Genetic characteristics of Jiaji Duck by whole genome re-sequencing.

Jiaji Duck (JJ) is a Muscovy duck species that possesses many superior characteristics, and it has become an important genetic resource in China. However, to date, its genetic characteristics and genetic relationship with other duck breeds have not been explored yet, which greatly limits the utilization of JJ. In the present study, we investigated the genome sequences of 15 individual ducks representing five different duck populations, including JJ, French Muscovy duck (FF), mallard (YD), hong duck (HD) and Beijing duck (BD). Moreover, we investigated the characteristics of JJ-specific single nucleotide polymorphisms (SNPs) and compared the genome sequences of JJ vs. YD and JJ vs. BD using integrated strategies, including mutation detection, selective screening, and Gene Ontology (GO) analysis. More than 40 Gb of clean data were obtained for each population (mean coverage of 13.46 Gb per individual). A total number of 22,481,367 SNPs and 4,156,829 small insertion-deletions (Indels) were identified for the five duck populations, which could be used as molecular markers in breeding and utilization of JJ. Moreover, we identified 1,447,932 JJ-specific SNPs, and found that genes covering at least one JJ-specific SNP mainly involved in protein phosphorylation and dephosphorylation, as well as DNA modification. Phylogenetic tree and principal components analysis (PCA) revealed that the genetic relationship of JJ was closest to FF, while it was farthest to BD. A total of 120 and 111 genes were identified as positive selection genes for JJ vs. BD and JJ vs. YD, respectively. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the positive selection genes for JJ vs. BD ducks mainly involved in pigmentation, muscle contraction and stretch, gland secretion, and immunology, while the positive selection genes obtained from JJ vs. YD ducks mainly involved in embryo development, muscle contraction and stretch, and gland secretion. Taken together, our findings enabled us to better understand the characteristics of JJ and provided a molecular basis for the breeding and hybrid utilization of JJ in the future.

1. Introduction

Jiaji Duck (JJ), a Muscovy duck variety, is initially raised at Jiaji Town, Qionghai County, Hainan Province. JJ duck is covered by black and white plumage and has been believed to be originated from Southeast Asia. JJ has been raised at Jiaji Town for more than 200 years, and it has become an important genetic resource in China. JJ duck has various superior characteristics, such as fast growth, heat resistance, strong adaptability, high lean meat rate, and good meat quality. However, only few studies on JJ have been reported, while none of them is related to the genetic characteristics of JJ and genetic relationship between JJ and other duck breeds. This greatly limits the breeding and hybrid utilization of JJ. The approach of whole genome re-sequencing (WGRS) has been proved to be a very powerful tool in genetic evaluation and genetic relationship exploration of various animals, including pigs [1], chickens [2], cattle [3], sheep [4], dogs [5] and rabbits [6]. For ducks, the genome sequences have been constructed in 2013 [7], providing a platform for the research of ducks using WGRS method and promoting the related exploration of ducks. Xu et al. have explored the genetic signatures of artificial selection using genome WGRS data [8]. Zhang et al. have indicated the genomic variation in Peking duck populations using WGRS data [9]. Zhou et al. have identified the genes associated with body size and plumage color in ducks using WGRS and genome-wide association study (GWAS) technologies [10]. However, no report has investigated the genetic characteristics of JJ and its relationship with other duck populations using WGRS method.

In this study, we selected five duck populations that are different in a couple of characteristics. The first one was JJ, which has been introduced as above-mentioned. Similar to JJ, French Muscovy duck (FF) is another Muscovy duck population that has been introduced into Hainan Province since 1990’s. However, FF grows faster than JJ, and it is fully covered by white plumage. Mallard (YD), widely accepted as the ancestor of domestic duck [11], is used to confirm whether JJ and FF are also derived from YD. Beijing duck (BD) has been subjected to intensive selection to provide the raw material for Beijing roast duck dishes. Hong duck (HD) is the hybrid offspring of Beijing duck and Sheldrake, which has higher quality of meat relative to Beijing duck and faster growth rate compared with Sheldrake. Four out of the five duck populations (JJ, FF, HD and BD) have been recorded by Animal Genetic Resources in China Poultry [12]. To characterize the genetic background of JJ duck and explore its genetic relationship with the above-listed duck breeds, we selected 15 ducks representing the five duck populations with three individuals for each population, and the whole genome of each duck was individually sequenced. Moreover, we, for the first time, investigated the genetic characteristics of JJ through analyzing JJ-specific single nucleotide polymorphisms (SNPs) and compared the genomic sequences of these five duck populations. Collectively, our results provided basis for the reasonable utilization of JJ in the future.

2. Materials and methods

2.1. Sample preparation

In the present study, 15 adult female ducks representing five different duck populations were selected, with three ducks for each population. Genomic DNA was extracted from about 5 mL venous blood for each individual as previously described [13]. After blood collection, the birds were housed for further analysis. JJ and FF ducks, obtained from Hainan Chuanwei Muscovy Duck Breeding Co., Ltd., were sampled. BD and HD ducks were developed by Institute of Animal Science, Chinese Academy of Agricultural Sciences. YD was raised by the Ji’ao Austrian Agricultural Science and Technology Co., Ltd. in Fenghua, Zhejiang Province.

2.2. Library construction and sequencing

Libraries were generated for each individual using standard Illumina sequencing protocols. The constructed libraries were sequenced as 150-bp paired-end reads on Illumina sequencing platform (HiSeqTM 4000), and more than 13 Gb clean data were generated for each individual (Table 1).

Table 1

The re-sequencing and mapping results.

Items	Clean Reads (M)	Clean Bases (Gb)	Mapped Bases (Gb)	Mapping Rate (%)	1Dup. Rate (%)	2Uniq. Rate (%)	Mean Depth	3Cov. Rate (%)
BD1	88.67	13.30	12.51	94.04	25.38	94.37	12.09	96.51
BD2	90.83	13.63	12.79	93.86	24.93	93.88	12.39	96.85
BD3	91.35	13.70	12.85	93.80	25.04	93.86	12.45	96.85
FF1	90.35	13.55	12.45	91.84	23.53	93.96	12.32	94.10
FF2	91.14	13.67	12.48	91.29	23.54	93.65	12.43	94.01
FF3	90.41	13.56	12.48	92.06	23.99	93.93	12.33	94.09
HD1	90.78	13.62	12.82	94.13	7.81	94.41	12.38	97.00
HD2	90.07	13.51	12.91	95.52	24.99	95.23	12.28	96.51
HD3	84.46	12.67	12.01	94.76	24.29	94.52	11.52	96.80
JJ1	90.21	13.53	12.41	91.71	24.40	93.60	12.30	94.11
JJ2	90.26	13.54	12.35	91.24	23.06	93.61	12.31	94.02
JJ3	89.62	13.44	12.18	90.61	23.70	92.97	12.22	94.13
YD1	89.14	13.37	12.57	93.98	25.15	94.42	12.15	96.50
YD2	88.75	13.31	12.51	93.94	25.49	94.29	12.10	96.46
YD3	89.99	13.50	12.72	94.25	24.95	94.51	12.27	96.50
Means	89.74	13.46	12.54	93.14	23.35	94.08	12.24	95.63
Total	1346.03	201.90	188.03				195.78

1 Duplication rate

2 Unique rate

3 The coverage rate, referring to the percentage of the mapped bases divide by the total bases of duck genome, the mapped bases means bases that covered by cleans bases more than 1 X. BD1- BD3, Beijing duck, individual 1-individual 3; FF1- FF3, French Muscovy duck, individual 1- individual 3; HD1- HD3, Hong duck, individual 1-individual 3; JJ1- JJ3, Jiaji Duck, individual 1-individual 3; YD1- YD3, mallard, individual 1- individual 3.

2.3. Read mapping

Sequencing adaptors were first removed using Trim Galore version 0.3.7 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Subsequently, the trimmed data (clean data) were mapped to the duck genome assembly (ensemble version: BGI_duck_1.0) using the Burrows-Wheeler Alignment tool (BWA) [14] with default settings. The uniquely mapped reads were used for downstream analysis.

2.4. Detection and annotation of SNPs and small insertion–deletions (Indels)

For each individual of a breed, the genome analysis toolkit (GATK) was used to detect SNPs and Indels (1–50 bp) with GATK best practices method using RealignerTargetCreator, IndelRealigner, HaplotypeCaller and GenotypeGVCFs [15]. Then SelectVariants was used to separate SNPs, Indels and other variants [16, 17]. The SNPs and Indels from the three individuals were merged to form the SNP and Indel sets of this breed. The merged SNPs and Indels were filtered using the parameters recommended by the GATK mentor, and those variants with ultra-high (> 500) or ultra-low (< 4) coverage were discarded. The remaining variants were used for downstream analysis.

SnpEff [18] was used to annotate SNPs and Indels against the reference genome annotation to identify the effects of sequence variants on gene functionality.

2.5. The genetic characteristics of JJ obtained through analyzing JJ-specific SNPs and the related genes

If an SNP was only found in JJ population, it was considered as JJ-specific SNP without considering whether it was a homozygous or heterozygous SNP. To enhance the sufficiency, a JJ-specific SNP must be found in two of the three individuals of JJ ducks. According to these criteria, the JJ-specific SNPs were identified using a homemade pearl script. The SNPs that were located on the exons of duck genes were then selected and then classified into different types based on the annotation of SNPs, such as synonymous mutation SNPs, nonsynonymous mutation SNPs, stop-gain SNPs and stop-loss SNPs. Finally, the genes covering at least one JJ-specific SNP that was located on exons of genes were picked out, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed as described in section 2.8.

2.6. Genetic structure analysis of five duck populations

The genetic relationships of the five duck populations were inferred through phylogenetic tree analysis and principal components analysis (PCA). SNPs for each duck population with a minor allele frequency (MAF) ≤ 0.1 were removed, and the remaining SNPs were used for phylogenetic tree analysis and PCA. For phylogenetic tree analysis, NJ method of PHYLIP software was used to construct phylogenetic tree [19] with the default settings, and Newick Utilities software was used to generate phylogenetic tree graphics [20]. For PCA, EIGENSOFT software [21] was used to infer genetic relationships among the five duck populations, and the first two principal components were used to construct the PCA figure.

2.7. Analysis of selective sweeps, positive selection genes

Selective sweeps occur when the frequency of beneficial genetic variants is increased as a result of positive selection along with nearby linked genetic variants [9]. Generally, positive selection can lead to reduced heterozygosity (Hp) of the selected population and increased fixation index (Fst) between populations around the selected site [6]. In order to define candidate regions undergoing positive selection, outlier approach in combination with Hp estimates was used for the five sequenced duck populations with estimates of Fst between duck populations. Since the current duck reference genome was assembled in sub-chromosomal scaffolds [22], only scaffolds>150 kb (1,226 scaffolds) were used for the downstream analysis to ensure the accuracy of our calculation of Hp and Fst, which was similar to studies on rabbits [6] and chickens [2]. In addition, to reduce the number of false positive windows, windows with > 20 SNPs were used in downstream analysis. The Hp values and Fst values of the genomic regions were calculated using a 100 kb-sliding window (step = 50 kb), Z-transform Hp (ZHp) and FST (ZFst) as previously described [5]. Windows with both their ZHp and ZFst values falling in the top 5% were identified as the positive selection regions. Genes that were overlapped with or harbored by positive selection regions were defined as positive selection genes and were used in downstream analysis.

2.8. GO and KEGG analyses of positive selection genes

The duck GO annotation information from the Ensembl Genome Browser was used to perform GO enrichment analysis of genes under positive selection, and the whole genome set of protein-coding genes in the annotation was used as the background. For each query, represented GO terms were tested against the background using the GOstats Bioconductor R package (https://www.r-project.org/) [23]. To further understand the biological functions of the positive selection genes, KEGG [24] was used to perform pathway enrichment analysis.

2.10. Funding

This work was financially supported by Key Research and Development Programs of Hainan Province (Grant no. ZDYF2019053, ZDYF2018224), Chinese Modern Technology System of Agricultural Industry (Grant no. CARS-42-50), and Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (Grant no. 1630032017034).

2.11. Ethics statement

The animal-related handling and sampling procedures were approved by the Animal Care and Use Committee of Chinese Academy of Tropical Agricultural Sciences (CATAS), and great efforts were made to minimize the suffering of animals in accordance with recommendations of European Commission (1997). All methods were conducted in accordance with relevant guidelines. Venous blood was kindly collected to ameliorate the suffering of ducks.

3. Results and discussion

3.1. A number of variants are found in duck genome

WGRS data were obtained from five duck populations with three samples of each population (JJ1, JJ2, JJ3; BD1, BD2, BD3; FF1, FF2, FF3; HD1, HD2, HD3; YD1, YD2, YD3) using HiSeq TM 4000 system. After quality control [25], averagely 13.46 Gb (12.24 X) data were obtained for each individual, > 40 Gb (> 36 X) data were obtained for each duck population, and a total of 201.90 Gb (195.78 X) high-quality data remained (Table 1). Subsequently, the high-quality data were mapped to the A. platyrhynchos genome (BGI_duck_1.0), resulting in an average mapping rate of 93.14% and an average coverage rate of 95.63% for each individual (Table 1). The average mapping rate of this study (93.14%) was higher compared with our previous report (89.71%) [8], indicating a better quality of sequencing data used in this study.

In this study, SNPs and Indels of each duck population were identified using GATK according to previous studies [26, 27]. The SNPs and Indels for codons with multiple alleles were removed. Since the duck genome only contains scaffolds without chromosomes, we could not compare the genome differences among the five duck populations at the chromosomal level. For JJ duck, a total number of 22,481,367 SNPs and 4,156,829 Indels were identified after filtration. For SNPs, ~50% of total SNPs were located in intergenic regions (11,042,847 SNPs, 49.12%), and 45.31% of total SNPs were located in intronic regions (10,186,307). SNPs located in exonic regions (303,498) only accounted for 1.35% of the total SNPs (Fig 1 and Table 2). Similar to SNPs, 50.02% of total Indels (2,079,246) were located in intergenic regions, 44.02% of total Indels (1,874,314) were located in intronic regions, and only 1.44% of total Indels (59,858) were harbored by exonic regions (Fig 1 and Table 2). These results indicated that the majority of the identified mutations were located in non-coding sequences (intergenic regions or intronic regions), and only a small proportion of them fell in exon sequence, showing that only very few variants along with duck genome could exert functional effects on protein function. Our results were consistent with those of Xu et al. [8] in ducks and Carneiro et al. in rabbits [6].

Fig 1

The percentage of SNPs and Indels of JJ located in different regions of duck genome.

(A) The percentage of SNPs which fell in different regions of duck genome; (B) The percentage of Indels which fell in different regions of duck genome.

Table 2

Annotation of identified SNPs and Indels for JJ duck population.

Items	SNPs		Indels
	Number	Percent (%)	Number	Percent (%)
Total	22,481,367	100	4,156,829	100
UTR5	123,648	0.55	17,459	0.42
UTR3	312,491	1.39	60,274	1.45
exonic	303,498	1.35	59,858	1.44
splicing	2,248	0.01	1,247	0.03
exonic; splicing	0	0	0	0
upstream	249,543	1.11	46,141	1.11
downstream	245,047	1.09	59,443	1.43
upstream	13,489	0.06	2,494	0.06
intronic	10,186,307	45.31	1,829,836	44.02
intergenic	11,042,847	49.12	2,079,246	50.02
ncRNA_UTR3	0	0	0	0
ncRNA_UTR5	0	0	0	0
ncRNA_exonic	0	0	0	0
ncRNA_splicing	0	0	0	0
ncRNA_intronic	2,248	0.01	831	0.02
other	0	0	0	0

SNPs mean single nucleotide polymorphisms; Indels mean short insertions and deletions.

3.2. The JJ-specific SNPs

The specific SNPs are usually the basic genetic characteristics of a species. A high throughput SNP marker can offer strong accuracy for genotyping, which can be used in marker-assisted selection [28, 29]. In this study, we screened the JJ-specific SNPs and found 1,447,932 specific SNPs, of which only 56,651 SNPs (accounting for 4% of the total specific SNPs) were located in the exons of duck genes. Among these SNPs located in the exons, there were 36,517 synonymous mutation SNPs (64.46%), suggesting that the majority of the SNPs were random mutations that did not lead to the changes of amino acids. In addition, there were 19,817 non-synonymous mutation SNPs, which could lead to the changes of amino acids. Moreover, we detected 285 stop-gain SNPs and 11 stop-loss SNPs, indicating that SNP mutations resulting in alteration or loss of gene function were rare events, which was consistent with previous reports in rabbits [6], pigs [30], and chickens [2].

The genes covering at least one JJ-specific non-synonymous SNP could lead to functional differences of these genes for JJ compared with other duck populations, and herein might represent the specificity of JJ. In this study, we selected genes covering at least one JJ-specific non-synonymous SNP, and performed GO and KEGG analysis on these genes to explore JJ-specific genetic characteristics. The biological process (BP) of GO analysis demonstrated that genes covering at least one JJ-specific non-synonymous SNP mainly played roles in protein phosphorylation and dephosphorylation, as well as DNA modification (Fig 2A and S1 Table). We also carried out KEGG analysis and constructed the significant enrichment graphics of q-values via the 20 most significant pathways (Fig 2B and S2 Table). The results indicated that Fanconi anemia pathway was the most significant pathway, while the metabolic pathway covered the most genes. These findings might reflect the specificity of JJ in Fanconi anemia and metabolism.

Fig 2

The functional enrichment graphics for genes covering at least one JJ-specific non-synonymous SNP.

(A) Directed acyclic graph of GO enrichment analysis. Colors from light to dark represent significant levels from weak to strong, respectively. (B) The significant enrichment graphics of q-values using the 20 most significant pathways.

3.3. Genetic relationships between JJ and other duck populations

A phylogenetic tree is a branching diagram or “tree” showing the inferred evolutionary relationships among various biological species or subspecies based on the similarities and differences of their genetic characteristics [8]. In this study, we constructed the phylogenetic tree to illustrate the genetic relationships among the five duck populations (Fig 3A). The results showed that the individuals of JJ were firstly clustered into a class, followed by FF, YD, HD, and BD sequentially, indicating that FF was the closest population genetically related to JJ, followed by YD, HD and BD. Interestingly, the genetic relationship between JJ and YD was closer than that between YD and HD or BD. Since YD (mallard) was considered as the ancestor of HD and BD, we hypothesized that JJ and FF were differently originated from HD and BD. In addition, we used PCA to infer genetic relationships among the five duck populations in another aspect (Fig 3B). The PCA results revealed that individuals of JJ and FF were clustered into one group, suggesting the closest genetic relationship of JJ and FF, which was consistent with the results of phylogenetic tree analysis. Individuals in the remaining three populations were clustered into three separate groups.

Fig 3

Population genetic structure of five duck populations.

(A) Results of phylogenetic tree analysis. A kind of color represents a duck population, and a big dot represents a duck individual. (B) PCA plot of duck populations. Eigenvector 1 and 2 explained 83.56% and 2.23% of the observed variance, respectively.

3.4. Many genomic regions and genes associated with positive selective between JJ and other duck populations are found

Selective sweeps occur when the frequency of beneficial genetic variants is increased as a result of positive selection along with nearby linked genetic variants [9]. Given that the current duck reference genome is assembled in sub-chromosomal scaffolds [22], only scaffolds>150 kb (1,226 scaffolds) were used for the downstream analysis in our study to ensure the accuracy of Hp and Fst calculation, which was similar to studies on rabbits [6] and chickens [2]. We calculated the Hp and Fst values of the genomic regions using a 100 kb-sliding window (step = 50 kb). Windows with both their ZHp and ZFst values in the top 5% were identified as the positive selection regions. Since individuals in JJ and FF were clustered into one group, and genetic structure analysis suggested that HD was very close to BD in genetics, we only explored the positive selection regions in two comparisons, namely JJ vs. YD and JJ vs. BD. We selected the longest 100 scaffolds to construct the ZHp and ZFst figures to show the basic information. For JJ vs. YD comparison, 79 positive selection regions were obtained, which covered 111 genes (Fig 4 and S3 Table). For JJ vs. BD comparison, 76 positive selection regions were obtained, which covered 120 genes (Fig 5 and S4 Table).

Fig 4

Selective sweep analysis of the duck genome of JJ-YD.

(A), Distribution of ZFst in JJ-YD, ZHp in JJ, and ZHp in YD for all 100-kb windows. Bins of ZFst and ZHp are presented along the x axes. (B), The positive end of the ZFst(JJ-YD) distribution and the negative end of the ZHp(JJ) distribution.

Fig 5

Selective sweep analysis of the duck genome of JJ-BD.

(A), Distribution of ZFst(JJ-BD), ZHp(JJ) and ZHp(BD) for all 100-kb windows. Bins of ZFst and ZHp are presented along the x axes. (B), The positive end of the ZFst(JJ-BD) distribution and the negative end of the ZHp(JJ) distribution.

The GO project is a collaborative effort to develop and use ontology to support biologically meaningful annotation of genes and their products [31]. In this study, we performed GO analysis to describe the properties of positive selection genes of JJ vs. BD ducks and those of JJ vs. YD ducks. For positive selection genes of JJ vs. BD ducks, 232 significant GO terms were enriched (S5 Table). Of which, the 20 most significant terms (Fig 5) were mainly involved in pigmentation (terms included melanocyte differentiation, pigment cell differentiation, developmental pigmentation), muscle contraction and stretch (muscle contraction, muscle system process, detection of muscle stretch, Wnt signaling pathway [32, 33], skeletal muscle thin filament assembly), and immunology (negative regulation of mast cell apoptotic process, mast cell apoptotic process, regulation of mast cell apoptotic process, mast cell homeostasis) [34, 35]. The results were consistent with the differences in feather color, athletic ability, and immunity between JJ and YD. For positive selection genes of JJ vs. YD ducks, 152 significant GO terms were obtained (S6 Table). The positive selection genes of JJ vs. YD ducks were mainly involved in embryo development because eight GO terms were related to embryo development in the 20 most significant terms, including wing disc development, instar larval or pupal development, larval development, imaginal disc-derived wing morphogenesis, wing disc morphogenesis, imaginal disc morphogenesis, post-embryonic appendage morphogenesis, and instar larval or pupal morphogenesis. Subsequently, terms of muscle contraction and stretch (muscle contraction, response to muscle stretch, and detection of muscle stretch) were also enriched (Fig 6). These results highlighted the differences in embryo development and muscle contraction and stretch between JJ and YD.

Fig 6

The 20 most significant annotation pathways of genes located in the top 5% ZFst and ZHp.

(A) for JJ vs. BD ducks, (B) for JJ vs. YD ducks. The x-axis shows the -log10(P-value) of each pathway.

KEGG is a "computer representation" of the biological system [24], and the KEGG database can be utilized for modeling, simulation, browsing and retrieval of data. In this study, the significant pathways (P<0.05) for JJ vs. BD ducks and JJ vs. YD ducks were selected (Table 3). The results showed that the pathways related to gland secretion were significantly enriched for JJ vs. BD and JJ vs. YD. For example, five out of eight significantly enriched pathways were associated with gland secretion for JJ vs. BD, while five out of 11 significantly enriched pathways for JJ vs. YD ducks were related with gland secretion. The results of KEGG analysis showed that gland secretion was the main difference between JJ and BD as well as YD.

Table 3

Significantly enriched KEGG pathways for JJ vs. BD ducks and JJ vs. YD ducks.

JJ vs. BD
pathway names	p-value	genes
Hypertrophic cardiomyopathy (HCM)	0.002826	gene4589, gene16244, gene13399
Salivary secretion	0.003638	gene13401, gene6509, gene12554
Insulin secretion	0.005433	gene14642, gene6509, gene12554
Pancreatic secretion	0.006374	gene13401, gene6509, gene12554
Bile secretion	0.03918	gene14642, gene12554
Dilated cardiomyopathy	0.0404	gene4589, gene13399
Gastric acid secretion	0.0404	gene13401, gene12554
PI3K-Akt signaling pathway	0.042055	gene3589, gene9998, gene16244
JJ vs. YD
Salivary secretion	0.002882	gene13401, gene6509, gene12554
Insulin secretion	0.004314	gene14642, gene6509, gene12554
Pancreatic secretion	0.005068	gene13401, gene6509, gene12554
Aldosterone-regulated sodium reabsorption	0.010621	gene9288, gene12554
Jak-STAT signaling pathway	0.015062	gene9287, gene3589, gene9286
Ubiquitin mediated proteolysis	0.024208	gene12556, gene12904, gene9290
Hypertrophic cardiomyopathy (HCM)	0.028686	gene4589 gene13399
Bile secretion	0.03379	gene14642, gene12554
Cytokine-cytokine receptor interaction	0.034	gene9287, gene3589, gene9286
Dilated cardiomyopathy	0.034851	gene4589, gene13399
Gastric acid secretion	0.034851	gene13401, gene12554

Grey represents the comparison of JJ vs. BD and the secretion related pathways in the significant enriched KEGG pathways. Light green means the comparison of JJ vs. BD and the secretion related pathways in the significant enriched KEGG pathways.

Collectively, we, for the first time, explored the genetic characteristics of JJ and its genetic relationship with other four duck breeds using WGRS data. The findings of this study would benefit our understanding of the genetic characteristics of JJ and provide valuable references for the utilization of JJ breeds in the future.

The BP of GO analysis for genes covering at least one JJ-specific non-synonymous SNP.

(XLSX)

Click here for additional data file.

The KEGG analysis for genes covering at least one JJ-specific non-synonymous SNP.

(XLSX)

Click here for additional data file.

Positive selection genomic regions and genes for JJ vs. YD ducks.

(XLSX)

Click here for additional data file.

Positive selection genomic regions and genes for JJ vs. BD ducks.

(XLSX)

Click here for additional data file.

Significantly enriched GO terms for positive genes of JJ vs. BD ducks.

(XLSX)

Click here for additional data file.

Significantly enriched GO terms for positive genes of JJ vs. YD ducks.

(XLSX)

Click here for additional data file.

15 Oct 2019

PONE-D-19-18411

Genetic characteristics of Jiaji Duck as revealed by genome resequencing data

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: This paper involves the sequencing of several individuals (3 per breed) spanning 5 duck breeds for the purpose of identify SNPs specific to the Jaiji duck (JJ) and to then broadly characterize the genes that are under positive selection.

The basis of the paper is sound and overall the science seems reasonable with the caveats below.

However there are two major issues that need to be corrected:

1) Numerous issues with grammar that range from trivial to humorous to obscuring the meaning of the sentence. I would recommend edited specifically for correct use of English.

2) The description of how the SNPs were called and how JJ specific snps were identified is incomplete. For example, it is not clear if SNPs are called on each individual or on the pooled reads per breed (or something else). This makes understanding the methodology difficult because the parameter space is not very clear. The authors describe that JJ specific SNPs were identified using a homemade perl script but neither the approach nor the criteria used in the script were described at all. Combined, these issues would make replicating the study impossible. It also leaves to the imagination what a JJ specific SNP would be. Are they homozygous and found in all three individuals or is it sufficient if they are in one individual? What about SNPs on the sex chromosomes? This should be associated with a discussion of what categories of SNPs would or would not be identified given this approach. I also feel that this homemade script should be provided.

Some other more more minor points:

1) line 50 and 63 : please explicitly define utilization in these contexts. Sure information can be useful but used to do what? There is no reason for mystery here but the vagueness of the statement is quite noticeable.

2) Figure 1 does not really show anything nor does it indicate which breeds are in panel a or b. Table 2 should probably show SNP counts/frequencies for every breed. I presume that table 2 only shows the JJ breed data.

3) Figure 2b - label the x-axis

4) Figure 3a - there is no metric on the tree; Figure 3ab - these really don't show much - a Venn diagram (or equivalent) showing the number of SNPs per breed would be a much better way of demonstrating this information

5) Figure 4 & 5 and associated text - what portion of the genome is covered by the sweep analysis? Its not clear why a 5% cutoff was set for sweeps - this seems to imply that there WERE selective sweeps but what if there wasn't a sweep? there should probably be a supplemental table that explicitly defines these regions.

Reviewer #2: In this study, the authors selected five duck populations with three individuals of each population to sequence the whole genome of each duck. Using these resequencing data, the authors explored out 1,447,932 JJ-specific SNPs which can be used as molecular markers, performed the genetic characteristics research of JJ and the genetic relationship between JJ and other duck breeds. Overall, the results of this paper are very interesting and it is an excellent paper. However, there are still some minor problems that should be modified.

1. The results of genetic relationship among these five duck populations should be mentioned in the abstract;

2. The tables presented in this paper should be changed to three-line tables;

3. please list the age and sex for the sequencing duck;

4. The description of sequencer is inconsistent in this paper;

5. The language should be improved by a native English speaker in order to eliminate grammatical and spelling errors and to conform to correct scientific English. I listed some of these errors below.

Line 30-33: “However, to date, the genetic characteristics and genetic relationship with other duck breeds has not been explored yet,” is not clear. The authors should refer to whose “genetic characteristics and genetic relationship with other duck breeds”;

Line 91: “other” should be changed to “the”;

Line 102: A comma should be placed behind “JJ and FF ducks”;

Line 251-252: The authors should delete the “on these genes”;

Line 275: should add a comma between FF and YD;

Line 298:should add blank space between the number and KB.

Reviewer #3: In my view, the manuscript is meaningful for breeding and genetics in duck. But , there are several shortcomings.

L8： What is the ‘d’ means?

L55-63：Jiaji Duck, if it is a definite breed, I think you should provide the information of variety approval, like the record of Breeds of Domestic Animal and Poultry in China or other published researches. So should other breeds you used in your research.

L64-75：The introduction of technical methods, NGS or others, is not the point. You can review the research progress in duck sequencing.

Why you choose these four breeds (France muscovy ducks, mallard, hong duck, and beijing duck) and did a phylogenetic tree for the all five breeds?

Results and discussion：Only basic and necessary results were showed.

What puzzles me is this, you list your results and say they are meaningful, but I can not find any deep discussion. After your sequencing and analysis, some specific SNPs and Indel, GO and KEGG project can illustrate what? I think readers are look forward to in-depth discussions and the specific and functional point.

Tables：All your tables do not meet the standards (Three-line table) , required notes missing. In Table 3, what is the meaning of different background notes?

Reference ：Format is also nonstandard. Journal Title, abbreviation, PubMed Central PMCID and so on.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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A: We have provided the volume of the blood samples collected for use in our study and marked in red in unmarked manuscript (LINE 94-95). That is “Genomic DNA was extracted from about 5 mL venous blood for each individual as previously described [13]”.

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A: We inserted the sentence “After blood collection, the birds were housed for further analysis” in the revised manuscript (LINE 96) and marked in red.

4. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing.

A: According to PLOS requirements, we comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. We then deposit the data obtained in this study to the Short Read Archive (SRA). The statements in revised manuscript were “2.9. Data availability

Duck genomic resequencing datasets from JJ, FF, YD, BD and HD were uploaded to the Short Read Archive (SRA) under the accession number of PRJNA549423” (LINE 174-177).

5) Thank you for stating the following in the Competing Interests section:

[The authors have declared that no competing interests exist.].

We note that one or more of the authors are employed by a commercial company: Hainan Chuanwei Muscovy breeding Ltd

i. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

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If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

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Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests) . If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.

Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

A: We have provided the Funding Statement and Competing Interests Statement in our cover letter according to the journal requirements.

Response to reviewers

Response to Reviewer #1

This paper involves the sequencing of several individuals (3 per breed) spanning 5 duck breeds for the purpose of identify SNPs specific to the Jaiji duck (JJ) and to then broadly characterize the genes that are under positive selection.

The basis of the paper is sound and overall the science seems reasonable with the caveats below.

However there are two major issues that need to be corrected:

1) Numerous issues with grammar that range from trivial to humorous to obscuring the meaning of the sentence. I would recommend edited specifically for correct use of English.

A: The revised manuscript was polished by Kyle Schachtschneider, a post doctor student from the University of Illinois in America. The additions and changes are marked in red in the revised text.

2) The description of how the SNPs were called and how JJ specific snps were identified is incomplete. For example, it is not clear if SNPs are called on each individual or on the pooled reads per breed (or something else). This makes understanding the methodology difficult because the parameter space is not very clear. The authors describe that JJ specific SNPs were identified using a homemade perl script but neither the approach nor the criteria used in the script were described at all. Combined, these issues would make replicating the study impossible. It also leaves to the imagination what a JJ specific SNP would be. Are they homozygous and found in all three individuals or is it sufficient if they are in one individual? What about SNPs on the sex chromosomes? This should be associated with a discussion of what categories of SNPs would or would not be identified given this approach. I also feel that this homemade script should be provided.

A: We appreciate the reviewer to point out the defects in calling the SNPs and identifying the JJ-specific SNPs. In the revised manuscript, we added the necessary information in calling the SNPs and identified the JJ-specific SNPs. The revised contents are listed as follow.

For SNPs calling, “2.4. Detection and annotation of SNPs and small insertion–deletions (Indels)

For each individual of a breed, the genome analysis toolkit (GATK) was used to detect SNPs and Indels (1–50 bp) with GATK best practices method using RealignerTargetCreator, IndelRealigner, HaplotypeCaller and GenotypeGVCFs [15]. Then SelectVariants was used to separate SNPs, Indels and other variants [16, 17]. The SNPs and Indels from the three individuals were merged to form the SNP and Indel sets of this breed. The merged SNPs and Indels were filtered using the parameters recommended by the GATK mentor, and those variants with ultra-high (> 500) or ultra-low (< 4) coverage were discarded. The remaining variants were used for downstream analysis.” (LINE 112-121)

For the identification of JJ-specific SNPs, “2.5. The genetic characteristics of JJ obtained through analyzing JJ-specific SNPs and the related genes

If an SNP was only found in JJ population, it was was considered as JJ-specific SNP without considering whether it was a homozygous or heterozygous SNP. To enhance the sufficiency, a JJ-specific SNP must be found in two of the three individuals of JJ ducks. According to these criteria, the JJ-specific SNPs were identified using a homemade pearl script. The SNPs that were located on the exons of duck genes were then selected and then classified into different types according to the annotation of SNPs, such as synonymous mutation SNPs, nonsynonymous mutation SNPs, stop-gain SNPs and stop-loss SNPs. Finally, the genes covering at least one JJ-specific SNP that was located on exons of genes were picked out, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed as described in section 2.8 of this study.”. (LINE 124-136)

In the current paper, we performed our analysis based on duck genome assembly (ensemble version: BGI_duck_1.076). In this version of duck genome assembly, there are only scaffolds without chromosomes. Therefore, we did not explore the SNPs information on the sex chromosomes. We added the correlated discussion of what categories of SNPs would or would not be identified in our revised manuscript. We listed the related context. “Since the duck genome only contains scaffolds without chromosomes, we could not compare the genome differences among the five duck populations at the chromosomal level.” (LINE 213-215).

Some other more minor points:

1) line 50 and 63 : please explicitly define utilization in these contexts. Sure information can be useful but used to do what? There is no reason for mystery here but the vagueness of the statement is quite noticeable.

A: This is a constructive suggestion. We changed “utilization” in line 50 and 63 of the initial manuscript to “breeding and hybrid utilization” in revised manuscript and marked in red (LINE 47 and 60).

2) Figure 1 does not really show anything nor does it indicate which breeds are in panel a or b. Table 2 should probably show SNP counts/frequencies for every breed. I presume that table 2 only shows the JJ breed data.

A: We think the suggestion is very suitable. Therefore, we replaced the data by the JJ breed data in Table 2. Correspondingly, the figure 1 was drawn using the SNPs and Indels frequencies of JJ duck in the revised manuscript. Figure 1A shows the distribution of SNPs along with duck genome, and Figure 1B shows the distribution of Indels. In addition, the related context that descripted Table 2 and Figure 1 was changed correspondingly. The changed items were listed as follow.

Table 2. Annotation of identified SNPs and Indels for JJ duck population

Items SNPs Indels

Number Percent (%) Number Percent (%)

Total 22,481,367 100 4,156,829 100

UTR5 123,648 0.55 17,459 0.42

UTR3 312,491 1.39 60,274 1.45

exonic 303,498 1.35 59,858 1.44

splicing 2,248 0.01 1,247 0.03

exonic;splicing 0 0 0 0

upstream 249,543 1.11 46,141 1.11

downstream 245,047 1.09 59,443 1.43

upstream 13,489 0.06 2,494 0.06

intronic 10,186,307 45.31 1,829,836 44.02

intergenic 11,042,847 49.12 2,079,246 50.02

ncRNA_UTR3 0 0 0 0

ncRNA_UTR5 0 0 0 0

ncRNA_exonic 0 0 0 0

ncRNA_splicing 0 0 0 0

ncRNA_intronic 2,248 0.01 831 0.02

other 0 0 0 0

Note: SNPs mean single nucleotide polymorphisms; Indels mean short insertions and deletions.

The related context that descripted Table 2 and Figure 1. “In this study, SNPs and Indels of each duck population were identified using GATK according to previous studies [26, 27]. The SNPs and Indels for codons with multiple alleles were removed. Since the duck genome only contains scaffolds without chromosomes, we could not compare the genome differences among the five duck populations at the chromosomal level. For JJ duck, a total number of 22,481,367 SNPs and 4,156,829 Indels were identified after filtration. For SNPs, ~50% of total SNPs were located in intergenic regions (11,042,847 SNPs, 49.12%), and 45.31% of total SNPs were located in intronic regions (10,186,307). SNPs located in exonic regions (303,498) only accounted for 1.35% of the total SNPs (Fig. 1 and Table 2). Similar to SNPs, 50.02% of total Indels (2,079,246) were located in intergenic regions, 44.02% of total Indels (1,874,314) were located in intronic regions, and only 1.44% of total Indels (59,858) were harbored by exonic regions (Fig. 1 and Table 2). These results indicated that the majority of the identified mutations were located in non-coding sequences (intergenic regions or intronic regions), and only a small proportion of them fell in exon sequence, showing that only very few variants along with duck genome could exert functional effects on protein function. Our results were consistent with those of Xu et al. [8] in ducks and Carneiro et al. in rabbits [6].”

3) Figure 2b - label the x-axis

A: We labeled the x-axis for Figure 2b in the revised manuscript, which was listed as follow.

4) Figure 3a - there is no metric on the tree; Figure 3ab - these really don't show much - a Venn diagram (or equivalent) showing the number of SNPs per breed would be a much better way of demonstrating this information

A: We have added the metric on the tree (Fig 3A, we listed the revised Fig 3 at the end of question). Fig 3, including two panels (Fig 3A and Fig 3B), were used to illustrate the genetic relationships between JJ and other duck populations. Fig 3A, the results of phylogenetic tree analysis, indicated the evolutionary relationships of the five duck populations obviously. Fig 3B, the results of principal components analysis (PCA), also clearly showed the genetic relationships of these five duck populations. Therefore, we think Fig 3 can clearly illustrate the genetic relationships between JJ and other duck populations.

In our opinion, a Venn diagram can clearly show the SNPs number per breed which is really a better way to demonstrate specific SNP number and shared SNP number for the five duck populations. Thus, we demonstrated the genetic relationships between JJ and other duck populations using Fig 3 of this paper without using a Venn diagram.

5) Figure 4 & 5 and associated text - what portion of the genome is covered by the sweep analysis? Its not clear why a 5% cutoff was set for sweeps - this seems to imply that there WERE selective sweeps but what if there wasn't a sweep? there should probably be a supplemental table that explicitly defines these regions.

A: Previous studies have found that "selection on a single advantageous mutation affects patterns of genetic variation at nearby loci, causing (i) a reduction in heterozygosity, (ii) a skewed allele frequency distribution and (iii) an excess of high frequency derived alleles across a region surrounding the selected allele (Stephan, W. Philos T R Soc B 365, 1245-1253)". Hence a reduction in heterozygosity (Hp) and an increase in fixation index (Fst) were accordingly used as signatures to define regions underwent natural or artificial selection. This method has been well accepted and widely adopted. For example, in the study of the polygenic basis for phenotypic change during domestication of rabbit, the authors defined selection regions using an Fst-H outlier approach and set Fst > 0.35 and H< 0.05 as the criteria to define a selected region (Carneiro et al. Science, 2014, 345(6200):1074-1079). In addition, the authors of the study of dog’s adaptation to a starch rich diet defined selection regions using a ZFst (and/or ZHp) threshold of 5 (Axelsson et al. Nature, 2013, 495(7441):360-364.). In the current paper, we applied the Fst-H outlier approach with setting both ZHp values and ZFst values falling in the top 5% to identify the positive selection regions (those mean selective sweeps). Focusing on those regions, and by analyzing the function of their associated genes, we can get a clear idea on which aspects of biological functionality are among the most preferred targets of selection.

As a result, we provided the positive selection genomic regions and genes for JJ vs. YD ducks and for JJ vs. BD ducks as the supplemental Table 3 and supplemental Table 4.

References：

Stephan W. Genetic hitchhiking versus background selection: the controversy and its implications[J]. Philosophical Transactions of the Royal Society B Biological Sciences, 2010, 365(1544):1245-1253.

Carneiro M, Rubin CJ, Di Palma F et al. Rabbit genome analysis reveals a polygenic basis for phenotypic change during domestication[J]. Science, 2014, 345(6200):1074-1079.

Axelsson E, Ratnakumar A, Arendt M L, et al. The genomic signature of dog domestication reveals adaptation to a starch-rich diet [J]. Nature, 2013, 495(7441):360-364.

Response to Reviewer #2

In this study, the authors selected five duck populations with three individuals of each population to sequence the whole genome of each duck. Using these resequencing data, the authors explored out 1,447,932 JJ-specific SNPs which can be used as molecular markers, performed the genetic characteristics research of JJ and the genetic relationship between JJ and other duck breeds. Overall, the results of this paper are very interesting and it is an excellent paper. However, there are still some minor problems that should be modified.

1. The results of genetic relationship among these five duck populations should be mentioned in the abstract;

A: We added the results of genetic relationship analysis in the abstract which is listed as “Phylogenetic tree and principal components analysis (PCA) revealed that the genetic relationship of JJ was closest to FF, while it was farthest to BD.” in the revised manuscript (LINE 37-39).

2. The tables presented in this paper should be changed to three-line tables;

A: We changed all the tables in the paper to three-line tables.

3. please list the age and sex for the sequencing duck;

A: We added “adult female” in “Sample preparation” in the revised manuscript (LINE 93).

4. The description of sequencer is inconsistent in this paper;

A: We read through our paper again and found the sequencer was written as “HiSeqTM 4000” in line 109 of initial version, while “HiSeq 4000” in Line 199. Therefore, we changed the “HiSeq 4000” in Line 199 to “HiSeqTM 4000” in revised version(LINE 195).

5. The language should be improved by a native English speaker in order to eliminate grammatical and spelling errors and to conform to correct scientific English. I listed some of these errors below.

Line 30-33: “However, to date, the genetic characteristics and genetic relationship with other duck breeds has not been explored yet,” is not clear. The authors should refer to whose “genetic characteristics and genetic relationship with other duck breeds”;

A: We have added “of JJ” behind the genetic characteristics and genetic relationship in the revised version. The modified sentence in revised manuscript is as follow: “However, to date, its genetic characteristics and genetic relationship with other duck breeds have not been explored yet, which greatly limits the utilization of JJ.” (LINE 22-24).

Line 91: “other” should be changed to “the”;

A: We have changed “other” to “the” in the revised version and marked in red.

Line 102: A comma should be placed behind “JJ and FF ducks”;

A: We have added a comma behind “JJ and FF ducks” in the revised manuscript.

Line 251-252: The authors should delete the “on these genes”;

A: We have deleted the “on these genes”.

Line 275: should add a comma between FF and YD;

A: We have added a comma between FF and YD.

Line 298:should add blank space between the number and KB.

A: We have added a blank space between the number (100) and KB.

Response to Reviewer #3

In my view, the manuscript is meaningful for breeding and genetics in duck. But , there are several shortcomings.

L8： What is the ‘d’ means?

A: The ‘d’ means nothing. We deleted the ‘d’ in the revised manuscript.

L55-63：Jiaji Duck, if it is a definite breed, I think you should provide the information of variety approval, like the record of Breeds of Domestic Animal and Poultry in China or other published researches. So should other breeds you used in your research.

A: This is a good suggestion. Mallard (YD) was widely accepted as the ancestor of domestic duck [11], which was indicated in the initial manuscript. The other four duck populations were all recorded by Breeds of Domestic Animal and Poultry in China. Therefore, we added a sentence as “Four out of the five duck populations (JJ, FF, HD and BD) have been recorded by Breeds of Domestic Animal and Poultry in China [12].” in the revised manuscript (LINE 81-83).

11. Li HF, Zhu WQ, Song WT, Shu JT, Han W, Chen KW. Origin and genetic diversity of Chinese domestic ducks. Mol Phylogenet Evol. 2010;57(2):634-640. doi: 10.1016/j.ympev.2010.07.011. PubMed PMID: 20674751.

12. Chinese animal and poultry genetic resources commission. Breeds of Domestic Animal and Poultry in China. 2010.

L64-75：The introduction of technical methods, NGS or others, is not the point. You can review the research progress in duck sequencing.

A: We agree with the opinion of the reviewer. Therefore, we deleted the NGS introduction and reviewed the research progress in duck sequencing which was listed as follow in revised version (LINE 60-71). “The approach of whole genome re-sequencing (WGRS) has been proved to be a very powerful tool in genetic evaluation and genetic relationship exploration of various animals, including pigs [1], chickens [2], cattle [3], sheep [4], dogs [5] and rabbits [6]. For ducks, the genome sequences have been constructed in 2013 [7], providing a platform for the research of ducks using WGRS method and promoting the related exploration of ducks. Xu et al. have explored the genetic signatures of artificial selection using genome WGRS data [8]. Zhang et al. have indicated the genomic variation in Peking duck populations using WGRS data [9]. Zhou et al. have identified the genes associated with body size and plumage color in ducks using WGRS and genome-wide association study (GWAS) technologies [10]. However, no report has investigated the genetic characteristics of JJ and its relationship with other duck populations using WGRS method.”.

Why you choose these four breeds (France muscovy ducks, mallard, hong duck, and beijing duck) and did a phylogenetic tree for the all five breeds?

A: Actually, we chose five duck populations. We reread our paper again and found the vague statement about the duck populations. For example, we wrote that “In this study, we selected five duck populations that differ in a couple of characteristics. Similar to JJ, France muscovy ducks (FF) is another muscovy duck population that is introduced into Hainan province since 1990’s. However, FF grows faster than JJ and is fully covered by white plumage. Mallard (YD), widely accepted as the ancestor of domestic duck [11], is used to confirm whether JJ and FF are also derived from YD. Beijing Duck (BD) has been subjected to intensive selection to provide the raw material of Beijing roast duck dishes. Hong duck (HD) is the hybrid offspring of Beijing duck and sheldrake with higher quality of meat relative to Beijing duck and faster growth rate compared to sheldrake.” in the last paragraph of introduction. Statements like the above always lead to vagueness and deems only four duck populations. Therefore, we added the sentence that “The first one was JJ, which has been introduced as above-mentioned.” before the sentence of “Similar to JJ, French muscovy duck (FF) is another muscovy duck population that has been introduced into Hainan Province since 1990’s” in the revised version (LINE 73).

Results and discussion：Only basic and necessary results were showed.

What puzzles me is this, you list your results and say they are meaningful, but I can not find any deep discussion. After your sequencing and analysis, some specific SNPs and Indel, GO and KEGG project can illustrate what? I think readers are look forward to in-depth discussions and the specific and functional point.

A: The reviewer provides a constructive suggestion. According to the reviewer, we reread the paper again and added the followed section.

1. The 3.1 section of results and discussion, we changed the statements in the revised version like as follow, “These results indicated that the majority of the identified mutations were located in non-coding sequences (intergenic regions or intronic regions), and only a small proportion of them fell in exon sequence, showing that only very few variants along with duck genome could exert functional effects on protein function. Our results were consistent with those of Xu et al. [8] in ducks and Carneiro et al. in rabbits [6].” (LINE 222-227).

2. The 3.3 section of results and discussion, we inserted that “Interestingly, the genetic relationship between JJ and YD was closer than that between YD and HD or BD. Since YD (mallard) was considered as the ancestor of HD and BD, we hypothesized that JJ and FF were differently originated from HD and BD.”(LINE 272-275).

Tables：All your tables do not meet the standards (Three-line table) , required notes missing. In Table 3, what is the meaning of different background notes?

A: We changed all tables to Three-line tables and added the required notes in the revised manuscript. The added notes are “BD1- BD3, Beijing duck, individual 1-individual 3; FF1- FF3, French muscovy duck, individual 1- individual 3; HD1- HD3, Hong duck, individual 1-individual 3; JJ1- JJ3, Jiaji duck, individual 1-individual 3; YD1- YD3, mallard, individual 1- individual 3.” for Table 1, “SNPs mean single nucleotide polymorphisms; Indels mean short insertions and deletions.” for Table 2, and “Grey represents the comparison of JJ vs. BD and the secretion related pathways in the significant enriched KEGG pathways. Light green means the comparison of JJ vs. BD and the secretion related pathways in the significant enriched KEGG pathways.” for Table 3.

Reference ：Format is also nonstandard. Journal Title, abbreviation, PubMed Central PMCID and so on.

A: A: We have changed the style of our manuscript to meet PLOS ONE's style requirements according to http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf, including the format of reference.

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28 Jan 2020

Genetic characteristics of Jiaji duck by whole genome re-sequencing

PONE-D-19-18411R1

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30 Jan 2020

PONE-D-19-18411R1

Genetic characteristics of Jiaji Duck by whole genome re-sequencing

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