Morphology Of Single-Petaled/Double-Petaled Flower Traits In 'Malus' Through Anatomical And Transcriptome Analyses

Double-flower ornamental crabapples display eye-catching morphologies in comparison to single flower,but the genetic basis of double-flower development is not yet well known in apples.These are often preferred by consumers because of larger,more floriferous,and showier look than single flowers.Several double flowered plants have been observed,which are a result of homeosis.The genetic mechanisms of these double flowers have been studied in core eudicots and other species,under several flowering models.Many mutations have been discovered and classified into groups and families.MADSbox genes are the main regulatory genes reported to be involved in regulation of double flowering in many horticultural crops(including apple)studied so far.Thus a detailed study focusing on mechanism of these genes is needed.In the present study,different phenotypes of crabapple were studied comparatively.Morphological analysis through paraffin sectioning and light microscopy was carried out for Malus Dolgo and Malus Kelsey.In double flowers,the stamens were found converted to petals or petal like structures.These data provided quantitative support for the observations and suggested the morphological signs of the differences among single and double flowers due to homeosis.In this study,we have focused to understand the molecular mechanism of MADS-box genes in double flowering by comparing with single flowers.In order to comprehensively understand the molecular basis of these genes involved in double flower,the transcriptome of double flower crabapples M.Kelsey,Malus Micromalus,Malus Royalty,and a single flower cultivar M.Dolgo were compared by RNA-sequencing.The results showed that there were 1854 genes in overlapped DEGs among all sample comparisons in apple single and double flower varieties.A large number of MADS-box and hormone related DEGs were recognized on the basis of GO and KEGG annotations,and most of the genes were found to be down-regulated in double flowers.We performed qRT-PCR to confirm the expression of randomly selected genes and to verify the RNA-seq data.Among 1854 DEGs,975 transcription factors encoding genes with flower organ development association were included.Weighted gene co-expression network analysis(WGCNA)was carried out followed by cytoscape analysis to find out the key genes involved in double flowering.A large number of development and hormone related DEGs were also recognized on the basis of GO and KEGG annotations,and most of the genes were found to be down-regulated in double flowers.Particularly,an AGL24-MADS-box gene(MD08G1196900)and an auxin responsive gene(MD13G1137000)were putatively key candidate genes in the development of double flower.The study provides insights into the complex molecular mechanism underlying the development of the double flower in apple.