Karyotype Analysis Based On Fluorescence In Situ Hybridization In Chrysanthemum Indicum L.

Chrysanthemum indicum is an important wild germplasm of the genus Chrysanthemum in the Compositae family. At present, the investigation on Chrysanthemum mainly focused on ploidy, chromosome banding, karyotype, meiosis behavior and in situ hybridization. Karyotype analysis is an important method for classification and genetic study of plant. Karyotype analysis of C. indicum was mainly investigated with the conventional pressing plate method according to the chromosome morphology and size of chromosome, which is difficult to accurately distinguish all of the chromosomes, especially for chromosome high similarity of species. However, Fluorescence In Situ Hybridization(FISH) is based on the principle of complementarity, which can be used in obtaining the precise position of modified nucleotide numerator probe on chromosomes, and could offer recognized stamps for karyotype analysis. There was little research on karyotype analysis using FISH, for one thing it is hard to access to high quality chromosome preparation, for another there is lack of good labeled probe and the development of marker method in genus Chrysanthemum. In this paper we optimized the chromosome sectioning of C. indicum, developed labeled probe through genome sequencing and established the FISH system. 45 S rDNA, 5S r DNA and repetitive sequence YJ223-5 was distributed on metaphase chromosomes and the karyotypes of C. indicum was analyzed based on the FISH results of three probes. The main results showed as follows:1、The problem of rooting difficultly in autumn and winter was solved by using cutting and tissue culture, they were all proved to be served as good materials through adjusting the chromosome spreading method.2、The best chromosome spreading condition was selected by adjusting the process of the pre-processing conditions and the time of enzyme hydrolysis: 8-hydroxyquinoline treating for 3 hours or mixture of ice and water for 16 hours, mixed enzymes(cellulose: pectinase =2:1) for 90~100 min.3、We used the Helianthus annuus and Lactuca sativa var.romana as reference to estimate the genome size of C. indicum, the result of flow cytometry showed the genome size of C. indicum is~6.36 Gb.4、6Gb data was obtained by Genome sequencing of C. indicum. The graph-based methods for similarity-based clustering of reads were used to reveal the genome structure of C. indicum, and we found that approximately 68.61% of the C. indicum genome is composed of repeat sequences. The proportion of LTR retrotransposon, a dominant repeat element, is 42.3% and tandem repeat sequence is 26.3% in C. indicum genome, respectively.5、Nine 45 S rDNA signals were detected on metaphases chromosome, which were all located at terminal regions of chromosomes 3,6 and 8; Three 5S rDNA signals were detected, which were all located at submetacentric regions of chromosomes 4; Eight YJ333-5 signals were detected on metaphase chromosome 2,4 and 8, both terminal of two chromosomes showed signals. The karyotype of C. indicum was analysed according to the number and location of signal sites and combing with karyotype parameters. The karyotype formula of C. indicum is 2n=4x=36=24m(2SAT) +12sm, which belongs to 2A type, a primitive type in evolution.