Cloning, structural characterization, and phylogenetic analysis of flower MADS-box genes from crocus (Crocus sativus...

Crocus (Crocus sativus L.) is a crop species cultivated for its flowers and, more specifically, for its red stigmas. The flower of crocus is bisexual and sterile, since crocus is a triploid species. Its perianth consists of six petaloid tepals: three tepals in whorl 1 (outer tepals) and three tepals in whorl 2 (inner tepals). The androecium consists of three distinct stamens and the gynoecium consists of a single compound pistil with three carpels, a single three-branched style, and an inferior ovary. The dry form of the stigmas constitutes the commercial saffron used as a food additive, in the coloring industry, and in medicine. In order to uncover and understand the molecular mechanisms controlling flower development in cultivated crocus and its relative wild progenitor species, and characterize a number of crocus flower mutants, we have cloned and characterized different, full-length, cDNA sequences encoding MADS-box transcription factor proteins involved in flower formation. Here we review the different methods followed or developed for obtaining these sequences involving conventional 5 inverted exclamation markä 3 inverted exclamation markä RACE, as well as newly developed methods from our group, named Rolling Circle Amplification C RACE (RCA-RACE) and its modification named familyRCA-RACE (famRCA-RACE). Furthermore, the characteristics of the protein structure and their common and specific domains for each type of MADS-box transcription factors in this lower nongrass monocot belonging to the Iridaceae family are described. Finally, a phylogenetic tree of all the MADS-box sequences available in our lab is presented and discussed in relation to other data from studies of species of the Iridaceae group and closely related families from an evolutionary perspective. The structural and phylogenetic analyses are based on both published and unpublished data.