Genome Gene Expression Profiling Analysis Reveals Fur Development in Rex Rabbits (Oryctolagus cuniculus) Journal: Genome Manuscript ID gen-2017-0003.R2 Manuscript Type: Article Date Submitted by the Author: 31-Jul-2017 Complete List of Authors: Zhao, Bohao; Yangzhou University Chen, Yang; Yangzhou University Yan, Xiaorong ; Yangzhou University Hao, Ye; YangzhouDraft University Zhu, Jie; Yangzhou University Weng, Qiiaoqing; Zhejiang Yuyao Xinnong Rabbit Industry Co., Ltd. Wu, Xinsheng; Yangzhou University, College of Animal Science and Technology Is the invited manuscript for consideration in a Special This submission is not invited Issue? : Keyword: Chinchilla rex rabbit, fur development, key gene, transcriptome https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 138 Genome 1 Gene Expression Profiling Analysis Reveals Fur Development in Rex 2 Rabbits ( Oryctolagus cuniculus ) 3 BoHao Zhao 1, Yang Chen 1, XiaoRong Yan 1, Ye Hao 1, Jie Zhu 1, QiaoQing Weng 2, and 4 XinSheng Wu 1* 5 1 The Key Laboratory of Animal Genetics & Breeding and Molecular Design of Jiangsu Province, 6 College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R. 7 China. ; 8 2 Zhejiang Yuyao Xinnong Rabbit Industry Co., Ltd., Yuyao, Zhejiang 315400, China 9 *Corresponding author E-mail: [email protected] 10 Draft 1 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 138 11 Abstract 12 Fur is an important economic trait in rabbits. The identification of genes that 13 influence fur development and knowledge regarding the actions of these genes 14 provides useful tools for improving fur quality. However, the mechanism of fur 15 development is unclear. To obtain candidate genes related to fur development, the 16 transcriptomes of tissues from backs and bellies of Chinchilla rex rabbits were 17 compared. Of the genes analyzed, 336 showed altered expression in the two groups 18 (285 upregulated and 51 downregulated), P≤0.05, fold-change≥2 or ≤0.5). Using GO 19 and KEGG to obtain gene classes that were differentially enriched, we found several 20 genes to be involved in manyDraft important biological processes. In addition, we 21 identified several signaling pathways involved in fur development, including the Wnt 22 and MAPK signaling pathways, revealing mechanisms of skin and hair follicle 23 development, and epidermal cell and keratinocytes differentiation. The obtained rabbit 24 transcriptome and differentially expressed gene profiling data provided 25 comprehensive gene expression information for SFRP2, FRZB, CACNG1, SLC25A4 26 and SLC16A3. To validate the RNA-seq data, the expression levels of eight 27 differentially expressed genes involved in fur development were confirmed by 28 qRT-PCR. The results of rabbit transcriptomic profiling provide a basis for 29 understanding the molecular mechanisms of fur development. 30 Keywords 31 Chinchilla rex rabbit, fur development, key gene, transcriptome 32 2 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 138 Genome 33 Introduction 34 The Chinchilla rex rabbit is an important rabbit breed with varied natural coat 35 colors; consumers highly appreciate the properties of rex furs, such as beauty, softness, 36 color, lightness, and warmth retention (Pan et al. 2015). The characteristics of 37 Chinchilla rex rabbit fur differs between the back and belly, especially the length and 38 diameter of the wools (Tao 2010). 39 In recent years, many studies have revealed the mechanisms of skin and fur 40 development. RNA-seq was used to explore the mechanisms of keratinocyte 41 development in mouse skin, and transcription factor (TF) p63 was found to be highly 42 expressed in stratified epithelia, whichDraft affected the epidermal phenotype (Rizzo et al. 43 2014). Many genes involved in skin development, including those for transcription 44 factors and growth factors, have been identified in rex rabbits with the plaice 45 phenotype (Pan et al. 2015). In cashmere goats, genes related to hair follicle 46 development and cycling were identified in anagen, catagen and telogen stages by 47 transcriptomic investigation of fur development (Geng et al. 2013). It is generally 48 known that fur development is influenced by many factors, including the proliferation 49 of keratinocytes, development of the epidermis and hair follicle (HF) morphogenesis 50 (Danilenko et al. 1995). Multiple genes involved in HF morphogenesis, regulation of 51 proliferation, differentiation and migration of skin are controlled by members of the 52 Wnt signaling pathway, such as the frizzled and secreted frizzled-related protein 53 (SFRP) families (Ehrlund et al. 2013; Kim and Yoon 2014). Epidermal growth factor 54 is regulated by the MAPK/ERK pathway and plays a vital role in the animal skin 3 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 138 55 development, enhancing epidermal growth and keratinization, directly stimulating the 56 proliferation of epidermal cells and promoting keratinocyte proliferation and 57 migration. However, fur characteristics are different at different parts of an animal, 58 and the mechanism of fur development regulation is still unclear in rabbits. 59 Rabbit genome sequencing has been used to study the polygene-related phenotypic 60 changes during rabbit domestication (Carneiro et al. 2014) and differential gene 61 expression in animal skin between anagen and telogen was shown by transcriptome 62 sequencing (Xu et al. 2013). In this study, the skin from the backs and bellies of 63 Chinchilla rex rabbits was collected, and gene expression profiling was used to obtain 64 the differentially expressed genesDraft related to the fur development. After functional 65 annotation, enrichment analysis and assessment of biological functions, candidate 66 genes were identified. These key genes were verified by quantitative real-time PCR. 67 The results obtained serve to improve our understanding of fur development and the 68 differential expression profiles of the candidate genes enable us to clarify the 69 mechanisms of fur development, providing a valuable theoretical basis for further 70 research on the hair and fur of animals. 71 72 Materials and methods 73 Ethics statement 74 All animal experiments were reviewed and approved by the Institutional Animal 75 Care and Use Committee of the School of Animal Science and Technology, Yangzhou 76 University, and performed in accordance with the Regulations for the Administration 4 https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 138 Genome 77 of Affairs Concerning Experimental Animals (China, 1988) and the Standards for the 78 administration of experimental practices (Jiangsu, China, 2008). All surgery was 79 performed according to recommendations proposed by the European Commission 80 (1997), and all efforts were made to minimize suffering of the animals. 81 Tissue collection 82 The Chinchilla rex rabbits used in the experiments were obtained from Zhejiang 83 Yuyao Xinnong Rabbit Co., Ltd. During our experiments, rabbits were raised in a 84 controlled environment and had free access to water and food. All rabbits were housed 85 in a suitable, clean and disease free environment, and a secure cage. The health of the 86 rabbits were monitored twice a Draftday (7 am and 6 pm) and recorded. Three healthy, 87 20-day-old rabbits with the same fur traits were evaluated. Fur on the back (B group) 88 and belly (F group) were different. Three biological samples were taken for each of 89 the two groups (one sample of back and belly fur from each rabbit) to ensure the same 90 genetic background and fur phenotype in each group. After transfer to the laboratory, 91 skin tissue samples (1.5 cm 2) were collected from the back and belly of each rabbit. 92 Animals were anesthetized with by injection with 0.7% sodium pentobarbital solution 93 into the ear vein of the rabbits; in order to prevent bacterial infection iodine solution 94 was smeared on the resultant lesion. Fur was removed from the surface, and then the 95 skin was cut into pieces. The pieces were placed in tubes containing RNase, 96 immediately preserved in liquid nitrogen and stored at -70°C until use in subsequent 97 experiments. 98 RNA extraction, cDNA library construction, and Illumina sequencing 5 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 6 of 138 99 Total RNA was extracted following the manufacturer’s instructions using the 100 mirVana™ miRNA isolation kit (Ambion); the integrity of the RNA was determined 101 with an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US) to 102 obtain a RNA Integrity Number (RIN). An RNeasy micro kit (Cat#74004, QIAGEN, 103 GmBH, Germany) was used to further purify the qualified total RNA, and DNA was 104 removed with the RNase-Free DNase set (QIAGEN, GmBH, Germany). RNA quality 105 was monitored using NanoDrop ND-1000 and Agilent Bioanalyzer 2100. After RNA 106 extraction and purification, 3 µg RNA was used for construction of the back and belly 107 cDNA libraries. Ribosomal RNA (rRNA) was depleted from the total RNA and the 108 remaining RNA was subsequentlyDraft fragmented. These steps were followed by first and 109 second cDNA strand synthesis, end repair, 3'-end adenylation, adapter ligation, and 110 enrichment of the cDNA templates. Finally, the library concentration was determined 111 using a Qubit® 2.0 fluorometer and a Qubit™ dsDNA HS kit (Invitrogen). Cluster 112 generation was completed the sample library, and the first primers hybridized to cBot 113 matched the Illumina HiSeq 2500 platform. After cluster generation, the sequencing 114 reagent was prepared according to the HiSeq 2500 user guide using paired-end 115 technology. Sequencing was controlled by data collection software (Illumina, San 116 Francisco, USA) and the data were analyzed in real time. 117 Transcriptome mapping and analysis of differentially expressed genes 118 The cDNA library was sequenced using an Illumina HiSeq 2500 sequencing 119 platform. Original image files were obtained, and bases were called and filtered, after 120 which the results were stored in fastq format. The original sequencing reads were used 6 https://mc06.manuscriptcentral.com/genome-pubs Page 7 of 138 Genome 121 for transcriptome sequencing analysis.