| Sweet orange [Citrus sinensis(L.) Osbeck] is the most widely cultivated Citrus varieties in the word, and of great economic value because of its unique flavor, rich nutrient, and wide variety. Sweet orange originates from interspecific hybridization of orange and pomelo, and its chromosomes are highly heterozygous. ?Valencia‘ orange occured chromosome reciprocal translocation, and these features of very late-ripening, less seeds and high yield make it be a good parent in Citrus seedless breeding.With the development of sequencing technology, cytogenetic mapping is a kind of important tool in quantitative trait loci mapping, map-based cloning and molecular marker assisted breeding, which has important significance in the breeding and genetic research. Predecessors obtained doubled haploid by anther in vitro culture, and whole genome sequencing was carried out. This study on the basis of predecessors using fluorescence in situ hybridization with double fluorescent staining techniques, created molecular cytogenetic research platform of a diploid and its doubled haploid, and expanded to the Citrus and its related genera. The results of this study are as follows:1) Characteristics of fragile sites in sweet orange.Chromosome fragment phenomenon was found in the conventional chromosome preparation of root meristematic in sweet orange, and telomere-FISH and CMA/DAPI proved the lightly stained regions are the fragile sites of sweet orange which resembled that of human beings and ryegrass. This study confirmed that there were two fragile sites in the chromosomes of sweet orange for the first time, one of which proximally located on B-type chromosome and the other one terminally located on D-type chromosome. Ag-NOR showed fragile sites located in regions with transcription activity of 45 S r DNA, and 5-m C immune-detection indicated fragile sites were highly methylated in mitosis metaphase, which accounted for that the fragile site expression is in epigenetic regulation.2)Analysis of karyotype and fragile site characteristics in Citrus and its related genera.Conbining the previous karyotype analysis and adding other species such as Fortunella hindsii, karyotype analysis of Citrus and its related genera found that the fragile site steadily existed in Citrus and its related genera, although the number and the position of 45 S r DNA loci in different kinds of plants are not identical. The locations of the fragile sites also had a polymorphism, but the numbers of fragile sites in diploid plants have two fragile sites except that pomelo has three fragile sites. Fragile sites in the formation of doubled haploid and somatic hybrids were stable inheritance, which may be related to chromosome variation and species evolution.3) Chromosome reciprocal translocation in ?Valencia‘ orangeChromosome characteristics analysis of ?Valencia‘ orange and its doubled haploid found that two B-type chromosomes were non-homologous chromosomes and they are homologous chromosomes with other two D-type chromosmes. The result revised predecessors‘ description of the chromosome characteristics and further confirmed the high heterozygosity of orange chromosomes from the level of cytology. Our study speculated that this kind of B-type chromosome heterozygosity may come from chromosome reciprocal translocation occurred in fragile site and observation of meiosis of ?Valencia‘ orange pollen mother cells was back up this view.4) Construction cytogenetic mapping of sweet orange.High tandem repeat sequence CL, moderate repeat sequences r DNA and single copy BAC obtained by genomic analysis were used as probes in fluorescence in situ hybridization combining with CMA/DAPI technique to paint cytogenetic map of sweet orange. Six linkage groups(L1, L2, L4, L6, L7, L8) anchored the corresponding chromosomes(D, D, C, Df, B, Bf), and the remaining F-type chromosomes could be identified according to the size of chromosomes and the length of the linkage groups. |
PDF Full Text Request