Whole comparative genomic hybridization (W-CGH) is a new technique that reveals cryptic differences in highly repetitive DNA sequences, when different genomes are compared using metaphase or interphase chromosomes. W-CGH provides a quick approach to identify differential expansion of these DNA sequences at the single-chromosome level in the whole genome. In this study, we have determined the frequency of constitutive chromatin polymorphisms in the centromeric regions of human chromosomes using a whole-genome in situ cross-hybridization method to compare the whole genome of five different unrelated individuals. Results showed that the pericentromeric constitutive heterochromatin of chromosome 6 exhibited a high incidence of polymorphisms in repetitive DNA families located in pericentromeric regions. The constitutive heterochromatin of chromosomes 5 and 9 was also identified as highly polymorphic. Although further studies are necessary to corroborate and assess the overall incidence of these polymorphisms in human populations, the use of W-CGH could be pertinent and of clinical relevance to assess rapidly, from a chromosomal viewpoint, genome similarities and differences in closely related genomes such as those of relatives, or in more specific situations such as bone marrow transplantation where chimerism is produced in the recipient.
Novel approaches to control diseases and pests in citrus involving the use of transgenic plants with pathogenic-related (PR) genes and viral sequences are explored. Genetic transformation of sour orange, Mexican lime and Volkamer lemon with oryzacystatin I gene, chitinase/glucanase genes and viral sequences (untranslatable p27 and coat protein genes from Mexican isolates of Citrus tristeza virus ), has been achieved, using Agrobacterium tumefaciens strains EHA 105 and LBA4404 and A. rhizogenes A4. Plasmids pCAMBIA 2301 and 1301 were used as transformation vectors. Regenerated shoots and roots representing independent transgenic events were assayed histochemically for GUS activity. The presence of transgenes was confirmed by PCR assays. Apical portions of GUS-positive regenerated shoots were shoot-tip grafted in vitro onto sour orange. Under greenhouse controlled conditions, shoots of 0.5-1.0 cm in length were grafted onto virus-free, 1-yr-old Volkamer lemon rootstocks. Nontransformed scions were also grafted as controls. Challenges with mild and severe CTV isolates will be done in plants containing oryzacystatin I gene and viral sequences. Plants expressing chitinase/glucanase genes will be challenged with severe strains of Fusarium spp. Recovery of plants showing different degrees of tolerance is expected and will be selected for further analysis.
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