Study On Effect Of Heavy Ion Irradiation And Mechanism Of Thread Art Mutation On Leaf In Cymbidium

Cymbidium is a traditional Chinese orchid with important ornamental,cultural and economic value and broad market prospect.Heavy ion irradiation breeding has the advantages of simple operation,high mutagenesis rate,short breeding period,which had been widely used in ornamental plant breeding.However,there were only a few reports focused on orchid.In this study,the rhizomes of Cymbidium 17-33,C.‘12g-5’,C.‘Xiao Feng’and protocorm like-bodies（PLBs）of C.‘Gong Fen Jia Ren’were used as test materials to investigate the effect of heavy ion radiation on survival,differentiation and induced mutation of intermediate propagules.C.‘Jun Hao’was employed to explore internal reference genes for gene expression analysis in root,pseudobulb and leaf,and to find candidate genes related to thread art mutation on leaf by using of transcriptome sequencing and q RT-PCR methods.The main findings are as follows:1.The effect of heavy ion irradiation on survival,differentiation and mutation of intermediate propagules in Cymbidium.Heavy ion irradiation caused the death of rhizomes of C.17-33,C.‘12g-5’,C.‘Xiao Feng’and PLBs of C.‘Gong Fen Jia Ren’,their final death rates of 40 Gy irradiation were 100%,96.15%,92.00%and 95.19%,respectively.The rhizomes irradiated with 20 Gy in C.17-33 and PLBs irradiated with 40 Gy in C.‘Gong Fen Jia Ren’were recoved to grow after cultured for 160 days and 240 days,respectively.The results of SSR marker analysis showed that no polymorphic bands were amplified in the rhizomes of 0 Gy irradiation in C.17-33 and the PLBs of 0 and 40 Gyirradiation in C.‘Gong Fen Jia Ren’,while two polymorphic bands were produced in the rhizomes of 20 Gy irradiation in C.17-33,with the polymorphism rate being 2.0%.2.Using ge Norm,Norm Finder and Best Keeper software,the expression stability of Rps3,GAPDH,ACT,EF1α,Ch ACT,Ch TUB,Ch UBQ,Ch EF-1a,Chrpo B,Rps19,18S,Rpo B,TUB,26S and Psa B genes were evaluated.The results indicated that Rps19 and Ch UBQ genes could be used as internal reference genes for gene expression analysis in root,pseudobulb and leaf of C.‘Jun Hao’and its mutants.3.Using Illumina Hiseq X technology,transcriptome sequencings of leaves and mixed samples of roots and pseudobulbs of C.‘Jun Hao’and C.‘Xian Yi Jun Hao’were performed,a total of 75,985 unigenes were obtained,among which the number of differentially expressed genes（DEGs）was 13059,with 4034 upregulated genes and 9025downregulated genes.There were 8808 DEGs in leaf,among which 2415 genes were upregulated and 6393 genes were downregulated.KEGG analysis showed that DEGs in leaf enriched in 124 biological metabolic pathways,among them there were five pathways related to thread art mutation on leaf,which were anthocyanin synthesis,photosynthesis-antenna protein,porphyrin and chlorophyll metabolism,flavonoid biosynthesis and photosynthetic pathway.There were eight,seven,three,sixteen and fourteen DEGs in the above five pathways,respectively.4.Twenty DEGs were chosed from the pathways related to thread art mutation on leaf and subjected for q RT-PCR analysis.The results suggested that CYP76B10 and PSBO genes were possibly involved in thread art mutation on leaf in C.‘Jun Hao’.Through the above research,the effect of¹²C⁶⁺heavy ion radiation on the survival,differentiation and mutation of intermediate propagule in Cymbidium were preliminarily clarified,the candidate gene caused the occurrence of thread art mutation on leaf in C.‘Jun Hao’were found.These lay a foundation of further study on molecular mechanism of mutation induced by heavy ion irradiation,the establishment of effecient mutation breeding technological system by heavy ion irradiation and creation of breakthrough cultivars in Cymbidium.