Genome-wide association analysis identified molecular markers and candidate genes for flower traits in Chinese orchid (Cymbidium sinense)

The orchid,the champagne of f lowers,brings luxury,elegance,and novelty to nature.Cymbidium sinense is a symbol of gigantic floral variability on account ofwavering shapes and sizes of f loral organs,althoughmarker-trait association(MTA)has not been studied for its f loral traits.We evaluated markers associated with 14 f loral traits of C.sinense through a genome-wide association study(GWAS)of 195 accessions.A total of 65318522 single-nucleotide polymorphisms(SNPs)and 3906176 insertion/deletion(InDel)events were identified through genotyping-by-sequencing.Among these,4694 potential SNPs and 477 InDelswere identified as MTAs at−log10 P>5.The genes related to these SNPs and InDels were largely associated with f loral regulators,hormonal pathways,cell division,and metabolism,playing essential roles in tailoring f loral morphology.Moreover,20 candidate SNPs/InDels linked to 11 genes were verified,8 of which were situated on exons,onewas located in the 5-UTR and twowere positioned in introns.Here,themultitepal trait-related gene RABBIT EARS(RBE)was found to be the most crucial gene.We analyzed the role of CsRBE in the regulation of flower-related genes via efficient transient overexpression in C.sinense protoplasts,and found that the floral homeotic genes CsAP3 and CsPI,as well as organ boundary regulators,including CsCUC and CsTCP genes,were regulated by CsRBE.Thus,we obtained key gene loci for important ornamental traits of orchids using genome-wide association analysis of populations with natural variation.The findings of this study can do a great deal to expedite orchid breeding programs for shape variability.

Transcriptome Analysis Reveals Clues into leaf-like flower mutant in Chinese orchid Cymbidium ensifolium

The floral morphology of Cymbidium ensifolium,a well-known orchid in China,has increasingly attracted horticultural and commercial attention.However,the molecular mechanisms that regulate flower development defects in C.ensifolium mutants are poorly understood.In this work,we examined a domesticated variety of C.ensifolium named‘CuiYuMuDan',or leaf-like flower mutant,which lacks typical characteristics of orchid floral organs but continues to produce sepal-to leaf-like structures along the inflorescence.We used comparative transcriptome analysis to identify 6234 genes that are differentially expressed between mutant and wild-type flowers.The majority of these differentially expre ssed genes are involved in membrane-building,anabolism regulation,and plant hormone signal transduction,implying that in the leaf-like mutant these processes play roles in the development of flower defects.In addition,we identified 152 differentially expre ssed transcription factors,including the bHLH,MYB,MIKC,and WRKY gene families.Moreover,we found 20 differentially expressed genes that are commonly involved in flower development,including MADS-box genes,CLAVATA3(CLV3),WUSCHEL(WUS),and PERIANTHIA(PAN).Among them,floral homeotic genes were further investigated by phylogenetic analysis and expression validation,which displayed distinctive spatial expression patterns and significant changes between the wild type and the mutant.This is the first report on the C.ensifolium leaf-like flower mutant transcriptome.Our results shed light on the molecular regulation of orchid flower development,and may improve our understanding of floral patterning regulation and advance molecular breeding of Chinese orchids.

Complete Genome Sequence Analysis of Guangdong Isolates of Cymbidium Mosaic Virus

[Objectives]The paper was to explore the genetic information and evolution of CymMV,and to provide an important scientific basis for monitoring and early warning of orchid virus disease and anti-virus genetic engineering of orchid in Guangdong Province.[Methods]RT-PCR and DASELISA were used to detect and identify CymMV from leaves with suspected virus disease of Cymbidium sinense collected from Guangzhou area.The genome sequence assembly,annotation,phylogeny and selection pressure analysis of CymMV isolates were performed with related molecular biology software.[Results]Two CymMV isolates(GZV013 and ZC29)were found in Guangdong Province for the first time in this study.The genome of both GZV013 and ZC29 were 6227 nt in length,encoding 5 functional proteins.The similarity analysis of the full sequence showed that the nucleotide sequence identity of GZV013 and Taiwan isolate M2 was 97.03% and that of ZC29 and Nanjing isolate NJ-1 was 97.11%.The complete genome sequence identity among CymMV isolates ranged from 86.85% to 98.31%,and the differentiation of diverse populations was closely related to host species and geographical isolation.Each region of CymMV genome was affected by negative selection and conformed to the neutral evolution model.The genes encoding RdRp,TGB1 and TGB2 had the highest mutation rates in the genome.[Conclusions]GZV013 was most closely related to Taiwan isolate M2,and ZC29 was most closely related to Nanjing isolate NJ-1,belonging to the same branch of a family.