Limitation of nitrogen source facilitated the production of nonmeiotic recombinants in Aspergillus nidulans.

Aspergillus nidulans is a fungal model organism extensively used in genetic approaches. It may reproduce sexually and asexually, with a well-defined parasexual cycle. The current paper demonstrates that the limitation of nitrogen source facilitates the production of A. nidulans's nonmeiotic recombinants directly from heterokaryons, without the recovery of the diploid phase. Heterokaryons formed between master strains were inoculated in sodium nitrate-low (basal medium [BM]) and sodium nitrate-rich media (minimal medium [MM]). All mitotic segregants produced by the heterokaryons were tested for their mitotic stability in the presence of benomyl, the haploidizing agent. Only mitotically stable haploid segregants were selected for subsequent analysis. Phenotypic analyses of such haploids favored the characterization of nonmeiotic recombinants. As the number of such recombinants was higher in BM than in MM, nitrogen limitation may have facilitated the isolation of nonmeiotic recombinants from heterokaryons by stimulating nuclear fusion still inside the heterokaryotic mycelium as a survival strategy.

Use of oval conidia as a tool to assess the genetic transfer among Colletotrichum sublineolum mutants

The aim of this study was to assess oval conidia production in mutants for the reductase nitrate gene in C. sublineolum and verify the possibility of using them as a tool to transfer the genetic material in this species. The mutants used in the present study lost the ability to form falcate conidia but all produced oval conidia. The number of nuclei by conidium was evaluated. Oval conidia were efficient in the heterokaryon formation and these heterokaryons in liquid culture medium they also produced oval conidia in abundance. Recombinants were obtained and the genetic exchanges were confirmed by the RAPD analyses.

O fungo Colletotrichum sublineolum agente causal da antracnose em sorgo mostra dimorfismo conidial, produzindo conídios falcados ou ovais dependendo das condições de cultivo. Conídios falcados têm sido usados para obter heterocários entre mutantes complementares. Muitas linhagens de C. sublineolum não produzem conídios falcados ou produzem em quantidades insuficientes para analises. O objetivo do presente estudo foi avaliar a produção de conídios ovais em mutantes para o gene da nitrato redutase e verificar a possibilidade de sua utilização como ferramentas na transferência de material genético nesta espécie. Os mutantes usados neste estudo perderam a capacidade de produzir conídios falcados, porém todos produziram conídios ovais. O número de núcleos por conídio foi determinado. Conídios ovais foram eficientes na formação de heterocários e os heterocários quando cultivados em meio líquido também produziram conídios ovais em abundância. Recombinantes foram obtidos e as trocas genéticas foram confirmadas pelas análises de RAPD.

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.

Genetic characterization of somatic recombination in Trichoderma pseudokoningii

Crossing experiments via hyphal anastomosis between two strains contrasting for auxotrophic markers of Trichoderma pseudokoningii were conducted to characterize the somatic recombination process in this specie. Four crossings were made and a total of 1052 colonies obtained from conidial suspensions of the heterokaryotic colonies were analyzed. Sixty-eight recombinant colonies, from four growing generations, were analyzed for the auxotrophic markers. Of the 68 colonies analyzed, 58 were stable after four generations and the remainders were unstable, reverting to one of the parentals. Most of the recombinant colonies were unstable through subculture and after four growing generations they showed the leu ino met markers (auxotrophic for leucin, inositol and metionin respectively). The unstable recombinant colonies showed irregular growing borders, sparse sporulation and frequent sector formation. The results suggest the occurrence of recombination mechanisms in the heterokaryon (somatic recombination), different from those described for the parasexual cycle or parameiosis. Therefore, we proposed the ocurrence of nuclei degradation from one parental (non prevalent parental) in the heterokaryon and that the resulting chromosomal fragments may be incorporated into whole nuclei of the another parental (prevalent parental). However the parameiosis as originally described cannot be excluded.

Com o objetivo de se caracterizar o processo de recombinação somática em Trichoderma pseudokoningii, foram realizados cruzamentos via anastomose de hifas entre duas linhagens desta espécie, contrastantes para os marcadores de auxotrofia. Foram realizados quatro cruzamentos, sendo analisadas um total de 1052 colônias obtidas a partir de suspensões de conídios provenientes das colônias heterocarióticas. Foram analisadas sessenta e oito colônias recombinantes em quatro gerações de crescimento, sendo verificados os marcadores de auxotrofia. Das 68 colônias, 58 se mostraram estáveis após quatro gerações de crescimento e as colônias restantes permaneceram instáveis, revertendo para as marcas de uma das linhagens parentais. A maioria das colônias se mostrou instável através das gerações de subcultivo, e após quatro gerações de crescimento estas apresentaram as marcas de auxotrofia leu ino met (auxotrofia para leucina, inositol e metionina, respectivamente). As colônias recombinantes instáveis apresentaram bordas irregulares de crescimento, esporulação esparsa e a freqüente formação de setores. Os resultados sugerem a ocorrência de mecanismos de recombinação no heterocário (recombinação somática), diferente daqueles descritos para o ciclo parassexual ou parameiose, sendo proposto a ocorrência da degradação no heterocário de núcleos provenientes de uma das linhagens parentais (parental não prevalente) e a incorporação de fragmentos cromossômicos nos núcleos íntegros da outra linhagem parental (parental prevalente). No entanto a parameiose, como originalmente descrita, não pode ser excluída.