Development Of Microsatellite Loci Primers In Chrysanthemum And Genetic Diversity Analysis

Chrysanthemum(Chrysanthemum morifolium Ramat) belongs to Compositae(Asteraceae), Chrysanthemum(Chrysanthemum L.) perennial flowers, originating in China, one of china ten-famous flower and the world four major cut flowers. Wild resources and cultivars of chrysanthemum in our country are very rich, the petal type and flower type are also ample. At present, China chrysanthemum resources is lack of system research, and their genetic background is lack of knowledge,its breeding is limited seriously. In recent years molecular marker technologies has been gradually applied to the study of chrysanthemum genetic relationship, assessment of its genetic diversity, species identified, genetic markers。 Microsatellites or simple sequence repeats (SSR) are a number of tandem repeats of1-6bp, widely distributed in the chromosome, it is one type of molecular markers based on PCR amplification, considered to be an ideal molecular markers for higher polymorphism compared to other DNA molecular markers. However, it is blank about the study of chrysanthemum microsatellite markers.In this study, with the magnetic beads enriched method144and118positive clones containing microsatellite sites were obtained using the probe T3and probe T2libraries respectively. Sequencing result showed there are183microsatellite sites; In the T3library microsatellite sequences are mainly (CT)n,(GA)n,(TG)n (GA)n,(CT)n and (CA)n (5≤n≤40); In the T2library microsatellite sequences are mainly (CAT)n,(TCA)n and (GAT)n (4≤n≤18).142primer pairs were designed with DNAman software according to88microsatellite sequences.In order to obtain the optimal SSR-PCR system for Chrysanthemum moriforlium, an orthogonal diagram L16(45) experimental design was employed to evaluate five factors (template DNA、Mg2+、dNTP、primer and Taq DNA polymerase) at four different levels. The fully random single factor experiment was used to select the optimal level for each factor to optimize the SSR-PCR amplification system. An optimal SSR-PCR system for chrysanthemum was obtained as following:60ng DNA template,2.0mmol·L-1Mg2+,0.1mmol·L"1dNTP,0.3μmol·L-1primer,1U Taq DNA polymerase in25μL reaction system. The optimal annealing temperature for SSR-PCR reaction system was determined as53.1℃by gradient PCR. The suitable thermal cycling conditions with initial melting at95℃for5min, followed by36cycles at94℃for50s,53.1℃for50s,72℃for50s; then keep the reaction mixture at4℃after a final extension step of72℃for8min. 54primer pairs were firstly screened from142primer pairs with three chrysanthemum varieties (’Ziruyi’,’Jinkuixiangyang’,’Cailongzhua’) leading to amplified product bands clear and legible. Further10available primer pairs were obtained from the above54primer pairs with8varieties (’Ziruyi’,’Jinkuixiangyang’,’Cailongzhua’,’Tiannvgequ’,’Huangyueshan’,’Ziyun’,’Laoswngyi’,’Laomohe) for higher polymorphism, they are applied to SSR later.With the10SSR primer pairs3118bands were obtained by PCR amplification, including2630polymorphic bands, average polymorphism rate is74.12%; There are42specific bands, its percentage of total bands is1.35%. The similarity coefficient is0.5322-0.8750among88materials; the highest similarity coefficient is0.8750between two large-flower varieties ’Guohuajindashe’and’Guohuaqiangda’, their petal and flower type are same. According to the data on the SSR, using the clustering analysis of the UPGMA, the results were shown as following:88materials were clustered two groups. Group I includes7small-flower chrysanthemum varieties and2chrysanthemum related genus, C. vestitum, C. zawadskii and Ajania pallasiana belong to subgroup I; The other5small-flower chrysanthemum varieties and Opisthopappus longilobus belong to subgroup II, the genetic relationship of C. morifolium ’Hangbaiju’, C. morifolium’Jinzhugongju’and C. morifolium’Quanjiaochuju’ is more closer, but genetic background of C. morifolium’Boju’is more similar to that of O. longilobus and C. mongolicum. Group Ⅱincludes79large-flower varieties,52varieties is from China,27varieties is from Japan; Subgroup I consists of4valve-type varieties; The other75varieties is clustered subgroup II. Petal type can be classification criterion. There is fewer correlation between the clustering result and flower colour. There is no correlation between genetic relationship and variety origination.