Physiological And Molecular Basic Research Of Light Green Stripe Mutant In Oncidium ‘Milliongolds'

Oncidium spp.is a kind of perennial herb,belonging to Oncidium genus,Orchidaceae family.As cut flower and pot culture,it is famous for its special shapes and bright colors in flower.Nowadays,the ornamental study on oncidium is mainly focused on its colours and types of flowers,but little about leaf colour.Mutation in leaf colour is a major way for obtaining new germplasm of oncidium hybridum.Leaf colour mutant is an important material for exploring photosynthetic mechanism,chlorophyll metabolism and chloroplast development,and it has high application value in the aspect of breeding.The photosynthetic pigments and chlorophyll synthesis precursors contents,ultra-structure of chloroplasts and chlorophyll fluorescence parameters were analysed in light green stripe mutant of Oncidium ‘Milliongolds'.Capillary Electrophoresis-Amplified Fragment Length Polymorphism(CE-AFLP)was used to determine the genome variation frequency.Furthermore,RNA-seq was used to obtain transcript derived fragment which were connected with leaf color,aiming to explore the mechanism of leaf mutation of oncidium.The results are as follows:The measurement of chloroplasts ultra-structure,photosynthetic pigments and chlorophyll synthesis precursors contents and chlorophyll fluorescence parameters indicated that the number of chloroplast reduced and there were some abnormal chloroplasts with less grana lamellas,as well as more osmioplilic granules and vesicae in mutants.The contents of chlorophyll a(Chl a),chlorophyll b(Chl b),carotenoids(Caro)and total chlorophyll(Chl)in mutant leaves revealed a remarkably decreased tendency,to decreased by 37.1%,34.0%,30.8% and 36.3% respectively,and the biosynthesis of chlorophyll in mutant was blocked at the reaction from prophobilinogen(PBG)to uroporphyrinogen(Urogen ?).The minimal fluorescence(Fo)of mutant was 39% higher than that of normal plant,the maximal fluorescence(Fm),maximum quantum yield of PSII(Fv/Fm),photochemical efficiency of PS II in the light(Fv'/Fm')and actual photochemical efficiency of PSII(?PS II)reduced significantly in mutant,while the photochemical quenching(qP)and non photochemical quenching coefficient(NPQ)of mutant were not significantly different from that of normal plant.The CE-AFLP results showed that a total of 596 fragments about ranging from 100 to 1000 bp were generated from 28 AFLP primers,of which 192 fragments were polymorphic.The total degree of gene polymorphism was 32.2%,and the variation frequency were ranged from 12.3% to 19.9% between different mutants and normal plant.The genetic similarity coefficient of mutants(N1-N10)and normal plant(CK)varied from 0.8132 to 0.8838.When the similarity coefficient was 0.85,N1,N5,N7,N9,N4,N2 and CK were clustered to group I;N3,N6,N8 and N10 were clustered to group II.It was concluded that genetic differences were found between the 10 mutants and the normal plant,which indicated different degree of variation in DNA level.By RNA-Seq,raw data was de novo assembled into 46 058 unigenes.Of all the unigenes,31 059(67.4%)was annotated to database.A total of 2 252 genes,963 up-regulated genes and 1 289 down-regulated genes,were found differently expressed in oncidium.Furthermore,20 964 unigenes were assigned into 51 GO functional classifications,the functions of 841 differentially expressed unigenes were closely related to metabolic processes,organelle and membrane.10 590 unigenes could be assigned into 25 COG functional categories,the functions of 692 differentially expressed unigenes were closely related to general fuction prediction only,replication,recombination and repair and transcription.KEGG pathway analysis showed that 6 983 unigene were assigned into 97 pathways,692 differentially expressed unigenes were involved in plant hormone signal transduction,protein processing in endoplasmic reticulum and phenylpropanoid biosynthesis.Furthermore,we selected the differentially expressed genes which were connected with chlorophyll metabolism,chloroplast development and photosynthesis.These genes are NOL?CHLH?RP-S10?LHCB?POD?F3H?HTC?UGT?ISF?GIOH?RPL16?NDHC?RPIA?GAPDH?F2KP?AOS?HSP20?TPT?Fd.RNA-Seq data were validated using Real-time RT-PCR techniques,the results showed that the expression patterns of 18 genes were similar to thosed observed in the RNA-Seq,whereas the GAPDH showed a different expression pattern.In general,the Real-time RT-PCR data was consistent with the RNA-Seq data,suggesting that the RNA-Seq data are reliable.In conclusion,The leaf color variation of Oncidium hybridum might be directly attributed to the obstruction in chlorophyll biosynthesis pathway and chloroplast dysplasia.Therefore,we speculate that CHLH and NOL gene directly affect the synthesis of chlorophyll,while LHCB,Fd and RP-S10 may play an important role in the growth of chloroplast.