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Ovary Transcriptome Profiling of Coilia Nasus During Spawning Migration

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Ovary Transcriptome Profiling of Coilia Nasus During Spawning Migration Marine Genomics 21 (2015) 17–19 Contents lists available at ScienceDirect Marine Genomics journal homepage: www.elsevier.com/locate/margen Genomics/technical resources Ovary transcriptome profiling of Coilia nasus during spawning migration stages by Illumina sequencing☆ Jin-Rong Duan b,Yan-FengZhoub,Dong-PoXub, Min-Ying Zhang b, Kai Liu b,YingShia, Qi-wei Wei a,⁎, Di-An Fang b,c,⁎⁎ a Key Lab of Freshwater Biodiversity Conservation Ministry of Agriculture, Yangtze River Fisheries Research Institute, CAFS, Wuhan 4302231, China b Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi, Shanshui Road 9, 214128, China c Wuxi Fisheries College, Nanjing Agricultural University, Wuxi, Xuejiali 69, 214128, China article info abstract Article history: Coilia nasus is an anadromous kind of small to moderate size fish species, and limited transcriptomics research Received 22 January 2015 has been performed. In the present study, we performed Illumina sequencing to produce a 22,996,612 clean Received in revised form 9 February 2015 reads representing with a total of 4,599,079,601 (4.5 Gb) nucleotides comprehensive transcript dataset for Accepted 9 February 2015 ovary of C. nasus. Over 20 million total reads were assembled into 63,141 unigenes, and 27,027 annotated Available online 27 February 2015 genes were predicted by Blastx and ESTScan, respectively. Applying Blast analysis and functional annotation (e.g., GO, COG, Swissprot and KEGG), we have sampled an extensive and diverse expressed gene catalog for Keywords: Coilia nasus C. nasus representing a large proportion of the genes expressed in the ovary development process. This approach Spawning migration will assist in the discovery and annotation of novel genes that play key roles in anadromous fish spawning migration Ovary transcriptome process. Illumina sequencing © 2015 Elsevier B.V. All rights reserved. 1. Introduction 2014b). Mechanism study of the ovary development and transcriptome information is identified as one of the most valid methods to resolve fish Coilia nasus is an important anadromous fishery resource in germplasm resources exhaustion (Bissonnette et al., 2009; Chalmel Clupeiformes, Engraulidae (Liu et al., 2014a; Yang et al., 2011). It runs et al., 2007). In this study we present a comprehensive analysis of the several kilometers up the rivers and spawns in the fresh water, and C. nasus ovary transcriptome and provided a general view of the poten- then the spherical eggs float down and hatch near the river bed (Jiang tial molecular mechanisms involving in female spawning migratory for et al., 2012; Zhong et al., 2007). Every year it migrates from the sea to this species. the middle lower reaches of the Yangtze River and its affiliated lakes in China (Li et al., 2010). C. nasus reaches sexual maturity at 2–3year- old age and lays eggs from April to October, spawning once every year (Cheng et al., 2008; Jiang et al., 2012). However, excessive fishing and changes in aquatic ecology have almost caused this species quantity de- cline sharply in the middle reaches of the Yangtze River (He et al., 2008; Zhong and Power, 1996). Nowadays the threat to C. nasus resources has not been alleviated (Liu et al., 2014a; Zhong and Power, 1996)although a number of research projects have been done involving in artificial spawning and larval rearing techniques in recent years (Liu et al., ☆ Ethics statement: All fish experimental procedures were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved and authorized by the State Council of People's Republic of China. ⁎ Corresponding author. Fax: +86 510 85390025. ⁎⁎ Correspondence to: D.-A. Fang, Wuxi Fisheries College, Nanjing Agricultural University, Wuxi, Xuejiali 69, 214128, China. Fax: +86 510 85390025. E-mail addresses: [email protected] (J.-R. Duan), [email protected] (Y.-F. Zhou), [email protected] (D.-P. Xu), [email protected] (M.-Y. Zhang), [email protected] (K. Liu), Fig. 1. Sampling distribution in the middle and lower reach of the Yangze River. Black shiy@yfi.ac.cn (Y. Shi), [email protected] (Q. Wei), [email protected], [email protected] dot display the sampling site. AQ: Anqing; DT: Dangtu; ZJ: Zhenjiang; JJ: Jingjiang; NT: (D.-A. Fang). Nantong; CM: Chongming, TH: Taihu lake; HZH: hongzehu lake; BYH: Boyanghu lake. http://dx.doi.org/10.1016/j.margen.2015.02.005 1874-7787/© 2015 Elsevier B.V. All rights reserved. 18 J.-R. Duan et al. / Marine Genomics 21 (2015) 17–19 Table 1 2.3. Ovary transcriptome assembling Summary of the annotated transcriptome. Name Total Data Total Read GC Content Q20 ratio Q30 ratio As shown in Table 1, Illumina high-throughput second generation sequencing produced 22,996,612 clean reads representing with a total Numbers 4,599,079,601 22,996,612 48.25% 90.46% 85.16% of 4,599,079,601 (4.5 Gb) nucleotides. Average read size, Q20 percent- Annotated databases All sequence ≥300 bp ≥1000 bp age, Q30 percentage and GC content were 98 bp, 90.46% 85.16% and COG 6347 2289 3218 48.25%, respectively. And 63,141 unigenes were obtained, with a medi- GO 18,110 7405 6433 an length of 700 bp, total 27,026 unigenes (42.8% of all unigenes) were KEGG 10,821 4354 3865 functionally annotated via EST scan analysis. 57.2% of the unigenes Swissprot 24,540 10,339 8396 could not be matched to known genes. Altogether we obtained an in- nr 26,624 11,321 8631 valuable resource for further studies of gene functions, protein products All 27,026 11,492 8658 and comparative genomics. 2. Data description 2.4. Functional unigene annotation 2.1. Fish collection Distinct gene sequences were first searched with Blastx against the NCBI nr, SwissProt, GO, COG, KEGG database (Conesa et al., 2005). A Healthy fish samples from six geographical populations (Anqing total of 63,141 gene ontology definitions were identified. The biological (AQ); Dangtu (DT); Zhenjiang (ZJ); Jingjiang (JJ); Nantong (NT), and processes class was the most highly represented (89.21%), followed Chongming (CM), seen in Fig. 1, total 48 individuals) were collected by molecular function (77.66%) and cellular component (35.19%; during the anadromous period (from March to July), respectively. All in- Table S2). Functional annotation using the COG database classified dividual ovaries were removed surgically and immediately stored at 8570 unigenes into 25 categories (Fig. 2). Among the total annotated −80 °C until used. Ovary tissues of different individuals were selected 27, 026 unigenes, 12,358 unigenes were associated with 234 predicted from different waters for RNA extraction and then all pooled as one KEGG metabolic pathways (Table S3). These predicted pathways are sample for the transcriptomics construction. likely to be useful in future investigations focusing on their functions in C. nasus spawning migration. In the ovary transcriptome of C. nasus described here, both gene 2.2. Sample preparation and Illumina sequencing annotation and pathway analysis helped predict potential genes and their likely specific roles at the whole transcriptome level. Enrich- Total RNA was extracted using Trizol Lysis Reagent and then purified ment analyses of GO functions and KEGG pathways lend support on RNA easy kit (Invitrogen, Beijing, China) as the manufacturer's in- to the biological significance of transcriptome profiles derived structions. Equal total RNA purified from each ovary was pooled and from short-read sequencing technology. This approach will assist then the mRNA was isolated using the Oligotex mRNA Kit (Invitrogen, in the discovery and annotation of novel genes that play key roles Beijing, China). The paired-end library was synthesized using the Geno- in the anadromous fish reproductive biology. mic Sample Prep kit (Illumina, Beijing, China). Short fragments were pu- rified and resolved with EB buffer for end reparation and adding of poly 2.5. Data deposition (A). After agarose gel electrophoresis, suitable fragments were selected as templates for PCR amplification. A mixed cDNA sample representing The raw sequence data from C. nasus ovaries were deposited in different anadromous stages undergoing rapid development of ovary the Sequence Read Archive (SRA) database with accession numbers tissues were prepared and sequenced using the Illumina HiSeq™ 2000 SUB799315. The full data set is also available from Di-an Fang on request sequencing technology. ([email protected]). Fig. 2. Clusters of orthologous groups (COG) classification of consensus sequence. J.-R. Duan et al. / Marine Genomics 21 (2015) 17–19 19 Supplementary data to this article can be found online at http://dx. References doi.org/10.1016/j.margen.2015.02.005. Bissonnette, N., Lévesque-Sergerie, J.-P., Thibault, C., Boissonneault, G., 2009. Spermatozo- al transcriptome profiling for bull sperm motility: a potential tool to evaluate semen quality. Reproduction 138, 65–80. Competing interests Chalmel, F., Rolland, A.D., Niederhauser-Wiederkehr, C., Chung, S.S.W., Demougin, P., et al., 2007. The conserved transcriptome in human and rodent male gametogenesis. Proc. Natl. Acad. Sci. 104, 8346–8351. The authors declare that they have no competing interests. Cheng, Q., Cheng, H., Wang, L., Zhong, Y., Lu, D., 2008. A preliminary genetic distinctness of four Coilia fishes (Clupeiformes: Engraulidae) inferred from mitochondrial DNA se- quences. Russ. J. Genet. 44, 339–343. Conesa, A., Götz, S., García-Gómez, J.M., Terol, J., Talón, M., et al., 2005. Blast2GO: a univer- Authors' contributions sal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, 3674–3676. Di-An Fang was responsible for data mining and writing the manu- He, W., Li, Z., Liu, J., Li, Y., Murphy, B.R., et al., 2008. Validation of a method of estimating age, modelling growth, and describing the age composition of Coilia mystus from the script. Jin-Rong Duan, Yan-Feng Zhou, Dong-Po Xu, Min-Ying Zhang Yangtze Estuary, China.
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