Development And Application Of SSR Markers In Chrysanthemum Based On Full-length Transcriptome Sequence

Chrysanthemum(Chrysanthemum x morifolium Ramat.)is an important ornamental plant which originated in China and widely cultivated all over the world.It has large varietal characteristics,diverse flower type and color and rich genetic variation etc.,which makes difficult to study the variety classification and genetic diversity of chrysanthemum.SSR markers as an important molecular marker technology,have been widely used in the study of plant genetic diversity.With the rise of transcriptome sequencing technology,the design of SSR primers based on transcriptome is easy to operate which can be used to screen SSR loci in batches and association with specific traits of species.Most of the cultivated chrysanthemums are hexaploid.Because of the complexity of their genome,the results of genome sequencing have not been reported so far.Therefore,it is of great practical significance to develop SSR markers based on the transcriptome sequencing data of chrysanthemum.The full-length transcriptome of traditional chrysanthemum varieties‘Jinbeidahong','Hechengxinghuo' and‘Quanxiangshuichang'was analyzed in the early stage of our research group.In this study,SSR markers were developed based on the results of full-length transcriptome sequencing.Using a variety of chrysanthemum varieties as experimental materials,the distribution of SSR loci in transcripts,the regional preference of SSR amplification polymorphism,correlation between specific SSR marker amplification and chrysanthemum characters were studied by PCR amplification and bioinformatics analysis.The changes of SSR loci in 5-azaC or EMS treated materials were analyzed with the established SSR markers.The results are as follows1.The third generation single molecule real-time sequencing technology was used to sequence the full-length transcriptome of chrysanthemum varieties‘Jinbeidahong',‘Hechengxinghuo'and'Quanxiangshuichang'.All SSR loci contained in the high-quality non redundant FLNC searched by software MISA6 were analyzed.Based on the analysis of the SSR distribution regions developed by the full-length transcriptome of chrysanthemum 'Jinbeidahong',the number of SSR loci in the 5'UTR region was the most(28,651),followed by the 3,UTR region(19,903),and the CDS region was the least(15,972)Based on SSR loci in the full-length transcriptome of chrysanthemum 'Jinbeidahong',SSR primers were designed in three different regions(5'UTR,3'UTR and CDS regions).In total,28 SSR markers were screened and the effective amplification rate was 29.79%.Using 30 chrysanthemum varieties as materials,PCR polymorphisms polymorphisms of 28 SSR markers were detected The results showed that the highest PCR polymorphism was found in 5'UTR region(80.78%),followed by 3'UTR region(76.87%)and CDS region(74.16%)with lowest2.Among the SSR markers developed by the floral full-length transcriptome of chrysanthemum'Hechengxinghuo'and 'Quanxiangshuichang',16 SSR markers on the genes related to flower characters were screened.Total of 83 chrysanthemum varieties were used as materials to verify the correlation between SSR marker polymorphism and flower characters.The results showed that the SSR markers of 2PB-9,3PB-6,2PB-10,2PB-1 and 2PB-12,which were located in VDE,TCP2,MADS box protein CMBI and MADS6 genes respectively,and their polymorphism could be cluster with the flower color and petal type characters of chrysanthemum3.Total of 8 SSR markers were screened out from the full-length transcriptome sequence of chrysanthemum 'Hechengxinghuo'.DNA and cDNA from petal tissue of 117 chrysanthemum varieties with different flower color traits were used as materials.The results showed that CHS and CHI-3 SSR markers located in CHS and CHI genes in anthocyanin synthesis pathway respectively,and LCYE-1,PSY and VDE SSR markers located in LCYE,PSY and VDE genes in carotene metabolism pathway respectively,and their polymorphism could be clustered together with the flower color characters of chrysanthemum4.The effects of 5-azaC on SSR marker polymorphism were analyzed in the leaves of Chrysanthemum 'Quanxiangshuichang' and 'Jinsihuangju'cuttings treated with different concentrations of 5-azaC.The results showed that SSR marker polymorphisms were more easy to change when the chrysanthemum was treated with 300 ?mol/L 5-azaC for 5 days5-azaC(100 ?mol/L,3 d)was used to treat 12 different varieties of chrysanthemum cutting,and the petals of the treated lines were used as materials to analyze the changes of SSR Marker polymorphism at the level of genome and transcriptome.The results showed that the SSR polymorphism of‘Zhuzipanlong',‘Tangyutianju'and‘Pushuiliubing'were more easy to change by 5-azaC treatment5.In order to detect whether the selected SSR Marker Polymorphism is affected by the methylation level of chrysanthemum,the leaves of 'Zijingling' tissue culture seedlings,'Zijingling'MET1-RNAi and wild-type tissue culture seedlings treated with 5-azac were used as materials to analyze the changes of SSR Marker polymorphism in these materials.The results showed that the polymorphisms of six SSR markers,2PB-7,2PB-11,2PB-16,2PB-17,3PB-3 and 3PB-15,might be affected by DNA methylation6.The effect of EMS treatment on SSR marker polymorphism and plant growth and development were analyzed in the leaves of G1 strain of chamomile.The results showed that EMS treatment had affected SSR marker polymorphism.The longer EMS treatment time and the higher the concentration resulted in the lower survival rate of the tissue culture seedlings of the G1 strain of chamomile while plant height was also affected by different EMS treatment methods.Most of the samples were dwarfIn summary,SSR markers in the 5'UTR region of the full-length transcriptome of chrysanthemum have the highest amplification polymorphism,followed by 3,UTR region and lowest in CDS region.SSR markers located in TCP2,MADS box protein CMBI and MADS6 genes were correlated with some flower type characters of chrysanthemum.SSR markers located in CHS,CHI,LCYE,PSY and VDE genes were correlated with some flower color characters of chrysanthemum.The polymorphism of SSR markers in regenerated plants and tissue cultured plantlets of chrysanthemum cuttings was affected by 5-azaC or EMS.