Seleção de genes de referência e perfis de expressão gênica da família LAV durante a embriogênese somática em cafeeiro

Abstract

In order to contribute to the understanding of the induction process of somatic embryogenesis, critical to the development of more effective regeneration protocols, this work aimed to identify, characterize, and analyze the expression patterns of LAV gene family during indirect somatic embryogenesis of Coffea arabica L. through the RT-qPCR data normalization with suitable reference genes. The correlation of the gene expression with the embryogenic potential was performed comparatively with the aid of histological analyses and somatic embryo regeneration in two independent lineages of cell suspension. First, the stability of the expression of twelve candidates to reference genes (24S, ACT, GAPDH, CYCL, EF1a, TUB, PP47, PP2A, RPL39, APRT, UBQ, 14-3-3) was analyzed in different tissues and developmental stages related to coffee somatic embryogenesis. Analyses have been performed using the RefFinder tool which compiles geNorm, NormFinder, BestKeeper, and Delta-Ct statistical algorithms. Results suggest that there is no universal reference gene suitable to all experimental variables. The most stable expression in non-embryogenic callus, embryogenic callus, and cell suspension on different cultivation periods corresponds to the genes UBQ, ACT, and APRT, respectively. The RPL39 gene presented the highest stability on analysis of embryogenic and non-embryogenic calli. Meanwhile, in seedlings and embryos in different stages, PP2A exhibited the highest expression stability. The analyses of all samples together indicated that 24S and PP2A are the most suitable reference genes for RT-qPCR data normalization. With the aid of bioinformatics tools, only three possible VAL2 (C15, SEA1, and SIC1) orthologs were identified among LAV gene family. The expression profiles confirmed in vivo differed from those obtained in silico, and the data showed variable quantitative relative expression of C15 and SIC1 in all analyzed samples and lack of SEA1 expression in all tissues. No relations at the expression level of C15 and SIC1 with the embryogenic potential have been found. The lineages of cell suspension presented the same histological pattern and increased regeneration rate due to the cultivation time. The successful development of seedlings obtained using the protocol may be a consequence of the high expression of VAL2 in cotyledonary embryos. The knowledge of VAL2 expression gives a chance to improve the propagation via somatic embryogenesis in order to ensure the conversion of embryos to normal seedlings.