Mu et al. BMC Genomics (2021) 22:349 https://doi.org/10.1186/s12864-021-07688-x RESEARCH Open Access Transcriptome analysis of ovary tissues from low- and high-yielding Changshun green-shell laying hens Ren Mu1, Yi-yin Yu1, Tuya Gegen2, Di Wen1, Fen Wang1, Zhi Chen1* and Wen-bin Xu3,4* Abstract Background: Changshun green-shell laying hens are unique to Guizhou Province, China, and have high egg quality. Improving egg production performance has become an important breeding task, and in recent years, the development of high-throughput sequencing technology provides a fast and exact method for genetic selection. Therefore, we aimed to use this technology to analyze the differences between the ovarian mRNA transcriptome of low and high-yield Changshun green-shell layer hens, identify critical pathways and candidate genes involved in controlling the egg production rate, and provide basic data for layer breeding. Results: The egg production rates of the low egg production group (LP) and the high egg production group (HP) were 68.00 ± 5.56 % and 93.67 ± 7.09 %, with significant differences between the groups (p < 0.01). Moreover, the egg weight, shell thickness, strength and layer weight of the LP were significantly greater than those of the HP (p < 0.05). More than 41 million clean reads per sample were obtained, and more than 90 % of the clean reads were mapped to the Gallus gallus genome. Further analysis identified 142 differentially expressed genes (DEGs), and among them, 55 were upregulated and 87 were downregulated in the ovaries. KEGG pathway enrichment analysis identified 9 significantly enriched pathways, with the neuroactive ligand-receptor interaction pathway being the most enriched. GO enrichment analysis indicated that the GO term transmembrane receptor protein tyrosine kinase activity, and the DEGs identified in this GO term, including PRLR, NRP1, IL15, BANK1, NTRK1, CCK, and HGF may be associated with crucial roles in the regulation of egg production. Conclusions: The above-mentioned DEGs may be relevant for the molecular breeding of Changshun green-shell laying hens. Moreover, enrichment analysis indicated that the neuroactive ligand-receptor interaction pathway and receptor protein tyrosine kinases may play crucial roles in the regulation of ovarian function and egg production. Keywords: Changshun green-shell laying hens, Egg production, Ovary, Transcriptome analysis * Correspondence: [email protected]; [email protected] 1College of Biological Science and Agriculture, Qiannan Normal University for Nationalities Duyun, Jianjiang Road 5, 558000 Duyun, China 3College of Animal Sciences, Zhejiang University, 310058 Hangzhou, China Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Mu et al. BMC Genomics (2021) 22:349 Page 2 of 11 Introduction Changshun green-shell chickens are native breeds Chicken eggs are an important food resource for found in Guizhou province, China. They are dual- humans as they contain high-quality protein, essential purpose egg and meat-type chickens, and their eggs have vitamins and minerals, and are inexpensive. Global egg extremely high economic value, owing to their appear- consumption has tripled in the past 40 years and this ance, higher protein content, better amino acid compos- trend is predicted to continue [1]. The question of how ition, and lower fat content [9]. The Hartz unit of to increase egg production has consequently become a Changshun green shell eggs is 76.39 ± 2.76 (Level AA), critically important question for the egg industry. which is extremely significantly better than that of white Improving the genetic potential of chickens is one of shell eggs (p < 0.01), and the calcium and phosphorus the most important strategies utilized to increase egg content are significantly higher, while the crude fat con- production. However, conventional breeding techniques tent is significantly lower than that of white shell eggs based on long-term selection that use egg numbers and (p < 0.01) [10]. Our previous study also found similar re- laying rate are usually laborious and time-consuming sults (data unpublished). However, as an indigenous [2]. Currently, various high-throughput techniques that chicken breed, Changshun green-shell chicken shows can identify genes at the genomic and transcriptomic relatively low egg production [11]. Thus, the purpose of levels have been increasingly employed for studying this study was to investigate the mechanisms that affect poultry reproduction. For instance, in goose ovaries, the egg production of Changshun green-shell chickens. twenty-six genes were identified that may be related to Towards this end, we conducted high-throughput RNA the egg-laying process by using suppression subtractive sequencing in the ovaries of Changshun green-shell hybridization and reverse dot-blot analysis [3]. Using a chickens, to (1) determine the differences in the ovary large-scale transcriptome sequencing technique, five mRNA transcriptomes between the low- and high- genes in the ovarian tissues of Anser cygnoides were yielding Changshun green-shell laying hens, and (2) identified that may play important roles in determining identify the critical pathways and candidate genes in- high reproductive performance [4]. In chicken, nine volved in controlling the egg production rate. genes (BDH, NCAM1, PCDHA, PGDS, PLAG1, PRL, SAR1A, SCG2, and STMN2) related to high egg produc- Materials and methods tion levels in the hypothalamus and pituitary gland were Ethics statement identified using a cDNA chip [5]. In another study, the This study was carried out in accordance with the rec- hypothalamus and pituitary expression profiles in high- ommendations in the Guide for the Care and Use of La- and low-yield laying chickens were analyzed by RNA-seq boratory Animals of the Ministry of Science and [6]. Seven and 39 differentially expressed genes (DEGs) Technology of the People’s Republic of China, and were identified in the hypothalamus and pituitary, re- followed the Regulations for the Administration of Af- spectively, and were associated with the amino acid me- fairs Concerning Experimental Animals, Qiannan Nor- tabolism, glycosaminoglycan biosynthesis, and estrogen mal University for Nationalities (Guizhou, China). and in negative feedback systems. Using cDNA microarray ana- compliance with ARRIVE 2.0 guidelines. The animal lysis, TXN, ACADL, ING4, and ANXA2 were reported protocol was approved by the Animal Ethics Committee to express at higher levels in the ovarian follicles of of the Qiannan Normal University for Nationalities. high-yielding chicken [7]. By comparing the transcrip- tomes in ovarian tissues of chickens that showed greater Animal and sample preparation and lesser egg-producing capacity, five candidate genes A total of 80 Changshun green-shell layers raised in the were identified to related to egg production, including poultry breeding farm of Qiannan Normal University for ZP2, WNT4, AMH, IGF1, and CYP17A1 [8]. In addition, Nationalities were used in this study. At the beginning Mishra et al. [2] performed RNA-seq to explore the of the study (age of 240 days), the layers had similar chicken transcriptome in the hypothalamic-pituitary- body weights of 1.36 ± 0.14 kg. All layers were housed ovarian (HPO) axis. Their results showed that 414, 356 individually in the battery cages (36 cm-width × 48 and 10 DEGs were identified in the pituitary gland, length × 38 height) with the same feeding and manage- ovary, and hypothalamus, respectively, between high and ment conditions throughout the study period. The room low-yielding chickens. These DEGs were involved in the temperature was maintained at 22 ± 2℃. The light re- regulation of the mTOR and Jak-STAT signaling path- gime was 16 L:8D. Layers were allowed ad libitum access ways, tryptophan metabolism and PI3K-Akt signaling to diet and water. Diet was supplemented three times pathways at the HPO axis. High throughput techniques daily (7:00, 13:00, and 19:00). Egg number and egg provide a fast and exact method for genetic selection weight were recorded every day (16:00). At 290 days of and have the potential to be an appealing alternative to age, four high-yield (high egg production group, HP) conventional breeding techniques. and four low-yield individuals (low egg production Mu et al. BMC Genomics (2021) 22:349 Page 3 of 11 group, LP) were selected from the batch of laying hens was assessed using principal component analysis (PCA) according to their laying rates. The eggshell thickness in R (v4.0.2). and strength were evaluated daily. In the early morning at the age of 300 days, the chickens were anesthetized using sodium pentobarbital after weighing, and ovarian Identification of differentially expressed genes samples were obtained after slaughter. All samples were The differentially expressed genes (DEGs) were identi- immediately frozen in liquid nitrogen and stored at fied using the DESeq2 package (v1.28.1) in R (v4.0.2). ≤ -80 °C until analysis. The layers had fasted overnight be- Genes with an adjusted p-value 0.05 and |Log2Fold ≥ fore being sampled.