Transgene Excision And Evaluation Of Codon Biology In Citrus And Related Genera

Pollen and seed mediated transgene flow is a serious concern of the scientific community. To address this problem, the GM-gene-deletor technology was developed using loxP-FRT fusion as recognition sites for FLP recombinase. In this technology, all functional foreign genes were automatically eliminated with a high efficiency from pollen and seeds of transgenic tobacco. It can be particularly significant to vegetatively propagated plants that are important to horticulture industry. Citrus (Rutaceae) is vegetatively propagated important fruit tree crop and their juvenile period ranges from5to12years, which has been an important limitation to traditional breeding and genetic studies. Like herbaceous model species (Arabidopsis, tobacco, tomato, etc.), short juvenile Citrus genotypes such as precocious trifoliate orange(Poncirus trifoliata [L.] Raf) and Hongkong kumquat (Fortunella hindsii swingle.), are good candidate for studying expression patterns of pollen and seed specific promoters by genetic transformation, since fruits can be obtained in a much shorter time (1-2years). In present study, the GM-gene-deletor technology was evaluated in Citrus model species precocious trifoliate orange and Hongkong kumquat to produce transgene free pollen and seeds. The efficacy of the GM-gene-deletor was exceptionally high in herbaceous model species tobacco, but in present investigation it was observed that the transformation efficiency of precocious trifoliate orange was only0.17%and no shoot was generated in Hongkong kumquat. These results suggested that, this technology requires to be standardized in accordance with the Citrus species because herbaceous model system has different genetic background, expression pattern, and regulatory mechanism.Citrus, as one of the globally important fruit trees, has been an object of interest for understanding genetics and evolutionary process in fruit crops. Meta-analyses of19Citrus species, including four globally and economically important Citrus sinensis, Citrus clementina, Citrus reticulata, and one Citrus relative Poncirus trifoliata, were performed. Present analyses were restercited to Citrus genera to develop resource information for Citrus species. It was observed that codons ending with A-or T-at the wobble position were preferred in contrast to C-or G-ending codons, indicating a close association with AT richness of Citrus species and P. trifoliata. The present study postulates a large repertoire of a set of optimal codons for the Citrus genus and P. trifoliata and demonstrates that GCT and GGT are evolutionarily conserved optimal codons. The present investigation and observations suggested that mutational bias is the dominating force in shaping the codon usage bias (CUB) in Citrus and P. trifoliata. Correspondence analysis (COA) revealed that the principal axis (axisl; COA/RSCU) contributes only a minor portion (～10.96%) of the recorded variance. In all analysed species, except P. trifoliata, Gravy and aromaticity played minor roles in resolving CUB. Compositional constraints were found to be strongly associated with the amino acid signatures in Citrus species and P. trifoliata. This analysis postulates compositional constraints in Citrus species and P. trifoliata and plausible role of the stress with GC3and coevolution pattern of amino acid. GC3-rich and-poor genes and their association with stress were analysed, and these results provided novel insights into stress evolution of stress adaptation in Citrus species and P. trifoliata in accordance with the GC3biology. GC3and evolution were correlated using Hamming distance parameter to identify suggestive evolutionary pairs of genes. Co-orthologous genes matrix identified using the alignment ratio suggested close association of C. clementina, C. sinensis and C. reticulata, as they belong to the same phylogenetic clade. The present findings are important for all Citrus species, as it could facilitate understanding genome dynamics and evolution in Citrus and P. trifoliata, transformation of interested target genes, for designing multi-targeting gene systems, and genetically improving these important fruit crops.