University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications: Department of Entomology Entomology, Department of 2017 Insect and plant-derived miRNAs in greenbug (Schizaphis graminum) and yellow sugarcane aphid (Sipha flava) revealed by deep sequencing Haichuan Wang University of Nebraska-Lincoln, [email protected] Chi Zhang University of Nebraska-Lincoln, [email protected] Yongchao Dou University of Nebraska-Lincoln, [email protected] Bin Yu University of Nebraska-Lincoln, [email protected] Yunfeng Liu University of Nebraska-Lincoln See next page for additional authors Follow this and additional works at: http://digitalcommons.unl.edu/entomologyfacpub Part of the Entomology Commons Wang, Haichuan; Zhang, Chi; Dou, Yongchao; Yu, Bin; Liu, Yunfeng; Heng-Moss, Tiffany; Lu, Guoqing; Wachholtz, Michael; Bradshaw, Jeffery D.; Twigg, Paul; Scully, Erin; Palmer, Nathan; and Sarath, Gautam, "Insect and plant-derived miRNAs in greenbug (Schizaphis graminum) and yellow sugarcane aphid (Sipha flava) revealed by deep sequencing" (2017). Faculty Publications: Department of Entomology. 509. http://digitalcommons.unl.edu/entomologyfacpub/509 This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Authors Haichuan Wang, Chi Zhang, Yongchao Dou, Bin Yu, Yunfeng Liu, Tiffany Heng-Moss, Guoqing Lu, Michael Wachholtz, Jeffery D. Bradshaw, Paul Twigg, Erin Scully, Nathan Palmer, and Gautam Sarath This article is available at DigitalCommons@University of Nebraska - Lincoln: http://digitalcommons.unl.edu/entomologyfacpub/ 509 Gene 599 (2017) 68–77 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene Research paper Insect and plant-derived miRNAs in greenbug (Schizaphis graminum)and yellow sugarcane aphid (Sipha flava) revealed by deep sequencing Haichuan Wang a,⁎, Chi Zhang b, Yongchao Dou b,BinYub, Yunfeng Liu b, Tiffany M. Heng-Moss c, Guoqing Lu d, Michael Wachholtz d, Jeffery D. Bradshaw c,PaulTwigge,ErinScullyf,NathanPalmerg, Gautam Sarath g a Department of Agronomy and Horticulture, University of Nebraska Lincoln, Lincoln, NE 68583, United States b School of Biological Sciences, University of Nebraska Lincoln, Lincoln, NE 68503, United States c Department of Entomology, University of Nebraska Lincoln, Lincoln, NE 68583, United States d Department of Biology, University of Nebraska Omaha, Omaha, NE 68182, United States e Department of Biology, University of Nebraska Kearney, Kearney, NE 68849, United States f USDA-ARS, Stored Product Inset and Engineering Research Unit, Center for Grain and Animal Health Research, Manhattan, KS 66502, United States g USDA-ARS, Wheat, Sorghum, and Forage Research Unit, Lincoln, NE 68583, United States article info abstract Article history: Schizaphis graminum (green bug; GB) and Sipha flava (yellow sugarcane aphid; YSA) are two cereal aphid species Received 7 September 2016 with broad host ranges capable of establishing on sorghum (Sorghum bicolor) and several switchgrass (Panicum Received in revised form 2 November 2016 virgatum) cultivars. Switchgrass and sorghum are staple renewable bioenergy crops that are vulnerable to dam- Accepted 7 November 2016 age by aphids, therefore, identifying novel targets to control aphids has the potential to drastically improve yields Available online 9 November 2016 and reduce losses in these bioenergy crops. Despite the wealth of genomic and transcriptomic information avail- able from a closely related model aphid species, the pea aphid (Acyrthosiphon pisum), similar genomic informa- Keywords: fi Next generation sequencing tion, including the identi cation of small RNAs, is still limited for GB and YSA. Deep sequencing of miRNAs mirDeep2 expressed in GB and YSA was conducted and 72 and 56 miRNA candidates (including 14 and eight novel) miRNAs were identified, respectively. Of the identified miRNAs, 45 were commonly expressed in both aphid species. Fur- Cereal-aphids ther, plant derived miRNAs were also detected in both aphid samples, including 13 (eight known and five novel) Sorghum sorghum miRNAs and three (novel) barley miRNAs. In addition, potential aphid gene targets for the host plant- Barley derived miRNAs were predicted. The establishment of miRNA repertoires in these two aphid species and the de- tection of plant-derived miRNA in aphids will ultimately lead to a better understanding of the role of miRNAs in regulating gene expression networks in these two aphids and the potential roles of plant miRNAs in mediating plant-insect interactions. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Mead et al., 2012). Oral feeding of synthetic miRNA mimics in Helicoverpa armigera induced a 40% increase in larval mortality and a MicroRNAs (miRNAs) are a class of small (~22 nucleotide) non-cod- 70% reduction in fecundity, indicating the potential use of miRNAs as ing RNAs that regulate gene expression in animals, plants, and viruses. agents in integrated pest insect management programs (Jayachandran MiRNAs were originally discovered in Caenorhabditis elegans in 1993 et al., 2013). Further, ingestion of plant-derived miRNAs by aphids and (Lee et al., 1993), and thereafter, many more miRNAs were identified other phytophagous insects during feeding has the potential to modu- in other organisms, such as insects (Asgari and Sullivan, 2010; Asgari, late expression of insect genes, indicating that plants may produce 2013; Ge et al., 2013; Lucas and Raikhel, 2013) and plants miRNAs that negatively impact insect fitness. While the precise targets (Jones-Rhoades et al., 2006; Kettles et al., 2013). MiRNAs are known of these plant-produced miRNAs have yet to be identified in many to play important roles in the regulation of gene expression networks cases, evidence for the presence of plant miRNAs in animal small RNA (Shukla et al., 2008; Cai et al., 2009) and are involved in a variety of de- datasets, including those of the pea aphid (Acyrthosiphon pisum), have velopmental processes (Aravin et al., 2003; Chawla and Sokol, 2011; been previously reported (Zhang et al., 2012b), confirming that they are ingested during feeding. Additionally, other studies have revealed that exogenous plant-derived miRNA can impact expression of genes Abbreviations: GB, green bug; YSA, yellow sugar cane aphid; miRNAs, MicroRNAs. and proteins involved herbivore metabolism, revealing the importance ⁎ Corresponding author at: Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln, NE, 68583-0915, United States. of exogenous miRNAs in modulating herbivore-plant interactions and E-mail address: [email protected] (H. Wang). co-evolution (Vaucheret and Chupeau, 2012; Zhang et al., 2012a). A http://dx.doi.org/10.1016/j.gene.2016.11.014 0378-1119/© 2016 Elsevier B.V. All rights reserved. H. Wang et al. / Gene 599 (2017) 68–77 69 recent study (Guo et al., 2014) demonstrated that plant-generated arti- the manufacturer's instructions. The quality and quantity of the RNA ficial small RNAs produced by transgenic Nicotiana tabacum cv. Xanti samples were evaluated on 1% agarose gels and using a NanoDrop- (tobacco) could significantly reduce the expression of the central ner- 1000 spectrophotometer (Thermo Fisher Scientific). vous system enzyme acetylcholinesterase 2 (AchE2) in Myzus persicae Small RNAs were fractionated from the total RNAs on a 16% TBU gel (green peach aphid). Although these data suggest a putative role for (16% acrylamide and 42% urea in 0.5X TBE buffer) as described by Ren et miRNAs in mediating insect-plant interactions, the impacts of endoge- al. (2012).Briefly, 10 μg total RNA (in ~10 μl nuclease free water) was nous plant miRNAs expressed at physiological levels on herbivore mixed with 10 μl 2x formamide (98%) loading dye. Samples were heat- gene expression networks and the precise roles of many plant-derived ed at 65 °C for 5 min, placed on ice, and briefly centrifuged prior to load- miRNAs in regulating herbivore gene expression still remain unclear ing on the gel. An RNA ladder (Zymo Research small-RNA ladder Cat. at a functional level. R1090) was used to determine approximate sizes of the gel-separated The inventory of miRNAs expressed by insects is aided by the avail- RNA. Gels were stained with 1x TBE containing ethidium bromide for ability of annotated reference genomes (Asgari and Sullivan, 2010; 2 min or longer until bands of interest were visible. Gel regions corre- Asgari, 2013; Etebari and Asgari, 2014; Rebijith et al., 2014; Calla and sponding to an approximate size range between 17 and 30 nt were ex- Geib, 2015). However, miRNAs are generally conserved among closely cised. Small RNAs were extracted from the gel by crushing the gel pieces related insects (Willmann and Poethig, 2007; Nozawa et al., 2010), sug- in 200 μl 0.3 M NaCl followed by incubation overnight at 4 °C. The gel gesting that reference genomes from close relatives could be exploited mix was passed through Costar Spin-X 0.25 μm filters (Costa to annotate and predict miRNAs in insects lacking reference genomes. Cat.8163), and small RNAs in the supernatant were ethanol precipitated For example, Ge et al. (2013) were able to successfully identify 97 and and finally redissolved in 11 μl nuclease free water (Ambion Cat. 91 miRNAs in H. armigera and Spodoptera litura, respectively, using ge- AM9932).