Screening and Functional Prediction of Differential miRNAs Associated with Broodiness in Black Muscovy Duck (Cairna moschata)
LI Li1,2, ZHANG Lin-Li2, Nemat O. KEYHANI3, XIN Qing-Wu2, MIAO Zhong-Wei2, ZHU Zhi-Ming2, QIU Jun-Zhi1,*, ZHENG Nen-Zhu2,*
1 College of Life Science, Fujian Agriculture and Forestry University, Fuzhou 363000, China; 2 Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China; 3 Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA
Abstract：The black Muscovy duck (Cairna moschata) is excellent lean meat ducks that is drought-tolerant and rough-fed, which has important value and special status in the poultry industry. However, the black Muscovy duck has strong broodiness and low fecundity, which restricts rapid development of the industry. The ovarian tissues of 3 black Muscovy ducks at the laying and broody stages were collected, respectively. The TRIzol method was used to extract total RNA from the ovarian tissues, the library was constructed and high-throughput sequencing technology was used to analyze the differential expression of miRNA between the 2 groups, then the known, novel miRNAs were predicted for target genes, and the functions of the target genes were enriched and analyzed by GO and KEGG, at last the high-throughput sequencing results were verified by qPCR. The results showed that the raw reads produced by sequencing exceeded 11 099 443 pieces in both groups, and the proportion of clean reads after filtering was all higher than 96.15%, which suggested the data could be used for subsequent analysis. A total of 344 miRNAs were identified, including 275 known miRNAs and 69 newly discovered miRNAs. Sixteen significantly differently expressed miRNAs were screened, including 5 down-regulated and 11 down-regulated in broody ducks with 440 predicted target genes, in which growth hormone secretagogue receptor (GHSR), follistatin (FST) and other target genes were related to reproduction and ovarian development. Thus, it was speculated that the corresponding gga-mir-16c-5p, gga-mir-146a-3p and novel_ 247、novel_ 325 and other miRNAs might be related to broodiness of black Muscovy duck. GO enrichment analysis showed that chromosome organization, nuclear chromosome, spindle microtubule, DNA binding, and nucleotide kinase activity were related to reproduction and development. KEGG pathway annotation placed 234 target genes into 101 signaling pathways, including Wnt and insulin signaling pathways which might be related to germ cell differentiation and development. qPCR confirmed that relative expression levels of up-regulated and down-regulated miRNAs were consistent with the high-throughput sequencing results. This study screened key miRNAs related to broodiness in Muscovy ducks, providing basic material for analyzing broodiness mechanism of Muscovy duck from transcriptional or post transcriptional regulatory level and accelerating the breeding of new high-yield strains.
[1] 陈杰, 卢立志, 陈伟华, 等. 1991. 四季鹅繁殖周期中血浆某些生殖激素水平的变化[J]. 南京农业大学学报, 14(4): 76-80. (Chen J, Lu L Z, Chen W H, et al.1991. Changes of some reproductive hormone levels in plasma during the reproductive cycle of Siji geese[J]. Journal of Nanjing Agricultural University, 14(4): 76-80.) [2] 黄潘, 龚炎长, 彭秀丽, 等. 2010. 鸡胚性分化早期性别差异表达miRNAs的表达谱分析[J]. 农业生物技术学报, 18(6): 1149-1155. (Huang P, Gong Y C, Peng X L, et al.2010. The expression profile analysis of sexual dimorphism miRNAs expressed in chicken embryo at the early stages of sex differentiation[J]. Journal of Agricultural Biotechnology, 18(6): 1149-1155.) [3] 黄正洋, 黎寿丰, 王钱保, 等. 2019. miR-26基因家族进化分析及其在鸡不同组织中表达研究[J]. 中国家禽, 41(17): 15-19. (Huang Z Y, Li S F, Wang Q B, et al. 2019. Molecular evolution of miR-26 gene family and its expression analysis in chicken[J]. China Poultry 41(17): 15-19.) [4] 李昂, 王宏, 朱明霞, 等. 2004. 番鸭就巢期生殖激素水平的变化规律[J]. 畜牧兽医学报, 35(5): 522-525. (Li A, Wang H, Zhu M X, et al.2004. Changes of reproductive hormone levels in Muscovy ducks during the nesting period[J]. Acta Veterinaria et Zootechnica Sinica, 35(5): 522-525.) [5] 李丽, 缪中纬, 辛清武, 等. 2017. 半番鸭与番鸭精巢组织差异表达转录组测序分析[J]. 中国农业科学, 50(18): 3608-3619. (Li L, Miao Z W, Xin Q W, et al.2017. Transcriptome analysis of differential gene expression associated with testis tissue in Mule duck and Muscovy duck[J]. Scientia Agricultura Sinica, 50(18): 3608-3619.) [6] 刘然, 张惠子, 云皓琪, 等. 2015. miRNA-34基因家族进化分析及其在鹅生殖周期内的表达变化[J]. 畜牧兽医学报, 47(2): 260-267. (Liu R, Zhang H Z, Yun H Q, et al.2015. Molecular evolution analyzing of miRNA-34 gene family and its expression in goose reprodutive cycle[J]. Acta Veterinaria et Zootechnica Sinica, 47(2): 260-267.) [7] 潘爱銮, 彭先文, 杜金平. 2001. 禽类就巢性的研究进展[J]. 甘肃畜牧兽医, 31(1): 35-36. (Pan A L, Peng X W, Du J P.2001. Advances in the study of nesting in birds[J]. Gansu Animal Husbandry and Veterinarian, 31(1): 35-36.) [8] 施振旦, 陈峰, 毕英佐. 2000. 禽类就巢发生和调控研究进展[J]. 黑龙江动物繁殖, 8(3): 37-41. (Shi Z D, Chen F, Bi Y Z.2000. Research progress on nesting occurrence and regulation in birds[J]. Animal Breeding in Heilongjiang, 8(3): 37-41.) [9] 施振旦, 郭日红, 邢光东. 2012. 催乳素调控禽类繁殖活动的研究现状与展望[J]. 中国家禽, 34(4): 1-4. (Shi Z D, Guo R H, Xing G D.2012. Research status and prospect of prolactin regulating poultry reproductive activities[J]. China Poultry, 34(4): 1-4.) [10] 斯托凯P D. 1982. 禽类生理学[M]. 北京: 科学出版社, pp. 238-309. (Sturkie P D.1982. Poultry Physiology[M]. Science Press, Beijing, China, pp. 238-309.) [11] 王光瑛. 1999. 番鸭养殖新技术[M]. 福州: 福建科学技术出版社, pp. 2-3. (Wang G Y.1999. New Technology of Muscovy Duck Breeding[M]. Fujian Science and Technology Publishing Press, Fuzhou, China, pp. 2-3.) [12] 吴旭, 严美姣, 刘丽平, 等. 2012. 促性腺激素释放激素基因(GnRH)和生长激素基因(GH)对番鸭产蛋性能的遗传效应分析[J]. 农业生物技术学报, 20(3): 289-295. (Wu X, Yan M J, Liu L P, et al. A.2012. Genetic effects of gonadotropin-releasing hormone (GnRH) and growth hormone (GH) genes on the egg performance in Muscovy duck (Cairina moschata)[J]. Journal of Agricultural Biotechnology, 20(3): 289-295.) [13] 辛清武, 缪中纬, 郑嫩珠, 等. 2013. 黑番鸭VIPR-1基因单核苷酸多态性与就巢性状的关联性分析[J]. 浙江农业学报, 25(6): 1202-1206. (Xin Q W, Miao Z W, Zheng N Z, et al.2013. Correlation analysis between nucleotide polymorphism of the VIPR-1 and broodiness in black Muscovy duck[J]. Acta Agriculturae Zhejiangensis, 25(6): 1202-1206.) [14] 辛清武, 郑嫩珠, 缪中纬,等. 2014. 黑番鸭高产优质专门化品系的选育研究[J]. 家畜生态学报, 35(2): 21-24. (Xin Q W, Zheng N Z, Miao Z W, et al.2014. A study on the breeding of high-yield, high-quality and specialized strain of black Muscovy duck[J]. Journal of Domestic Animal Ecology, 35(2): 21-24.) [15] 张涛, 刘贺贺, 罗俊, 等. 2016. 鸭输卵管壳腺部参与绿壳蛋性状形成的miRNAs富集与分析[J]. 农业生物技术学报, 25(1): 133-141. (Zhang T, Liu H H, Luo J, et al.2017. Enrichment and analysis of miRNAs involve in the formation of blue shell-egg trait in duck (Anas platyrhynchos) oviduct shell gland[J]. Journal of Agricultural Biotechnology, 25(1): 133-141.) [16] 郑炜, 赵宗胜, 李青峰, 等. 2014. Solexa测序技术分析鸡与鹌鹑属间杂交雌性和雄性胚胎的差异microRNAs[J]. 中国兽医学报, 34(1): 116-122. (Zheng W, Zhao Z S, Li Q F, et al.2014. Analysis of difference microRNAs in female and male embryos of chicken-quail hybrid by solexa sequencing[J].Chinese Journal of Veterinary Science, 34(1): 116-122.) [17] 周敏, 和俊, 谢袖娟, 等. 2012. VIPR-1基因5'调控区多态位点与鸡就巢性的关联分析[J]. 畜牧兽医学报, 43(3): 343-352. (Zhou M, He J, Xie X J, et al.2012. Association of polymorphisms in the 5' regulator region of the VIPR-1 gene with chicken broodiness[J]. Acta Veterinaria et Zootechnica Sinica, 43(3): 343-352.) [18] Baley J, Li J.2012. MicroRNAs and ovarian function[J]. Journal of Ovarian Research, 5(1): 8. [19] Bhatt R, Youngren O, Kang S, et al.2003. Dopamine infusion into the third ventricle increases gene expression of hypothalamic vasoactive intestinal peptide and pituitary prolactin and luteinizing hormone β subunit in the turkey[J]. General and Comparative Endocrinology, 130(1): 41-47. [20] Chaiseha Y, Youngren O, Al-Zailaie K, et al.2003. Expression of D1 and D2 dopamine receptors in the hypothalamus and pituitary during the turkey reproductive cycle: Colocalization with vasoactive intestinal peptide[J]. Neuroendocrinology, 77(2): 105-118. [21] Chen F, Li J, Zhang H, et al.2014. Identification of differentially expressed known and novel miRNAs in broodiness of goose[J]. Molecular Biology Reports, 41(5): 2767-2777. [22] Huang Z Y, Yuan X Y, Wu N Z, et al.2016. Molecular cloning of the SMAD4 gene and its mRNA expression analysis in ovarian follicles of the Yangzhou goose (Anser cygnoides)[J]. British Poultry Science, 57(4): 515-521. [23] Johnson A, Woods D C.2009. Dynamics of avian ovarian follicle development: Cellular mechanisms of granulosa cell differentiation[J]. General and Comparative Endocrinology, 163(1-2): 12-17. [24] Kansaku N, Shimada K, Ohkubo T, et al.2001. Molecular cloning of chicken vasoactive intestinal polypeptide receptor complementary DNA, tissue distribution and chromosomal localization[J]. Biology of Reproduction, 64(5): 1575-1581. [25] Kenneth J L, Thomas D S.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods, 25(4): 402-408. [26] Kineman R D, Kamegai J, Frohman L A.1999. Growth hormone (GH)-releasing hormone (GHRH) and the GH secretagogue (GHS), L692, 585, differentially modulate rat pituitary GHS receptor and GHRH receptor messenger ribonucleic acid levels[J]. Endocrinology, 140(8): 3581-3586. [27] Li H F, Shu J T, Du Y F, et al.2013. Analysis of the genetic effects of prolactin gene polymorphisms on chicken egg production[J]. Molecular Biology Reports, 40: 289-294. [28] Li Q, Hu S Q, Wang Y, et al.2019. mRNA and miRNA transcriptome profiling of granulosa and theca layers from geese ovarian follicles reveals the crucial pathways and interaction networks for regulation of follicle selection[J]. Frontiers in Genetics, 10: 988. [29] Liu L B, Xiao Q H, Elizabeth R G, et al.2018. Whole-transcriptome analysis of atrophic ovaries in broody chickens reveals regulatory pathways associated with proliferation and apoptosis[J]. Scientific Reports, 8(1): 1-14. [30] McBride D, CarréW, Sontakke S D, et al.2012. Identification of miRNAs associated with the follicular-luteal transition in the ruminant ovary[J]. Reproduction, 144(2): 221-233. [31] Murchison E P, Stein P, Xuan Z, et al.2007. Critical roles for dicer in the female germline[J]. Genes & Development, 21(6): 682-693. [32] Wilkanowska A, Mazurowski A, Mroczkowski S, et al.2014. Prolactin (PRL) and prolactin receptor (PRLR) genes and their role in poultry production traits[J]. Folia Biologica, 62(1): 1-8. [33] Yang C, Xiong X, Jiang X, et al.2020. Novel miRNA identification and comparative profiling of miRNA regulations revealed important pathways in Jinding duck ovaries by small RNA sequencing[J]. 3 Biotech, 10(2): 38. [34] Yu D B, Jiang B C, Gong J, et al.2013. Identification of novel and differentially expressed microRNAs in the ovaries of laying and non-laying ducks[J]. Journal of Integrative Agriculture, 12(1): 136-146. [35] Zhou M, Lei M, Rao Y, et al.2008. Polymorphisms of vasoactive intestinal peptide receptor-1 gene and their genetic effects on broodiness in chickens[J]. Poultry Science, 87(5): 893-903.
