INVESTIGATION Genome-Wide Investigation of MicroRNAs and Their Targets in Response to Freezing Stress in Medicago sativa L., Based on High-Throughput Sequencing Yongjun Shu,1 Ying Liu, Wei Li, Lili Song, Jun Zhang, and Changhong Guo1 Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province, College of Life Science and Technology, Harbin Normal University, 150025, China ABSTRACT Winter damage, especially in northern climates, is a major limitation of the utilization of perennial KEYWORDS forages such as alfalfa. Therefore, improving freezing tolerance is imperative in alfalfa genetic breeding. However, Medicago sativa freezing tolerance is a complex trait that is determined by many genes. To understand the complex regulation cold acclimation mechanisms of freezing tolerance in alfalfa, we performed small RNA sequencing analysis under cold (4°)and freezing stress freezing (28°) stress. The sequencing results revealed that 173 known, and 24 novel miRNAs were microRNA expressed, and that the expression of 35 miRNAs was affected by cold and/or freezing stress. Meanwhile, degradome 105 target genes cleaved by these miRNAs were characterized by degradome sequencing. These targets sequencing were associated with biological regulation, cellular processes, metabolic processes, and response to stress. Interestingly, most of them were characterized as transcription factors (TFs), including auxin response factors, SBP, NAC, AP2/ERF, and GRF, which play important roles in plant abiotic responses. In addition, important miRNAs and mRNAs involved in nodulation were also identified, for example, the relationship between miR169 and the TF CCAAT (also named as NF-YA/HAP2), which suggested that nodulation has an important function in freezing tolerance in alfalfa. Our results provide valuable information to help de- termine the molecular mechanisms of freezing tolerance in alfalfa, which will aid the application of these miRNAs and their targets in the improvement of freezing tolerance in alfalfa and related plants. Lack of tolerance to freezing is a major environmental limitation of the physiological modifications occur, including accumulation of soluble survival, productivity, and ecological distribution of plants. However, sugars, free amino acids, and the expression of cold-regulated (COR) freezing tolerance is a complex trait that is determined by numerous genes, which potentially improving freezing tolerance in plants. To factors from plants and the environment. Among these factors, cold date, the identification and characterization of C-repeat (CRT)- acclimation, the exposure of plants to low, subfreezing temperatures, binding factors (CBFs) has shown that they play critical roles in the plays an important role in conferring freezing tolerance (Thomashow cold acclimation process (Gilmour et al. 1998; Thomashow 2010), by 2010). During the cold acclimation process, several biochemical and regulating downstream functional genes, such as COR genes (Hajela et al. 1990; Thomashow 2010). Regulation of COR genes by CBFs Copyright © 2016 Shu et al. constitutes the central component of cold signaling pathways that doi: 10.1534/g3.115.025981 confer freezing tolerance on plants. In addition, the CBF signaling Manuscript received December 11, 2015; accepted for publication January 16, pathway is also regulated by other factors, for example, ICE1 (In- 2016; published Early Online January 20, 2016. ducer of CBF Expression 1) (Chinnusamy et al. 2003; Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/ 2005; Miura and Hasegawa 2008), and HOS1 (High Expression of licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction Osmotically Responsive Gene 1) (Jung and Park 2013; Lee et al. in any medium, provided the original work is properly cited. 2001), which are important components in the plant cold acclimation Supporting information is available online at www.g3journal.org/lookup/suppl/ process. doi:10.1534/g3.115.025981/-/DC1 1 MicroRNAs are a class of noncoding small RNAs of approximately Corresponding authors: College of Life Science and Technology, Harbin Normal – University, No.1 of Shida Road, 150025, Heilongjiang, China. E-mails: syjun2003@ 21 24 nt, which bind to complementary sequences in the mRNAs of 126.com and [email protected] target genes (Mallory and Vaucheret 2006). This binding results in the Volume 6 | March 2016 | 755 n Table 1 Overview of small RNA sequences in three alfalfa libraries Control Cold Freezing Data Type Total Reads Unique Reads Total Reads Unique Reads Total Reads Unique Reads Clean reads 10,823,011 4,415,425 10,833,023 4,746,716 10,781,132 4,296,444 Rfam 229,925 18,696 400,604 28,495 1,947,224 38,460 Transcriptome 4,933,169 1,201,107 4,509,305 1,232,313 3,892,472 1,219,439 Known miRNA 1,597,215 24,334 1,287,494 27,292 987,515 24,278 Novel miRNA 172,273 1355 82,573 1121 62,290 944 regulation of gene expression at the posttranscriptional level by MATERIALS AND METHODS cleavage-induced degradation of the mRNA, or suppression of its Plant growth and treatment translation (Mallory and Vaucheret 2006). In recent years, many Seeds of M. sativa (cv. Zhaodong), kindly provided by Prof. Hong Li miRNAs have been demonstrated to have important regulatory (IAH-HLJ, China), were germinated and transferred onto a mix of perlite functions in plant growth, development, and stress responses and sand (3:1, v/v). All seedlings were grown in a growth chamber (Babar et al. 2008; Khraiwesh et al. 2012; Sunkar et al. 2012). A ° number of miRNAs are involved in the cold response process in (Conviron E15, Canada), at a temperature between 18 (night) and 24° (day), with humidity ranging from 60% to 80%, and a light period Arabidopsis (Liu et al. 2008; Sunkar and Zhu 2004; X. Zhou et al. – 2008), poplar (Chen et al. 2012; Lu et al. 2008), Brachypodium of 14 hr/10 hr (daytime, 06:00 20:00). The seedlings were irrigated with distachyon (Zhang et al. 2009), rice (Lv et al. 2010), and wheat half-strength Hoagland solution onceeveryotherday,andafter8wk (Tang et al. 2011, 2012), including miR156/157, miR169, miR393, they were randomly divided into three groups for stress treatments. For miR396, miR394, and miR398 (Rajwanshi et al. 2014). With the the control group (untreated, A group), the seedlings continued to grow ° ° development of high-throughput sequencing technology, numer- at 18 (night) to 24 (day). For cold stress (B group) and freezing stress ousmiRNAshavebeenidentified and characterized in plants. (C group), the seedlings were transferred into another chamber with the ° 2 ° MiRNAs biological functions have been deduced by the identifi- temperature set at 4 or 8 , respectively. According to our previous cation and characterization of their target genes. Degradome se- research (Shu et al. 2015), all seedlings were harvested at 3 hr after stress fi quencing, also termed parallel analysis of RNA ends (PARE), was treatments; ve whole seedlings from each group were bulked separately. 2 ° developed based on high-throughput sequencing technology for All samples were frozen in liquid nitrogen, and stored at 80 until use. the genome-wide identification of miRNA target genes (Folkes et al. 2012). Using high throughput sequencing methods, many Small RNA library construction and sequencing new miRNA–mRNA target pairs have been identified successfully Small RNAs were extracted from samples from the three treatments in Arabidopsis (Addo-Quaye et al. 2008), rice (Sun et al. 2015), (control, cold, and freezing) using the TRIzol method (Invitrogen, Carls- ’ wheat (Chen et al. 2015a), soybean (Shamimuzzaman and Vodkin bad, CA), according to the manufacturer s instructions. Small RNAs were 9 9 2012), maize (Zhao et al. 2013), and grapevine (Pantaleo et al. ligated sequentially to 5 -and3- RNA/DNA chimeric oligonucleotide fi 2010), which has helped lucidate the regulatory relationships be- adaptors; the resulting ligation products were gel-puri ed by 15% de- tweenmiRNAsandtheirtargetgenes. naturing PAGE, and reverse-transcribed to produce cDNAs. The cDNAs Alfalfa (Medicago sativa L.), which is grown worldwide, is a were sequenced using a Genome Analyzer IIx System, according to the ’ highly productive perennial forage species with the capacity for manufacturer s instructions (BGI-Shenzhen Co. Ltd., Shenzhen, China). biological fixation of atmospheric nitrogen. However, because of insufficient freezing tolerance, hard winters (with extremely Identification of conserved and novel miRNAs low temperatures) are a major limitation to alfalfa production Raw data were first processed by filtering out low-quality reads, trim- (Castonguay et al. 2013; Pennycooke et al. 2008). Thus, improve- mingtheadaptors,andremovingothernoisereads,toobtaincleanreads. ment of freezing tolerance is an important breeding aim for high The clean reads were then aligned to the Rfam database to remove other yield and longer production periods in alfalfa, especially in north- noncoding RNAs, including rRNA, tRNA, and snRNA. The remaining ern climates, for example, in the United States, Canada, and China reads were mapped to assembled transcriptome sequences from (Chen et al. 2015b). M. sativa L. cv. Zhaodong was domesticated M. sativa cv. Zhaodong (as described in the section below on tran- and bred from wild M. sativa by the Institute of Animal Hus- scriptome sequencing, RNA-seq library construction, sequencing and bandry of Heilongjiang Province