| Grape ranks as the world's second fruit,and seedless grape has been preferred by fresh consumption and raisin making industry,making it a goal for grape breeding to meet the requirement of market and industry development.Thus study on seedless grape is of great value.So far,the mechanism of grape seedlessness hasn't been clear,and key genes related to seedlessness remain to be identified.Here,we used progeny derived from the seeded maternal parent ‘Red Globe' and the seedless paternal parent ‘Centennial seedless' to conduct ovule transcriptome analysis at three key developmental stages.Moreover,geneome sequencing was conducted with bulked segregant analysis of seeded and seedless progeny,as well as their seeded maternal parent ‘Red Globe' and seedless paternal parent ‘Centennial seedless'.Mechanisms of grape seedlessness has been explored by combining transcriptome and genome analysis.Systematical study on grape MADS-box gene family has been carried out in both evolution and expression perspective.Two differentially expressed genes(DEGs)related to grape seedlessness have been identified and cloned,and their potential function has been preliminary verified by gene overexpression in tomato.The main results obtained are described as followings:1.Ovules of seeded and seedless grape progeny derived from the seeded maternal parent ‘Red Globe' and the seedless paternal parent ‘Centennial seedless' were collected,and three key developmental staged were identified based on their weight changes during development.At each stage,ovules from 5 seedless progeny and 5 seeded progeny were mixed separately for RNA-Seq analysis,a total of 6,607 DEGs were identified and analyzed from multiple perspectives,including expression patterns,Gene Ontology(GO)annotations,pathway enrichment,inferred hormone influence and epigenetic regulation.Ovule development differences between seedless and seeded progeny were mainly focused on aspects of seed coat development,endosperm development,hormone balance,epigenetic regulation and complexes formation of seed identity genes.Hormone level measurement reveals that the content of CTK was higher in ovules of seeded grape progeny than in seedless ones,while IAA,GA and ABA higher in seedless progeny than in seeded ones.Based on both our results and previous studies of A.thaliana seed development,we generated network maps of grape seed-related DEGs,with particular reference to hormone balance,seed coat and endosperm development,and seed identity complexes.2.A large number of functional variants were identified by genome sequencing with bulked segregant analysis of leaves from seeded and seedless progeny derived from the seeded maternal parent ‘Red Globe' and the seedless paternal parent ‘Centennial seedless'.By combining ovule transcriptome data of seeded and seedless progeny,DEGs containing functional variants were found to be factors related to seed development,including disease resistantance protein,protein kinase,transcription factors,Cytochrome P450,cellulose-related protein and calmodulin,theses DEGs were involved in biological processes like oxidation reduction and senescence.QTL regions associated with seedlessness were obtained based on SNP-index of two pool-seq.Expression pattern of all genes contained in the QTL regions were analyzed using transcriptome data,and 9 candidate genes were identified.Expression profiles analysis of these 9 candidate genes in different tissues as well as during ovule development of multiple seeded and seedless grape cultivars further reveals all these genes to have different expression patterns between seeded and seedless progeny.In combination with existing literature and gene function annotation,these genes mentioned were further identified as candidate genes related to seedlessness.3.A total of 54 grape MADS-box genes were identified in the grape genome and the nomenclature of putative grape MADS-box genes was assigned based on their chromosomal placement.Synteny analysis indicated that both segmental and tandem duplication events contributed to the expansion of the grape MADS-box family.Furthermore,synteny analysis between the grape and Arabidopsis genomes suggested that several grape MADS-box genes arose before divergence of the two species.Phylogenetic analysis and comparisons of exon-intron structures provided further insight into the evolutionary relationships between grape MADS-box genes,and grape MADS-box genes were divided into type I and type II subgroups.Tissue-specific expression analysis suggested roles in both vegetative and reproductive tissue development.Expression analysis of the MADS-box genes following gibberellic acid(GA3)treatment revealed that most do not respond significantly,and that seedlessness caused by GA3 treatment underwent a different mechanism from that of normal ovule abortion.Expression profiling of MADS-box genes from six cultivars suggests their function in ovule development,and 5 genes with different expression patterns in ovules of multiple seeded and seedless cultivars were identified as potential ovule identity genes involved in parthenocarpy.4.VvMADS28 and VvMADS39 were identified as two DEGs during ovule development of seeded and seedless cultivars based on both expression patterns and evolution analysis of grape MADS-box genes.Transcript level of VvMADS28 gene were significantly higher in ovules of seeded cultivars compared to seedless ones,and expression of VvMADS39 were up-regulated in ovules of seedless cultivars compared to seeded ones.After cloning and sequencing,the ORF of VvMADS28 was 744 bp,encoding 247 amino acids,and VvMADS39 741 bp,encoding 245 amino acids.Expression pattern analysis of theses 2 genes in different tissues and flower organs reveals they both high expressed in flowers.Ecotopic expression of VvMADS28 in tomato displayed smaller fruits with a tip in the bottom and seeds to be smaller in size but more in number compared with wild ones.In addition,ecotopic expression of VvMADS39 in tomato also displayed smaller fruits with abortion seeds.Expression patterns of tomato genes associated with fruit and seed development were found to be different in transgenetic tomato lines compared with wild ones,suggesting VvMADS28 and VvMADS39 genes may influence fruit and seed development of transgenetic tomato lines by regulating expression of these genes,which to some extent made a preliminary function verification of VvMADS28 related to seed development and Vv MADS39 related to seedlessness. |
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