uvs+ heterokaryons (B511//A757 and B511// A288). Parameiotic segregants originated through mitotic crossing-over and independent assortment of chromosomes.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Characters that differ between diploid and haploid honey bee (Apis mellifera) drones

Diploid males have long been considered a curiosity contradictory to the haplo-diploid mode of sex determination in the Hymenoptera. In Apis mellifera, 'false' diploid male larvae are eliminated by worker cannibalism immediately after hatching. A 'cannibalism substance' produced by diploid drone larvae to induce worker-assisted suicide has been hypothesized, but it has never been detected. Diploid drones are only removed some hours after hatching. Older larvae are evidently not regarded as 'false males' and instead are regularly nursed by the brood-attending worker bees. As the pheromonal cues presumably are located on the surface of newly hatched bee larvae, we extracted the cuticular secretions and analyzed their chemical composition by gas chromatograph-mass spectrometry (GC-MS) analyses. Larvae were sexed and then reared in vitro for up to three days. The GC-MS pattern that was obtained, with alkanes as the major compounds, was compared between diploid and haploid drone larvae. We also examined some physical parameters of adult drones. There was no difference between diploid and haploid males in their weight at the day of emergence. The diploid adult drones had fewer wing hooks and smaller testes. The sperm DNA content was 0.30 and 0.15 pg per nucleus, giving an exact 2:1 ratio for the gametocytes of diploid and haploid drones, respectively. Vitellogenin was found in the hemolymph of both types of imaginal drones at 5 to 6 days, with a significantly lower titer in the diploids.

R- and G-band patterns in Astyanax scabripinnis paranae (Pisces, Characiformes, Characidae)

The absence of longitudinal bands in fish chromosomes has been associated with technical problems in chromosome preparations or the absence of a structural compartmentalization in the fish genome. In the present study, a R-banding pattern was obtained using a replication banding technique by in vivo treatment with 5-bromodeoxyuridine (5-BrdU). G-banding patterns were obtained after trypsin treatment and also after chromosome cleavage by in situ treatment with the restriction endonuclease BamHI. A similar G-banding pattern was also obtained after cleavage with the endonuclease HinfI. Presence of a resolute R- and G-banding patterns shows that Astyanax scabripinnis paranae chromosomes could present an isochore-like structure similar to that found in other vertebrates.

Flow cytometry analysis of cell cycle in myosin II-deficient yeast

OBJECTIVE: To determine whether cell cycle changes can be detected in myosin II-deficient cells using flow cytometry techniques. BACKGROUND: Although the primary role of myosin II (Myo1p) in the yeast Saccharomyces cerevisiae is in cytokinesis we have reported that this conventional myosin also appears to inuence the regulation of cell wall metabolism as indicated by increases in the expression of chitin metabolizing enzymes in a null mutant of the MYO1 gene. The expression of these enzymes is known to be regulated in the cell cycle suggesting that cell cycle changes may alter their expression. METHODS: Flow cytometry was employed to assess the nuclear DNA content of logarithmic yeast cell cultures as a means of determining changes in the cell cycle of Myo1p-deficient cells. RESULTS: Significant changes were observed in the Myo1p-deficient strain suggesting that these cells are arrested in G2/M-phase of the cell cycle. CONCLUSIONS: Based on the results of this preliminary study, we propose a model in which the increased activity of chitin metabolizing enzymes may be explained by a mitotic arrest in these cells.

Estudo do DNA na previsäo de resposta à quimioterapia por M-VAC em câncer infiltrativo de bexiga / DNA ploidy as a possible predictor of response to M-VAC chemotherapy for locally invasive bladder cancer

Em 20 pacientes com CCTB localmente infiltrativo realizou-se pesquisa do conteúdo do DNA em amostras de tumores obtidos por ressecçäo ou biópsia endoscópica, para avaliar a possibilidade de previsäo de resposta àquimioterapia por M-VAC. Histologicamente, 30 por cento dos tumores eram diferenciados e 70 por cento pouco diferenciados. Os tumores eram diplóides em 5 por cento, tetraplóides em 5 por cento e aneuplóide em 90 por cento. Observou-se resposta completa à quimioterapia em 55 por cento e resposta negativa em 45 por cento dos casos. A avaliaçäo da haploidia do DNA näo contribuiu para a detecçäo de neoplasias responsivas ao esquema M-VAC

Haploid karyotype analysis of Chelymorpha variabilis Boheman (Coleoptera chrysomelidae) with microspreading techniques

A independência do nucléolo e dos cromossomos sexuais Xyp no coleóptero Chelymorpha variabilis Boheman foi demonstrada em trabalho anterior. No presente trabalho foram aplicadas técnicas ópticas e de "microspreading" para espermatócitos desta espécie. Resultados de técnicas TEM também säo apresentados. Cariótipos haplóides também mostraram a presença de uma regiäo organizadora do nucléolo no 5§ paquímetro bivalente. os eixos dos cromossomos sexuais X e Y foram sempre encontrados näo-pareados e sem nenhuma substância em torno dos mesmos, diferindo do Xyp típico já observado, Postiglioni and Brum-Zorrilla (Genética 77:137-141, 1988). A facilidade de acesso aos espermatócitos para cariotipagem sugere o uso de técnicas de "microspreading" para estudar o cariótipo haplóide dos cromossomos dos insetos. Nossos resultados apoiam a associaçäo "non-chiasmatic" dos cromossomos sexuais X e Y

Micro-and macrospermatid production induce by meiotically unstable B-chromosomes in two species of grasshoppers (Acrididae)

Os efeitos dos cromossomos B na formaçäo de esperma anormal (no haplóide) foram investigados em duas espécies de gafanhotos, Dichroplus pseudopunctulatus e Sinipta dalmani. Um pequeno cromossomo B telocéntrico foi estudado em machos da primeira espécie. Quando o B se encontrava em dose simples, ele apresentava um comportamento muito irregular que incluia retardo nas primeira e segunda divisöes meióticas. Como resultado, foram observados grandes números de microespermátides portadoras do B atrasado nos machos B. Um macho que possui dois cromossomos B näo mostrou um aumento significativo na produçäo de microespermátides, fato este que está correlacionado com o comportamento meiótico regular do bivalente B. Näo foi observado aumento na produçäo de macroespermátides nos machos portadores do B, e é evidente que um mecanismo de eliminaçäo age sobre o cromossomo B desta espécie. Em S.dalmani foram encontrados dois pequenos cromossomos B; um deles induzia divisäo celular defeituosa, provavelmente através do seu comportamento meiótico falho, resultando em grande proporçäo de espermátides diplóides (cerca de 50%), com consequente reduçäo de fertilidade. Os efeitos dos cromossomos B na freqüência de quiasmas foram também registrados. Os efeito do B de D.pseudopunctulatus aumentava significativamente a freqüência média de quiasmas por célula, enquanto que, inesperadamente, os cromossomos B de S.dalmani pareciam diminuir a freqüência de quiasmas significativamente. A conseqüência da produçäo incrementada de esperma anormal e a modificaçäo das freqüências de quiasmas através da presença de cromossomos B säo avaliadas em termos de seus resultados evolutivos e citológicos

Chromosome formulae of neotropical freshwater fishes

É apresentada uma resenha dos números cromossômicos haplóides e/ou diplóides de 433 espécies de peixes das divisöes primária e secundária (sensu Myers., 1938) da Regäo Neotropical, compreendendo 145 gêneros e 33 famílias. Para 11,3% dessas espécies mais de um número cromossômico foi descrito; 5,8% apresentam cromossomos sexuais morfologicamente diferenciados e 2,3% apresentam cromossomos supranumerários. A comparaçäo dos números cromossômicos modais dentro dos sub-grupos da Super-Ordem Ostariophysi mostra uma tendência para o aumento de número de cromossomos nesse grupo