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Random amplified polymorphic DNA (RAPD) markers were used to detect chimerism of citrus cultivars. Polymerase chain reaction conditions suitable for discriminating citrus chimeras were determined. Primers that produced consistent and repeatable bands that differed between the parental cultivars were chosen to create discriminating band patterns. Our results show that selected 12-mer primers can be useful for identifying the four citrus chimeras tested using RAPD technology.
Interspecific hybridizations by electrofusion of embryogenic callus cells from `Seminole' tangelo (Citrus reticulata Blanco × C. paradisi Macf.), `Hazzara (Abohar)', or `Ohta' ponkan (C. reticulata Blanco) and leaf cells from `Lisbon' lemon [C. limon (L.) Burm. f.] or rough lemon (C. jambhili Lush.), respectively, were performed. Electrofusion of `Seminole' tangelo and `Lisbon' lemon, `Hazzara (Abohar)' and rough lemon, and `Ohta' ponkan and rough lemon resulted in 33, 43, and 36 plants, respectively. Seven to 10 plants in each combination were selected randomly and used to investigate nuclear and cytoplasmic genomes. Regenerated plants derived from electrofusion of `Seminole' tangelo and `Lisbon' lemon, and `Hazzara (Abohar)' and rough lemon possessed the same restriction fragment pattern for nuclear rDNA as that of the mesophyll parents and the same mitochondrial DNA (mtDNA) restriction pattern as that of the respective suspension parents, indicating that they were cybrids. In contrast, all the plants resulting from a combination between `Ohta' ponkan and rough lemon were confirmed by nuclear rDNA and mtDNA analysis to be somatic hybrids. The analysis of chromosome number supported the results of Southern blot hybridization. The results suggest that specific cell lines, parental combinations, or both can increase the efficiency of inducing cybrids in Citrus by electrofusion.
Randomly amplified polymorphic DNA (RAPD) analysis was used to investigate the histogenic structure of leaf and fruit tissues in four graft chimeras, two intentional chimeras that were produced in combination with `Hamlin' orange [Citrus sinensis (L.) Osbeck] and `Satsuma' mandarin (C. unshiu Marc.), and two naturally occurring periclinal chimera cultivars, Kobayashi Mikan (a graft chimera of C. unshiu and C. natsudaidai Hayata), and Kinkoji Unshu (a graft chimera of C. unshiu and C. obovoidea hort. ex Takahashi). RAPD profiles of the lamina epidermis and the mesophyll cells of specific individuals indicated that the four graft chimeras were interspecific monekto chimeras, whose outermost layer (histogenic layer L-1) of the shoot apical meristem consisted of a species that was different from that in the inner layers (histogenic layers L-2 and L-3). Moreover, juice vesicles, which develop from the inside cells of the pericarp and become the main edible parts of Citrus fruit, were a mixture of the cells from both parental source cultivars. Therefore, the vesicles were at least composed of L-1 and subepidermal inner L-2 cells. This determination of interspecific chimeral construction (which was made possible by molecular techniques) is a valuable finding, in terms of improving Citrus through intentional use of periclinal chimerism.
A cDNA library was constructed from satsuma mandarin (Citrus unshiu Marc.) fruit tissues during the rapid cell enlargement phase. A total of 950 individual cDNA clones was partially sequenced and compared with GenBank databases for characterizing the gene repertoire expressed during this developmental phase. Among these, 426 cDNA clones (44.8%) showed sequence identity with previously characterized genes with optimized (OPT) scores of ≥200, while 524 clones (55.2%) resulted in low OPT scores (<200) and did not show any significant sequence identity with previously published genes. Based on nucleotide sequence, most clones with OPT scores of ≥200 were assumed to be transcription-, translation-, cell-wall-metabolism-, and stress-response-related genes. Other clones showed homology with published sequences related to housekeeping and stress-response-related genes, including metallothionein-like proteins, late-embryogenesis-abundant (LEA) proteins, and heat-shock proteins. These results suggested that Citrus fruit during rapid cell enlargement were metabolically active and expanding in response to biotic and abiotic stress. For clones with low nucleotide sequence homology, the recurrence was evaluated by aligning nucleotide sequences of each clone and generating contig maps. Expressed sequence tags (ESTs) of 162 clones with OPT scores <200 have not been reported for any other organism. Collectively, randomly sequenced cDNA clones described in this study provided information on types of genes expressed during the rapid cell enlargement phase in Citrus fruit. These genes should be used as candidates for Citrus genome mapping projects.