## 4 Discussion  
miRNAs act as important regulators in transcriptional and post-transcriptional gene silencing and play essential roles in many plant physiological processes, such as plant growth and development, morphogenesis, embryogenesis, stress tolerance, and metabolism [2, 41, 42, 43, 44]. In recent years, accompanied by the rapid development of sequencing technology, a greater number of studies have focused on the identification of novel miRNAs using the next-generation sequencing technology [45, 46, 47, 48, 49, 50, 51]. Nevertheless, up to now, no information related to miRNAs has been reported in wax gourd (_Benincasa bispida_). In this study, we firstly discovered and characterized the miRNAs of wax gourd using high-throughput sequencing from five tissues (root, stem, leaf, flower, and fruit). The major size distributions of the total small RNA reads in the five libraries ranged from 20-25 nt, in which 24 nt small RNAs were most abundant and followed by 23 nt small RNA. Similar results were also reported in other species, such as cucumber [52], persimmon [49], and Lonicera japonica [50], but different from that of radish [48], grapevine [53], Populus euphratica [54]. Furthermore, when compared with the other four libraries, the length distribution of small RNAs in the root library showed a significant difference, which implied the presence of tissue specificity of small RNAs in wax gourd. The above results suggested that the distribution of small RNAs might vary among plant species as well as tissues. It is documented that 24 nt small RNA function in heterochromatin modification, especially for genomes with a high content of repetitive sequences [55, 56]. As more than 75.5% of wax gourd genome was constituted by repetitive sequences [33], it's possible that the dominant 24 nt small RNAs might play diverse functions in different processes of wax gourd. Deep sequencing of small RNA libraries from different tissues is of great importance for comprehensively detecting the populations and roles of miRNAs [47]. Based on the sequences in the miRBase database and the reference genome of wax gourd, a total of 255 (leaf), 146 (root), 264 (stem), 219 (flower) and 213 (fruit) known miRNAs together with 407 (leaf), 381 (root), 408 (stem), 408 (flower), and 405 (fruit) novel miRNAs were identified. For both the known and novel miRNAs, the read number varied significantly. Most of the novel miRNAs existed at low copies in all five tissues (Table S2). Although presented as low levels, these miRNAs may play particular roles in specific development and cellar processes as well, such as plant development, fruit ripening, biotic and abiotic stress responses [40, 47, 48, 54, 57]. In our present study, a large number of differentially expressed miRNAs between tissues were detected by pairwise comparisons and those miRNAs with similar expression patterns were clustered into the same profile by trend analysis. miRNAs in many profiles, such as profile 13 and profile 19, had more expression in wax gourd fruit. miR164-x is a member of profile 13 and it had the highest expression in fruit than any other tissues. MiR164 was previously reported involved in fruit ripening. For instance, miR164 was highly expressed during fruit ripening of sweet orange [58]. What is more, miR164 also affects fruit development. During prickly pear cactus fruit development, miR164 expressed in all fruit development stages and homogenously expressed in fruit-related organs [59]. miR164 deficient mutant tomato slmir164 aCR possessed smaller fruit than wild type indicating its role in regulating fruit development [29]. Mostly, miR164 regulates fruit development by targeting NAC transcription factors, such as _CUP-SHAPED COTYLEDON2 (CUC2)_. MiR164-CU2 module regulates fruit enlargement and therefore affects fruit size in _Arabidopsis_[60]. Evolutionary analysis revealed that most plant miR164s are relatively conserved and miR164-x of wax gourd is the same as miR164a of melon.