3.13 target genes per miRNA,respectively (Table S7). miRNAs and their corresponding targets at different We viewed that the known miRNAs targeted more genes stages of vernalization (4C for 0,2,4 and 6 weeks), on average compared to novel miRNAs,and this find- 11 vernalization-related miRNAs (9 known and 2 ing suggested that the known miRNAs could function novel miRNAs)and 8 targets were randomly selected in a broader range of biological process than the novel for qPCR analysis.The expression profiles were dis- miRNAs.In addition,no targets for 116 known and played in Figs.4 and 5,and the primers were listed 322 novel miRNAs were found,possibly due to the tar- in Table S9.As Fig.4 shown,there exists a similar gets expression levels below the detection level dur- tendency between the qPCR and sRNA sequencing ing the process of vernalization.Additionally,114 tar- results of the selected miRNAs expressions,suggesting gets related to vernalization were isolated as the tar- that the results of sRNA sequencing were reliable.Nine gets of 33 vernalization-related miRNAs(11 known and miRNAs (bra-miR157,bra-miR159a,bra-miR393b 22 novel miRNAs).These targets were related to a bra-miR5020b, bra-miR5054, bra-miR5072 wide variety of biological processes,and several targets bra-miR5077,bra-miR5368 and bra-miRn429)expres- were transcription factors.For instance,MYBs(targeted sions were increased and two miRNAs (bra-miR397a by bra-miR159a)and SPLs (targeted by bra-miR157a), and bra-miRn93)expressions were reduced.The results which were in accord with the previous research in rice suggested that the levels of the tested miRNAs vary and Chinese cabbage (Sunkar et al.2008,Wang et al. significantly during the process of vernalization.Further- 2011).Additionally,many hypothetical and unknown more,some miRNAs showed stage-specific expression, function genes were targeted,indicating possible new probably involved in vernalization. functions for these miRNAs in B.rapa. Additionally,we also validated the expression pat- To functionally analyzed the annotation,the targets terns of eight corresponding targets.The qRCR results of vernalization-related miRNAs were subjected to showed that different targets of one miRNA expressed GO enrichment analysis by Blast2 GO program with varied during the process of vernalization and in differ- default parameters.GO analysis indicated that these ent tissues in B.rapa,and that most targets expression predicted targets could be classified into five molecular were relatively higher at flower or leaf tissues(Fig.5).For functions,16 biological processes and nine cellular example,NEDD1-Bra009155 exhibited highest expres- components (Fig.3).Most targets,which were clas- sion level in flower,while SAP130B-Bra034172 was sified as the binding category,encode transcription highly expressed in leaf,and both of them are targeted factors.Since transcription factors play an important by bra-miR5077.These results showed that different tar- role in gene expression,these targets are thought to gets of the same vernalization-related miRNA may have be the major targets of miRNAs (Eldem et al.2013). different function.In addition,there are inverse corre- Interestingly,we found that most targets were enriched lations between the expression levels of the selected in hormone-mediated signaling pathway,including miRNAs and those of their targets,suggesting that these GAs-mediated signaling pathway (Fig.S1).To further miRNAs might negatively regulate their targets.The rela- investigate the biological pathways influenced by vernal- tionships between miRNAs and their targets were shown ization in B.rapa,KEGG pathway enrichment analysis in Supplementary Table S10.These findings suggested was carried out.We found 106 pathways enriched with that miRNA-mediated silencing of their potential tar- targets of significantly differentially expressed miRNAs, get genes occur during the process of vernalization in and each target was assigned to at least one KEGG B.rapa. annotation.Among these pathways,plant hormone signal transduction involved the most miRNA targets (Bra0329.54,Bra003518,Bra007720.Bra026953and Bra016754,all targeted by bra-miR393b)(Table S8).The Endogenous hormone measurements functional annotations of these genes showed that they As mentioned above,GO and KEGG results suggested all acted in auxin signal pathway.The GO and KEGG that plant hormones may function in the process of B results suggested that plant hormone (GA and auxin) rapa vernalization.To further investigate the roles of signal transduction has vital influences on the process endogenous hormones in the vernalization process,the of B.rapa vernalization. IAA and GA contents were measured in B.rapa.Sam- ples were collected from the leaves before and after ver- qPCR analysis of miRNAs and their targets nalization.As Fig.6 shown,the IAA level decreased and the GA level increased after vernalization,indicating To confirm the deep sequencing data and quan- that the IAA and GA signaling might be involved in the tify the expression patterns of both the identified process of B.rapa vernalization. Physiol.Plant.20183.13 target genes per miRNA, respectively (Table S7). We viewed that the known miRNAs targeted more genes on average compared to novel miRNAs, and this find￾ing suggested that the known miRNAs could function in a broader range of biological process than the novel miRNAs. In addition, no targets for 116 known and 322 novel miRNAs were found, possibly due to the tar￾gets expression levels below the detection level dur￾ing the process of vernalization. Additionally, 114 tar￾gets related to vernalization were isolated as the tar￾gets of 33 vernalization-related miRNAs (11 known and 22 novel miRNAs). These targets were related to a wide variety of biological processes, and several targets were transcription factors. For instance, MYBs (targeted by bra-miR159a) and SPLs (targeted by bra-miR157a), which were in accord with the previous research in rice and Chinese cabbage (Sunkar et al. 2008, Wang et al. 2011). Additionally, many hypothetical and unknown function genes were targeted, indicating possible new functions for these miRNAs in B. rapa. To functionally analyzed the annotation, the targets of vernalization-related miRNAs were subjected to GO enrichment analysis by Blast2 GO program with default parameters. GO analysis indicated that these predicted targets could be classified into five molecular functions, 16 biological processes and nine cellular components (Fig. 3). Most targets, which were clas￾sified as the binding category, encode transcription factors. Since transcription factors play an important role in gene expression, these targets are thought to be the major targets of miRNAs (Eldem et al. 2013). Interestingly, we found that most targets were enriched in hormone-mediated signaling pathway, including GAs-mediated signaling pathway (Fig. S1). To further investigate the biological pathways influenced by vernal￾ization in B. rapa, KEGG pathway enrichment analysis was carried out. We found 106 pathways enriched with targets of significantly differentially expressed miRNAs, and each target was assigned to at least one KEGG annotation. Among these pathways, plant hormone signal transduction involved the most miRNA targets (Bra032954, Bra003518, Bra007720, Bra026953 and Bra016754, all targeted by bra-miR393b) (Table S8). The functional annotations of these genes showed that they all acted in auxin signal pathway. The GO and KEGG results suggested that plant hormone (GA and auxin) signal transduction has vital influences on the process of B. rapa vernalization. qPCR analysis of miRNAs and their targets To confirm the deep sequencing data and quan￾tify the expression patterns of both the identified miRNAs and their corresponding targets at different stages of vernalization (4∘C for 0, 2, 4 and 6 weeks), 11 vernalization-related miRNAs (9 known and 2 novel miRNAs) and 8 targets were randomly selected for qPCR analysis. The expression profiles were dis￾played in Figs. 4 and 5, and the primers were listed in Table S9. As Fig. 4 shown, there exists a similar tendency between the qPCR and sRNA sequencing results of the selected miRNAs expressions, suggesting that the results of sRNA sequencing were reliable. Nine miRNAs (bra-miR157, bra-miR159a, bra-miR393b, bra-miR5020b, bra-miR5054, bra-miR5072, bra-miR5077, bra-miR5368 and bra-miRn429) expres￾sions were increased and two miRNAs (bra-miR397a and bra-miRn93) expressions were reduced. The results suggested that the levels of the tested miRNAs vary significantly during the process of vernalization. Further￾more, some miRNAs showed stage-specific expression, probably involved in vernalization. Additionally, we also validated the expression pat￾terns of eight corresponding targets. The qRCR results showed that different targets of one miRNA expressed varied during the process of vernalization and in differ￾ent tissues in B. rapa, and that most targets expression were relatively higher at flower or leaf tissues (Fig. 5). For example, NEDD1-Bra009155 exhibited highest expres￾sion level in flower, while SAP130B-Bra034172 was highly expressed in leaf, and both of them are targeted by bra-miR5077. These results showed that different tar￾gets of the same vernalization-related miRNA may have different function. In addition, there are inverse corre￾lations between the expression levels of the selected miRNAs and those of their targets, suggesting that these miRNAs might negatively regulate their targets. The rela￾tionships between miRNAs and their targets were shown in Supplementary Table S10. These findings suggested that miRNA-mediated silencing of their potential tar￾get genes occur during the process of vernalization in B. rapa. Endogenous hormone measurements As mentioned above, GO and KEGG results suggested that plant hormones may function in the process of B. rapa vernalization. To further investigate the roles of endogenous hormones in the vernalization process, the IAA and GA3 contents were measured in B. rapa. Sam￾ples were collected from the leaves before and after ver￾nalization. As Fig. 6 shown, the IAA level decreased and the GA3 level increased after vernalization, indicating that the IAA and GA signaling might be involved in the process of B. rapa vernalization. Physiol. Plant. 2018