Isolation and characterization of MADS-box genes in Medicago truncatula: gene duplications and subfunctionalization in the euAGAMOUS lineage 
  
  Angiosperms show great diversity in the development and morphology of inflorescences, flowers and fruits, although it has been established that these processes have a common genetic basis. This diversity demands an analysis of these processes in model plants different from Arabidopsis thaliana or Antirrhinum majus. In this study, we have used Medicago truncatula, which has the appropriate characteristics to serve as a model system for legumes.
  We have isolated and characterized MADS-box family genes in M. truncatula. These genes are transcription factors that are involved in processes such as control of flowering time, determination of the identity of the floral meristem or development of floral organs, fruits and seeds. We have studied 11 MADS-box genes: two B-class genes (MtTM6 and MtNMH7), three C-class genes (MtSHP, MtAGa and MtAGb), one E-class gene (MtSEP) and five genes that do not belong to the ABC(DE) model (MtAGL6, MtAGL6-like, MtSOC1a, MtSOC1b and MtSOC1-like). We have paid special attention to the functional characterization of the C-class genes. The expression patterns of the isolated genes were studied by Northern blot and in situ hybridization, finding that all of them are expressed in different floral tissues and/or different floral development stages, although several of them are also expressed in vegetative tissues, perhaps perfoming a broader range of functions than just regulation of flower development events. These results are the basis for determining the functions of these genes in the development of inflorescences, flowers and fruits of M. truncatula.
  It has been described that many Arabidopsis duplicated genes are represented by a single gene in M. truncatula and that this legume presents duplication of genes that are unique in Arabidopsis. The latter happens with C-class genes. We have isolated three MADS-box genes belonging to this class in M. truncatula: MtSHP, MtAGa and MtAGb. The first is member of the PLE lineage and the last two are members of the euAG lineage. Duplication in the euAG lineage appears to be specific of leguminous species. For this reason, we investigated if MtAGa and MtAGb paralog genes play roles in the floral organ identity specification and/or in the floral meristem determination, described functions for C-class genes. M. trucatula transgenic plants with RNA interference constructs and a loss of C-function mutant labelled by Tnt1 retrotransposon were analysed. In addition, using VIGS technology, plants of M. truncatula and Pisum sativum (phylogenetically close legume) with loss of function of MtAGa/MtAGb and PsAGa/PsAGb genes, respectively, were obtained. Also, we carried out experiments of gain of function through constitutive expression in the heterologous system A. thaliana.
  The results of this study indicate that MtAGa and MtAGb are C-class genes that have a high degree of functional redundancy, and furthermore, they subfunctionalized distributing the ancestral C-function between both paralogs, in such a way that MtAGa has a priority role in the floral meristem determination, while MtAGb plays a key role in the specification of the identity of floral reproductive organs.