Avaliação fisiológica e aplicação de ddPCR (differential display PCR) em genótipos diplóides (AA) de bananeira (Musa ssp.) submetidos ao estresse salino

Abstract

The Northeast of Brazil, major national producer of bananas, presents as a major limiting factor the stretch of saline soil. The urgency in the development of tolerant cultivars to salinity has led to breeding programs with the aimed to differ the bananas cultivars in diploid genotypes tolerant and sensitive, to be considered viable genetic material for pollination of triploid and tetraploid cultivars. Generally, the present study aimed to characterize diploid genotypes belonging to the AA banana genomic group on salinity and identify genes differentially expressed in contrasting genotypes. At first, this research assessed the growth and accumulation of inorganic ions in 19 diploid genotypes (AA) of bananas subjected to salt stress. The genotypes were grown in a greenhouse and submitted to irrigation with no saline water (0 mM NaCl) or with saline water (100 mM NaCl) for a period of 21 days, when the experiment was collected. To the physiological evaluation, were considered the parameters of growth:leaf area, height, number of leaves, diameter of the pseudostem, weight of the tax burden of fresh and dry, while the chemical assessment observed the concentration of sodium ions, potassium, chloride, magnesium and calcium in limbo leaf, and root pseudostem / subroot. The addition of NaCl to the cultivar solution has, in general, reduced the growth expressed by height, formation of new leaves, leaf area, diameter of the pseudostem and production of fresh and dry materials. This reduction in growth probably due to factors such as: the toxic effect of ions that have been absorbed, the low osmotic and water potential of the cells as well as the use of metabolic energy in the process of osmotic adjustment. In assessing chemical factors, it was possible to observe that the ions concentration has been preferentially presented in root tissue, showing that it is a culture moderately tolerant to salinity.The sodium and chlorine ions increased significantly with the salt increase in differentparts of the plant, while potassium suffered reduction in the leaf lamina and the pseudostem probably because it is associated to the competition with the sodium ion leading to the conclusion that the differential accumulation of ions potentially toxic (sodium and chlorine) and the maintenance of potassium, contribute to the tolerance to salinity in banana. From the physiological analysis and by means of the toxicity symptoms caused by NaCl, it was possible to observe that the effects were less intense in Birmania and Khai Nai On genotypes, than in Sowmuk, its indicates the possibility of use of this plants in programmes for cultivars improvement as tolerant and sensitive, respectively. Seven genotypes were selected for characterization by means of molecular markers RAPD, with the aim to link the physiological responses to salt stress with the formation of groups. We tested 16 random primers. The resultsshow a broad molecular genetic variability among seven genotypes studied. The formation of groups, in part is related to the data obtained in the physiological assessment, keeping in the same group the Birmania and Khai Nai On genotypes.These genotypes that showed higher tolerance to salt stress, when compared to the more salt-sensitive genotype (Showmuk), became distant genetically, so, it can be noticed the possibility of use of this molecular marker in the study of genetic diversity for this species. For the genome functional study, or transcriptoma, this study aimed the detection of possible changes in the pattern of genes expressed in the three diploid genotypes (AA) of banana, Khai Nai On, Burma and Sowmuk with contrasting results when in the absence and presence of high salt concentrations. The Differential Display PCR - ddPCR technique was used to identify and compareregions of bands from fragments of cDNA on agarose gel. A total of 43 fragments of differentially expressed cDNA was generated from the combination of four primers anchors and six random primers. Among the transcripts, 30 were once expressed by the genotype Burma and Kha Nai On, and 13 only by Sowmuk genotype. Theregions of bands with greater consistency of formation, is between 4000 bp and 150 bp. By the results of this research, it was possible to identify some fragments potentially involved to the condition of salinity in banana. The isolation, purification and sequencing of these - Expressed sequence tags - ESTs can assist in the development of new varieties of banana, more adapted to salt stress, in addition to enriching the database of public functional sequences of genomes banks.