MicroRNA Transcriptomic Analysis Of Heterosis During Maize Seed Germination

Maize is an important food, economic and forage crop, and heterosishas been utilized widely in maize breeding for a long time, but the molecular mechanismresponsible for heterosis is far from clear. Therefore, to clarify the molecular mechanismof heterosis will play an important role on the molecular breeding in maize.MicroRNAs (miRNAs) is newly identified class of20-24nt non-protein-codingand endogenous small RNAs. This non-protein-coding RNAs are highly conserved in allkinds of animals, plant and microbial species. MicroRNA regulate gene expressionmainly at the transcriptional and post-transcriptional level, which plays an important rolein regulating plant growth, development and response to environmental stresses in plants.Therefore, it is urgent to search for important miRNAs correlating with heterosis duringmaize seed germination and preliminarily reveal the molecular mechanism of heterosis.In previous study, we find that maize germination is affected less by externalenvironment, and express a strong heterosis, so it is a good direction to study heterosisin maize. In this study, one hybrid maize Yuyu22and its two parents87-1and Zong3were utilized for dissecting the genetic basis of heterosis with miRNA regulation, whichis an elite hybrid widely cultivated in China that exhibits high heterosis for grain yield.The significant miRNAs expressed differently amount between the hybrid and itsparental lines were got through Solexa high throughput sequencing, and the target geneswere predicted by bioinformatics methods. The important candidate miRNAs and targetgene which related to the heterosis were analyzed and validation with Real-time PCR.The main results of this study were as follows:Ten million reads small RNA were got by Solexa sequencing (reads per million). Intotal,107known miRNAs belonging to21miRNA families were co-detected in thehybrid and its two parental lines, and most (94/107) of the miRNAs were differentially(non-additively) expressed in the germinating embryos between the hybrid and theinbred lines.There were1068small RNAs in the inbred line87-1that did not match conservedmiRNAs, but was located within a stem-loop structure with their flanking sequence. TheMFEI (minimal folding free energy index) of all1068miRNA candidates werecalculated and searched in other samples to confirm whether they were true miRNAs.Thirty-four miRNAs were most likely true novel miRNAs, which have not yet beenreported.The target genes of these34newly defines miRNAs were predicted by the targetgene prediction software online-psRNATarget. A total of408unique miRNA targets were detected. These target genes were further analyzed by GO (Gene Ontology), andmost of them were concentrated on transcriptional and translational regulators. Some ofthem were involved in the metabolic pathways processes, such as transcriptionregulation, substance and energy metabolism, stress response, and signal transductionthrough further function prediction.Expression profiles were established based on real-time RT-PCR analysis of sixselected conserved miRNAs and eight novel miRNAs, as well as real-time RT-PCRanalysis of their target genes. The differential expression of miRNAs showed that thesequencing data accorded well with the real-time RT-PCR reactions, which show thereliability of the sequencing results.Combined present results with previous studies on the model plant and analyzed ofdifferentially expressed microRNAs, it was speculated that the differentially expressedmicroRNAs regulated the expression of their target genes between hybrid and parentallines to affect heterosis during maize seed germination. So, the facts that most of themiRNAs were repressed in the hybrid compared to the parental inbred line, whichpossibility be one of the causes of its heterosis performance.