Genome-wide identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis

Background: A long juvenile period between germination and flowering is a common characteristic among fruit trees, including Malus hupehensis (Pamp.) Rehd., which is an apple rootstock widely used in China. microRNAs (miRNAs) play an important role in the regulation of phase transition and reproductive growth processes. Results: M. hupehensis RNA libraries, one adult and one juvenile phase, were constructed using tree leaves and underwent high-throughput sequencing. We identified 42 known miRNA families and 172 novel miRNAs. We also identified 127 targets for 25 known miRNA families and 168 targets for 35 unique novel miRNAs using degradome sequencing. The identified miRNA targets were categorized into 58 biological processes, and the 123 targets of known miRNAs were associated with phase transition processes. The KEGG analysis revealed that these targets were involved in starch and sucrose metabolism, and plant hormone signal transduction. Expression profiling of miRNAs and their targets indicated multiple regulatory functions in the phase transition. The higher expression level of mdm-miR156 and lower expression level of mdm-miR172 in the juvenile phase leaves implied that these two small miRNAs regulated the phase transition. mdm-miR160 and miRNA393, which regulate genes involved in auxin signal transduction, could also be involved in controlling this process. The identification of known and novel miRNAs and their targets provides new information on this regulatory process in M. hupehensis, which will contribute to the understanding of miRNA functions during growth, phase transition and reproduction in woody fruit trees. Conclusions: A comprehensive study on M. hupehensis miRNAs related to the juvenile to adult phase transition was performed. The combination of sRNA and degradome sequencing can be used to better illustrate the profiling of hormone-regulated miRNAs and miRNA targets involving complex regulatory networks, which will contribute to the understanding of miRNA functions during growth, phase transition and reproductive growth in perennial woody fruit trees. In June, ‘Adult phase’ (A) leaves were collected as mixed samples from the tops of 18 6-year-old M. hupehensis (Pamp.) Rehd. trees and ‘Juvenile phase’ (J) leaves were collected from the base.And were used for small RNA and degradome sequencing for identification of vegetative phase transition-associated microRNAs and target predictions using degradome sequencing in Malus hupehensis.

背景：种子萌发至开花之间存在漫长的幼年期，是包括湖北海棠（Malus hupehensis (Pamp.) Rehd.）在内的果树的普遍特征，该树种是我国广泛应用的苹果砧木。微小RNA（microRNAs, miRNAs）在植物相转变与生殖生长过程中发挥重要调控作用。结果：本研究以湖北海棠的叶片为材料，构建了成年期与幼年期两个RNA文库，并进行高通量测序（high-throughput sequencing）。共鉴定得到42个已知miRNA家族以及172个新的miRNA。通过降解组测序（degradome sequencing），我们分别鉴定出25个已知miRNA家族的127个靶基因，以及35个独特新miRNA的168个靶基因。所鉴定的miRNA靶基因可归类为58个生物学过程，其中已知miRNA的123个靶基因与相转变过程相关。京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）分析显示，这些靶基因参与淀粉与蔗糖代谢以及植物激素信号转导通路。对miRNA及其靶基因的表达谱分析表明，miRNA在相转变过程中发挥多种调控功能。幼年期叶片中mdm-miR156的高表达与mdm-miR172的低表达提示，这两种小分子RNA参与调控相转变过程。调控生长素信号转导相关基因的mdm-miR160与miR393，同样可能参与该过程的调控。已知miRNA与新miRNA及其靶基因的鉴定，为湖北海棠中的该调控过程提供了新的研究信息，有助于理解木本果树生长、相转变与生殖过程中miRNA的功能。结论：本研究针对湖北海棠幼年期向成年期相转变相关的miRNA开展了系统性研究。小RNA（small RNA, sRNA）测序与降解组测序的联合应用，可更清晰地阐释激素调控miRNA及参与复杂调控网络的miRNA靶基因的表达谱，这将有助于理解多年生木本果树生长、相转变与生殖生长过程中miRNA的功能。本研究于6月采集18株6年生湖北海棠植株顶部的混合叶片作为成年期（A）样品，从植株基部采集幼年期（J）叶片样品，将其用于小RNA测序与降解组测序，以鉴定湖北海棠营养相转变相关的miRNA，并通过降解组测序进行靶基因预测。