Integrated microRNA and mRNA expression profiling reveals a complex network regulating pomegranate ( Punica granatum L.) seed hardness

The breeding of new soft-seeded pomegranate cultivars provides new products for the market and increases farmers’ incomes, yet the genetic architecture mediating seed hardness is largely unknown. Here, the seed hardness and hundred-seed weights of 26 cultivars were determined in 2 successive years. We conducted miRNA and mRNA sequencing to analyse the seeds of two varieties of Punica granatum : soft-seeded Tunisia and hard-seeded Sanbai, at 60 and 120 d after flowering. Seed hardness was strongly positively correlated with hundred-seed weight. We detected 25 and 12 differentially expressed miRNA–mRNA pairs with negative regulatory relationships between the two genotypes at 60 and 120 d after flowering, respectively. These miRNA–mRNA pairs mainly regulated seed hardness by altering cell wall structure. Transcription factors including NAC1, WRKY and MYC, which are involved in seed hardness, were targeted by differentially expressed mdm-miR164e and mdm-miR172b. Thus, seed hardness is the result of a complex biological process regulated by a miRNA–mRNA network in pomegranate. These results will help us understand the complexity of seed hardness and help to elucidate the miRNA-mediated molecular mechanisms that contribute to seed hardness in pomegranate.