Study On Technique Of Molecular Marker-assisted Flower Color Selection And Identification Of Flower Colour And Fragrance Gene In Cymbidium

Cymbidium is a traditional famous flower in China,which has high cultural value and unique ornamental value,and a broad market prospect.Flower fragrance and color are important ornamental characters of Cymbidium,but their genetic mechanism are not clear so far.Therefore,identification of genes related to fragrance and color and establishment of molecular marker-assisted flower color selection technical system are important for further investigations on genetics and breeding of flower fragrance and color in Cymbidium.In this paper,the C.sinense’Qi Jian Bai Mo’,C.sinense’Wu Zi Cui’,C.’Huang Hua 1 Hao’,C.’Huang Hua 2 Hao’,C.’Gong Fen Jia Ren’,C.’Xiao Feng’and the progenies of C.’Xiao Feng’and C.sinense’Wu Zi Cui’were used as materials to find the genes related to Cymbidium fragrance and color by transcriptome sequencing and q RT-PCR analysis.According to the results of transcriptome sequencing,SSR markers were developed,and the markers closely linked to Cymbidium color were sceened.The experiment of light and spraying sucrose,Fe³⁺,Al³⁺affecting on the flower color of C.’Gong Fen Jia Ren’was also carried out.The main results are as follows:1.The dissolution and standard curves of eighteen genes including18S,26S,ACT,EF1α,Rpo B,TUB,GAPDH,Psa B,RPS3,RPS19,Ch18Sr RNA,Ch28Sr RNA,Ch ACT,Ch EF1α,Chrpo B,Ch TUB,Ch UBQ and Ch TUA were analyzed by q RT-PCR technology,and six candidate reference genes were screened out,which were RPS3,RPS19,EF1a,GAPDH,Ch TUA and Ch TUB.The expression stability of the candidate reference genes was evaluated by ge Norm,Norm Finder and Best Keeper software.The results showed that RPS3,EF1a and GAPDH could be used as reference genes for the gene expression analysis of flower color and fragrance in Cymbidium.2.The sepals and petals of C.’Huang Hua 1 Hao’and C.’Huang Hua 2 Hao’were subjected for transcriptome sequencing by Illumina Hi Seq^TM.A total of 65512 unigenes were obtained,among them,8965 unigenes were differentially expressed.KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched on metabolic pathways including photosynthesis-antennal protein,photosynthesis,phenylpropane biosynthesis,monoterpene biosynthesis,flavonoid biosynthesis,diterpenoid biosynthesis,cyanide amino acid metabolism,cutin,amber and wax biosynthesis,carotenoid biosynthesis,α-linolenic acid metabolism,peroxisome and phenylalanine metabolism.The results of q RT-PCR analysis indicated that 4CL-7-5,TPS10-1 andα-TPS-2were the candidate genes of flower fragrance.3.According to the results of transcriptome sequencing of vegetative and reproductive organs in C.sinense’Qi Jian Bai Mo’and sepals and petal of C.’Huang Hua 1 Hao’and C.’Huang Hua 2 Hao’as well as q RT-PCR analysis,the five genes,Cy CHS-1,Cy CHS-2,Cy HYB,Cy PDS and CCD7,were the candidate genes related to flower color.4.According to the results of transcriptome sequencing of vegetative and reproductive organs in C.sinense’Qi Jian Bai Mo’,forty-four new SSR markers were developed by Primer Premier 5.0.C.’Xiao Feng’and C.sinense’Wu Zi Cui’were subjected for markers polymorphism analysis,the results indicated that among three hundred and sixty-two SSR markers employed,two hundred and ninety-two SSR markers（80.7%）generated clear bands,one hundred and eight SSR markers（37.0%）produced polymorphic bands between C.’Xiao Feng’and C.sinense’Wu Zi Cui’.C.’Xiao Feng’and C.sinense’Wu Zi Cui’and their forty progenies were employed to screen the SSR marker closed-linked to Cymbidium flower color,the results proved among one hundred and eight polymorphic SSR markers,eight SSR markers were able to distinguish variegated-color flower from pure-yellow-green-color flower,which were CQX5,CQX31,CQX59,CQX145,CQX165,CQX185,CQX235 and CQX243,respectively.5.The flower color of C.’Gong Fen Jia Ren’was different under different light intensities,the flower color of C.’Gong Fen Jia Ren’was redder under stronger light intensity.The results of q RT-PCR analysis demonstrated that differential expression of Cy CHS-2 and Cy PDS were related to the change of flower color in C.’Gong Fen Jia Ren’under strong light.Through the above research,three genes related to Cymbidium flower fragrance,five genes related to Cymbidium flower color and two genes related to the change of flower color in C.’Gong Fen Jia Ren’were identified,and eight SSR markers distinguishing the pure-yellow-green-color flower of Cymbidium were screened out.These results lay a foundation of further study on the genetic basis of flower fragrance and color and establishment of molecular marker-assisted flower color selection technical system in Cymbidium.