RNA-Seq of Rice Yellow Stem Borer, Scirpophaga Incertulas Reveals Molecular Insights During Four Larval Developmental Stages
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G3: Genes|Genomes|Genetics Early Online, published on July 21, 2017 as doi:10.1534/g3.117.043737 1 RNA-seq of Rice Yellow Stem Borer, Scirpophaga incertulas reveals molecular insights 2 during four larval developmental stages 3 4 Renuka. P,* Maganti. S. Madhav,*.1 Padmakumari. A. P,† Kalyani. M. Barbadikar,* Satendra. K. 5 Mangrauthia,* Vijaya Sudhakara Rao. K,* Soma S. Marla‡, and Ravindra Babu. V§ 6 *Department of Biotechnology, ICAR-Indian Institute of Rice Research, Hyderabad, India, 7 †Department of Entomology, ICAR- Indian Institute of Rice Research, Hyderabad, India, 8 ‡Division of Genomic Resources, ICAR-National Bureau of plant Genomic Resources, New 9 Delhi, India, 10 §Department of Plant Breeding, ICAR- Indian Institute of Rice Research, Hyderabad, India. 11 1Corresponding author 12 Correspondence: 13 Maganti Sheshu Madhav, 14 Principal Scientist 15 Department of Biotechnology, 16 Crop Improvement Section, 17 ICAR-Indian Institute of Rice Research, 18 Rajendranagar, Hyderabad 19 Telangana State-500030, India. 20 Telephone-+91-40-24591208 21 FAX-+91-40-24591217 22 E-mail: [email protected] 23 1 © The Author(s) 2013. Published by the Genetics Society of America. 24 ABSTRACT 25 The yellow stem borer (YSB), Scirpophaga incertulas is a prominent pest in the rice cultivation 26 causing serious yield losses. The larval stage is an important stage in YSB, responsible for 27 maximum infestation. However, limited knowledge exists on biology and mechanisms 28 underlying growth and differentiation of YSB. To understand and identify the genes involved in 29 YSB development and infestation, so as to design pest control strategies, we performed de novo 30 transcriptome at 1st, 3rd, 5th and 7th larval developmental stages employing Illumina Hi-seq. High 31 quality reads of ~229 Mb were assembled into 24,775 transcripts with an average size of 1485bp. 32 Genes associated with various metabolic processes i.e. detoxification mechanism (CYP450, 33 GSTs and CarEs), RNAi machinery (Dcr-1, Dcr-2, Ago-1, Ago-2, Sid-1, Sid-2, Sid-3 and sid-1 34 related gene), chemoreception (CSPs, GRs, OBPs and ORs) and regulators (TFs and hormones) 35 were differentially regulated during the developmental stages. Identification of stage specific 36 transcripts made possible to determine the essential processes of larval development. 37 Comparative transcriptome analysis revealed that YSB has not much evolved in detoxification 38 mechanism but showed presence of distinct RNAi machinery. Presence of strong specific visual 39 recognition coupled with chemosensory mechanisms supports the monophagous nature of YSB. 40 Designed EST-SSRs will facilitate accurate estimation of genetic diversity of YSB. This is the 41 first report on characterization of YSB transcriptome and identification of genes involved in key 42 processes which will help researchers and industry to devise novel pest control strategies. This 43 study opens a new avenue to develop next generation resistant rice using RNAi or genome 44 editing approaches. 45 46 2 47 Key words: 48 Insect, Scirpophaga incertulas, de novo transcriptome, RNAi, growth and development, 49 detoxification mechanism 50 INTRODUCTION 51 Rice Yellow Stem Borer (YSB), Scirpophaga incertulas (Walker) (Lepidoptera: Crambidae) is 52 the most destructive insect pest of rice found in diverse ecosystems across the world. It has been 53 reported to be the dominant pest in Asia (Listinger 1979), South East Asia (Banerjee and 54 Pramanik 1967) and India in particular (Chelliah et al. 1989). It is predominantly a 55 monophagous pest and there are no reports that it can successfully complete its life cycle on any 56 other plant outside the species, Oryza (Pathak and Khan 1994). YSB attacks the rice plant from 57 seedling to maturity stage resulting into formation of dead hearts and white ears. Yield losses due 58 to YSB damage are significant and one per cent each of dead hearts and white ears led to 2.5 and 59 4.0 per cent yield loss respectively (Muralidharan and Pasalu 2006). Application of chemical 60 insecticides at seedling and reproductive stages of rice is an effective and therefore, widely 61 adopted practice in the management of YSB. However, continuous use of pesticides causes 62 health and environmental hazards (Rath 2001). Hence, there is a need to search for alternative 63 strategies to combat this deadly pest. To control YSB, it is essential to understand its 64 biochemical and molecular mechanisms associated with its life cycle. Process of exploring pest 65 genomic information for designing control strategies has been progressive, but limited to only 66 some pests. Genome-wide expression of genes during the different developmental stages of 67 insect will provide a molecular insight on life cycle. This would eventually aid in identifying the 68 key insect transcripts involved in infestation and growth which can be targeted for developing 69 resistance against this pest (Kola et al. 2015). 3 70 With the advent of next generation sequencing platforms, providing biological 71 perspectives to the cellular processes have become a reality. Transcriptome analysis through 72 RNA-seq can be utilized efficiently to identify the temporal and unique gene expression 73 patterns in an organism (Ozsolak and Milos 2011). Specifically, it provides novel opportunities 74 for expression studies in organisms lacking genome or transcriptome sequence information 75 (Haas and Zody 2010; Wolf 2013). RNA-seq has been used immensely in insect pests for 76 revealing the biological phenomenon (Ou et al. 2014), gene expression profiles and gene 77 discovery (Vogel et al. 2014). It has also been used for identifying RNAi targets to develop pest 78 control strategies (Wang et al. 2011). However, developmental transcriptome studies in insect 79 pests have provided better understanding of the transition phases in insect life cycle. The RNA- 80 seq data also offers great opportunity to develop genomic resources viz., the expressed 81 sequenced tags (EST)-single sequence repeats (SSRs) leveraged by the function of the 82 transcripts. Such functional markers act as a repository of genomic resources for the insect 83 species (Stolle et al. 2013). The larval stage is the important developmental stage in YSB, being 84 responsible for economic damage. In the present study, with an aim to understand and identify 85 the temporal and unique gene expression patterns during the larval stages in YSB, we 86 performed RNA-seq at 1st, 3rd, 5th, 7th instars. A total of 236 million reads were generated and de 87 novo assembled into 24,775 transcripts of YSB. The differentially expressed transcripts were 88 analyzed by comparing gene expression profiles and further validated using qRT-PCR. We also 89 performed comparative transcriptome analysis with related insect pest species for better 90 understanding of behavioral and evolutionary aspects of YSB. Functional EST-SSR markers 91 were also developed for YSB, which will serve as a rich genetic resource for diversity studies. 92 These findings would facilitate the molecular studies in understanding the biology of YSB and 4 93 related insects. The YSB specific functionally important transcripts can be used as target genes 94 for RNAi to develop YSB resistance in rice. 95 MATERIALS AND METHODS 96 Insect rearing and sample collection 97 The YSB adults were collected from Indian Council of Agricultural Research-Indian 98 Institute of Rice Research ICAR-IIRR paddy fields in wet season 2013. The insects were 99 released on rice plants (TN1variety) maintained under controlled conditions at 25±2 0C 100 temperature and 80 per cent relative humidity for oviposition. Egg masses laid on the leaf blades 101 were collected and placed in glass vials for hatching. Neonate larvae were reared on cut stems of st rd th th 102 rice plants (TN1variety). Different larval instars viz., 1 , 3 , 5 and 7 (L1, L3, L5 and L7 103 respectively) were identified on the basis of mandibular width and visual observation of exuvae 104 (Padmakumari et al. 2013). Each larval instar was collected at 2-4 h after moulting in three 105 replications and stored in RNA later (Invitrogen, USA) at -80 0C for total RNA isolation. 106 RNA sequencing 107 Total RNA was isolated using the SV total RNA isolation system (Promega, USA) as per 108 manufacturer’s protocol. The RNA was pooled from three replicates of each instar. The yield and 109 purity of RNA were assessed by measuring the absorbance at 260 and 280 nm and quality was 110 checked on formaldehyde gel using MOPS buffer. The RNA integrity number (RIN) was 111 checked by running the RNA Nano chip on Bioanalyzer (Agilent technologies, USA) and 112 samples with RIN value more than 7.5 were processed further. As required for Illumina 113 sequencing, mRNA was isolated from 5 μg of total RNA and pair end (2×100 bp) cDNA 5 114 sequencing libraries were prepared using Illumina TruSeq® RNA Library Preparation Kit as per 115 manufacturer's protocol (Illumina®, San Diego, CA) and sequenced on HiSeq 2000 sequencing 116 system at Nucleome Informatics Ltd., Hyderabad, India. The schematic representation of YSB de 117 novo transcriptome analysis is given in Figure 1. 118 Pre-processing of raw data and read mapping 119 The YSB raw reads were filtered to obtain high-quality (HQ) reads by removing low- 120 quality reads and adaptors using Trimmomatic v0.32 with default parameters. The resulting HQ 121 reads from all the four samples were assembled using Trinity v2.0.0 with default parameters 122 (http://trinityrnaseq.sourceforge.net/, Haas et al. 2013). The redundancy of the assembly was 123 reduced through processing by Cd-hit (Li et al. 2006), TGICL (Pertea et al. 2003) and Evigene 124 (http://arthropods.eugenes.org /Evidential Gene/about/Evidential Gene_trassembly _pipe.html). 125 The de novo assembled transcripts were named as YSB_LS as prefixed by the number emanated 126 from Trinity assembler.