Molecular Mechanism Of Dormancy Release Of Liliumpumbulbs Based On Transcriptome And MiRNA Sequencing

Lilium pumilum with strong resistance to cold is a good wild lily parent material for breeding.Lily bulbs have the characteristics of natural dormancy,which can only be transferred from the vegetative growth stage to the reproductive growth stage only under suitable environmental conditions.Therefore,the study of the mechanism of bulb dormancy has been paid much attention.Generally,low temperature treatment is one of the effective methods to break the dormancy of lily bulbs.The study of dormancy was mainly focused on morphological and physiological and biochemical changes during the dormancy release process of cultivated species,but relatively few studies on the molecular mechanism of dormancy in wild species.In addition,the use of molecular breeding methods to shorten the dormancy time of lilies and accelerate the application and promotion of its production is of great significance for the annual supply and quality assurance of Lilium.Previous studies showed that the 90d of lily bulbs could be completely relieving dormancy at 5? low temperature.In order to explore the molecular mechanism of the dormancy release of lily bulbs,with the Lilium pumilum as the experimental material,on the basis of the ultrastructure of the bud growth point and the external morphological observation of the bulb,this research treated lily bulbs at 5? and with Od,30d,60d,and 90d after treatment respectively to dissolve the dormancy process.A large number of transcriptional and miRNA information were obtained by using the Illumina/Solexa Hiseq 2000 technology platform to analyze the transcriptional sequence of the bulbs at different stages.The main results were as follows:1,During the low temperature storage,the mitochondria of most Lilium tenuifolia were spherical,the Golgi vesicle layer increased and starch granule content decreased significantly.Low temperature storage accompanied by dormancy release and flower development,and the flower bud differentiation was divided into 4 periods,that was,the formation period of the flower primordia,the stage of the primordia differentiation of the outer ring flower,the stage of the differentiation of the inner ring,and the stage of the differentiation of the pistil and pistil primordia.2,Transcriptome sequencing analysis showed a total of 62.04 Gb high quality data.Differential gene analysis showed that amylase related enzymes and sucrose synthesis genes were almost up-regulated expressed,and the pentose pathway of lily bulb responded to cold induction.The difference of gene analysis involved in plant hormone signal transduction pathway showed that antagonism between ABA and GA levels during low temperature storage might promote the release of bulb dormancy,while plant hormone ethylene coding genes which promoting dormancy release was up-regulated,and genes encoding SA and JA were down regulated,and they were closely related with defense mechanism in plants stress underlying.3,Ascorbic acid glutathione cycle might be used to cope with increased oxidative stress in Lilium pumilum.The transcription factor,ARF-like gene and MADS-box gene expression were significantly up-regulated,and DREB-like gene expression decreased in the later period,and the genes MYB-like and WRKY-like differential genes had significant enrichment.4,4 miRNA sequencing libraries were constructed,and 14 miRNA families information were annotated to participate in growth and development(miR166,miR168 and miR396),hormone signal transduction(miR159,miR160,miR164,miR536),and the process of abiotic stress(miR394,miR395,miR398,miR168,etc.).The expression of miR166 and miR168 were up-regulated and significantly enriched.Lpu-1,miR408 and Lpu-12 targeted the key enzymes of antioxidant reaction,and all decreased expression.The accumulation of low temperature might activate the activity of antioxidant enzymes and remove the oxidizing substances produced by the stress process.5,MiR159 and miR536 targeted MYB and ABI3 transcription factors to affect ABA/GA content.The target gene MYB transcriptional factor down-regulated and weakened the ABA synthesis signal.This negative feedback regulation mechanism might be crucial to the development of the bulb from dormancy to dormancy release.MiR160 and miR164 participated in IAA signal transduction process,interacted with ABA and participated in the regulation of developmental pattern.MiR398 and miR857 were continue to decline during low temperature storage,and their potential target genes might be activated to participate in energy metabolism and scavenging oxidant.The research found that promoter binding protein(SPL)was the target gene of miR395.6,Unconserved miRNA,Lpu-5 target gene encoded major DNA methylation enzymes,histone lysine-N methyltransferase and DNA(cytosine-5)-methylase,which had high level of expression during low temperature storage.It was regulated by negative feedback of target gene to reduce the level of cell methylation,consistent with the results of transcriptional data,the decrease of DNA methylation might respond to the release of dormancy at the later period of low temperature storage,which indicated that epigenetics was one of the important mechanisms regulating the dormancy release of bulbs.