Xác định, đặc trưng và phân tích chức năng của các RNA không mã hóa dài ở cơ quan sinh dục trong một loài cá lưỡng tính chuyển giới protogynous, cá chình lúa (Monopterus albus)

Springer Science and Business Media LLC - Tập 23 - Trang 1-17 - 2022
Zhi He1, Lijuan Ye1, Deying Yang1, Zhijun Ma1, Faqiang Deng1, Zhide He2, Jiaxiang Hu3, Hongjun Chen1, Li Zheng1, Yong Pu1, Yuanyuan Jiao1, Qiqi Chen1, Kuo Gao1, Jinxin Xiong1, Bolin Lai1, Xiaobin Gu4, Xiaoli Huang1, Shiyong Yang1, Mingwang Zhang1, Taiming Yan1
1College of Animal Science and Technology, Sichuan Agricultural University, Chengdu, China
2Luzhou Municipal Bureau of Agriculture and Rural Affairs, Luzhou, China
3Sichuan Water Conservancy Vocational College, Chengdu, China
4College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China

Tóm tắt

Số lượng ngày càng tăng của RNA không mã hóa dài (lncRNA) đã được phát hiện có vai trò quan trọng trong việc phân biệt giới tính và phát triển tuyến sinh dục bằng cách điều chỉnh biểu hiện gen ở cấp độ di truyền biểu sinh, phiên mã và sau phiên mã. Cá chình lúa, Monopterus albus, là một loài cá lưỡng tính chuyển giới protogynous, trải qua một quá trình chuyển đổi giới tính tuần tự từ cái sang đực. Tuy nhiên, vai trò của lncRNA trong quá trình chuyển giới vẫn chưa rõ ràng. Trong nghiên cứu này, chúng tôi đã thực hiện giải trình tự RNA để phân tích các kiểu biểu hiện lncRNA ở năm giai đoạn phát triển khác nhau của M. albus nhằm nghiên cứu vai trò của lncRNAs trong quá trình chuyển giới. Tổng cộng đã xác định 12.746 lncRNAs (1.503 lncRNAs đã biết và 11.243 lncRNAs mới) và 2.901 lncRNAs được biểu hiện khác nhau (DE-lncRNAs) trong các tuyến sinh dục. Các gen mục tiêu của DE-lncRNAs bao gồm foxo1, foxm1, smad3, foxr1, camk4, ar và tgfb3, chủ yếu được làm phong phú trong các con đường tín hiệu liên quan đến phát triển sinh dục như con đường tín hiệu insulin, con đường tín hiệu MAPK, và con đường tín hiệu canxi. Chúng tôi đã chọn 5 DE-lncRNAs được biểu hiện cao (LOC109952131, LOC109953466, LOC109954337, LOC109954360 và LOC109958454) để khuếch đại chiều dài đầy đủ và xác minh mẫu biểu hiện. Tất cả đều được biểu hiện ở mức cao hơn trong buồng trứng và tuyến sinh dục lưỡng tính so với tinh hoàn, và thể hiện biểu hiện phụ thuộc vào thời gian cụ thể trong mô buồng trứng được ương với hormone kích thích nang trứng (FSH) và gonadotropin huyết thanh người (hCG). Kết quả của phân tích PCR theo thời gian thực định lượng (qRT-PCR) và thử nghiệm dual-luciferase cho thấy rằng znf207, như là gen mục tiêu của LOC109958454, được biểu hiện trong nhiều mô và giai đoạn phát triển sinh dục của M. albus, và biểu hiện của nó cũng bị ức chế bởi hormone FSH và hCG. Những kết quả này cung cấp cái nhìn mới về vai trò của lncRNAs trong sự phát triển tuyến sinh dục, đặc biệt liên quan đến sự chuyển đổi giới tính tự nhiên ở cá, điều này sẽ hữu ích cho việc nâng cao hiểu biết của chúng tôi về lưỡng tính tuần tự và sự chuyển giới ở cá chình lúa (M. albus) và các loài cá xương khác.

Từ khóa

#RNA không mã hóa dài #phát triển sinh dục #cá chình lúa #chuyển giới giới tính #lưỡng tính

Tài liệu tham khảo

Novikova IV, Hennelly SP, Tung CS, Sanbonmatsu KY. Rise of the RNA machines: exploring the structure of long non-coding RNAs. J Mol Biol. 2013;425(19):3731–46. https://doi.org/10.1016/j.jmb.2013.02.030. Ulitsky I, Bartel DP. lincRNAs: genomics, evolution, and mechanisms. Cell. 2013;154(1):26–46. https://doi.org/10.1016/j.cell.2013.06.020. Derrien T, Johnson R, Bussotti G, Tanzer A, Guigó R. The GENCODE v7 catalog of human long noncoding RNAs: analysis of their gene structure, evolution, and expression. Genome Res. 2012;22(9):1775–89. https://doi.org/10.1101/gr.132159.111. Guo CJ, Ma XK, Xing YH, Zheng CC, Xu YF, Shan L, et al. Distinct processing of lncRNAs contributes to non-conserved functions in stem cells. Cell. 2020;181(3):621–36. https://doi.org/10.1016/j.cell.2020.03.006. Tian B, Manley JL. Alternative polyadenylation of mRNA precursors. Nat Rev Mol Cell Biol. 2016;18(1):18–30. https://doi.org/10.1038/nrm.2016.116. Csorba T, Questa JI, Sun Q, Dean C. Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization. Proc Natl Acad Sci. 2014;111(45):16160–5. https://doi.org/10.1073/pnas.1419030111. Rosa S, Duncan S, Dean C. Mutually exclusive sense-antisense transcription at FLC facilitates environmentally induced gene repression. Nat Commun. 2016;7:13031. https://doi.org/10.1038/ncomms13031. Yap KL, Li S, Cabello AMM, Raguz S, Zeng L, Mujtaba S, et al. Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a. Mol Cell. 2010;38(5):662–74. https://doi.org/10.1016/j.molcel.2010.03.021. Holdt LM, Hoffmann S, Sass K, Langenberger D, Scholz M, Krohn K, et al. Alu elements in ANRIL non-coding RNA at chromosome 9p21 modulate atherogenic cell functions through trans-regulation of gene networks. PLoS Genet. 2013;9(7):e1003588. https://doi.org/10.1371/journal.pgen.1003588. Song FB, Wang LM, Zhu WB, Dong ZJ. Long noncoding RNA and mRNA expression profiles following igf3 knockdown in common carp, Cyprinus carpio. Sci Data. 2019;6:190024. https://doi.org/10.1038/sdata.2019.24. van Werven FJ, Neuert G, Hendrick N, Lardenois A, Buratowski S, van Oudenaarden A, et al. Transcription of two long noncoding RNAs mediates mating-type control of gametogenesis in budding yeast. Cell. 2012;150(6):1170–81. https://doi.org/10.1016/j.cell.2012.06.049. Herrera L, Ottolenghi C, Garcia-Ortiz JE, Pellegrini M, Manini F, Ko MSH, et al. Mouse ovary developmental RNA and protein markers from gene expression profiling. Dev Biol. 2005;279(2):271–90. https://doi.org/10.1016/j.ydbio.2004.11.029. Laiho A, Kotaja N, Gyenesei A, Sironen A. Transcriptome profiling of the murine testis during the first wave of spermatogenesis. PLoS One. 2013;8(4):e61558. https://doi.org/10.1371/journal.pone.0061558. Zhu YY, Liu XL, Wang YK, Li W, Hong XY, Zhu XP, et al. Screening and preliminary analysis of lncRNA and mRNA related to sex regulation in yellow-throated turtle (Mauremys mutica). J Fish China. 2020;44(12):1960–75 (in chinese). https://doi.org/10.11964/jfc.20200312187. Rastetter RH, Smith CA, Wilhelm D. The role of non-coding RNAs in male sex determination and differentiation. Reproduction. 2015;150(3):R93–107. https://doi.org/10.1530/REP-15-0106. Song XH, Kyi-Tha-Thu C, Takizawa T, Naing BT, Takizawa T. 1700108J01Rik and 1700101O22Rik are mouse testis-specific long non-coding RNAs. Histochem Cell Biol. 2018;149(5):517–27. https://doi.org/10.1007/s00418-018-1642-4. Mao HG, Xu XL, Cao HY, Dong XY, Zou XT, Xu NY, et al. Comparative transcriptome profiling of mRNA and lncRNA of ovaries in high and low egg production performance in domestic pigeons (Columba livia). Front Genet. 2021;12:571325. https://doi.org/10.3389/fgene.2021.571325. Guo SY, Zhong Y, Zhang Y, Zhu YF, Guo J, Fu YS, et al. Transcriptome analysis provides insights into long noncoding RNAs in medaka gonads. Comp Biochem Physiol part D genomics. Proteomics. 2021;100842. https://doi.org/10.1016/j.cbd.2021.100842. Ma X, Cen SS, Wang LM, Zhang C, Wu LM, Tian X, et al. Genome-wide identification and comparison of differentially expressed profiles of miRNAs and lncRNAs with associated ceRNA networks in the gonads of Chinese soft-shelled turtle, Pelodiscus sinensis. BMC Genomics. 2020;21(1):443. https://doi.org/10.1186/s12864-020-06826-1. Cai J, Li L, Song L, Xie L, Luo F, Sun S, et al. Effects of long term antiprogestine mifepristone (RU486) exposure on sexually dimorphic lncRNA expression and gonadal masculinization in Nile tilapia (Oreochromis niloticus). Aquat Toxicol. 2019;215:105289. https://doi.org/10.1016/j.aquatox.2019.105289. Shapiro DY. Plasticity of gonadal development and protandry in fishes. J Exp Zool. 1992;261(2):194–203. https://doi.org/10.1002/jez.1402610210. Cheng HH, He Y, Zhou RJ. Swamp eel (Monopterus albus). Trends Genet. 2021;37(12):1137–8. https://doi.org/10.1016/j.tig.2021.09.007. Cheng H, Guo Y, Yu Q, Zhou R. The rice field eel as a model system for vertebrate sexual development. Cytogenet Genome Res. 2003;101(3-4):274–7. https://doi.org/10.1159/000074348. He Z, Deng FQ, Xiong S, Cai YP, He ZD, Wang XY, et al. Expression and regulation of smad2 by gonadotropins in the protogynous hermaphroditic ricefield eel (Monopterus albus). Fish Physiol Biochem. 2020;46(3):1155–65. https://doi.org/10.1007/s10695-020-00778-9. Wang Z, Yang Y, Li S, Li K, Tang Z. Analysis and comparison of long non-coding RNAs expressed in the ovaries of Meishan and Yorkshire pigs. Anim Genet. 2019;50(6):660–9. https://doi.org/10.1111/age.12849. Liu Y, Li MX, Bo XW, Li T, Ma LP, Zhai TJ, et al. Systematic analysis of long non-coding RNAs and mRNAs in the ovaries of duroc pigs during different follicular stages using RNA sequencing. Int J Mol Sci. 2018;19(6):1722. https://doi.org/10.3390/ijms19061722. Liu Y, Qi B, Xie J, Wu XQ, Ling YH, Cao XY, et al. Filtered reproductive long non-coding RNAs by genome-wide analyses of goat ovary at different estrus periods. BMC Genomics. 2018;19(1):866. https://doi.org/10.1186/s12864-018-5268-7. Yang H, Wang F, Li FZ, Ren CF, Pang J, Wan YJ, et al. Comprehensive analysis of long non-coding RNA and mRNA expression patterns in sheep testicular maturation. Biol Reprod. 2018;99(3):650–61. https://doi.org/10.1093/biolre/ioy088. Fatica A, Bozzoni I. Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet. 2014;15(1):7–21. https://doi.org/10.1038/nrg3606. Taylor DH, Chu TE, Spektor R, Soloway PD. Long non-coding RNA regulation of reproduction and development. Mol Reprod Dev. 2015;82(12):932–56. https://doi.org/10.1002/mrd.22581. Mercer TR, Dinger ME, Mattick JS. Long non-coding RNAs: insights into functions. Nat Rev Genet. 2009;10(3):155–9. https://doi.org/10.1038/nrg2521. Qin L, Huang CC, Yan XM, Wang Y, Li ZY, Wei XC. Long non-coding RNA H19 is associated with polycystic ovary syndrome in Chinese women: a preliminary study. Endocr J. 2019;66(7):587–95. https://doi.org/10.1507/endocrj.EJ19-0004. Li K, Zhong SS, Luo YS, Zou DF, Li MZ, Li YH, et al. A long noncoding RNA binding to QKI-5 regulates germ cell apoptosis via p38 MAPK signaling pathway. Cell Death Dis. 2019;10(10):699–715. https://doi.org/10.1038/s41419-019-1941-2. Zhang J, Yu P, Zhou QY, Li XL, Ding SQ, Su SP, et al. Screening and characterisation of sex differentiation-related long non-coding RNAs in Chinese soft-shell turtle (Pelodiscus sinensis). Sci Rep. 2018;8(1):8630–9. https://doi.org/10.1038/s41598-018-26841-3. Yang XL, Ikhwanuddin M, Li XC, Lin F, Wu QY, Zhang YL, et al. Comparative transcriptome analysis provides insights into differentially expressed genes and Long non-coding RNAs between ovary and testis of the mud crab (Scylla paramamosain). Mar Biotechnol (NY). 2018;20(1):20–34. https://doi.org/10.1007/s10126-017-9784-2. Yuan CC, Chen KR, Zhu YF, Yuan YM, Li MY. Medaka igf1 identifies somatic cells and meiotic germ cells of both sexes. Gene. 2018;642:423–9. https://doi.org/10.1016/j.gene.2017.11.037. Kanai Y, Hiramatsu R, Matoba S, Kidokoro T. From SRY to SOX9: mammalian testis differentiation. J Biochem. 2005;1:13–9. https://doi.org/10.1093/jb/mvi098. He Z, Wu YS, Xie J, Wang TX, Zhang LH, Zhang WM. Growth differentiation factor 9 (Gdf9) was localized in the female as well as male germ cells in a protogynous hermaphroditic teleost fish, ricefield eel Monopterus albus. Gen Comp Endocrinol. 2012;178(2):355–62. https://doi.org/10.1016/j.ygcen.2012.06.016. Yuan J, Tao WJ, Cheng YY, Huang BF, Wang DS. Genome-wide identification, phylogeny, and gonadal expression of fox genes in Nile tilapia, Oreochromis niloticus. Fish Physiol Biochem. 2014;40(4):1239–52. https://doi.org/10.1007/s10695-014-9919-6. Lau MT, Ge W. Cloning of smad2, smad3, smad4, and smad7 from the goldfish pituitary and evidence for their involvement in activin regulation of goldfish FSHbeta promoter activity. Gen Comp Endocrinol. 2005;141(1):22–38. https://doi.org/10.1016/j.ygcen.2004.10.019. Wu JY, Ribar TJ, Cummings DE, Burton KA, Means AR. Spermiogenesis and exchange of basic nuclear proteins are impaired in male germ cells lacking Camk4. Nat Genet. 2000;25(4):448–52. https://doi.org/10.1038/78153. Wu JY, Gonzalez-Robayna IJ, Richards JS, Means AR. Female fertility is reduced in mice lacking Ca2+/calmodulin-dependent protein kinase IV. Endocrinology. 2000;141(12):4777–83. https://doi.org/10.1210/en.141.12.4777. Zhao ZQ, Qiao L, Dai ZN, He QJ, Lan X, Huang SY, et al. LncNONO-AS regulates AR expression by mediating NONO. Theriogenology. 2020;145:198–206. https://doi.org/10.1016/j.stheriogenology.2019.10.025. O'Hara L, Smith LB. Androgen receptor roles in spermatogenesis and infertility. Best Pract Res. 2015;29(4):595–605. https://doi.org/10.1016/j.beem.2015.04.006. Droździk M, Kaczmarek M, Malinowski D, Broś U, Kazienko A, Kurzawa R, et al. TGFβ3 (TGFB3) polymorphism is associated with male infertility. Sci Rep. 2015;5:17151. https://doi.org/10.1038/srep17151. Jackowska M, Kempisty B, Woźna M, Piotrowska H, Antosik P, Zawierucha P, et al. Differential expression of GDF9, TGFB1, TGFB2 and TGFB3 in porcine oocytes isolated from follicles of different size before and after culture in vitro. Acta Vet Hung. 2013;61(1):99–115. https://doi.org/10.1556/AVet.2012.061. Yang J, Zhang Y, Xu XT, Li J, Yuan FF, Bo SM, et al. Transforming growth factor-β is involved in maintaining oocyte meiotic arrest by promoting natriuretic peptide type C expression in mouse granulosa cells. Cell Death Dis. 2019;10(8):558. https://doi.org/10.1038/s41419-019-1797-5. Long XR, Song KQ, Hu H, Tian Q, Wang WJ, Dong Q, et al. Long non-coding RNA GAS5 inhibits DDP-resistance and tumor progression of epithelial ovarian cancer via GAS5-E2F4-PARP1-MAPK axis. J Exp Clin Cancer Res. 2019;38(1):345–60. https://doi.org/10.1186/s13046-019-1329-2. Zhou H, Chen A, Lu WQ. Corticotropin-releasing hormone reduces basal estradiol production in zebrafish follicular cells. Mol Cell Endocrinol. 2021;527(3):111222. https://doi.org/10.1016/j.mce.2021.111222. Xu KK, Yang WJ, Tian Y, Wu YB, Wang JJ. Insulin signaling pathway in the oriental fruit fly: the role of insulin receptor substrate in ovarian development. Gen Comp Endocrinol. 2014;216:125–33. https://doi.org/10.1016/j.ygcen.2014.11.022. Abdou HS, Gabrielle V, Tremblay JJ. The calcium signaling pathway regulates Leydig cell steroidogenesis through a transcriptional Cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein. Endocrinology. 2013;154(1):511–20. https://doi.org/10.1210/en.2012-1767. Lefèvre B, Nagyova E, Pesty A, Testart J. Acquisition of Meiotic Competence is Related to the functionality of the phosphoinositide/calcium signaling pathway in the mouse oocyte. Exp Cell Res. 1997;236(1):193–200. https://doi.org/10.1006/excr.1997.3720. He Z, Deng FQ, Yang DY, He ZD, Hu JX, Ma ZJ, et al. Crosstalk between sex-related genes and apoptosis signaling reveals molecular insights into sex change in a protogynous hermaphroditic teleost fish, ricefield eel Monopterus albus. Aquaculture. 2022;552:737918. https://doi.org/10.1016/j.aquaculture.2022.737918. Mizuta H, Mushirobira Y, Nagata J, Todo T, Hara A, Reading BJ, et al. Ovarian expression and localization of clathrin (Cltc) components in cutthroat trout, Oncorhynchus clarki: evidence for Cltc involvement in endocytosis of vitellogenin during oocyte growth. Comp Biochem Physiol A Mol Integr Physiol. 2017;212:24–34. https://doi.org/10.1016/j.cbpa.2017.06.021. Morini M, Lafont AG, Maugars G, Baloche S, Dufour S, Asturiano JF, et al. Identification and stable expression of vitellogenin receptor through vitellogenesis in the European eel. animal. 2020;1-10. https://doi.org/10.1017/S1751731119003355. Lubzens E, Young G, Bobe J, Cerdà J. Oogenesis in teleosts: how eggs are formed. Gen Comp Endocrinol. 2010;165(3):367–89. https://doi.org/10.1016/j.ygcen.2009.05.022. Schulz RW, França LRD, Lareyre JJ, Gac FL, Chiarini-Garcia H, Nobrega RH, et al. Spermatogenesis in fish. Gen Comp Endocrinol. 2010;165(3):390–411. https://doi.org/10.1016/j.ygcen.2009.02.013. Wu YS, He Z, Zhang LH, Jiang H, Zhang WM. Ontogeny of immunoreactive Lh and Fsh cells in relation to early ovarian differentiation and development in protogynous hermaphroditic ricefield eel Monopterus albus. Biol Reprod. 2012;86(3):93. https://doi.org/10.1095/biolreprod.111.095646. Putra W, Mulah A. Combination effects human chorionic gonadotropin hormon and ovaprim distribution on the time latency, percentage of fertilization, hatching and survival of silver pompano (Trachinotus blochii) larve fish. IOP Conf Ser: Earth Environ Sci. 2019;278(1):012063. https://doi.org/10.1088/1755-1315/278/1/012063. Ohta H, Tanaka H. Relationship between serum levels of human chorionic gonadotropin (hCG) and 11-ketotestosterone after a single injection of hCG and induced maturity in the male Japanese eel, Anguilla japonica. Aquaculture. 1997;153(1-2):123–34. https://doi.org/10.1016/S0044-8486(97)00020-3. Yang H, Ma JY, Wang ZB, Yao XL, Zhao J, Zhao XY, et al. Genome-wide analysis and function prediction of Long noncoding RNAs in sheep pituitary gland associated with sexual maturation. Genes (Basel). 2020;11(3):320. https://doi.org/10.3390/genes11030320. Heidi K, Marion A, Jenni MJ, Pauliina D, Damdimopoulos AE, Juha K, et al. The Hydroxysteroid (17β) dehydrogenase family gene HSD17B12 is involved in the prostaglandin synthesis pathway, the ovarian function, and regulation of fertility. Endocrinology. 2016;10:3719–30. https://doi.org/10.1210/en.2016-1252. Yung Y, Ophir L, Yerushalmi GM, Baum M, Hourvitz A, Maman E. HAS2-AS1 is a novel LH/hCG target gene regulating HAS2 expression and enhancing cumulus cells migration. J Ovarian Res. 2019;12(1):21. https://doi.org/10.1186/s13048-019-0495-3. Liu Y, Ao X, Jia Z, Bai XY, Xu Z, Hu G, et al. FOXK2 transcription factor suppresses ERα-positive breast Cancer cell growth through Down-regulating the stability of ERα via mechanism involving BRCA1/BARD1. Sci Rep. 2015;5(4):8796. https://doi.org/10.1038/srep08796. Thepsuwan T, Rungrassamee W, Sangket U, Whankaew S, Sathapondecha P. Long non-coding RNA profile in banana shrimp, Fenneropenaeus merguiensis and the potential role of lncPV13 in vitellogenesis. Comp Biochem Physiol A. 2021. https://doi.org/10.1016/j.cbpa.2021.111045. Cassandri M, Smirnov A, Novelli F, Pitolli C, Agostini M, Malewicz M, et al. Zinc-finger proteins in health and disease. Cell Death Dis. 2017;3:17071. https://doi.org/10.1038/cddiscovery.2017.71. Hajikhezri Z, Darweesh M, Akusjrvi G, Punga T. Role of CCCH-type zinc finger proteins in human adenovirus infections. Viruses. 2020;12(11):1322. https://doi.org/10.3390/v12111322. Sun QY, Hao QY, Prasant KV. Nuclear long noncoding RNAs: key regulators of gene expression. Trends Genet. 2018;34(2):142–57. https://doi.org/10.1016/j.tig.2017.11.005. Wan YH, Zheng XB, Chen HY, Guo YX, Jiang H, He XN, et al. Splicing function of mitotic regulators links R-loop–mediated DNA damage to tumor cell killing. J Cell Biol. 2015;209(2):235–46. https://doi.org/10.1083/jcb.201409073. Liu C, Banister CE, Buckhaults PJ. Spindle assembly checkpoint inhibition can Resensitize p53-null stem cells to Cancer chemotherapy. Cancer Res. 2019;79(1):2392–403. https://doi.org/10.1158/0008-5472.CAN-18-3024. Zhang Y, Zhang WM, Yang HY, Zhou WL, Hu CQ, Zhang LH. Two cytochrome P450 aromatase genes in the hermaphrodite ricefield eel Monopterus albus: mRNA expression during ovarian development and sex change. J Endocrinol. 2008;199(2):317–31. https://doi.org/10.1677/JOE-08-0303. Ghosh S, Chan CK. Analysis of RNA-seq data using topHat and cufflinks. Methods Mol Biol. 2016;1374:339–61. https://doi.org/10.1007/978-1-4939-3167-5_18. Kong L, Zhang Y, Ye ZQ, Liu XQ, Zhao SQ, Wei L, et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res. 2007;35:W345–9. https://doi.org/10.1093/nar/gkm391. Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL. Transcript-level expression analysis of RNA-seq experiments with HISAT. StringTie and Ballgown Nat Protoc. 2016;11(9):1650–67. https://doi.org/10.1038/nprot.2016.095. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11(2):R14. https://doi.org/10.1186/gb-2010-11-2-r14. Mao XZ, Cai T, Olyarchuk JG, Wei LP. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics. 2005;21(19):3787–93. https://doi.org/10.1093/bioinformatics/bti430. Hu Q, Guo W, Gao Y, Tang R, Li DP. Reference gene selection for real-time RT-PCR normalization in rice field eel (Monopterus albus) during gonad development. Fish Physiol Biochem. 2014;40(6):1721–30. https://doi.org/10.1007/s10695-014-9962-3.