Studies On Molecular Mechanism Of Pear Dormancy

Bud dormancy is a complex developmental process that exhibits a dynamic continuum during the activity-dormancy-activity cycle. With global warming, many fruit trees, including pears, growing in warm areas have suffered from inadequate winter chill, which are unfavorable for sustainable pear production. In recent years, with the development of the facility cultivation of pear, understanding the characteristics of bud dormancy will greatly assist programs to select cultivars and determine the heating time of the facility cultivation. Pear is one of the most important deciduous fruit trees in the world, but the mechanisms regulating bud dormancy in this species are unknown. Studies of bud dormancy have focused mainly on the physiological level, while few studies at the molecular level have been conducted. Therefore, the characteristics of pear dormancy were analyzed for these cultivars ’Suli’(Pyrus pyrifolia white pear group),’Cuiguan’(P. pyrifolia),’Wonhwang’(P. pyrifolia),’Cuiyu’(P. pyrifolia), and ’Xueqing’(P. pyrifolia). Meanwhile, to better understand the molecular mechanisms, a dynamic view of transcriptomic variation was conducted during bud dormancy in pear using RNA-Seq technology. The main results were as follows:There were some differences on the status of bud dormancy depending on cultivars, ecological conditions, even reproductive and vegetative buds of the same cultivar. Endodormancy-release of reproductive and vegetative buds occurred on the same time for ’Wonhwang’ and ’Xueqing’. However, the time of endodormancy-release of reproductive buds appeared prior to vegetative buds for ’Suli’,’Cuiguan’, and’Cuiyu’. Furthermore, there were largely differences on the depth of dormancy of reproductive and vegetative buds for’Cuiguan’under different kinds of ecological conditions. No deep dormancy was observed for reproductive buds of’Cuiguan’in different regions. However, except for in Jintang, vegetative buds of’Cuiguan’had a certain time of deep dormancy for the rest regions studied. The time of deep dormancy of vegetative buds was longer than that of reproductive buds for ’Suli’,’Wonhwang’,’Cuiyu’, and ’Xueqing’.There were some differences on chilling requirement (CR) depending on cultivars, ecological conditions, reproductive and vegetative buds. The same CR of reproductive and vegetative buds showed for ’Wonhwang’ and ’Xueqing’. However, the CR of reproductive buds was lower than that of vegetative buds for ’Suli’.’Cuiguan’, and’Cuiyu’. Furthermore, there were largely differences on the CR of reproductive and vegetative buds for’Cuiguan’under five kinds of ecological conditions. The range of CR of reproductive buds was205-486CH (chilling hour),229-565CU (chill unit),20-29CP (chill portion), while vegetative buds559-1239CH,555-1097CU,26-61CP by7.2℃model, Utah model, and dynamic model, respectively. Based on differences in range of change, dynamic model showed the least change among the regions, followed by Utah model and7.2℃model.We performed transcriptome assembly and digital gene expression profiling analyses of’Suli’pear using the RNA-Seq technology. RNA-Seq generated approximately100M high-quality reads mapped back to the apple genome reference to identify continuous gene regions. Then de novo transcriptome assembly,69,393Unigenes (mean length=853bp) were obtained, including14,531Clusters and34,194Singletons. A total of51,448(74.1%) Unigenes were annotated using public protein databases with a cut-off E-value above10"5. Of the Unigenes,36,717,39,973, and29,500were categorized into60Gene Ontology terms,25Clusters of Orthologous Groups categories, and128Kyoto Encyclopedia of Genes and Genomes pathways, respectively.Differences in gene expression at four times during pear bud dormancy were examined, and DEGs were identified by pairwise comparisons of the four libraries. Although lateral flower buds sampled on Nov.15and Dec.15were both in the endodormant stage,1,978genes were significantly differentially expressed between these libraries. Of these,1,228were down-regulated and750were up-regulated. Between the Dec.15and Jan.15libraries, there were1,024DEGs, with443down-regulated and581up-regulated. A total of3,468DEGs were detected between the Jan.15and Feb.15libraries, with794down-regulated and2,674up-regulated, although lateral flower buds sampled on both dates were in the endodormant-released stage. Of these DEGs,599,343,793were significantly enriched in15,6,17pathways in the Nov.15-VS-Dec.15, Dec.15-VS-Jan.15, and Jan.15-VS-Feb.15comparison respectively. Hierarchical clustering analysis arranged190significantly differentially-expressed genes into seven groups. Seven genes were randomly selected to confirm their expression levels using quantitative real-time PCR (Q-PCR). The Q-PCR data for these genes were basically consistent with the RNA-Seq results of the four samples. Linear regression analysis showed a highly significant correlation (R=0.7533**) which indicated good reproducibility between transcript abundance assayed by RNA-Seq and the expression profile revealed by Q-PCR data.PpMADSl and PpMADS2contained well-conserved MIKC-motifs of MADS-box. A phylogenetic analysis revealed that PpMADSl was closely related to PpMADSl3-1of Japanese pear, whereas PpMADS2was similar to PpMADSl3-2of Japanese pear. The pear DAM genes formed an independent subclade and were closely related to those of Prunus spp. The expression patterns of PpMADSl and PpMADS2were similar with one peak in three cultivars during the period of dormancy. However, the timing of gene expression peak differed among three cultivars. The PpMADSl and PpMADS2expression peak occured on the November15th in’Cuiguan’pear, while the PpMADSl expression peak on the December30th, and the PpMADS2expression peak on the December15th in’Wonhwang’pear. The PpMADSl and PpMADS2expression peak appeared prior to the release of endodormancy, and their expression levels were gradually down-regulated after the turn over of endodormancy and kept at a lower level during the period of ecodormancy in three cultivars. These results indicated that PpMADS1and PpMADS2might play an important role in the regulation of bud endodormancy-release in pear.