Identification Of Metaphase Chromosome In Brassica Oleracea With Blocking Of B Genome By FISH

The genus Brassica is one of the most important plants in Cruciferae, which include many major crops with great economic value and wildly cultivated at home and abroad. With initiation of Multinational Brassica Genome Project in 2004, the study of molecular cytogenetic in Brassica species has become a new hotspot. Brassica oleracea is one of the three basic species in Brassica, and play an important role in providing lots of vegetable for our daily life. As one of the most important researchful objects of Multinational Brassica Genome Project, constructing the high-density physical map, combining genetic map with physical map, and genes localization has become the main content of molecular cytogenetic in Brassica oleracea. Whereas, the precondition of these content is to identify different chromosomes exactly and fastly.Because chromosomes in Brassica oleracea are shorter and more homogeneous condensation in cell division phase than other plants, and the traditional technique of banding couldn't distinguish from different chromosomes, so far, there is no any reports about identifying chromosomes in Brassica oleracea by systemically using the technique of Fluorescence in situ Hybridizatione after block of B Genome. In this paper we try to identify metaphase chromosome from root tip in Brassica oleracea by Fluorescence in situ Hybridization, the study results were as follows:1. Root tip of Brassica oleracea (K172-1) was used as experimental material, the high-efficient technique system on metaphase chromosome preparation of Brassica oleracea was found through the comparative study of correlative parameters.(1)When the root length is 0.5-0.6cm, the root has more metaphase cells than other length.(2)Good shape of chromosomes could be obtained by pretreating with 0.002mol/L 8-Hydroxyquinoline at 20℃for 2 hours.(3)The chromosome sample shoud be treated by digestion of 2.5% cellulose and 2.5% pectolyase at 37℃for 2 hours, which could refrain from the background interference of cytoplasm and cell wall.2. The analysis of karyotype of chromosome at metaphase in Brassica oleracea (K172-1) showed that: the somatic chromosome numbers were 2n=18, and the karyotype formula was K(2n)=18=16m+2Sm(SAT). chromosome 2, 3,4, 5,6,7, 8 and 9 are all metacentric chromosome, and chromosome 1 with satellites was sub-metacentric chromosome. The relative length of the chromosomes were between 8.304% and 14.198%, the length ratio of the longest and shortest chromosome was 1.710. The chromosome type of Brassica oleracea (K172-1) belonged to 2A type, a symmetrical karyotype.3. Using double enzyme digestion of Genomic DNA in Brassica oleracea as probe, as well as Genomic DNA in Brassica nigra as Block reagent, in this research, identification on metaphase chromosome of Brassica oleracea by the technique of Fluorescence in situ Hybridization, we could identify 1,4, 7 and 9 respectively by good diagnostic signal. This method successfully realized preliminary study on exactly identification of metaphase chromosome in Brassica oleracea.4. Comparative analysis between karyotype and FISH identification indicated that karyotype could only recognise a few chromosome with specific morphological characteristics, while FISH identification could recognise more chromosome with cytogenetics characteristics .These study results will contribute to genes localization on metaphase chromosome, the high-density physical Map construction, As well as the new method on comparative genetic groups between Brassica oleracea and Arabidopsis thaliana, Some new insights also be brought into the origin and evolution of Brassica oleracea.