Autochton landraces characterization: proteomic and genomic approach

The results reported in the thesis are related with the study of characterization of two autochthonous lentil (Lens culinaris M.) landraces of Molise region and of a new approach for genotyping of local varieties. In particular the work has been focused on three major issues: 1)use of proteomics to investigate natural variation within and between lentil populations, 2) response of lentil to salt stress and 3) use of the loop-mediated isothermal DNA amplification (LAMP) method to amplify SSR. To accomplish the first objective investigations have been focused on the proteomic analysis of mature seed of lentil. The use of 2DE couple to the MS/MS allowed the identification of 135 well resolved spots which represent the first lentil seed proteome reference map. In addition the multivariate statistical analyses carried out on spots, resulted differentially expressed between different lentil populations, led to detection of the proteins which were essential for population discrimination, as shown in the paper Scippa et al., 2010. The diversity of two lentil landraces (Capracotta and Conca Casale) from Molise was studied using a combination of morphological, genetic and proteomic analyses, as shown by results published in the paper by Viscosi et al., 2010.Moreover, a further study has been carried out to deepen the knowledge about proteomic characterization of Capracotta and Conca Casale. The data obtained were elaborated by uni-and multivariate statistical analysis to identify the proteins that characterize the lentil ecotypes, mainly involved in abiotic and biotic stress responses. On the base of aforementioned results, the PhD project proceeded in analyzing the physiological and proteomic response of two lentil landraces from Molise and five commercial varieties to salt stress. In particular it has been investigated how salt stress affected seed germination and caused changes in expression of proteins. Furthermore, the multivariate statistical analysis was performed using quantitative data of spots in order to identify important proteins involved in saline stress response. The third aim of the PhD thesis was the detection of a new molecular approach for genotyping of local varieties. Work has been carried out by incorporation rice simple sequence repeat (SSR) motif within a LAMP assay. It has been demonstrated that results were consistent with analysis performed by PCR. In addition, LAMP assay was characterized by high sensitive being able to amplify from near single copy of target. The work reported in the PhD thesis represents a novelty in the field of proteome of lentil seed and it surely shows that proteomics is a powerful tool for analysis of biodiversity in ecotypes of a single plant species. Furthermore, the work confirms that proteomics studies can significantly contribute to understand the complex mechanisms involved in the plant response to salt stress. The originality of the PhD thesis is also represented by novel use of the loop-mediated isothermal DNA amplification method to amplify SSR.