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Genome-wide analysis of the role of DNA methylation ininbreeding depression of reproduction in Langshan chicken

Wei Han, Qian Xue, Guohui Li, Jianmei Yin, Huiyong Zhang,Yunfen Zhu, Weijie Xing, Yuxia Cao, Yijun Su, Kehua Wang,Jianmin Zou

PII: S0888-7543(19)30466-5

DOI: https://doi.org/10.1016/j.ygeno.2020.02.007

Reference: YGENO 9469

To appear in: Genomics

Received date: 23 July 2019

Revised date: 1 February 2020

Accepted date: 7 February 2020

Please cite this article as: W. Han, Q. Xue, G. Li, et al., Genome-wide analysis of therole of DNA methylation in inbreeding depression of reproduction in Langshan chicken,Genomics (2019), https://doi.org/10.1016/j.ygeno.2020.02.007

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© 2019 Published by Elsevier.

Original Article

Genome-wide analysis of the role of DNA methylation

in inbreeding depression of reproduction in Langshan

chicken

Wei Han †,Qian Xue

†, Guohui Li, Jianmei Yin, Huiyong Zhang, Yunfen Zhu, Weijie Xing, Yuxia

Cao, Yijun Su, Kehua Wang * and Jianmin Zou *

National Chickens Genetic Resources, Poultry institute, Chinese Academy of Agricultural Science, Yangzhou

225000, China; hanwei830@163.com † These authors contributed equally to this work and should be considered co-first authors

* Correspondence: sqbreeding@126.com (K.W.); jqszjm@163.com (J.Z.); Tel.: + 86-0514-8559-9078 (K.W);

+86-0514- 8559-9000 (J.Z.)

Abstract: Inbreeding depression of chicken reproduction is a major concern in the conservation of

chicken genetic resources . To investigate the potential DNA methylation sites involved in the

inbreeding depression of chicken reproduction, we carried out whole-genome b isulfite sequencing

(W GBS) of hypothalamus and ovary tissues from the strongly and weakly inbred Langshan chickens,

respectively. 5948 and 4593 differentially methylated regions (DMRs) were identified in the

hypothalamus and ovary between the strongly and weakly inbred Langshan chickens, respectively.

Large numbers of DMR-related genes (DMGs) were enriched in reproduction-related pathways. By

combin ing the WGBS and transcriptome data, two DMRs in SRD5A1 and CDC27 genes were inferred

as the most likely biomarkers of inbreeding depression of reproduction in Langshan chicken. Our study

provides the first systematic investigation of the DNA methylation changes in strongly inbred ch ickens,

and extends our understanding of the regulatory mechanisms underly ing inbreeding depression in

chicken reproduction.

Keywords: methylome; differentially methylated region; Langshan chicken; reproductive traits;

inbreeding depression

1. Introduction

‘Inbreeding’ refers to the mat ing of two genetically related indiv iduals. Negative and positive

effects can be induced by inbreeding [1-5]. The reduction in offspring fitness caused by inbreeding is

called ‘inbreeding depression’, which is a critical concern especially for the conservation of endangered

species [6]. According to past reports, inbreeding depression not only occurs in adaptive traits, such as

viability, fert ility, and disease susceptibility [3], but also in some production traits, such as egg, milk, and

meat production [7]. At present, two classic hypotheses explaining the genetic basis of inbreeding

depression have been proposed: the partial dominance hypothesis, which states that increased

homozygosity in inbred indiv iduals leads to the exposure of deleterious recessive alleles; and the

overdominance hypothesis, which suggests that heterozygotes are generally superior to homozygotes

across the genome [8]. Some other genetic theories, such as epistatic effects [9] and

pseudo-overdominance [10], have also been proposed to explain inbreeding depression in some cases.

However, none of these hypotheses conclusively explains all cases of inbreeding depression.

Inbreeding effects are reported to change with environmental stress [8,11-14]. Past studies have

shown that inbreeding depression appears more frequently in harsh environments [15,16] and slowly

disappears as stress is relieved [17,18]. The mechanis ms of environment-dependent inbreeding

depression can be explained in various ways [11,13], including the conditional expression of deleterious

alleles and the different levels of phenotypic plasticity present in inbred and outbred ind ividuals [19]. As

we know, the ep igenetic modification of DNA, which alters gene activ ity or regulates gene expression, is

also affected by environmental conditions and can modulate p lasticity [20]. Therefore, it has been

suggested that an epigenetic process is probably involved in the regulation of genes associated with

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inbreeding depression [21-23]. However, the epigenetic mechanism regulat ing inbreeding depression

has rarely been reported.

Ep igenetic changes are important variations in DNA that influence gene expression without directly

altering the DNA coding sequence [24]. One of the most widely investigated epigenetic modifications is

DNA methylation, which generally refers to the addition of a methyl group to the fifth carbon of cytosine

in a DNA sequence [25]. DNA methylation is involved in diverse bio logical processes [26] and plays a

critical ro le in regulating gene expression and maintaining genomic stability [27]. Several studies in

plants have shown that a close relationship exists between DNA methylation and inbreeding depression

[22,23,28]. Two studies, one in Scabiosa columbaria [23] and the other in Solanum chacoense [28], both

demonstrated that genome-wide DNA methylation was more extensive in inbred individuals than in

outbred individuals. One study in a vertebrate—Chinook salmon—also showed that methylation changes

in three specific genes were associated with inbreeding depression [29]. Despite all these findings, the

role of DNA methylat ion in inbreeding depression is not well understood. To the best of our knowledge,

no studies of epigenetically based inbreeding depression in poultry have been reported. With the

development of the next-generation sequencing technology, whole-genome bisulfite sequencing

(W GBS) provides an effective way to systematically analyze genome-wide changes in DNA methylation

with single-base resolution.

In this study, the Langshan chicken (a Chinese native chicken breed) was used as a material for

research. After the creation of strongly and weakly inbred chickens , obvious inbreeding depression was

detected in many traits, including v iability, fert ility, and some reproductive traits, in the strongly inbred

individuals. Because reproductive traits are ext remely important in poultry production, breeding, and

conservation, we collected tissues from the gonadal axis, including the hypothalamus and ovary, for

study. The WGBS technology was used to examine the DNA methylation patterns and to investigate the

changes in genome-wide DNA methylation between the strongly and weakly inbred Langshan chickens.

The aim of this study was to identify the regulatory mechanism involving DNA methylat ion that

underpins the inbreeding depression of reproduction in Langshan chicken. The results of this study

should provide a theoretical basis for future poultry breeding and the conservation of species resources.

2. Materials and Methods

2.1. Creation of Strongly and Weakly Inbred Chickens

A Chinese native chicken breed, the Langshan chicken, conserved at the National Chicken Genetic

Resources (Jiangsu, China), was selected as the model fo r study. Based on the conserved population, a

subpopulation was manually expanded according to the recorded pedigrees to create the F1 generation .

Then 160 individuals from 10 families were selected from the F1 generation according to their similar

reproductive performances (within ± 10% of the group mean value). 43 of the 160 individuals were used

for restriction-site-associated DNA sequencing (RAD-seq) (only one was sequenced to represent full

siblings). The molecular inbreeding coefficients (FIS) of these 43 individuals (ranging from −0.003 to

0.283) were then calculated based on the results of RAD-seq. FIS of the other indiv iduals was regarded

as the same as that of their fu ll sibling tested with RAD-seq. Individuals with FIS > 0.15 (58 indiv iduals

from six families) were crossed with full-sibling mating to produce the highly inbred F2 generation, and

individuals with FIS ≈ 0 (12 individuals) were selected and crossed, avoiding close relatives, to create the

weakly inbred F2 generation. All the chickens in this study were raised under the same standard

conditions in a closed chicken house with automat ic ventilation, temperature control and humid ity

adjustment, and fed ad libitum with commercial complete feed.

2.2. Estimating the Effects of Inbreeding on Reproductive Traits

Phenotypic data associated with reproductive performance were measured (including the t raits of

age at the first egg, weight of first egg, egg number at 300 days, and egg weight at 300 days) and recorded

as the F2 generations were raised. We observed obvious inbreeding depression in the strongly inbred

chicken group compared with the weakly inbred group. The inbreeding depression coefficients (IDCs)

were calculated as: IDC = (WL − WH)/WL, where WH and WL are the mean values for a particular trait in

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the strongly and weakly inbred chicken groups, respectively. The IDCs for age at first egg were

multip lied by −1 because high trait values indicate poor performance. The data are presented as the

means ± standard errors of the means (SEM), and a single-sample t test was used in the estimation of

differences. The traits with P < 0.05 were considered to be significantly depressed by inbreeding.

2.3. Individual Selection and Tissue Collection

Based on the estimate of inbreeding depression in the reproduction of the strongly inbred chicken

group, four healthy chickens with obviously depressed reproductive performance were selected from the

strongly inbred chicken group. Three chickens with reproductive traits around the group mean values were

selected from the weakly inbred chicken group. These seven chickens were immediately humanely

euthanized. The hypothalamus and ovary tissues were collected rapidly and snap-frozen in liquid nitrogen.

One of the hypothalamus tissue samples from the four highly inbred chickens was not collected due to

negligence, which must be noted here. Thus, a total of 13 tissue samples were collected and stored at −80

°C until DNA and RNA extraction.

All animal experiments were performed in accordance with the protocol of the Animal Use

Committee of the Chinese Ministry of Agriculture, and were approved by the Animal Care Committee of

Chinese Academy of Agricultural sciences, Beijing, China. All efforts were made to minimize animal

suffering.

2.4. DNA Isolation and WGBS

Genomic DNA was isolated from each of the collected tissues with the QIAamp Fast DNA Tissue Kit

(Qiagen, Dusseldorf, Germany), according to the manufacturer’s protocol, and quantified with an Agilent

2100 spectrophotometer (Agilent Technologies, Palo Alto, CA, USA). The DNA samples were fragmented

with sonication and subjected to bisulfite conversion. The Accel-NGS® Methyl-Seq DNA Library Kit

(Swift Biosciences, Ann Arbor, MI, USA) was used to attach adapters to the single-stranded DNA

fragments. The adapter-ligated DNA was enriched by eight cycles of PCR with the following thermal

profile: initial incubation at 95 °C for 2 min, eight cycles of 95 °C for 30 s, 65 °C for 20 s, and 72 °C for 45

s; and final incubation at 72 °C for 7 min. The reaction products were then purified with the Qiagen Gel

Purification Kit (Qiagen) and quantified with the Qubit dsDNA HS Assay Kit (Life Technologies,

Carlsbad, CA, USA). An Agilent 2100 spectrophotometer was used to test the integrity of the sequencing

library. We then performed paired-end 2 × 150 bp sequencing on the Illumina HiSeq 4000 platform

(Illumina, San Diego, CA, USA) housed at LC Sciences (Houston, TX, USA). A 30× sequence depth was

achieved for each library with WGBS.

2.5. Data Filtering and Identification of Methylated Cytosines

Cutadapt [30] and in-house perl scripts were used to remove the reads that contained adapter

contamination, low-quality bases, or undetermined bases. The sequence quality was then verified with

FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads that passed quality control

were mapped to the chicken reference genome

(ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/002/315/GCF_000002315.5_GRCg6a/GCF000002315.

5_GRCg6a_genomic.fna.gz) with WALT [31]. After alignment, the reads were further deduplicated with

samtool [32]. For each cytosine site, the DNA methylation level was determined as the ratio of the number

of reads supporting methylated C (mC) to the number of total reads (methylated and unmethylated) using

in-house perl scripts and MethPipe [33]. The binomial distribution was used to identify methylcytosines

with 0.01 false-discovery rate (FDR)-corrected P values. Each type of methylation context was considered:

cytosine–guanine (CG), CHG, or CHH (where H = adenosine [A], cytosine [C], or thymine [T]). We kept

the number of false-positive methylcytosine calls below 1% of the total number of methylcytosines

identified. The probability p in the binomial distribution B (n, p) was estimated from the number of cytosine

bases sequenced in the reference cytosine positions in the unmethylated mitochondrial genome (error rate:

nonconversion plus sequencing error frequency) [34,35]. All the identified methylcytosine sites were

required to have more than 3× sequence coverage. The methylcytosines presented in this study represent

the consensus data from the biological replicates.

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2.6. Identification of Differentially Methylated Regions (DMRs) and Functional Enrichment Analysis

DMRs were calculated with the R package-MethylKit [36]. A sliding-window approach (1000-bp

window size, 500-bp overlap) was used to screen for genomic regions that were differentially methylated

between the strongly and weakly inbred chicken groups. Only cytosine sites covered by at least three reads

were included in the analysis. Fisher’s exact test was performed for each window and the P values were

corrected for multip le tests with FDR. Windows with FDR < 0.05 and a d ifference of at least two -fold in the

methylation level (total mC/total C reads) between the strongly and weakly inbred chicken groups were

identified as DMRs. Integrative Genomics Viewer (IGV) was used to visualize the DMRs [37]. To explore

the function of the genes containing DMRs, called ‘DMR-related genes (DMGs)’, we annotated all of them

based on the Gene Ontology (GO) (http://www.geneontology.org/) and Kyoto Encyclopedia of Genes and

Genomes (KEGG) databases (http://www.genome.jp/kegg/pathway.html). A functional enrichment

analysis was performed on the basis of the annotated DMGs, with the GOseq R package [38]. GO terms

with P < 0.05 were considered significantly enriched. KEGG is a database resource for determining the

various biological pathways related to diverse gene functions. The pathways with P < 0.05 were considered

significantly enriched.

2.7. Total RNA Isolation and Transcriptome Analysis

The tissue samples used for WGBS were also used for a transcriptome analysis. Total RNA was

extracted with the TRIzol Total RNA Extraction Kits (Life Technologies, Carlsbad, CA, USA), according

to the manufacturer’s protocol. mRNA libraries were constructed with the TruSeq RNA Sample

Preparation Kit (Illumina, Inc., San Diego, CA, USA) and then sequenced in single lane with the HiSeq

X10 platform (Illumina, Inc.). After the raw data were filtered and their quality tested, the clean reads were

mapped to the reference genome with the TopHat software [39]. The mapped reads were used for further

transcript annotation and to calculate the expression levels using the “fragments per kilobase of exon per

million fragments mapped (FPKM)” method. The DESeq package was used to calculate the differences in

gene expression [40-42]. Genes with P < 0.05 and fold change ≥ 2 were considered to be differentially

expressed genes (DEGs).

2.8. Identification of Single Nucleotide Polymorphisms (SNPs)

Based on the mapping results of the reads to the reference genome, the SAMtools [43] software was

used to detect potential SNPs in the transcriptome lib raries. The accurate SNPs were identified when the

quality score (QC) of the variant calling not less than 20, the reads with more than 20× sequence depth

and the SNPs not within the range of 5bp around one gap. The annovarR was used to annotate the SNPs

in the genome.

2.9. Integrative Analysis of the Transcriptome Data and WGBS Data

Genes showing differential expression (fold change ≥ 2, P < 0.05) and differential methylation in the

promoter (P < 0.05) between the two chicken groups were picked out. Based on the expression levels and

methylation levels of each gene, correlation analysis was performed by calculating the Pearson correlation

coefficient (r value). Two-tailed t-test was used to test the significance of the r value. The correlation was

considered significant at P < 0.05.

3. Results

3.1. Effects of Inbreeding on Reproductive Traits in Langshan Chickens

As expected, the strongly inbred chicken group showed clearly reduced reproductive performance

compared with the weakly inbred chicken group. The IDCs for reproductive traits were calculated from

the data recorded. Inbreeding depression was observed for most reproductive traits, especially for age at

first egg, egg number at 300 days, and egg weight at 300 days (P < 0.01; Table1).

Table1. Inbreeding depression coefficients (IDCs) for different reproductive traits

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Traits IDC 1 (mean ± SEM)

Statistics

ttwo-tailed nfamilies P value

age at first egg 0.113±0.022 5.232 6 0.001

weight of first egg -0.007±0.029 -0.244 6 0.814

egg weight at 300 days 0.093±0.016 5.564 6 0.001

egg number at 300 days 0.150±0.043 3.513 6 0.010 1 Positive numbers indicate inbreeding depression.

3.2. Summary of WGBS Data

To investigate the role of DNA methylation in the inbreeding depression of chicken reproductive

traits and to characterize the DNA methylation profiles of the chickens’ gonadal axis tissues, the

hypothalamus and ovary tissues from the strongly and weakly inbred Langshan chickens were isolated

for W GBS. A total of 13 samples from four groups (three biological replicates from each group, except

that four biological replicates of the ovaries in the strongly inbred chicken group were used) were used

for bisulfite sequencing (BS-seq), which y ielded a sequencing depth of at least 30× and nearly 154.9

million clean reads per sample. On average, 69.14% of the clean reads were uniquely mapped to the

reference genome (Table S1). The mapped reads were used for the subs equent analysis. To confirm the

accuracy and reliability of the collected samples, a principal components analysis and correlation

analysis of the samples were performed. The results showed that the replicates from the same groups

clustered relatively closely (Figure S1a) and the samples from the same type of tissue correlated strongly

(hypothalamus, Pearson’s r = 0.956–0.997; ovary, Pearson’s r = 0.862–0.973) (Figure S1b), indicat ing

the high repeatability of our BS-Seq results.

3.3. DNA Methylation Profiles of Chicken Hypothalamus and Ovary

The global DNA methylation status of the two types of tissue in the strongly and weakly inbred

Langshan chickens was investigated. We found no significant differences between the two chicken

groups in either type of tissue. However, a comparison of the methylation levels in the two types of tissue

revealed that the global methylation levels in the hypothalamus (4.31% and 4.35% in the strongly and

weakly inbred chicken groups, respectively) were significantly higher than those in the ovary tissues

(3.49% and 3.48%, respectively; P < 0.05; Figure 1a). To determine the methylation status with

single-base resolution, the methylcytosine (mC) sites were then identified by scanning the whole

genomes. We detected an average of 6.15 and 10.32 million mCs in the hypothalamus and ovary tissues,

respectively, which represented about 2% and 3% of all the cytosines identified with sequencing (334.29

million) (Table S2). The classification of mC contexts (mCG, mCHH, or mCHG) for each type of tissue

showed that the proportion of mCGs was highest, followed by mCHHs and mCHGs (Figure 1b).

Interestingly, the proportion of non-mCG (mCHH and mCHG) in the hypothalamus (30.01%) was

approximately 2.5 times higher than that in the ovary (12.12%). Several past studies have also reported

greater proportions of non-mCG sites in some specific t issues, such as brain tissues and embryonic stem

cells [44,45], indicating the specific roles of non-mCG sites in these tissues.

By scanning the chromosomes of the chickens with a sliding-window approach, we determined the

distributions of mCG, mCHH, and mCHG sites in each chromosome. The same types of tissue from all

the individuals in this study showed similar distribution patterns (Figure S 2). The largest number of mCG

sites and only a few mCHH and mCHG sites were d istributed in each of chromosome, excluding the

mitochondrial DNA in the hypothalamus, in which the mCHH sites accounted for the highest proportion

of all mCs (Figure 1c,d). The global average DNA methylation levels of mCG, mCHG, and mCHH in the

whole genome were 51.3%, 1.15%, and 1.48% for the hypothalamus tissue, and 53.31%, 0.42%, and

0.46% for the ovary tissues in this study. These figures are consistent with the reports of previous studies

of the DNA methylation profiles of other vertebrates and other chicken t issues. This also indicates that

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mCG is the main type of mC and that this DNA methylation pattern is conserved in the vertebrates

[46-48].

We then divided the genome into different functional regions: promoter (−2.2 kb to 0.5 kb relative to

the transcription start site) [46, 49], exons, introns, and intergenic regions and calculated the methylation

levels in each (Figure 1e). We found no significant differences between the methylation levels of the

exonic and intronic regions (P > 0.05) in both the hypothalamus and ovary tissues. However, in the

ovary, the methylation levels of these two regions were significantly h igher than those of the promoter

and intergenic regions (P < 0.05). The promoter regions of the two tissues both had significantly higher

methylation levels (P < 0.05) than the intergenic regions. For all the genomic regions, the methylation

levels in the hypothalamus tissues were significantly higher than those if in the ovary tissues (P < 0.05),

which is consistent with the comparison of the global DNA methylation levels between the two types of

tissue in this study (Figure 1a).

Figure 1. DNA methylation status and distribution of methylcytosines (mCs) in the hypothalamus and

ovary tissues. (a) Global methylation status of the two types of tissue in strongly (Hinb) and weakly

(Linb) inbred chicken groups. (b) Proportion of mCs in the three contexts identified in the chicken

hypothalamus and ovary tissues. (c, d) Distribution of mCs in chromosomes of chicken hypothalamus

and ovary. ‘Linb_H1’ and ‘Linb_O1’ denote the hypothalamus and ovary tissues, respectively, from

individual ‘replicate 1’ in the weakly inbred chicken group. (e) Methylation levels in different genomic

regions. “*” and “**” indicate the difference is significant (P < 0.05) and extremely significant (P <

0.01), respectively.

3.4. DMRs in Strongly and Weakly Inbred Langshan Chickens

DMRs are important markers of ep igenetic changes, and are involved in the regulat ion of many

biological processes. In this study, to identify inbreeding-depression-related DMRs, the regions showing

significant differences in methylation (fold change ≥ 2 and FDR < 0.05) in strongly and weakly inbred

Langshan chickens were identified. A total of 5948 DMRs (3634 hyperDMRs and 2314 hypoDMRs) in

the hypothalamus and 4593 DMRs (2698 hyperDMRs and 1895 hypoDMRs) in the ovary were detected

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in the strongly inbred chickens relative to the weakly inbred chickens (Figure 2a, Tables S3, 4), although

the global methylation levels in the two types of tissue did not differ significantly between the groups.

The number of hyperDMRs was higher than the number of hypoDMRs in bo th the hypothalamus and

ovary. The same trend in methylation changes in these two types of tissue may suggest the potential role

of DNA methylat ion in the regulation of inbreeding depression in reproduction. Based on these DMRs,

we then identified 1245 and 766 DMGs in the hypothalamus and ovary, respectively (Figure 2b, Table

S5). Of all the DMGs identified, 182 were shared by the ovary and hypothalamus.

Figure 2. Differentially methylated regions (DMRs) and DMR-related genes (DMGs) in strongly and

weakly inbred Langshan chickens. (a) Numbers and distributions of hyper-DMRs (hypermethylated in

strongly inbred chickens relative to weakly inbred chickens) and hypo-DMRs (hypomethylated in

strongly inbred chickens relative to weakly inbred chickens) in the ovary and hypothalamus. (b) Venn

diagram of the numbers of DMGs in the ovary and hypothalamus.

3.5. Functional Enrichment Analyses of DMGs

To investigate the potential functions of the genes differentially methylated in the strongly and

weakly inbred Langshan chickens, functional enrichment analyses of the genes with DMRs were

performed based on their annotation in the GO and KEGG databases. In the chicken hypothalamus, 1112

and 830 of all the DMGs were annotated in the GO and KEGG databases, respectively. A GO enrichment

analysis showed that these genes were significantly enriched in a total of 175 GO terms (P < 0.05; Table

S6), many of which were associated with the bio logical processes of signal transduction and neurological

development, such as the regulation of dendritic spine development, positive regulation of synaptic

transmission, regulation of membrane potential, and dopamine metabolic process (Figure 3a). These

biological processes are closely associated with the neuroendocrine regulation of ch icken reproduction.

Many of the DMGs were also enriched in transcription-related terms, such as the biological processes of

transcription, regulation of transcription (DNA-templated), and negative regulation of NF-κB

transcription factor activity (Figure 3a), indicating that these changes in DNA methylat ion play

important roles in transcriptional regulation. The KEGG pathway analysis of the DMGs in the

hypothalamus showed that the signal transduction KEGG subclass was enriched in the highest number of

DMGs (Figure 3b). Among the top 20 pathways most enriched in DMGs, two pathways were related to

reproductive performance: the transforming growth factor β (TGF -β) signaling pathway and

progesterone-mediated oocyte maturation (Figure 3c). Another 15 reproduction-related pathways were

also enriched, including dopaminerg ic synapse, oocyte meiosis, oxytocin signaling pathway, and

gonadotropin releasing hormone (GnRH) signaling pathway. A total of 75 DMGs were detected in these

pathways (Table S7), including IGF-I, SMAD6, and SMAD7, which have been associated with animal

reproduction [50,51].

In the chicken ovary, 654 and 494 of the total DMGs were annotated in the GO and KEGG

databases, respectively. The GO enrichment analysis showed that 275 GO terms fo r biological processes

were significantly enriched (P < 0.05; Table S8). Among these GO terms, many involved neurological

system processes, such as the regulation of postsynaptic membrane potential, neuron development, ion

transport, positive regulation of neuron differentiat ion, and neuronal action potential (Figure 4a), which

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is consistent with the results for the chicken hypothalamus. More importantly, some biological processes

related to gonad development, sex hormone secretion, and germ cell development were also detected in

the enriched GO terms, including lutein izing hormone secretion, oocyte development, and female

genitalia development (Figure 4a). The KEGG enrichment analysis showed that a large number of genes

were enriched in the KEGG subclass signal transduction (Figure 4b), consistent with the results for the

hypothalamus. We found that 16 pathways associated with the regulation of reproduction were enriched

in the ovary, including circadian rhythm, oxytocin signaling pathway, and progesterone-mediated oocyte

maturation. A total of 45 genes were included in these pathways (Table S9).

Among the 17 and 16 reproduction-related pathways enriched in the hypothalamus and ovary,

respectively, we found that 11 were shared by both tissues (Figure 5a), which indicates that these

pathways may greatly affect the regulation of inbreeding depression in reproduction. However, only nine

of all the DMGs included in these reproduction-related pathways were shared by the hypothalamus and

ovary (Figure 5b). The DMRs in these nine genes observed in hypothalamus and ovary were visualized

using IGV and shown in Figures S3, 4, respectively. These results suggest that different sets of genes are

regulated by DNA methylation in the hypothalamus and ovary, and may collaboratively influence the

inbreeding depression of chicken reproduction in almost the same pathways.

Figure 3. Functional enrichment analyses of DMGs in the hypothalamus. (a) Significantly enriched Gene Ontology

(GO) terms for biological processes involved in signal transduction, neurological development, and transcriptional

regulation (P < 0.05). (b) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DMGs in

the hypothalamus. (c) Top 20 most-enriched pathways.

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Figure 4. Functional enrichment analysis of DMGs in the ovary. (a) Significantly enriched biological processes

involved in neurological system processes and regulation of reproduction (P < 0.05). (b) KEGG enrichment analysis

of DMGs in the ovary.

Figure 5. Shared reproduction-related pathways and DMGs in the chicken hypothalamus and ovary. (a) Shared

enriched pathways involved in the regulation of reproduction. (b) Shared DMGs included in these pathways.

3.6. Integrative Analysis of DNA Methylation and Gene Expression

To investigate the relationship between the DNA methylation status and gene expression, the

transcriptome data for the two types of tissue from the two chicken groups were generated with the

RNA-seq technology. A total of 96 and 298 DEGs (fold change ≥ 2, P < 0.05) were identified in the

hypothalamus and ovary, respectively, between the strongly and weakly inbred Langshan chickens

(Table S10). An integrative analysis of the transcriptome data and W GBS data showed that 80 and 167

DEGs in the hypothalamus and ovary, respectively, were differentially methylated (P < 0.05) in their

promoter regions. The methylation status of the promoter region is considered to be an important factor

regulating gene transcription. Therefore, based on these genes, a correlation analysis was performed

between the changes of their promoter methylation and their expression levels (Table S11, Figure 6).

And a total of 13 genes in hypothalamus were found to exh ibit significant correlations (P < 0.05)

between their promoter methylation and their expression levels , of which 6 showed highly negative

correlations (rmean = -0.872) and 7 showed highly positive correlations (rmean = 0.875) (Table S12). In the

ovary, we found 32 of the 167 genes showed significant correlations (P < 0.05) between their promoter

methylation and their expression, including 14 genes exhib iting highly negative correlations (rmean =

-0.90) and 18 genes exhib iting highly positive correlations (rmean = 0.89) (Table S12).. These results

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suggest that the influence of DNA methylation in the promoter region on gene expression is neither

exclusively positive nor negative, but may be determined by multiple factors.

In order to deepen our understanding on the role of DNA methylation in inbreeding depression of

Langshan chicken’s reproduction, we set three limiting factors for all the detected genes. The first factor

was that the gene should be a DEG between the strongly and weakly inbred chickens. The second was

that the gene was a DMR-related gene. The last condition was that the gene may be involved in the

regulation of chicken reproduction. Finally, two genes (SRD5A1 and CDC27) in the ovary were found to

meet the above three conditions. These two genes were both included in the pathways related to the

regulation of reproduction (Table S9). SRD5A1, one of the progesterone metabolizing enzyme genes, is

reportedly associated with female reproduction and fecundity defects [52]. Here, the expression of

SRD5A1 was significantly higher in the weakly inbred Langshan chickens than in the strongly inbred

chickens (Figure 7c). SRD5A1 also showed different methylation levels in its specific promoter reg ion

between the two groups (Figure 7a). The specific promoter region of CDC27 was hypermethylated in the

strongly inbred chickens than in the weakly inbred group (Figure 7b). Moreover, CDC27 showed

significantly higher expression in the strongly inbred chicken (Figure 7d). The results are shown in more

detail in Table 2. We predict that these two genes were strongly involved in the regulation of inbreeding

depression of reproduction in Langshan chicken.

Figure 6. The changes in methylation levels and expression levels of genes in chicken hypothalamus and ovary. (a)

The changes in the promoter methylation and expression levels of genes in chicken hypothalamus. (b) The changes

in the promoter methylation and expression levels of genes in chicken ovary . Blue and red dots represent the genes

exhibiting either inverse or equivalent relationships between their promoter methylation and their expression levels,

respectively. Jour

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Figure 7. Visualization of the DMRs in SRD5A1 and CDC27 genes using IGV (a, b) and their expression levels in

chicken ovary (c, d). Red and blue peaks indicate methylation levels in strongly (Hinb_O) and weakly (Linb _O)

inbred chickens, respectively.

Table 2. Two genes in ovary most likely involved in the regulation of inbreeding depression in chicken

reproduction.

Gene DMR

Hinb

methyl

level

Linb methyl

level

Genomic

feature1

Hinb

FPKM2

Linb

FPK

M

Fold-change

(Hinb/Linb)

SRD5A1

chr2(79732501

-79733500) 2.29% 3.06% promoter 0.9 2.19 0.41

CDC27

chr27(2835501

-2836500) 3.75% 3.10% promoter 64.96 22.77 2.85 1 ‘Genomic feature’ indicates the gene structure of the DMR area. 2 Fragments per kilobase of exon per

million fragments mapped (FPKM) were used to determine the differential expression of transcripts.

3.7. Statistical Analysis and Annotation of SNPs in Srongly and Weakly Inbred Langshan Chickens

We obtained an average of 2,335,633 and 2,442,507 SNPs from the hypothalamus transcriptome

lib raries of the strongly and weakly inbred Langshan chickens, respectively. And an average of

2,330,994 and 2,424,574 SNPs from the ovary transcriptome libraries were identified in the strongly

and weakly inbred Langshan chickens, respectively (Table S13). Thus, for the same chicken group,

almost the same number of SNPs were detected in the transcriptome libraries of two diffe rent tissues,

which indicated the identificat ions of the SNPs were reliab le. However, although the numbers of SNPs

in the weakly inbred chickens were slightly h igher than those in the strongly inbred chickens for the

both tissues, no significant differences (P ＞ 0.05) were found between them. We also analyzed the

distribution of the SNP types (Figure 8, Table S13). Of all the SNPs, the transition was 2.4 times the

transversion (Ti/Tv) and the heterozygosity rate was 0.42 on the average. And we found no significant

difference (P ＞ 0.05) on the quantity distribution of any SNP type between the two groups of chicken,

especially fo r the C>T SNPs which may be misidentified as unmethylated Cs after bisulfite conversion

of the DNA. Therefore , we inferred the SNPs within the population would not influence the results of

WGBS data analysis .

CDC27 and SRD5A1 genes were d ifferently expressed in the ovaries between the strongly and

weakly inbred Langshan chickens, which may be involved in the regulat ion of inbreeding depression of

chicken reproduction. To fu rther exp lore whether the SNPs in the two genes participated in the

regulation of inbreeding depression of reproduction in Langshan chicken, we detected the SNPs in the

two genes in the six ovary transcriptome lib raries. And we found an average of 183 and 191 SNPs in

CDC27 gene in the weakly and strongly inbred Langshan chickens, respectively, most of which were

located in the introns (Table S14). Generally, the SNPs in the 5’flank region and the non-synonymous

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SNPs in the exon may be more focused by researchers. In this study, for CDC27 gene, we detect only

one SNP in the 5’flank region (T3024224G) and a single non-synonymous SNP (G3042312A) in the

exon, which were the same in all the indiv iduals (Table S14). Thus, we inferred the two SNPs in

CDC27 may not be involved in the regulat ion of inbreeding depression of reproduction in Langshan

chicken. For SRD5A1 gene, we identified an average of 86 and 49 SNPs in the weakly and st rongly

inbred Langshan chickens, respectively (Table S15). The number of SNPs in SRD5A1 in the strongly

inbred individuals was significantly lower than that in the weakly inbred individuals (P < 0.05). We

observed no SNPs in the 5’flank reg ion of the gene in all the six indiv iduals. However, we found a

shared non-synonymous SNP (G80031632A) in the strongly inbred individuals, which was not present

in the weakly inbred group (Table S15).

Figure 8. Distribution of single nucleotide polymorphisms (SNPs) in hypothalamus (a) and ovary (b)

transcriptome libraries.4. Discussion

4. Discussion

Previous research has indicated that mCs not only occur in CpG dinucleotides but also in the

extensive non-CpG regions of the mammalian genome [53]. In this study, more than 10% and 30% of

mCs were found in non-CpG regions in the chicken ovary and hypothalamus, although the great majority

of mCs was observed in the CG context, which demonstrates that non-CpG can be considered an

alternative form of methylation. In part icular, we found a significantly higher proportion of non -mCG

sites in the chicken hypothalamus than in the chicken ovary. Past studies have shown that non -mCG sites

occur in h igher proportions in some tissues, such as the brain and embryonic stem cells [44,45], and

Kinde et al. reported that non-CpG methylation plays key ro les in the nervous system [54]. Our finding is

consistent with these reports, and indicates the specific ro le of non-mCG sites in the hypothalamus.

Therefore, mCs in all three contexts are considered to be potentially involved in inbreeding depression.

The global methylation levels in the strongly and weakly inbred Langshan chickens were calculated and

no significant differences were found between the two groups in either of the two t issue types tested.

Several previous studies have shown that DNA methylation is higher in inbred individuals than in

outbred individuals [23,28]. However, these studies all used the methylation-sensitive amplificat ion

polymorphis m (MSAP) technique, which can be insensitive at intermediate or low levels of methylation

[55], although it is a rapid and simple technique. Our study is the first to use an WGBS approach to study

the genome-wide DNA methylation changes in strongly and weakly inbred chickens, and its high

sensitivity allows the detection of differentially methylated sites. Although no significant differences in

the global methylation levels of the two chicken groups were detected, we found a large number of

DMRs between the two genomes in both tissue types. Importantly, the number of hyperDMRs was

higher than the number of hypoDMRs in both the hypothalamus and ovary tissues of the strongly inbred

chickens relative to the weakly inbred chickens, which is consistent to some extent with previous studies

[23,28, 29].

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We identified large numbers of DMR-related genes enriched in the biological processes of GO

terms involved in neurological development, signal transduction, and neurological system process,

which are necessary for the neuroendocrine regulation of chicken reproduction. Some biological

processes related to sex hormone secretion and germ-cell development were also significantly enriched

in the ovary (P < 0.05). These biological processes are closely related to the reproductive performance of

chickens. Therefore, a close relationship may exist between these DMRs and the inbreeding depression

of chicken reproduction. Moreover, 17 and 16 enriched pathways were related to the regulation of

chicken reproduction in the hypothalamus and ovary, respectively, which included 75 and 45

DMR-related genes in total, respectively. Interestingly, most of these pathways (11 pathways) were

affected in both the hypothalamus and ovary. Among the shared pathways, we found several pathways

related to sex hormones, such as the GnRH signaling, estrogen signaling, steroid hormone biosynthesis,

steroid biosynthesis, and oxytocin signaling pathways, which are essential fo r the regulation of

reproduction [56,57]. The pathways associated with the development of oocytes, including

progesterone-mediated oocyte maturation and oocyte meiosis, were also affected. Thyroid hormones not

only play key roles in promoting metabolis m in tissues and the development of organisms, but also have

an important function in maintaining the excitability of the nervous system [58]. Prev ious studies have

shown that thyroid disorders greatly affect the fertility in both sexes [59]. Dopamine is an excitatory

neurotransmitter that is related to sexual desire in animals. In this study, the thyroid hormone signaling

pathway and dopaminerg ic synapse were also enriched in DMR-related genes in both the hypothalamus

and ovary. Previous studies have suggested that the TGF-β signaling pathway is essential for oogenesis

[60,61], which was also affected by differential methylation in both the hypothalamus and ovary in this

study. These data suggest that these pathways may be regulated by DNA methylation and play important

roles in the regulation of inbreeding depression in chicken reproduction. However, only nine of all the

DMR-related genes included in these pathways were shared by the hypothalamus and ovary, which

implies that different sets of genes are regulated by DNA methylation in the hypothalamus and ovary,

and collaboratively influence the inbreeding depression of chicken reproduction in the same pathways.

According to previous reports, DNA methylation is an important mechanism regulating gene

transcription [62,63], especially DNA methylation in the promoter regions of genes [48]. In the present

study, many DMGs were enriched in transcription-related GO terms, confirming that changes in DNA

methylation play important roles in transcriptional regulation. However, the detailed regulatory

relationship between DNA methylation and gene expression has been controversial in many studies.

Most studies have shown a negative correlation between DNA methylation of the promoter region and

gene expression [48,63]. However, our data suggest that not only a negative but also a positive

correlation can exist between the DNA methylat ion of the promoter reg ion and gene expression. Liu et al.

reported a negative relat ionship between promoter CG methylation and gene expression levels that was

not observed for CHG or CHH methylation [64]. It has also been reported that methylation in various

genic regions is differentially associated with gene expression [64,65]. Therefore, the relationship

between DNA methylation in a specific reg ion in a particular sequence context and gene expression

remains to be clarified in future studies. Factors other than methylation, such as histone modificat ion and

long noncoding RNAs, may also influence gene expression [66,67], which may interfere with the results

of correlat ion analyses of DNA methylation and expression levels in some genes. Together, these data

suggest that the regulatory relationship between promoter methylation and gene expression is more

complex than previously thought.

After setting three limit ing factors, SRD5A1 and CDC27 genes were obtained by combin ing the

WGBS and transcriptome data. The expression level and promoter methylation of SRD5A1 were both

downregulated in the strongly inbred individuals relative to those in the weakly inbred indiv iduals. As

one of the progesterone-metabolizing enzyme, SRD5A1 reportedly participates in the regulation of

various psychophysiological phenomena [68]. SRD5A1 catalyzes the conversion of testosterone to the

more potent androgen, dihydrotestosterone, and is also associated with female reproduction and fetal loss

in mid gestation due to excessive estrogen [52]. CDC27 encodes one of the essential proteins required for

cell-cycle progression [69], and is suggested to be involved in cell proliferation and cell d ivision [70]. In

this study, both the promoter methylation and expression of CDC27 differed significantly between the

strongly and weakly inbred chicken groups. We infer that the DNA methylation and expression of the

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CDC27 gene might be involved in the inbreeding depression of chicken reproduction by regulating

oocyte meiosis and maturation. Therefore, we suggest that SRD5A1 and CDC27 are the DMR-related

genes most likely to be involved in the regulation of inbreeding depression in chicken reproduction. The

functional roles of DNA methylation in the inbreeding depress ion of chicken reproduction are highly

complex and d iverse. More research is required to determine the molecular mechanis ms in which these

DNA methylation sites are involved. This will be an enormous challenge for many years to come.

5. Conclusions

In summary, this study reports the genome-wide DNA methylat ion profiles in the hypothalamus

and ovary of inbred chickens, with single-base resolution. A distinctive methylation pattern was detected

in the hypothalamus. We identified numerous DMRs involved in the neuroendocrine regulat ion of

chicken reproduction and germ-cell development, which we infer p lay important roles in regulat ing the

inbreeding depression of reproduction in Langshan chicken. Our analyses also provide insight into the

complex relationship between the DNA methylation status of the promoter region and gene expression.

Two DMRs in the SRD5A1 and CDC27 genes were identified as likely biomarkers of inbreeding

depression of reproduction in Langshan chicken. These results of our study may supply some cues for

further investigation of the regulatory mechanisms of inbreeding depression in chicken reproduction.

Availability of data and materials: The raw data generated by BS-seq in this study have been submitted to NCBI

(SRA: SRP233032).

Authors’ contributions: Conceived the study and designed the project: WH, JZ, KW. Performed genetic and

bioinformatics analyses: QX, GL, YS. Contributed to collecting samples and measuring data: JY, HZ, YZ, WX, YC.

Wrote and revised the manuscript: QX, WH, JZ. All authors read and approved the final manuscript.

Competing interests: The authors declare no competing interests.

Acknowledgements: Thanks to every one making efforts for this study. This research was funded by the National

Natural Science Foundation of China (31572358), Six Talent Peaks Project in Jiangsu Province (NY-024) and the

Natural Science Foundation of Yangzhou (YZ2018098). This work was also funded by the National Broiler

Industrial and Technology System (CARS-41) and the Scientific Research Funds of Public Welfare Research Institutes in Jiangsu Province (BM2018026).

Abbreviations: WGBS, Whole-genome bisulfite sequencing; DMRs,Differentially methylated regions; RAD-seq,

Restriction-site-associated DNA sequencing; IDC, Inbreeding depression coefficient; TSS, Transcription start site;

DMGs, Differentially methylated genes; FPKM, Fragments per kilobase per million reads; BS-seq, Bisulfite

sequencing; MSAP, Methylation-sensitive amplification polymorphism; mC, Methylated cytosine.

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