Multi-color genomic in situ hybridization (MCGISH) was performed for mitotic cells of the somatic hybrids of Diospyros kaki (2n = 6x = 90) and D. glandulosa (2n = 2x = 30). Total DNA of D. kaki and D. glandulosa were isolated and labeled with biotin-16-UTP and digoxigenin (DIG)-11-UTP, respectively. The labeled DNAs were used as probes to differentiate parental chromosomes. The biotin-labeled probe was detected with avidin-rhodamine, and the DIG-labeled probe was detected with anti-DIG-FITC (fluorescein isothiocyanate). Ninety chromosomes from D. kaki that showed reddish-orange and 30 chromosomes from D. glandulosa that showed greenish-yellow were observed under a fluorescence microscope. Some chromosomes showed cross-hybridization with both probes at their terminal or other chromosome regions. These results indicated that MCGISH could be used to analyze genomes of Diospyros species whose chromosomes are small and numerous.
5S ribosomal DNA (rDNA) was visualized on the somatic metaphase chromosome of persimmon (Diospyros kaki) and ten wild Diospyros species by fluorescent in situ hybridization (FISH). The digoxigenin (DIG)-labeled 5S rDNA probe was hybridized onto the chromosomes and visualized by incubation with anti-DIG-fluorescein isothiocyanate (FITC). Strong signals of 5S rDNA probe were observed on several chromosomes of Diospyros species tested. Furthermore, multicolor FISH using 5S and 45S rDNA probes differently labeled with DIG and biotin, revealed separate localization of the two rDNA genes on different chromosomes of Diospyros species tested, suggesting that 5S and 45S rDNA sites can be used as chromosome markers in Diospyros. The number of 5S rDNA sites varied with the Diospyros species. More 5S rDNA sites were observed in four diploid species native to Southern Africa than in three Asian diploid species. The former had four or six 5S rDNA sites while the latter had two. Three Asian polyploidy species had four to eight 5S rDNA sites. Among the Asian species, the number of 5S rDNA sites seemed to increase according to ploidy level of species. These features of 5S rDNA sites were very similar to those of 45S rDNA sites in Diospyros. Phylogenetic relationship between D. kaki and wild species tested are discussed based on the number and chromosomal distribution of 5S and 45S rDNA.
To produce nonaploid Japanese persimmon (Diospyros kaki L.f.) by artificial hybridization, we surveyed the natural occurrence of unreduced (2n) pollen among hexaploid cultivars and sorted them from normal reduced (n) pollen. The sorted 2n pollen was crossed with a hexaploid female cultivar and the resultant embryos were rescued by in vitro culture techniques to obtain plantlets. Three out of six male-flower-bearing cultivars (2n = 6x = 90) produced 2n pollen at rates of 4.8% to 15.5% varying with the cultivar, which was estimated by both pollen size and flow cytometry. After sorting giant (2n) from normal pollen grains by using nylon mesh, they were crossed with a hexaploid female cultivar. The seeds obtained from pollination with normal pollen were perfect, but those obtained from pollination with giant pollen were mostly imperfect, with embryo growth being suspended at the globular stage. Although the rate of survival was very low, some embryos at the globular stage were rescued successfully and grown in vitro. Both flow cytometric analysis and chromosome counting proved that the plantlets obtained were nonaploid.
Eight new green fluorescent protein (GFP) binary vectors were developed by inserting gfp reporter gene cassettes into pGreen vectors. We chose one of them, pG52KF, with the nptII selection and gfp reporter gene and one recombinant construct, pG52KFp, for a preliminary evaluation in citrus using Agrobacterium-mediated transformation. High-transformation efficiency was observed, whereas green fluorescence greatly facilitated the early in vivo screening and categorizing of the transformants. These pGreen-derived GFP binary vectors, freely available on request, provide more and flexible options for genetic transformation in citrus and other woody plants.
As one of the Florida's state wildflowers, Coreopsis leavenworthii is highly desirable for roadside plantings in all parts of the state. Seeds of this species are being produced by growers. Where should seed be produced for different ecotypes? Where can the seed be used? These are among questions that have arisen in commercial seed production and distribution. To address these questions, it was necessary to assess the levels of genetic diversity. Eleven populations (242 total individuals) were collected from different parts of Florida, grown at one location in central Florida, and observed for morphological variations. North Florida natural populations had more complex leaves, while south Florida natural populations had smaller flowers. Principal component analyses revealed that two of the seven characteristics studied accounted for as much as 88% of the morphological variations observed. Molecular diversity was analyzed by using the fluorescent amplified fragment length polymorphism (AFLP) technique and the capillary sequencing system. Four primer combinations detected 320 AFLP fragments, of which 90.6% were polymorphic. The overall genetic diversity in the species was 0.2206 (estimated using AMOVA), of which 77.9% was within populations and 22.1% was among populations. The genetic distance among populations seemed to be loosely correlated with geographical distances. A high level of gene flow was found in several populations. Based on the results, a model has been developed to describe the genetic relationship of Coreopsis leavenworthii populations.