基于RNA-seq技术挖掘鹌鹑羽色自别雌雄相关基因

doi:10.3969/j.issn.1004-1524.2022.03.10

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (3): 498-506.DOI: 10.3969/j.issn.1004-1524.2022.03.10

基于RNA-seq技术挖掘鹌鹑羽色自别雌雄相关基因

王乾昆¹(), 张小辉¹^,², 庞有志¹^,²^,^*(), 祁艳霞¹^,², 雷莹¹^,², 白俊艳¹^,², 户运奇¹, 赵毅威¹, 苑志文¹, 王涛¹

1.河南科技大学动物科技学院,河南洛阳 471003
2.洛阳市动物遗传育种重点实验室,河南洛阳 471003

收稿日期:2021-07-12 出版日期:2022-03-25 发布日期:2022-03-30
通讯作者: 庞有志
作者简介:*庞有志,Email: pyzh2006@126.com
王乾昆(1995—),男,河南商丘人,硕士研究生,主要从事动物遗传育种与繁殖研究。E-mail: 1536971128@qq.com
基金资助:
河南省自然科学基金(202300410153);河南省科技攻关项目(202102110088)

Screening of genes related to auto-sexing on feather color based on RNA-seq technology

WANG Qiankun¹(), ZHANG Xiaohui¹^,², PANG Youzhi¹^,²^,^*(), QI Yanxia¹^,², LEI Ying¹^,², BAI Junyan¹^,², HU Yunqi¹, ZHAO Yiwei¹, YUAN Zhiwen¹, WANG Tao¹

1. College of Animal Science, Henan University of Science and Technology, Luoyang 471003, Henan, China
2. Luoyang Key Laboratory of Animal Genetic and Breeding, Luoyang 471003, Henan,China

Received:2021-07-12 Online:2022-03-25 Published:2022-03-30
Contact: PANG Youzhi

摘要/Abstract

摘要：

鹌鹑的羽色自别雌雄现象是由性染色体上的基因决定的,但具体的分子机制未知。本研究以北京白羽公鹌鹑和朝鲜栗羽母鹌鹑为研究对象,利用RNA-seq技术分析了F₁公鹑和F₁母鹑胚胎期第10天皮肤组织样品的转录组,以筛选调控鹌鹑羽色自别雌雄的关键基因,并利用qRT-PCR技术进行验证。结果表明:RNA-Seq共得到38.94 G的原始数据,平均每个样本获得了6.49 G的原始数据,6个库的Q30均在90%以上,GC含量平均值为49.7%,所测样品至少89%的reads比对到参考基因组上。通过数据库比对共得到16 013个基因,其中,上调基因69个,下调基因22个。GO富集分析发现有13 841个基因注释到GO数据库中,其中包含78个差异表达基因。KEGG富集结果显示,有22个差异基因富集到38条通路中。对全部差异基因进行筛选,得到了7个与羽色表型相关的基因,分别是DCT、MLANA、SLC45A2、TYRP1、TRPM1、FAM174A和KIT。qRT-PCR结果表明,候选基因均在栗羽鹌鹑中高表达,与RNA-seq结果一致,这7个候选基因可能与自别雌雄有关。

关键词: 鹌鹑, 羽色, 自别雌雄, 转录组, 基因表达

Abstract:

The phenomenon of auto-sexingby feather color in quail is determined by genes on the sex chromosomes, but the specific molecular mechanism is unknown. In this study, Beijing male white feather quail and female maroon feather quail were selected as research animals.The transcriptome of F₁ male and F₁ female quails at the 10th day of embryo stage were analyzed by RNA-seq technique, and the key genes associated with feather color were screened and verified by qRT-PCR. The results showed that a total of 38.94 G of raw reads were obtained in RNA-seq, with an average of 6.49 G for each sample. And the Q30 of the six libraries was above 90%, average of GC content was 49.7%.At least 89% of the reads from the tested sample were aligned to the reference genome.A total of 16 013 genes were obtained through database comparison, of which 69 were up-regulated genes and 22 were down-regulated genes.GO enrichment analysis revealed that 13 841 genes were annotated into the GO database, including 78 differentially expressed genes.KEGG enrichment results showed that 22 differentially expressed genes were enriched in 38 pathways. All differentially expressed genes were screened and seven genes related to feather color phenotype were obtained: DCT, MLANA, SLC45A2, TYRP1, TRPM1, FAM174A and KIT. According to the results of qRT-PCR, the candidate genes were highly expressed in maroon feather quail, which was consistent with RNA-seq results.The seven candidate genes obtained in this study might be related to auto-sexing.

Key words: quail, feather color, auto-sexing, transcriptome, gene expression

中图分类号:

S839

王乾昆, 张小辉, 庞有志, 祁艳霞, 雷莹, 白俊艳, 户运奇, 赵毅威, 苑志文, 王涛. 基于RNA-seq技术挖掘鹌鹑羽色自别雌雄相关基因[J]. 浙江农业学报, 2022, 34(3): 498-506.

WANG Qiankun, ZHANG Xiaohui, PANG Youzhi, QI Yanxia, LEI Ying, BAI Junyan, HU Yunqi, ZHAO Yiwei, YUAN Zhiwen, WANG Tao. Screening of genes related to auto-sexing on feather color based on RNA-seq technology[J]. Acta Agriculturae Zhejiangensis, 2022, 34(3): 498-506.

图/表 8

表1 实时荧光定量PCR引物信息

Table 1 Primer information of real-time fluorescent quantitative PCR

基因 Gene	上游引物 Forward primer(5'→3')	下游引物 Reverse primer(5'→3')	产物长度 Product length/bp	退火温度 Annealing temperature/℃
KIT	TCATTCAAGGGCTGCTACCA	GCTTGTACGCTGCTATCCAC	197	59
DCT	TGATGAGTGGATGAAGCGGT	GCAGGTCAATGGCATAGGTG	170	59
MLANA	AAGGACGCACCTATTTCACA	GTTGCTCCCTCACTCACCAC	178	57
FAM174A	CAGTGCGGCTCAGAAGAAAT	TTGAGTGCATTCTGTTCCGA	163	60
SLC45A2	AATGGTACGAGTAAGCCG	GGTAGCGATAATGGGATG	118	54
TYRP1	TGAGGGACCTGCTTTCGT	GATTTCTGCGGATGGGAC	299	56
TRPM1	AGCAGGTCTTAGTGCCTCTTAC	TCCTTTATAGTCTTGGCTTTCC	156	56
EIF4E	GACTGCGTCAAGCAATCG	CAGAAGTACAAGACAAAGGCG	139	56

表2 测序数据质量预处理结果与基因组比对率

Table 2 Reference of sequencing data quality pretreatment and statistical results of genome comparison rate

样本名称 Sample	原始数据 Raw reads/M	过滤后数据 Clean reads/M	分析数据 Clean bases/G	Q30/%	GC含量 GC content/%	总比对读段 Total mapped reads	多位点比对读段 Multiple mapped	唯一比对读段 Uniquely mapped
BF1	46.16	44.44	6.19	91.25	49.74	40 037 396(90.08%)	1 269 910(2.86%)	38 767 486(87.23%)
BF2	47.33	45.61	6.35	91.47	49.78	41 019 585(89.93%)	1 239 218(2.72%)	39 780 367(87.21%)
BF3	49.89	48.13	6.68	91.78	49.87	43 274 927(89.91%)	1 321 985(2.75%)	41 952 942(87.16%)
LF1	50.57	48.39	6.67	90.75	50.04	43 467 145(89.83%)	1 365 790(2.82%)	42 101 355(87.01%)
LF2	47.27	45.35	6.35	90.96	49.21	41 127 739(90.68%)	1 255 232(2.77%)	39 872 507(87.92%)
LF3	49.85	47.80	6.70	90.90	49.23	43 289 872(90.56%)	1 332 581(2.79%)	41 957 291(87.77%)

图1 基因表达水平箱线图

Fig.1 Box plot of gene expression level

图2 基因表达火山图灰色为非显著差异表达的基因,红色表示显著上调基因,绿色表示显著下调基因。

Fig.2 Volcanic maps of gene expression Gray represented genes with no significant difference,red indicated up-regulation genes, and green indicated down-regulated genes.

图3 差异表达基因GO水平分布图

Fig.3 Map of GO levels of differentially expressed genes

图4 差异表达基因KEGG Level2 水平分布图横轴是注释到各 Level2 通路的上调(下调)差异表达基因和所有注释到KEGG通路的上调(下调)差异表达基因总数的比值(%),纵轴表示 Level2 Pathway 的名称,柱子右边数字代表注释到该 Level2 Pathway 的上调(下调)差异表达基因数量。红色表示显著上调基因,绿色表示显著下调基因。

Fig.4 KEGG Level2 distribution map ofdifferentially expressed gene Horizontal axis was the ratio (%) of the total number of up-regulated (down-regulated) differentially expressed genes annotated to each Level2 pathway and all up-regulated (down-regulated) genes annotated to the KEGG pathway, vertical axis represented the name of Level2 pathway, and the number on the right side of the column represented the number of up-regulated (down-regulated) differentially expressed genes annotated to the Level2 pathway.Red indicated up-regulation genes and green indicated down-regulated genes.

表3 差异表达基因显著富集的KEGG通路

Table 3 KEGG pathway for differentially expressed genes

通路注释 Description	通路ID Pathway accession	基因数量 Gene numbers	基因名称 Gene name	P值 P-vaule
黑色素生成Melanogenesis	gga04916	3	TYRP1,DCT,KIT	0.001 717
酪氨酸代谢Tyrosine metabolism	gga00350	2	TYRP1,DCT	0.003 700
神经活性配体受体相互作用Neuroactive ligand-receptor interaction	gga04080	4	C3,GRP,CCK,HRH3	0.010 209
苯丙氨酸、酪氨酸和伤寒杆菌生物合成	gga00400	1	PAH	0.014 978
Phenylalanine, tyrosine and typtohan biosynthesis
MAPK信号通路MAPK signaling pathway	gga04010	3	CACNGS,AMHR2,KIT	0.028 778
苯丙氨酸代谢Phenylalanine metabolism	gga00360	1	PAH	0.037 033

图5 白羽鹌鹑和栗羽朝鲜鹌鹑不同发育阶段胚胎中候选基因相对表达水平 *和**分别表示在P<0.05 和P<0.01 水平差异显著。

Fig.5 Relative expression levels of candidate genes in embryos of white feather and maroon feather Korean quails at different developmental stages * and ** meant significant differences at the levels of P<0.05 and P<0.01, respectively.

参考文献 31

[1]	NG C S, LI W H. Genetic and molecular basis of feather diversity in birds[J]. Genome Biology and Evolution, 2018, 10(10): 2572-2586. DOI URL
[2]	陈黎, 沈军达, 李国勤, 等. 不同羽色斑嘴野鸭毛囊中Tyr, Tyrp1及C-kit基因的表达及调控分析[J]. 浙江农业学报, 2015, 27(5): 729-733.
	CHEN L, SHEN J D, LI G Q, et al. Expression and regulation of Tyr, Tyrp1 and C-kit gene in feather bulbs of spot-billed ducks[J]. Acta AgriculturaeZhejiangensis, 2015, 27(5): 729-733. (in Chinese with English abstract)
[3]	SHULTZ A J, BURNS K J. The role of sexual and natural selection in shaping patterns of sexual dichromatism in the largest family of songbirds (Aves: Thraupidae)[J]. Evolution; International Journal of Organic Evolution, 2017, 71(4): 1061-1074. DOI URL
[4]	MCQUEEN A, NAIMO A C, TEUNISSEN N, et al. Bright birds are cautious: seasonally conspicuous plumage prompts risk avoidance by male superb fairy-wrens[J]. ProceedingsBiological Sciences, 2017, 284(1857): 20170446.
[5]	YANG T, JOEL W. Automatic feather sexing of poultry chicks using ultraviolet imaging:US6396938 [P/OL].(2002-05-28) [2021-07-12]. https://www.freepatentsonline.com/6396938.html .
[6]	YANG C W, DU H R, ZHANG Z R, et al. Genetic and breeding progress analysis on five pure lines of dahen broiler[J]. Agricultural Biotechnology, 2018, 7(5): 130-132.
[7]	ZHANG L, XU H D, LENG Q Y, et al. A genetics laboratory class to analyze early and late feather traits of chicken[J]. Hereditas, 2018, 40(3): 250-256.
[8]	GALVÁN I, RODRÍGUEZ-MARTÍNEZ S. A negative association between melanin-based plumage color heterogeneity and intensity in birds[J]. Physiological and Biochemical Zoology: PBZ, 2019, 92(3): 266-273. DOI URL
[9]	刘坤举, 张小辉, 庞有志, 等. 朝鲜鹌鹑GNAS基因表达、克隆及其多态性与羽色的相关性[J]. 浙江农业学报, 2020, 32(8): 1369-1377.
	LIU K J, ZHANG X H, PANG Y Z, et al. Relationship of plumage color with expression and polymorphism of GNAS gene in Korean quail[J]. Acta AgriculturaeZhejiangensis, 2020, 32(8): 1369-1377. (in Chinese with English abstract)
[10]	张小辉, 庞有志, 雷莹, 等. 鹌鹑性别的分子鉴定方法研究[J]. 中国家禽, 2020, 42(5): 20-23.
	ZHANG X H, PANG Y Z, LEI Y, et al. Study on molecular sex identification in quail[J]. China Poultry, 2020, 42(5): 20-23. (in Chinese with English abstract)
[11]	BOLGER A M, LOHSE M, USADEL B. Trimmomatic: a flexible trimmer for Illumina sequence data[J]. Bioinformatics, 2014, 30(15): 2114-2120. DOI URL
[12]	KIM D, LANGMEAD B, SALZBERG S L. HISAT: a fast spliced aligner with low memory requirements[J]. Nature Methods, 2015, 12(4): 357-360. DOI URL
[13]	LOVE M I, HUBER W, ANDERS S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J]. Genome Biology, 2014, 15(12): 550. DOI URL
[14]	YU G C, WANG L G, HAN Y Y, et al. clusterProfiler: an R package for comparing biological themes among gene clusters[J]. Omics: a Journal of Integrative Biology, 2012, 16(5): 284-287. DOI URL
[15]	CHEN T Z, ZHAO B L, LIU Y, et al. MITF-M regulates melanogenesis in mouse melanocytes[J]. Journal of Dermatological Science, 2018, 90(3): 253-262.
[16]	岳丹, 刘兴能, 熊和丽, 等. 兰坪乌骨绵羊黑色素及其候选基因研究进展[J]. 家畜生态学报, 2021, 42(5): 1-6.
	YUE D, LIU X N, XIONG H L, et al. Research progress on melanin and its candidate gene of Lanping black-bone sheep(Ovisaris)[J]. Journal of Domestic Animal Ecology, 2021, 42(5): 1-6. (in Chinese with English abstract)
[17]	黄海艳, 杜娟, 张杰, 等. 紫铆素通过AMPK通路促进正常人黑素细胞黑素合成的机制研究[J]. 中国中西医结合皮肤性病学杂志, 2020, 19(5): 406-409.
	HUANG H Y, DU J, ZHANG J, et al. Butin promotes melanocyte cytochrome synthesis in PIG1 cells through AMPK pathway[J]. Chinese Journal of Dermatovenereology of Integrated Traditional and Western Medicine, 2020, 19(5): 406-409. (in Chinese with English abstract)
[18]	WU Y, ZHANG Y L, HOU Z C, et al. Population genomic data reveal genes related to important traits of quail[J]. GigaScience, 2018, 7(5): giy049.
[19]	SUN L, ZHOU T, WAN Q H, et al. Transcriptome comparison reveals key components of nuptial plumage coloration in crested ibis[J]. Biomolecules, 2020, 10(6): 905. DOI URL
[20]	SULTANA H, SEO D, CHOI N R, et al. Identification of polymorphisms in MITF and DCT genes and their associations with plumage colors in Asian duck breeds[J]. Asian-Australasian Journal of Animal Sciences, 2018, 31(2): 180-188. DOI URL
[21]	XI Y, LIU H, LI L, et al. Transcriptome reveals multi pigmentation genes affecting dorsoventral pattern in avian body[J]. Frontiers in Cell and Developmental Biology, 2020, 8: 560766. DOI URL
[22]	YAO L D, BAO A, HONG W J, et al. Transcriptome profiling analysis reveals key genes of different coat color in sheep skin[J]. PeerJ, 2019, 7: e8077. DOI URL
[23]	LE L, ESCOBAR I E, HO T, et al. SLC45A2 protein stability and regulation of melanosome pH determine melanocyte pigmentation[J]. Molecular Biology of the Cell, 2020, 31(24): 2687-2702. DOI URL
[24]	DU Z, HUANG K, ZHAO J, et al. Comparative transcriptome analysis of raccoon dog skin to determine melanin content in hair and melanin distribution in skin[J]. Scientific Reports, 2017, 7: 40903. DOI URL
[25]	LAI X L, WICHERS H J, SOLER-LOPEZ M, et al. Phenylthiourea binding to human tyrosinase-related protein 1[J]. International Journal of Molecular Sciences, 2020, 21(3): 915. DOI URL
[26]	LI J Y, BED'HOM B, MARTHEY S, et al. A missense mutation in TYRP1 causes the chocolate plumage color in chicken and alters melanosome structure[J]. Pigment Cell & Melanoma Research, 2019, 32(3): 381-390.
[27]	WENG Z X, XU Y J, LI W N, et al. Genomic variations and signatures of selection in Wuhua yellow chicken[J]. PLoS One, 2020, 15(10): e0241137. DOI URL
[28]	JIA Q, HU S X, JIAO D X, et al. Synaptotagmin-4 promotes dendrite extension and melanogenesis in alpaca melanocytes by regulating Ca²⁺ influx via TRPM1 channels[J]. Cell Biochemistry and Function, 2020, 38(3): 275-282. DOI URL
[29]	XU Q, LIU X M, CHAO Z, et al. Transcriptomic analysis of coding genes and non-coding RNAs reveals complex regulatory networks underlying the black back and white belly coat phenotype in Chinese Wuzhishan pigs[J]. Genes, 2019, 10(3): 201. DOI URL
[30]	NIE C S, ZHANG Z B, ZHENG J X, et al. Genome-wide association study revealed genomic regions related to white/red earlobe color trait in the Rhode Island Red chickens[J]. BMC Genetics, 2016, 17(1): 115. DOI URL
[31]	JONES M, SERGEANT C, RICHARDSON M, et al. A non-synonymous SNP in exon 3 of the KIT gene is responsible for the classic grey phenotype in alpacas (Vicugna pacos)[J]. Animal Genetics, 2019, 50(5): 493-500. DOI URL

基于RNA-seq技术挖掘鹌鹑羽色自别雌雄相关基因

Screening of genes related to auto-sexing on feather color based on RNA-seq technology

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