A citrus NAM, ATAF, and CUC (NAC)-like gene (CitNAC) was isolated from fruit tissues of Citrus sinensis Osbeck using complementary DNA (cDNA) amplified fragment length polymorphism and rapid amplification of cDNA ends techniques. Its full length was 988 bp in which 781 bp form the open reading frame, coding for a protein of 264 amino acids. Sequence comparison revealed that CitNAC possesses the general structural features at the N terminus of the NAC domains. Phylogenetic analysis results showed that CitNAC was closely related to AtNAP and PeNAP, which are involved in plant organ senescence. Gene expression analysis showed that the messenger RNA level of CitNAC was just detected in fruit peel and pulp during fruit ripening or senescence stage. The observed expression pattern of CitNAC along with the result of phylogenetic analysis suggested that CitNAC is related to fruit development and senescence.
Retro-transposons are common components of plant genomes, functional at transcription, translation and integration levels. Their abundance and ability to transpose render them good potential markers. Present study was undertaken with the view of IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism) based DNA marker analysis for genus Citrus and related taxa. Both the IRAP and REMAP exhibited a remarkable variation among the tested genotypes. As a whole, ISSR, IRAP and REMAP analysis generated 113 score able bands ranging from 250 to 2,000 bps. There were 94 polymorphic bands, with an average of eight polymorphic bands per primer combination. The level of polymorphism was found to be 84%. No bands were shared with the ISSR pattern in REMAP analysis. Genetic similarity analysis was performed based on the Dice coefficient, and dendrogram was constructed by using the average linkage methods from combined analysis of IRAP and REMAP. A cophenetic correlation coefficient was also calculated. The clustering approach revealed a good adjustment between matrixes, with correlation coefficient of 0.77. Average similarity for all the genotype pairs was used as a cutoff value for defining the clusters. UPGMA demonstrated eight different clusters. Citrus genus showed wide range of heterogeneity, specially the mandarin group. Genera Fortunella and Poncirus were placed in relatively divergent cluster.
Two deep-coverage Bacterial Artificial Chromosome (BAC) libraries of Citrus sinensis (L.) Osbeck 'Cara Cara' navel orange and Citrus reticulata (L.) Blanco 'Egan No. 1' Ponkan mandarin, which belong to the two most important species of the Citrus genus, have been constructed and characterized to facilitate gene cloning and to analyze variety-specific genome composition. The C. sinensis BAC library consists of 36 000 clones with negligible false-positive clones and an estimated average insert size of 126 kb covering ~4.5 x 109 bp and thus providing an 11.8-fold coverage of haploid genome equivalents, whereas the C. reticulata library consists of 21 000 clones also with negligible false-positive clones and an estimated average of 120 kb covering ~2.5 x 109 bp representing a 6.6-fold coverage of haploid genome equivalents. Both libraries were evaluated for contamination with high-copy vector, empty pIndigoBAC536 vector, and organellar DNA sequences. Screening has been performed by Southern hybridization of BAC filters, which results in <0.5% chloroplast DNA contamination and no mitochondrial DNA contamination in both libraries. Eight and five positive clones harboring the gene encoding Phytoene synthase (Psy (EC 2.5.1.32)) were identified from the C. sinensis and C. reticulata libraries, respectively, using the filter hybridization procedure. These results suggest that the two BAC libraries are useful tools for the isolation of functional genes and advanced genomics research in the two important species C. sinensis and C. reticulata. Resources, high-density filters, individual clones, and whole libraries are available for public distribution and are accessible at the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.
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