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Background  Zhengdan 958 (Zheng 58 × Chang 7–2), a commercial hybrid that is produced in a large area in China, is the result of the successful use of the heterotic pattern of Reid × Tang-SPT. The jointing stage of maize is the key period from vegetative to reproductive growth, which determines development at later stages and heterosis to a certain degree. MicroRNAs (miRNAs) play vital roles in the regulation of plant development, but how they function in the sixth leaf at the six-leaf (V6) stage to influence jointing stage heterosis is still unclear.    Result  Our objective was to study miRNAs in four hybrid combinations developed in accordance with the Reid × Tang-SPT pattern, Zhengdan 958, Anyu 5 (Ye 478 × Chang 7–2), Ye 478 × Huangzaosi, Zheng 58 × Huangzaosi, and their parental inbred lines to explore the mechanism related to heterosis. A total of 234 miRNAs were identified in the sixth leaf at the V6 stage, and 85 miRNAs were differentially expressed between the hybrid combinations and their parental inbred lines. Most of the differentially expressed miRNAs were non-additively expressed, which indicates that miRNAs may participate in heterosis at the jointing stage. miR164, miR1432 and miR528 families were repressed in the four hybrid combinations, and some miRNAs, such as miR156, miR399, and miR395 families, exhibited different expression trends in different hybrid combinations, which may result in varying effects on the heterosis regulatory mechanism.    Conclusions  The potential targets of the identified miRNAs are related to photosynthesis, the response to plant hormones, and nutrient use. Different hybrid combinations employ different mature miRNAs of the same miRNA family and exhibit different expression trends that may result in enhanced or repressed gene expression to regulate heterosis. Taken together, our results reveal a miRNA-mediated network that plays a key role in jointing stage heterosis via posttranscriptional regulation.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12870-020-02751-3.

MicroRNA transcriptomic analysis of the sixth leaf of maize (Zea mays L.) revealed a regulatory mechanism of jointing stage heterosis