|Integrative analysis of hypothalamic transcriptome and genetic association study reveals key genes involved in the regulation of egg production in indigenous chickens
|WANG Dan-dan1, ZHANG Yan-yan1, TENG Meng-lin1, WANG Zhang1, XU Chun-lin1, JIANG Ke-ren1, MA Zheng4, LI Zhuan-jian1, 2, 3, TIAN Ya-dong1, 2, 3, Kang Xiang-tao1, 2, 3, LI Hong1, 2, 3, LIU Xiao-jun1, 2, 3
|1 College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, P.R.China
2 Henan Key Laboratory for Innovation and Utilization of Chicken Germplasm Resources, Zhengzhou 450046, P.R.China
3 International Joint Research Laboratory for Poultry Breeding of Henan, Zhengzhou 450046, P.R.China
4 School of Life Science and Engineering, Foshan University, Foshan 528225, P.R.China
本研究首先通过对产蛋前期（15周龄）和产蛋高峰期（30周龄）卢氏绿壳蛋鸡（LS）下丘脑比较转录组分析，鉴定差异表达基因（DEGs）；然后利用Gene Ontology (GO)富集分析，筛选DEGs中参与繁殖调控生物学过程（BP）的基因；进而通过蛋白质互作网络（PPI）分析，筛选调控繁殖过程的潜在核心候选基因（PCCGs）。在此基础上，利用qRT-PCR对PCCGs在两个地方鸡品种产蛋前期（15周龄）和产蛋高峰期（30周龄）下丘脑中表达水平的变化趋势进行分析，进而对基因表达量与30周龄产蛋数（EN30w）和血液繁殖激素水平的相关性分析，筛选影响地方鸡产蛋性能的关键基因；最后，从这些关键基因中筛选单核苷酸多态性位点（SNPs），并与不同时期产蛋数进行关联分析，进一步确定这些关键基因中影响产蛋的潜在SNP位点。产蛋前期和产蛋高峰期LS下丘脑比较转录组分析共鉴定出518个DEGs。对这些DEGs功能富集分析发现，10个BP中包含的64个DEGs可能通过神经内分泌过程参与鸡繁殖调控。进一步的PPI分析发现，64个DEGs中有16个高连接度（Degree≥12）的基因，即PCCGs。对这16个PCCGs在LS和固始鸡（GS）产蛋前期和产蛋高峰期下丘脑中的表达模式检测发现，其中的11个PCCGs在两品种两个时期下丘脑的表达水平差异显著（P<0.05），且变化趋势相同。在上述11个基因中，有8个基因的表达量与EN30w和血清生殖激素浓度呈显著相关（P<0.05）。8个基因中筛选的SNP位点与产蛋性状的关联分析表明，这8个基因与不同阶段的产蛋量存在显著相关（P<0.05），是调控地方鸡产蛋性能的关键基因。本研究鉴定出参与地方鸡产蛋调控的8个关键基因，包括CNR1、AP2M1、NRXN1、ANXA5、PENK、SLC1A2、SNAP25和TRH。这些发现为进一步理解鸡产蛋性能调控机制提供了新见解，并为地方鸡繁殖性能选育提供了可能的分子标记。
Abstract Indigenous chicken products are increasingly favored by consumers due to their unique meat and egg quality. However, the relatively poor egg-laying performance largely impacts the economic benefits and hinders sustainable development of the local chicken industry. Thus, excavating key genes and effective molecular markers associated with egg-laying performance is necessary to improve egg production via genetic selection in indigenous breeds. In the present study, comparative hypothalamic transcriptome between pre-laying (15 weeks old) and peak-laying (30 weeks old) Lushi blue-shelled-egg (LBS) chicken was performed. A total of 518 differentially expressed genes (DEGs) were identified. Among the DEGs, 64 genes were enriched in 10 Gene Ontology (GO) terms associated with reproductive regulation via GO analysis and considered as potential candidate genes regulating egg-laying performance. Of the 64 genes, 16 showed high connectivity (degree≥12) by protein–protein interaction (PPI) network analysis and were considered as potential core candidate genes (PCCGs). To further look for key candidate genes from the PCCGs, firstly, the expression patterns of the 16 genes were examined in the hypothalamus of two indigenous breeds (LBS and Gushi (GS) chickens) between the pre-laying and peak-laying stages using quantitative real-time PCR (qRT-PCR). Eleven out of the 16 genes showed significantly differential expression (P<0.05) with the same changing trends in the two breeds. Then, correlations between the expression levels of the above 11 genes and egg numbers and reproductive hormone concentrations in serum were investigated in high-yielding and low-yielding GS chickens. Of the 11 genes, eight showed significant correlations (P<0.05) between their expression levels and egg numbers, and between expression levels and reproductive hormone concentration in serum. Furthermore, an association study on single nucleotide polymorphisms (SNPs) identified in these eight genes and egg production traits was carried out in 640 GS hens, and a significant association (P<0.05) between the SNPs and egg numbers was confirmed. In conclusion, the eight genes, including CNR1, AP2M1, NRXN1, ANXA5, PENK, SLC1A2, SNAP25 and TRH, were demonstrated as key genes regulating egg production in indigenous chickens, and the SNPs sites within the genes might be served as markers to provide a guide for indigenous chicken breeding. These findings provide a novel insight for further understanding the regulatory mechanisms of egg-laying performance and developing molecular markers to improve egg production of indigenous breeds.
Received: 30 March 2021
Accepted: 18 September 2021
|Fund: This work was supported by the Key Project of NSFC-Henan Province Joint Fund, China (U1704233), the Innovation Research Team of Ministry of Education, China (IRT-16R23), the Program for Innovative Research Team (in Science and Technology) in University of Henan Province, China (21IRTSTHN022), and the Key Scientific Research Project of Higher Education of Henan Province (21A230011).
|About author: WANG Dan-dan, E-mail: firstname.lastname@example.org; Correspondence LI Hong, E-mail: email@example.com; LIU Xiao-jun, E-mail: firstname.lastname@example.org
Cite this article:
WANG Dan-dan, ZHANG Yan-yan, TENG Meng-lin, WANG Zhang, XU Chun-lin, JIANG Ke-ren, MA Zheng, LI Zhuan-jian, TIAN Ya-dong, Kang Xiang-tao, LI Hong, LIU Xiao-jun.
Integrative analysis of hypothalamic transcriptome and genetic association study reveals key genes involved in the regulation of egg production in indigenous chickens. Journal of Integrative Agriculture, 21(5): 1457-1474.
| Alizadeh A, Zendehdel M, Babapour V, Charkhkar S, Hassanpour S. 2015. Role of cannabinoidergic system on food intake in neonatal layer-type chicken. Veterinary Research Communications, 39, 151–157.
Castañeda-Cabral J L, López-Ortega J G, Fajardo-Fregoso B F, Beas-Zárate C, Ureña-Guerrero M E. 2020. Glutamate induced neonatal excitotoxicity modifies the expression level of EAAT1 (GLAST) and EAAT2 (GLT-1) proteins in various brain regions of the adult rat. Neuroscience Letters, 735, 135237.
Chen Y, Zhao Y L, Jin W J, Li Y F, Zhang Y H, Ma X F, Sun G R, Han R L, Tian Y D, Li H, Kang X T, Li G X. 2019. MicroRNAs and their regulatory networks in Chinese Gushi chicken abdominal adipose tissue during postnatal late development. BMC Genomics, 20, 778.
Chowdhury V S, Sultana H, Furuse M. 2014. International perspectives on impacts of reproductive technologies for world food production in Asia associated with poultry production. Advances in Experimental Medicine & Biology, 752, 229–237.
Cottone E, Pomatto V, Bovolin P. 2013. Role of the endocannabinoid system in the central regulation of nonmammalian vertebrate reproduction. International Journal of Endocrinology, 2013, 941237.
Donna D, Cottone E, Aramu S, Campantico E, Guastalla A, Franzoni M F. 2006. Endocannabinoids and amphibian reproduction: An immunohistochemical study in the green frog. Accademia Delle Scienze di Torino, 140, 37–45.
Du Y L, Liu L L, He Y, Dou T F, Jia J J, Ge C R. 2020. Endocrine and genetic factors affecting egg laying performance in chickens: A review. British Poultry Science, 1, 1–12.
Erik H, Turi G F, Kalló I, Zsolt L. 2004. Expression of vesicular glutamate transporter-2 in gonadotropin-releasing hormone neurons of the adult male rat. Endocrinology, 145, 4018–4021.
Gotow T, Sakata M, Funakoshi T, Uchiyama Y. 1996. Preferential localization of annexin V to the axon terminal. Neuroscience, 75, 507–521.
He C, Holme J, Anthony J. 2014. SNP genotyping: The KASP assay. Methods in Molecular Biology, 1145, 75–86.
Helbig I, Lopez-Hernandez T, Shor O, Galer P, Ganesan S, Pendziwiat M, Rademacher A, Ellis C A, Hümpfer N, Schwarz N, Seiffert S, Peeden J, Shen J, Štěrbová K, Hammer T B, Møller R S, Shinde D N, Tang S, Smith L, Poduri A, et al. 2019. A recurrent missense variant in AP2M1 impairs clathrin-mediated endocytosis and causes developmental and epileptic encephalopathy. The American Journal of Human Genetics, 104, 1060–1072.
Ibrahim D, Goshu G, Esatu W, Cahaner A. 2019. Dual-purpose production of genetically different chicken crossbreeds in Ethiopia. 2. Egg and meat production of the final-crossbreed females and males. Poultry Science, 98, 3405–3417.
Iremonger K J, Constantin S, Liu X, Herbison A E. 2010. Glutamate regulation of GnRH neuron excitability. Brain Research, 1364, 35–43.
Irfan M, Gopaul K R, Miry O, Hökfelt T, Stanton P K, Bark C. 2019. SNAP-25 isoforms differentially regulate synaptic transmission and long-term synaptic plasticity at central synapses. Scientific Reports, 9, 6403.
Jeong W, Lim W, Kim J, Ahn S E, Lee H C, Jeong J W, Han J Y, Song G, Bazer F W. 2012. Cell-specific and temporal aspects of gene expression in the chicken oviduct at different stages of the laying cycle. Biology of Reproduction, 86, 172.
Jiang X F, Zhou H F, Shi M Q, Zhou B R, Liu B, Yuan Y Z, Shan J J, Xu J Y, Xie T. 2018. Bu-shen-zhu-yun decoction promotes synthesis and secretion of FSHβ and LHβ in anterior pituitary cells in vitro. Biomedicine & Pharmacotherapy, 102, 494–501.
Jiang X L. 2011. Study on five key genes of porcine hypothalamo-pituitary-thyroid axis. Ph D thesis, Zhejiang Unversity, China. (in Chinese)
Kanduri C, Kantojärvi K, Salo P M, Vanhala R, Buck G, Blancher C, Lähdesmäki H, Järvelä I. 2016. The landscape of copy number variations in Finnish families with autism spectrum disorders. Autism Research, 9, 9–16.
Kang L, Cui X X, Zhang Y J, Yang C H, Jiang Y L. 2013. Identification of miRNAs associated with sexual maturity in chicken ovary by Illumina small RNA deep sequencing. BMC Genomics, 14, 352.
Kang L, Yang C H, Wu H Z, Chen Q Y, Huang L B, Li X Y, Tang H, Jiang Y L. 2017. miR-26a-5p regulates TNRC6A expression and facilitates Theca cell proliferation in chicken ovarian follicles. DNA and Cell Biology, 36, 922–929.
Kawaminami M, Etoh S, Miyaoka H, Sakai M, Nishida M, Kurusu S, Hashimoto I. 2002. Annexin 5 messenger ribonucleic acid expression in pituitary gonadotropes is induced by gonadotropin-releasing hormone (GnRH) and modulates GnRH stimulation of gonadotropin release. Neuroendocrinology, 75, 2–11.
Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R. 2013. TopHat2: Accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biology, 14, R36.
Li X, Ji W, Sun G, Xiao W, Bian Y, Qing H. 2020. Cloning and expression analysis of PRL and PRLR genes in black Muscovy duck. British Poultry Science, 61, 92–96.
Luo P T, Yang R Q, Li Y, Yang N. 2007. Estimation of genetic parameters for cumulative egg numbers in a broiler dam line by using a random regression model. Poultry Science, 86, 30–36.
Meccariello R, Franzoni M F, Chianese R, Cottone E, Scarpa D, Donna D, Cobellis G, Guastalla A, Pierantoni R, Fasano S. 2008. Interplay between the endocannabinoid system and GnRH-I in the forebrain of the anuran amphibian Rana esculenta. Endocrinology, 149, 2149–2158.
Michael G C, Mark F G, Xie W, Petersen S L, Wetsel W C. 2005. Regulation of gonadotropin-releasing hormone secretion by cannabinoids. Endocrinology, 146, 4491–4499.
Mishra S K, Chen B L, Zhu Q, Xu Z G, Ning C Y, Yin H D, Wang Y, Zhao X L, Fan X L, Yang M Y, Yang D Y, Ni Q Y, Li Y, Zhang M W, Li D Y. 2020. Transcriptome analysis reveals differentially expressed genes associated with high rates of egg production in chicken hypothalamic-pituitary-ovarian axis. Scientific Reports, 10, 5976.
Nillni E A, Aird F, Seidah N G, Todd R B, Koenig J I. 2001. PreproTRH(178–199) and two novel peptides (pFQ7 and pSE14) derived from its processing, which are produced in the paraventricular nucleus of the rat hypothalamus, are regulated during suckling. Endocrinology, 142, 896–906.
NLPGRC (National Livestock and Poultry Genetic Resources Commission). 2011. Annals of Livestock and Poultry Genetic Resources of China: Annals of Poultry. China Agriculture Press, China. (in Chinese)
Pecori G F, Pesce S, Maroni P, Pagliardini L, Lasio G, Losa M, Cavagnini F. 2010. Inhibitory effect of prepro-thyrotrophin-releasing hormone (178–199) on adrenocorticotrophic hormone secretion by human corticotroph tumours. Journal of Neuroendocrinology, 22, 294–300.
Plant T M. 2015. The hypothalamo-pituitary-gonadal axis. Journal of Endocrinology, 226, T41–T45.
Royle S J, Lagnado L. 2010. Endocytosis at the synaptic terminal. Journal of Physiology, 553, 345–355.
Schreiner D, Scheiffele P. 2020. Neuroligins and neurexins. Synapse development and maturation. In: Rubenstein J, Rakic P, Chen B, Kwan K Y, Cline H T, Cardin J, eds., Comprehensive Developmental Neuroscience, Academic Press, San Diego, USA. pp. 193–212.
Supek F, Bošnjak M, Škunca N, Šmuc T. 2011. REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS ONE, 6, e21800.
Tanabe Y, Nakamura T, Tanase H, Doi O. 1981. Comparisons of plasma LH, progesterone, testosterone and estradiol concentrations in male and female chickens (Gallus domesticus) from 28 to 1 141 days of age. Endocrinologia Japonica, 28, 605–613.
Taylor J A, Goubillon M L, Broad K D, Robinson J E. 2007. Steroid control of gonadotropin-releasing hormone secretion: Associated changes in pro-opiomelanocortin and preproenkephalin messenger RNA expression in the ovine hypothalamus. Biology of Reproduction, 76, 524–531.
Terashima R, Saigo T, Laoharatchatathanin T, Kurusu S, Brachvogel B, Pschl E, Kawaminami M. 2020. Augmentation of Nr4a3 and suppression of fshb expression in the pituitary gland of female annexin A5 null mouse. Journal of the Endocrine Society, 4, bvaa096.
Ubuka T, Bentley G E. 2011. Neuroendocrine control of reproduction in birds. Hormones and Reproduction of Vertebrates, 4, 1–25.
Vega-Zuniga T, Marín G, González-Cabrera C, Planitscher E, Hartmann A, Marks V, Mpodozis J, Luksch H. 2016. Microconnectomics of the pretectum and ventral thalamus in the chicken (Gallus gallus). Journal of Comparative Neurology, 524, 2208–2229.
Walsh J P, Clarke I J. 1996. Effects of central administration of highly selective opioid μ, δ and κ-receptor agonists on plasma luteinizing hormone (LH), prolactin, and the estrogen-induced LH surge in ovariectomized ewes. Endocrinology, 137, 3640–3648.
Wang C, Ma W. 2019. Hypothalamic and pituitary transcriptome profiling using RNA-sequencing in high-yielding and low-yielding laying hens. Scientific Reports, 9,10285.
Wang D D, Xu C L, Wang T A, Li H, Li Y M, Ren J X, Tian Y D, Li Z J, Jiao Y P, Kang X T, Liu X J. 2016. Discovery and functional characterization of leptin and its receptors in Japanese quail (Coturnix japonica). General and Comparative Endocrinology, 225, 1–12.
Wang H B, Dey S K, Maccarrone M. 2006. Jekyll and hyde: Two faces of cannabinoid signaling in male and female fertility. Endocrine Reviews, 27, 427–448.
Wang W W, Wu K L, Jia M T, Sun S H, Kang L, Zhang Q, Tang H. 2018. Dynamic changes in the global MicroRNAome and transcriptome identify key nodes associated with ovarian development in chickens. Frontiers in Genetics, 9, 491.
Wieffer M, Maritzen T, Haucke V. 2009. SnapShot: Endocytic trafficking. Cell, 137, 381–383.
Wu G M, Zhu L L, Han X, Tang J G. 2020. Sequence variation of TRH gene coding region of Yaoshan chicken and its correlation with reproductive traits. Guizhou Agricultural Sciences, 48, 73–76. (in Chinese)
Wu H Z, Fan F T, Liang C Z, Zhou Y, Qiao X B, Sun Y, Jiang Y L, Kang L. 2019. Variants of pri-miR-26a-5p polymorphisms are associated with values for chicken egg production variables and affects abundance of mature miRNA. Animal Reproduction Science, 201, 93–101.
Ye Q, Xu J G, Gao X F, Ouyang H J, Luo W, Nie Q H. 2017. Associations of IGF2 and DRD2 polymorphisms with laying traits in Muscovy duck. PeerJ, 5, e4083.
Yonezawa T, Watanabe A, Kurusu S, Kawaminami M. 2015. Gonadotropin-releasing hormone is prerequisite for the constitutive expression of pituitary annexin A5. Endocrine Journal, 62, 1127–1132.
Yu S G, Wang G, Liao J, Tang M, Chen J. 2020. Identification of differentially expressed genes associated with egg production in black-boned chicken. British Poultry Science, 61, 3–7.
Yuan Z J, Chen Y X, Chen Q Y, Guo M, Kang L, Zhu G Y, Jiang Y L. 2016. Characterization of chicken MMP13 expression and genetic effect on egg production traits of its promoter polymorphisms. G3 Genesgenetics, 6, 1305–1312.
Zhang T, Chen L, Han K P, Zhang X Q, Zhang G X, Dai G J, Wang J Y, Xie K Z. 2019. Transcriptome analysis of ovary in relatively greater and lesser egg producing Jinghai yellow chicken. Animal Reproduction Science, 208, 106114.
Zhou M, Du Y, Nie Q, Liang Y, Luo C, Zeng H, Zhang X. 2010. Associations between polymorphisms in the chicken VIP gene, egg production and broody traits. British Poultry Science, 51, 195–203.
Zhu G Y, Kang L, Wei Q Q, Cui X X, Wang S Z, Chen Y X, Jiang Y L. 2014. Expression and regulation of MMP1, MMP3, and MMP9 in the chicken ovary in response to gonadotropins, sex hormones, and TGFB1. Biology of Reproduction, 90, 57.
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