Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (8): 1594-1605.doi: 10.3864/j.issn.0578-1752.2023.08.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Screening of Candidate Genes for Green Shell Egg Shell Color Traits in Chishui Black Bone Chicken Based on Transcriptome Sequencing

XIAO Tao1,3(), LI Hui1(), LUO Wei1, YE Tao1, YU Huan1, CHEN YouBo1, SHI YuShi1, ZHAO DePeng1, WU Yun2   

  1. 1 Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of Education/ Guizhou Provincial Key Laboratory of Animal Genetics, Breeding and Reproduction/College of Animal Science, Guizhou University, Guiyang 550025
    2 College of Biology and Agriculture Technology, Zunyi Normal University, Zunyi 563006, Guizhou
    3 Anshun City Animal Husbandary Technology Extension Station, Anshun 561000, Guizhou
  • Received:2021-12-13 Accepted:2022-11-16 Online:2023-04-16 Published:2023-04-23

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Abstract:

【Background】Green shell eggs are loved by consumers, and the green color of eggshell is an important reference index affecting the pricing and sales of green shell eggs in the market. The formation of green shell eggs is regulated by multiple genes, and the color of green shell varies. However, the molecular mechanism of green shell eggs is still unclear. In this study, transcriptomic sequencing was conducted on the eggshell gland tissue of Chishui black bone chicken, and the candidate genes and key signal pathways that regulated the depth and depth of eggshell color of green shell eggs were excavated, so as to explore the heritability of eggshell color, and to develop the seed selection and breeding of green shell eggs and improve economic benefits. 【Objective】The aim of this study was to investigate the genetic basis of Chishui black bone chicken and to identify and then screen them by SLCO1B3 genotyping, in order to provide new insights through molecular markers in the breeding planning of green-shelled hens, and to help control and improve the homogeneity of eggshell quality of Chishui black bone chicken in the later selection strategy. 【Method】A pure 280-day-old Chishui black bone chicken was used as the research object. Three hens were slaughtered to produce light green eggs (QL) and dark green eggs (SL), and the eggshell glands were collected and analyzed by RNA-SEQ technology. Selected differentially expressed genes (DEGs) closely related to eggshell color, and analyzed for GO and KEGG enrichment. Qrt-pcr was used to detect the transcriptional level changes of six candidate genes related to eggshell color to verify the reliability of transcriptomic data. 【Result】A total of 93 DEGs were screened in SL group and QL group, among which 59 genes were up-regulated and 34 genes were down-regulated in SL group. DEGs was annotated to GO database for comparison, and sodium ion transport, negative ion binding, and sarcoplasmic reticulum were mainly enriched significantly. KEGG analysis showed that aldosterone regulated sodium reabsorption, enriched mineral absorption, linoleic acid metabolism and other signal pathways. The results of QRT-PCR showed that the expression trend of these genes was consistent with the transcriptome sequencing results. 【Conclusion】By functional analysis, TF gene, SCNN1 gene, CYP450 gene, SLC gene and FAM gene, as well as the sodium reabsorption signaling pathway regulated by aldosterone, might be involved in eggshell pigment synthesis, transport and deposition. These genes and signal pathways might be candidate genes and key signal pathways affecting the different shades of green in eggshell of Chishui black bone chicken.

Key words: transcriptome sequencing, Chishui black bone chicken, green egg, eggshell color, qRT-PCR

Fig. 1

SLCO1B3 parting figure"

Table 1

Specific primers of differentially expressed genes for qRT-PCR"

基因
Gene		 引物序列
Primer sequence (5'-3')		 产物大小
Amplicon length (bp)		 退火温度
The annealing temperature (℃)
SLC34A2 F: TCGGTCCGTTCACTCTGTTG
R: GCCACGTTGCCTTTGTGATT		 164 60.0
SLC20A1 F: CAGAAAGGCGTCAAATGGTC
R: CACGGTGCAGGCGTAGAA		 180 54.0
CYP27A1 F: AGGACTTTCGTCTGGCTCT
R: CTCCGCATCGGGTATTT		 185 58.6
TF F: AGATGCCATTAGCTTGGAC
R: GGTTGTGGAGCCTTCAGTA		 109 60.0
FAM136A F: GCACGCTTCACTGCTCTGACA
R: ACCCGTCCCGCATCCTCTT		 153 57.0
SLC6A9 F: CGTACCTCTGCTACCGCAAT
R: CACGCGTCATGGACACAAAG		 261 60.0
β-actin F: CAGCAAGCAGGAGTACGATG
R: ATAAAGCCATGCCAATCTCG		 145 60.0

Table 2

Statistical analysis table of eggshell color"

样本 Sample △L* △a*
SL-1 56.48±0.52 -6.01±0.36B
SL-2 57.28±1.71 -6.40±0.88B
SL-3 55.45±2.32 -7.01±0.84B
QL-1 59.85±1.19 -3.93±0.29A
QL-2 59.18±2.85 -4.27±0.17A
QL-3 60.61±0.61 -4.16±0.07A

Table 3

Quality test information of eggshell gland RNA samples"

样本
Sample		 浓度
Concentration(ng·μL-1)		 体积
Volume(μL)		 总量
Total amount(μg)		 RIN
RIN		 OD260/280
OD260/280		 OD260/230
OD260/230		 等级
Level
SL-1 651.5 20 13.0 9.5 1.99 1.93 A
SL-2 1729.2 20 34.6 9.5 2.02 1.63 A
SL-3 487.0 20 9.70 9.4 2.00 1.83 A
QL-1 993.2 20 19.9 8.9 2.01 1.87 A
QL-2 850.8 20 17.0 8.5 2.02 1.67 A
QL-3 1002.0 20 20.0 9.0 1.98 1.72 A

Table 4

Statistical analysis for sequence quality of RNA-seq libraries"

样品
Sample		 原始读数
Raw reads		 质控后序列
Clean reads		 质控后序列量
Clean bases (G)		 Q20
(%)		 Q30
(%)		 GC
(%)
QL-1 57502212 56416296 7.75 97.26 92.45 47.99
QL-2 48412576 47722420 6.58 97.21 92.39 47.50
QL-3 42574086 42043646 5.79 97.41 92.83 47.56
SL-1 52836592 51864304 7.14 97.26 92.52 47.83
SL-2 57938312 57099094 7.87 97.23 92.39 47.07
SL-3 58071340 56266562 7.75 97.20 92.37 47.45

Table 5

Alignment information of RNA-seq libraries"

样品
Sample		 质控序列条数
Total reads		 比对率
Total mapped
(%)		 多比对率
Multiple mapped (%)		 单一比对率
Unique mapped (%)		 正链比对率
Reads map
to '+' (%)		 负链比对率
Reads map
to '-' (%)		 基因组全比对率
Non-splice
reads (%)		 外显子单比对率
Splice reads
(%)
QL-1 56416296 91.54 2.29 89.25 44.59 44.66 60.74 28.51
QL-2 47722420 92.65 1.84 90.81 45.37 45.44 60.99 29.82
QL-3 42043646 92.13 2.91 89.22 44.57 44.64 60.29 28.93
SL-1 51864304 91.82 2.35 89.47 44.68 44.79 61.17 28.30
SL-2 57099094 92.45 2.39 90.06 44.99 45.07 61.28 28.78
SL-3 56266562 91.72 2.05 89.68 44.81 44.87 61.88 27.80

Fig. 2

Volcanic map of SL VS QL expressed genes The abscissa represents the change of gene expression multiple in different experimental groups, and the ordinate represents the statistical significance of the change of gene expression level. The blue on the left represents significantly down-regulated genes, the red on the right represents significantly up-regulated genes, and the gray represents genes with no significant difference"

Fig. 3

Comparison of candidate differentially expressed genes The abscissa represents the gene name, and the ordinate represents the gene expression multiple"

Fig. 4

Histogram of the top 30 GO enrichment of SL vs. QL differentially expressed genes The abscissa is the Pvalue after processing -log10, and the items are arranged in ascending order according to the P value. BP represents biological process, MF represents molecular function, CC represents cell component, and ordinate represents enrichment function description"

Fig. 5

Bubble map of KEGG enrichment pathway of Slvs. QL differentially expressed gene The abscissa represents the enrichment factor corresponding to the pathway. The ordinate represents the pathway name, the P value calculated by the logarithmic function is represented by the color of the point, the smaller the point, the closer to red, and the number of differential genes contained in the pathway is represented by the scatter size"

Table 6

Correlation analysis table between candidate DEGs and eggshell color"

TF CYP27A1 CYP3A5 SLC6A9 SLC34A2 SLC20A1 FAM136A SCNN1A SCNN1G ESC(△a*)
TF 1
CYP27A1 0.599 1
CYP3A5 -0.219 -0.577 1
CLC6A9 0.099 0.738 0.557 1
SLC34A22 -0.511 -0.883* -0.44 -0.767 1
SLC20A1 -0.489 -0.904* -0.527 -0.776 0.665 1
FAM136A 0.277 0.899* 0.692 0.926** -0.907* -0.813* 1
SCNN1A -0.618 -0.921** -0.550 -0.609 0.655 0.943** -0.756 1
SCNN1G -0.370 -0.937** -0.610 -0.893* 0.913* 0.857* -0.964** 0.773 1
ESC (△a*) 0.561 0.953** 0.465 0.830* -0.820* -0.965** 0.882* -0.898* -0.938** 1

Table 7

SLCO1B3 expression"

基因名称Gene_id SL_readcount QL_readcount
SLCO1B3 6061.83 4436.93
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