Chinese Domestic Ducks Evolved from Mallard Duck (Anas platyrhynchos) and Spot-Billed Duck (A. zonorhyncha)

Simple Summary Domestic duck populations in China have abundant genetic resources, and this study is the first to evaluate domestic and wild Mallard populations. To explore the genetic diversity and ancestry of duck populations, we analyzed a sequence (619–667 bp) of mitochondrial DNA (mtDNA) in the control region. Two Muscovy duck breeds were included in this analysis, along with wild Mallard ducks, Eastern spot-billed ducks, and 22 domestic duck breeds. Significant genetic diversity was found among domestic duck breeds. These results not only provide valuable insights into the genetic makeup of domestic ducks but also provide clues to the possible origin of domestic ducks in China. Abstract China has a rich genetic resource in its 34 domestic duck breeds. In order to detect the genetic diversity and explore the origin of these indigenous duck populations, the mitochondrial DNA (mtDNA) control region was sequenced and analyzed for 208 individual ducks, including 22 domestic breeds, wild Mallards ducks, Eastern spot-billed ducks, White Muscovy ducks, and Black Muscovy ducks. The haplotype diversity (Hd) was 0.653 and the average nucleotide diversity (Pi) was 0.005, indicating moderate genetic diversity. Sixty haplotypes were detected, and the maximum-likelihood (ML) phylogenetic tree and median-joining (MJ) network were generated from the sequence analyses. In this study, haplotypes from the Mallard duck (Anas platyrhynchos) were detected in most of the Chinese domestic duck breeds. In addition, the Eastern spot-billed duck (A. zonorhyncha) H8 haplotype was detected in two duck breeds. Only two haplotypes were found in Muscovy ducks, suggesting low genetic diversity within this population. The sequence and haplotype analyses revealed that both A. platyrhynchos and A. zonorhyncha contributed to the evolution of domestic ducks in China.


Introduction
Waterfowl resources are rich in China, and the Genetic Resources Committee has officially recognized these resources [1,2]. There are many possible origins of indigenous duck breeds in China [2][3][4]. The ducks may have originated from the domestication of the wild Mallard ducks (Anas platyrhynchos), the interbreeding of domesticated Mallard ducks and domesticated Eastern spot-billed ducks (A. zonorhyncha), or the domestication of wild hybrids of A. platyrhynchos and Anas zonorhyncha [3]. Previous studies of genetic diversity using amplified fragment length polymorphism (AFLP) of genomic DNA or analyses of genetic distance and relationships determined that domestic duck breeds evolved from both A. platyrhynchos and A. zonorhyncha [5][6][7]. Using microsatellite markers of genomic DNA polymorphisms, Chen et al. determined that the contribution to domestic duck breeds was greater for A. platyrhynchos than for A. zonorhyncha [8]. He et al. also used microsatellite DNA markers from 449 domestic ducks and wild Mallard ducks to demonstrate that all domestic duck breeds in China evolved from a single origin [9]. Similarly, Sultana et al. analyzed six Southeast Asian duck populations and determined that the ducks were domesticated from A. platyrhynchos [10]. Overall, these studies support the evolution of Chinese domestic ducks from both A. platyrhynchos and A. zonorhyncha.
It was confirmed that the mitochondrial DNA (mtDNA) could more effectively evaluate the genetic diversity of poultry [11]. Unlike nuclear DNA, mtDNA acquires higher mutation rates that are 10 times than those of nuclear genomic DNA, and it is generally not recombined with the mtDNA of the parents [12]. Previous studies have confirmed that the control region has a higher mutation rate compared to the coding region [13]. In addition, it is estimated that mitochondrial control regions have a 2-5 fold higher evolution rate than mitochondrial protein-coding genes, making them more suitable for studying genetic variation among breeds [14]. In addition, a previous study using partial sequencing of mtDNA showed that nine domestic duck breeds along the Yangtze River evolved solely from A. platyrhynchos [15]. However, the major limitation of these genetic diversity studies was the limited number of domestic duck breeds and, more specifically, the lack of seven important duck breeds identified by the state, including the Fengtou duck (FT), Mawang duck (MW), Taiwan duck (TW), Zhongshan duck (ZS), Zongyang duck (ZY), White Muscovy duck (BF), and Black Muscovy duck (HF).
This study performed the first comprehensive evaluation of domestic duck populations and wild Mallard ducks in China. We analyzed a 619-667 bp sequence of the control region of the mitochondrial DNA (mtDNA) of 22 domestic duck breeds, in addition to wild Mallard ducks, Eastern spot-billed ducks, and two Muscovy duck breeds. Sequence and haplotype analyses of the region revealed a high genetic diversity among the domestic duck breeds evaluated in this study. In addition, the identification of haplotypes shared across duck species suggested that Chinese domestic ducks originated from A. platyrhynchos and A. zonorhyncha.

Ethics Approval
All animal experiments were performed in accordance with the Regulations for the Administration of Experimental Animals issued by the Ministry of Science and Technology (Beijing, China). All experiments were approved by the Animal Care and Use Committee of Yangzhou University (approval code: 151-2014.).

Collection of Blood Samples and DNA Extraction
According to the pedigree records, 208 blood samples were collected from unrelated duck individuals (Table 1, Figure 1). In addition, two Muscovy duck populations, the White Muscovy duck (n = 8) and the Black Muscovy duck (n = 8), were collected from Putian in Fujian Province and used as the reference group for the analysis. These blood samples (approximately 3 mL) were collected from the wing vein of the ducks in individual vacuum blood collection tubes. The genomic DNA was extracted using a genomic DNA extraction kit (TIANGEN, Beijing, China). RNA was purified to remove genomic DNA (gDNA), miRNA, and rRNA using the RNeasy Micro kit (Qiagen, Hilden, Germany), RNase-Free DNase kit (Qiagen), and RiboZero™ MagneticKit (Epicentre, Illumina, San Diego, CA, USA), respectively. Data collection and analysis were conducted at the College of Animal Science and Technology, Yangzhou University, Yangzhou, Jiangsu Province, China.

Data Analysis
Sequences of the mtDNA fragments were visualized by Chromas2 software and manually browsed to ensure the accuracy of the bases. Sequences were aligned by DNAStar-MegAlign V.7.10. The genetic diversity parameters of the populations, such as variable nucleotide sites, haplotype diversity, and nucleotide diversity, were calculated by DnaSP V.5.0 software [16,17]. The haplotype ratio was calculated as the ratio of the number of haplotypes to the sample size. The distances (Kimura 2-parameter) among all populations were calculated using Mega v.5.0 [18], and then the maximum-likelihood(ML) evolutionary tree was mapped out. Finally, the median-joining (MJ) network of the control region of the mtDNA haplotypes was drawn using Popart v.1.7 software [19].

Nucleotide Composition of the mtDNA Region of 26 Duck Populations
The data of 208 ducks were analyzed, and the average composition of the sequences was 25.01% A, 15.71% G, 34.23% C, and 25.05% T (50.06% A + T, 49.94% G + C), showing little base bias. A total of 99 polymorphic sites (10 singleton variable sites and 89 parsimonyinformative sites) were detected. As all insertions and deletions were removed from the analysis, the variable types were classified as transitions and transversions.

Phylogenetic Analysis of Chinese Duck Populations
The major H5 haplotype was shared by 61 ducks (30.77% of all ducks) from 18 domestic breeds and 3 wild Mallard ducks. The H8 haplotype was shared by one Mallard duck, one Zongyang duck, and one spot-billed duck ( Table 3). The ML phylogenetic tree (Figure 4) of the 26 Chinese duck populations and the median-joining network chart ( Figure 5) was constructed using all the mallards (A. platyrhynchos) and spot-billed ducks (A. zonorhyncha) in this study.

Phylogenetic analysis of Chinese Duck Populations
The major H5 haplotype was shared by 61 ducks (30.77% of all ducks) from 18 domestic breeds and 3 wild Mallard ducks. The H8 haplotype was shared by one Mallard duck, one Zongyang duck, and one spot-billed duck ( Table 3). The ML phylogenetic tree (Figure 4)

Discussion
Studies on the origin and evolution of livestock breeds through the different mtDNA sequences also contribute to the understanding of the domestication process of species [20]. For the identification of the genetic diversity and phylogeny of different breeds and populations, mtDNA control region sequence analysis has proven to be the most valuable tool [21]. Some studies support the finding that natural selection affects the genetic diversity of populations, which can be measured by haplotype diversity (Hd) and nucleotide diversity (Pi) [22][23][24]. It has been shown that evaluating the impact of natural selection on genetic diversity through haplotype diversity (Hd) and nucleotide diversity (Pi) can help us better understand natural selection and the trends in population genetic diversity trends. In this study, the Hd and Pi of 26 Chinese domestic duck populations were determined to be 0.653 and 0.005, respectively. These results indicate that the domestic duck populations have a relatively large number of haplotypes and exhibit fewer nucleotide sequence differences. In addition, these values were slightly higher than the detected diversity values of four domestic duck breeds in Fujian Province (Hd = 0.645, Pi = 0.001) [25], but they were much lower than those of A. platyrhynchos (Hd = 0.987 and Pi = 0.008 in Kulikova's study) [26]. In comparison with other animals, the Pi of Chinese ducks was higher than that of chickens (<0.001) [27][28][29] and swine (0.001) [30], but it was significantly lower than that of cattle (0.021) [31]. Haplotype and nucleotide diversity were highest in ZS ducks and lowest in SC ducks and BF Muscovy ducks.
Studies on the genetic diversity and genetic structure of species are important components of biodiversity conservation [32]. Previous studies have also found that the SC and BF duck breeds may have experienced a long duration of high selective pressure for particular traits, resulting in low genetic diversity [33][34][35]. By contrast, the ZS population was more recently discovered and was bred in closed regions (Guangdong Province) without any artificial selection. Interestingly, the higher selection pressure might have led to some genotypes being better suited to specific environments [34]. Therefore, this selection increased the frequency of certain genotypes in the population, thereby reducing genetic diversity and even leading to the extinction of some genotypes. In addition, the between-population analyses showed a lower average nucleotide difference (K) than the within-population analyses in this study. Previous studies have found low genetic diversity across 24 domestic Chinese duck breeds and concluded that efficient measures should be Animals 2023, 13, 1156 8 of 10 adopted to protect domestic duck resources, especially for the JX and LW breeds [15,35]. The results presented here suggest the SC and BF breeds require more immediate attention because of their low genetic diversity.
However, there is still no consensus in the field regarding the origin of domestic ducks in China [2,36]. In this study, the 192 domestic Chinese ducks presented 58 haplotypes, while the 16 Muscovy duck individuals conformed to two haplotypes. The H5 haplotype was shared by the majority of domestic and Mallard ducks, suggesting that most domestic duck breeds in China originated from A. platyrhynchos. The Eastern spot-billed ducks clustered into the H7-H10 haplotypes. Interestingly, the H8 haplotype was shared by one Mallard duck, one ZY duck, and one spot-billed duck, indicating that the partial domestic duck breeds in China might have originated from A. zonorhyncha. However, there were no other shared haplotypes between the domestic and spot-billed duck populations. Finally, our results suggested that domestic Chinese ducks evolved from A. platyrhynchos and A. zonorhyncha.

Conclusions
Sixty haplotypes were detected in most of the domestic Chinese duck breeds analyzed in this study. The sequence and haplotype analyses revealed that both A. platyrhynchos and A. zonorhyncha contributed to the evolution of domestic ducks in China. The Sichuan Ma duck (SC) and White Muscovy duck (BF) had the lowest Hd, K, and Pi. To summarize, their conservation requires more attention and prioritization of conservation funds in the future.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ani13071156/s1, Figure S1: High-resolution images of each representative duck population; Table S1: The proportion of individuals with each haplotype in duck populations.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this published paper.