| The genus Citrus is known worldwide for its economic importance, as well as for its taxonomic difficulties in species delimitation, mainly caused by weak reproductive barriers and nucellar polyembryony, associated with a long history of cultivation and somatic mutations. Citrus metaphase chromosomes basically all very small in size, their length is only between 1-4 μm, their morphological characteristics is very similar with each other. It is dificult to study the chromosomes due to similar appearance and absence of discernible markers.The nucleic acid sequences can be accurately positioned on chromosomes by using the technique of Fluorescence in situ hybridization (FISH). Combined with the traditional cytogenetics, it will provide a more detailed understanding of the structure of chromosomes at the molecular level for citrus and its related genera. Meanwhile, it also provides a fast, accurate, and effective method for further study on chromosome identification and gene mapping etc. In this paper, the 5SrDNA was used as a probe to research the 5SrDNA loci in citrus and its related genera by FISH. The obtained information is expected to study the systemic evolution of the citrus and provide more effective marker for recognition of chromosome in the citrus plants. In addition, the difference analysis of the 5SrDNA locus in some citrus diploid and natural tetraploid. Here are some main results:1.The procedure for citrus chromosome preparation and the system of chromosome in situ hybridization were further optimized. The process of chromosome preparation without fire drying, but natural drying,which for reducing the damage of chromosome. According to the characteristics of the probe, then reduce the time of hybridization and elution. This made the experiment quick, easy and efficient. Also, it saved time and effort.2. The number of 5S rDNA sites on 16 samples which belong to Citrus L. Fortunella Swingle and Poncirus Raf. was 1,2 and 6. Among them,13 samples had 2 sites. The presence of 5S rDNA sites located on chromosome 2,4,6,7,8 and 9, mostly on chromosome 7 (5 samples) and 8 (7 samples). The locations of all the samples with 2 sites were identified on homoeologous chromosome groups except C.reticuiata (red tangerine) and trifoliate orange.2 sites were detected among 11 samples belonging to Citrus L. with the exception of C.junos Sieb. ex Tanaka and C.limon (Eureka) which had 1 site. The signals were located in the end of short chromosome arms of a pair of homologous chromosomes which samples with 2 sites. However, there were differences between 2 sites of C.reticuiata (red tangerine), of which, one located in the end of short chromosome arms while the other one in the medium position. Both of 2 samples of Fortunella Swingle had 2 sites located on the end of short chromosome arms and no significant differences of signal were found. It showed that 6 sites were detected on the only one sample belonging to Poncirus Raf. which located on chromosome 2,3,8,9 respectively.3 of all the sites were identified on the end of short chromosome arms while 2 were near the centromeres and 1 was in the region of satellite chromosome.3. The comparison of 5S rDNA sites among 3 natural tetraploids belonging to Citrus L. and its corresponding diploid showed that 2 sites were found in diploid of C.sinensis (Hongjiangcheng) and C.sinensis (Huazhoucheng) while 4 sites in its tetraploids. Moreover,1 site was detected in the diploid of C.junos Sieb. ex Tanaka while 2 sites in its tetraploids. It implies that the ploidy of samples in this study is likely to be doubled directly from its corresponding diploid.The results reported here showed that the 5S rDNA sites of Citrus L. and its relatives were conserved. However, there was a closely relation between the number of 5S rDNA sites and the level of ploidy that was the number was increasing with the higher ploidy which provided more information for the mechanism of occurrence of polyploid. |
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