Starch plays an important role in non-fresh and fresh starchy fruit yield and quality.Banana is a potential excellent model for starch biosynthesis in fresh starchy fruit because of its relatively high starch content and large starch granules.However,no systematic studies focusing on starch biosynthesis in fresh starchy fruit are available.In the present research,we used a high-throughput sequencing technique(RNA-Seq)to reveal the molecular mechanisms involved in banana fruit starch biosynthesis.Six RNA-Seq libraries,representing two biological replicates from pulps at 0,20 and 80 days after inflorescence emergence from the pseudostem(DAF),were constructed and sequenced,resulting in > 4.4 billion raw reads for each library.By RPKM values,46,113 differentially expressed genes(FDR ≤0.001,fold change ≥2.0)were identified.The results were compared against public databases(Gene Ontology and Kyoto Encyclopedia of Genes and Genomes)to annotate the gene functions and to assign each to pathways.A large number of genes related to the metabolic pathways,biosynthesis of secondary metabolites,plant hormone signal transduction,carbohydrate catabolic process,starch metabolic process and starch biosynthetic process were strongly expressed at 0 DAF,20 DAF,and 80DAF.Interestingly,most metabolic pathways were down-regulated between 0 and 80 DAF.By further coexpression analysis,56 transcription factors and 30 candidate starch biosynthesis genes among all three time points were identified.We also analyzed chromosomal distribution and gene structures of candidate starch biosynthesis genes based on banana genome.Compared with the published starch biosynthesis pathways in cereal and duckweed,the starch biosynthesis pathway in banana fruit has features that are obviously different from that in non-freshstarchy fruits.This result provides a reference for systematic understanding the molecular mechanisms of starch biosynthesis in fresh starchy fruits.