Biological Characteristics Of ‘Century Seedless Pear' And Gene Mining Of Its Regulation Mechanism

In recent years,there has been an increasing number of reports on seedless fruits.However,the reports on seedless pear were rare.In this study,the seedless rate of 'Century Seedless Pear' was tested.And its parents were identified by SRAP molecular markers.We took 'Century Seedless Pear' and its parents 'Bartlett' and 'Yali' as materials to observe their similarities and differences of reproductive organs on morphological anatomy to identify the critical period of seed abortion.The contents of plant hormone were also detected to analyze the effects on abnormal seed development.Furthermore,from the transcriptional level,we analyzed the differentially expressed genes of 'Century Seedless Pear' compared with its parents using RNA-seq method.Two key genes were cloned and their overexpression vectors were constructed.Generally,this study laid the foundation for further revealing the seedless molecular mechanism of the 'Century Seedless Pear' and also provided gene resources for the breeding of other Rosaceaea seedless fruits.The main research results were as follows:1.In order to observe the occurrence of seed abortion in 'Century Seedless Pear',the seed abortion rate was investigated.Its parents were identified by SRAP molecular markers.The results showed that the seedless rate was 100%,and its parents were 'Bartlett' and'Yali'.In order to explore the causes of seed abortion of 'Century Seedless Pear',a series of observation on the pollination experiments,development of style and ovule were carried out.The results showed that 'Century Seedless Pear' was a self-incompatible variety.Meantime the stamen organ and pollen were normal.So the reason of seed abortion result from female organs.What's more,the pollinated varieties needed to be configured to ensure its fruit set.In addition,through the observation of morphological structure on the'Century Seedless Pear' and its parents,it was found that the ovules of 'Century Seedless Pear' began to abort at 8 DACP,with partially black in color and shriveling in volume.At 12 DACP with completely black in color and obviously shriveling in volume,the seed completely aborted.The flesh of young fruit 'Century Seedless Pear' and its parents 'Yali'and 'Bartlett' were taken as materials to detect the plant hormone.Compared with its parents,the flesh of 'Century Seedless Pear' had higher IAA and GA3,which was one of the causes of seed abortion.The seed can also produce plant hormone and is one of the sources of hormone,the higher levels of IAA and GA3 in the flesh of 'Century Seedless Pear' also make up the drawback of seedlessness thus to ensure the normal development of pear fruit.2.In order to explore the differences of the seedless variety‘Century Seedless Pear'and its parents seedy variety 'Bartlett' and 'Yali' from transcriptional level,three growth periods' ovule samples of 'Century Seedless Pear' were taken as materials,and the ovule samples of 'Bartlett' and 'Yali' at the same growth periods were taken as the control to make transcriptome study using RNA-seq method.Total clean reads,ranging from 26,658,800 to 39,250,026 were obtained from eighteen libraries which provided abundant data for the analysis of ovule abortion.About 79.81%-93.35%of clean reads could be uniquely mapped to the reference genome('Dangshansuli' and 'Bartlett').Based on the DEGs,seed,embryogenesis and hormone signal transduction related genes were selected as the candidate genes to be involved in the seed abortion.We speculated that several transcription factor families also play important roles in the ovule abortion such as MYB,bHLH,WRKY and NAC transcription factors.And the interaction of MYB and bHLH transcription factors were emphatically analyzed.A number of genes related to lipid metabolism and carbohydrate metabolism also showed enrichment as a result of seed abortion.In order to verify the reliability of the transcriptome data,14 differentially expressed genes were selected to do qRT-PCR.The results of qRT-PCR were almost consistent with the changes of transcriptional expression level.The results above further laid a foundation to reveal the molecular mechanism of the seed abortion.The RNA-seq study will also provide genetic resources for the molecular breeding of other Rosaceae seedless fruits.3.According to the RNA-seq and qRT-PCR results,a series of candidate genes were cloned to do further transgenic experiments.Up to now,two key genes PbrMYB108(Pbr038922.1)and PbrWRKY75(Pbr042883.1)were successfully cloned and the overexpression vectors pCAMBIA1301S-PbrWRKY75/PbrMYB108 were constructed.The work provided a basis for the subsequent transgenic experiments,and thus verify whether the two transcription factors regulate the abnormal development of seed.