Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (1): 204-215.doi: 10.3864/j.issn.0578-1752.2024.01.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Study of Key Genes and Signaling Pathways Regulating Dry Feather Traits in Yellow-Feathered Broiler Chickens Based on Transcriptome Analysis

JI GaiGe1(), CHEN ZhiWu2(), SHAN YanJu1, LIU YiFan1, TU YunJie1, ZOU JianMin1, ZHANG Ming1, JU XiaoJun1, SHU JingTing1(), ZHANG HaiTao3, TANG YanFei4, JIANG HuaLian4   

  1. 1 Key Laboratory of Poultry Genetics and Breeding of Jiangsu Province/Jiangsu Institute of Poultry Sciences, Yangzhou 225125, Jiangsu
    2 Guangxi Jinling Agriculture and Animal Husbandry Group Co., Ltd., Nanning 530000
    3 Jiangsu Lihua Animal Husbandry Stock Co., Ltd., Changzhou 213168, Jiangsu
    4 Guangxi Fufeng Farm Group Co., Ltd., Nanning 530024
  • Received:2023-05-09 Accepted:2023-10-31 Online:2024-01-01 Published:2024-01-10

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Abstract:

【Objective】 The study aimed to identify important candidate genes and signaling pathways associated with dry feather traits by comparing the morphological and gene expression differences between the feather follicles of dry and undried feathers. 【Method】 Three samples of skin tissue were selected from each of the undried and dried feathers. The histological examinations were used to compare the morphological differences between the feather follicles of dried and undried feathers. RNA-seq technology was employed to compare the differentially expressed genes (DEGs) between the two groups of skin samples. The accuracy of transcriptome results was validated by using the fluorescent quantitative PCR technique (RT-qPCR). 【Result】 By histological H.E staining, it was confirmed that the feather follicles of the undried feathers were in the growth phase, while the feather follicles of the dried feathers were in the resting phase. The feather follicle skin samples at the growth stage were used as controls, 942 DEGs were identified in resting feather follicle samples (|fold-change|>2 and P<0.05), including 384 significantly down-regulated DEGs and 558 upregulated DEGs. Gene ontology (Go) analysis suggested that the DEGs were significantly enriched in cell division, cycle regulation, and other related biological processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway results showed that the DEGs were related to MAPK, TGF-β, p53, and cell cycle-related signaling pathways. Protein-protein interaction (PPI) network was constructed and six hub genes were obtained by CytoHubba analysis, including CDK1, MAD2L1, BUB1, CCNB2, PLK1, and BUB1B. Gene Set Enrichment Analysis (GSEA) results indicated that the signaling pathways related to the tight junction, insulin, MAPK, TGF-β, and cell cycle-related pathways were significantly associated with the growth cycle of chicken feather follicles. The expression patterns of 8 DEGs detected by RT-qPCR were consistent with the RNA-seq results. 【Conclusion】 In summary, the dry feather traits of chickens were related to the development of feather follicle cycles. Signaling pathways such as MAPK and TGF-β might play important roles in feather growth and development by regulating the expression of cell cycle-related genes. The study provided clues for understanding the molecular regulatory mechanisms for dry feather traits in yellow-feathered broiler chickens.

Key words: chicken, dry feather, feather follicle, RNA-seq, gene, signaling pathway

Table 1

The qPCR primers were used in this study"

基因 Gene symbol 序列号 Accession number 引物序列 Primer sequence (5′→3′) 产物长度 Product length (bp)
β-actin NM_205518.2 F: GCTGAACTCCATCTTGGTTCCC
R: TGCTTCTAGGGTCACTCGCA		 150
BUB1 NM_001012870.1 F: GCTGAACTCCATCTTGGTTCCC
R: TGCTTCTAGGGTCACTCGCA		 112
BIRC5 NM_001012319.1 F:CTGGATAAAAAGCGGACCAA
R: AACCTAAGGGCCCATGTTCT		 246
CDK1 NM_205314.1 F:GGAACGCATGTCCAAAACTT
R: GCAGGCAGGCAAAGATAAAG		 236
NEK2 NM_001031050.2 F:ATGTCTTCCTGGATGGCAAG
R: TCTGCCAACTCCTTCTGGTT		 241
CDC6 XM_040691823.1 F:GTGTCCTCTCGGAGGTGTTT
R: AGGCACTCTGTCTGGTCCAC		 214
ANXA1 NM_206906.1 F:AAAAACTCCACCTGGCAATG
R: CCACATAAAGCGACCAGGAT		 193
BCL2 NM_205339.2 F: TGACACCTCGATCTCACAGG
R: CATGCAACACACTGGGATTC		 247
EDA2R NM_001083360.1 F: ACCCTCATCAACCGCATCCA
R: GCCTTGCGGTAAAACCCTGG		 89
FZD4 NM_204099.1 F:AATGTCACCAAGATGCCCAACC R:AGACCGAACAAAGGAAGAACTGGA 130

Fig. 1

Morphological changes of chicken back feathers at different developmental periods A: Undried feathers; B: Dried feathers"

Fig. 2

Histological H.E staining of chicken back feather follicles at different developmental stages A: Feather follicles of undried feathers; B: Feather follicles of dried feathers"

Table 2

Summary of sequencing reads statistics and mapping to the reference genome"

样品
Sample		 原始序列
Raw reads		 有效序列
Clean reads		 Q30
(%)		 GC
(%)		 比对率
Mapped on reference		 未比对率
Unmapped		 唯一位置比对
Uniquely mapped
H1-1 40968048 38142462 92.33 48.605 36255024
(95.05%)		 1887438
(4.95%)		 35196169
(92.28%)
H1-2 46296028 43781388 93.07 49.900 41547151
(94.9%)		 2234237
(5.1%)		 40274977
(91.99%)
H1-3 48737026 45301826 92.01 48.995 42668140
(94.19%)		 2633686
(5.81%)		 41786822
(92.24%)
H2-1 46250350 43867410 93.33 49.985 41559772
(94.74%)		 2307638
(5.26%)		 40597907
(92.55%)
H2-2 43917200 40983494 92.28 49.945 38739383
(94.52%)		 2244111
(5.48%)		 37804671
(92.24%)
H2-3 42372910 39985456 93.17 49.750 38040749
(95.14%)		 1944707
(4.86%)		 36991013
(92.51%)

Fig. 3

Volcano plots of DEGs between feather follicles at different developmental periods (growth and rest stage) in chicken skin samples"

Fig. 4

The top 30 significantly enriched GO biological processes (A) and KEGG enrichment pathways (B) for differentially expressed genes"

Fig. 5

Differentially expressed gene protein-protein interaction network Red represents upregulated genes and green represents downregulated genes"

Table 3

Top 10 genes identified in the protein-protein interaction (PPI) network by CytoHubba plug in 4 algorithms"

基因
Gene		 MCC 基因
Gene		 Degree 基因
Gene		 MNC 基因
Gene		 EPC
CDK1 5.11E+19 CDK1 56 CDK1 55 APITD1 85.564
BUB1 5.11E+19 PLK1 46 PLK1 46 AURKA 85.564
PLK1 5.11E+19 CCNB2 44 CCNB2 43 CDK1 85.564
CCNB2 5.11E+19 CENPE 43 CENPE 43 TPX2 85.564
NDC80 5.11E+19 BUB1 41 BUB1 41 BIRC5 85.564
CENPE 5.11E+19 NDC80 41 NDC80 41 MAD2L1 85.564
BUB1B 5.11E+19 BUB1B 37 BUB1B 37 BUB1 85.564
MAD2L1 5.11E+19 MAD2L1 34 KIF11 34 CCNB2 85.564
CENPF 5.11E+19 KIF11 34 MAD2L1 33 PLK1 85.564
INCENP 5.11E+19 AURKA 32 AURKA 31 BUB1B 85.564

Fig. 6

GSEA enrichment of DNA replication (A) and cell cycle (B) pathways up-regulated in feather follicle skin tissue at the growth phase"

Fig. 7

GSEA enrichment of MAPK (A) and TGF-β (B) pathways up-regulated in feather follicle skin tissue at the resting phase"

Fig. 8

RT-qPCR validation of differentially expressed genes"

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