High-density single nucleotide polymorphism chip-based conservation genetic analysis of indigenous pig breeds from Shandong Province, China

Objective Shandong indigenous pig breeds are important Chinese pig resources. Their progressive population decline in recent decades has attracted attention towards their conservation. Conservation genetics of these indigenous breeds are essential for developing a conservation and utilization scheme. Methods A high-density single nucleotide polymorphism (HD-SNP) chip-based comparative analysis of genetic characteristics was performed for seven Shandong indigenous pig breeds in the context of five Western commercial breeds. Results The results showed that Shandong indigenous pig breeds varied greatly in genetic diversity, effective population size, inbreeding level, and genetic distance with the Western commercial breeds. Specifically, Laiwu and Dapulian displayed low genetic diversity, and had a genetically distant relationship with the Western commercial breeds (average F statistics [FST] value of 0.3226 and 0.2666, respectively). Contrastingly, the other five breeds (Yantai, Licha, Yimeng, Wulain, and Heigai) displayed high genetic diversity within breed and had some extent of mixture pattern with the Western commercial breeds, especially Duroc and Landrace (FST values from 0.1043 to 0.2536). Furthermore, intensive gene flow was discovered among the seven Shandong indigenous breeds, particularly Wulian, Licha, and Heigai, as indicated by the large cluster formed in the principal component analysis scatterplot and small population differentiation (average of 0.1253) among them. Conclusion Our study advances the understanding of genetic characteristics of Shandong indigenous breeds and provides essential information for developing an appropriate conservation and utilization scheme for these breeds.


INTRODUCTION
Globally, China is the largest pork producing and consuming nation [1], with Shandong province, located in eastern China, being the fourth largest pigproducing region. His torically, several indigenous pig breeds have been developed by locals to meet their pork demands. In total, Shandong has seven officially authorized indigenous pig breeds (Laiwu, Dapulian, Licha Black, Yantai Black, Yimeng Black, Wulian Black, and Zaozhuang Heigai), six of which are recorded in national or provincial animal genetic resources [2,3] with the seventh (Zaozhuang Heigai) being newly approved (Announcement No. 168 of the Ministry of Agriculture and Rural Affairs of the People's Republic of China). These in digenous pigs are well adapted to local environments, and some of them display prominent characteristics. For example, Laiwu pigs have excessively high level of intramuscular fat content (~10% in Laiwu vs ~1.5% in Yorkshire or Duroc×Landrace×Yorkshire) [4,5]; Dapulian pigs display higher resistance to porcine repro ductive and respiratory syndrome than Duroc×Landrace× Yorkshire [6,7]; Licha Black is characterized by more vertebrae (1 to 2) compared with other Chinese native breeds [8].
Over the last few decades, more attention was paid to growth performance and lean meat content due to changes in consumer preference. Resultantly, Western commercial pig breeds (Duroc, Yorkshire, Landrace, Pietrain, and Berk shire) were introduced in China to increase productivity for these traits. This led to the progressive replacement and mar ginalization of Chinese indigenous pig breeds, some of which are currently close to extinction due to dwindling population numbers or hybridization with highly productive breeds [9]. Population decline of these indigenous breeds may lead to loss of allelic variation and reduced response to changing environments, especially to newly emerging pathogens [10,11]. Thus, it may finally result in the loss of valuable future breeding resources. In addition, these traditional breeds are usually associated with local forms of pig husbandry forms, and their meat is used to produce highquality products [5,12]. Efficient conservation and utilization of local breeds are needed to support the development of sustainable pig indus try. A comprehensive study of their genetic characteristics is a preliminary step for developing a conservation and utiliza tion scheme. The genetic diversities of Shandong indigenous pig breeds have previously been evaluated using microsatel lite markers [13] and mtDNA sequences [14]. Recent advances in highthroughput genotyping technologies, mainly high density single nucleotide polymorphism (HDSNP) chip and nextgeneration sequencing, have markedly facilitated studies on genetic characteristics at the genomic level, sig nificantly extending our understanding of major questions in conservation genetics, including genetic distance, effective population size (Ne), and inbreeding level. For instance, the Ne and inbreeding coefficient are of major interest in con servation genetics, and common estimation methods based on pedigree information are sometimes not feasible due to the frequent unavailability of pedigree information in the indigenous breeds. Highthroughput genotyping technolo gies allow the study of linkage disequilibrium (LD), and runs of homozygosity (ROH) [1517] which enable us to es timate Ne and inbreeding coefficient. These technologies have been used to study other indigenous pigs [1820], and studies have demonstrated their competency in assessing major questions in conservation genetics. Although high throughput genotyping technologies provide a useful tool for genetic study, only one study has been recently conducted to investigate genetic diversity in Shandong indigenous breeds by using specificlocus amplified fragment sequencing (SLAF seq) and a small sample size of seven to ten animals per breed [21]. Use of HDSNP chip has the advantages of lower cost and higher efficiency in genotyping and statistics compared to highthroughput sequencing. Thus, more samples per breed can be genotyped to obtain a suitable representation of each breed using the SNP chip.
Here, HDSNP BeadChips were employed to investigate the genetic characteristics of seven indigenous pig breeds from Shandong province, China, in the context of widely used Western commercial breeds. First, parameters for the genetic diversity and inbreeding within breeds were calcu lated. Second, the LD patterns and Ne were analyzed. Finally, individual clustering based on principal components (PCs) and historical admixture patterns were assessed to evaluate the structures among the analysed breeds, and genetic dif ferentiation and phylogenetic trees were conducted to assess the relationship among them. All these joint analyses would provide valuable information for developing conservation and utilization strategies.

Ethics statement
All animal procedures used were reviewed and approved by the Institutional Animal Care and Use Committee of Insti tute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences under permit number IACC20060101.

Sample collection and SNP genotyping
A total of 397 pigs from seven indigenous breeds (Laiwu, Dapulian, Licha Black, Yantai Black, Yimeng Black, Wulian Black, and Zaozhuang Heigai) and five Western commercial breeds (Yorkshire, Landrace, Duroc, Berkshire, and Pietrain) were used in the study. The sample sizes of each analysed breed are presented in Table 1, and characteristics and pic tures of the seven indigenous pig breeds are provided in the Supplementary File S1 of the supporting information. For each breed, all boars and unrelated sows (no common an cestry for three generations) in the conservation or breeding farms were sampled to obtain representative samples for each analysed breeds. The sample size per breed ranged from 26 for Heigai to 42 for Laiwu and Berkshire (Table 1). Genomic DNA was extracted from ear tissues using a routine phenol/ chloroform method [22], and was diluted to a final concen tration of 50 ng/mL. All animals were genotyped using Porcine SNP55K Bead Chips according to the manufacturer' s protocol. The BeadChip was designed and promoted by Compass Biotechnology Corporation, Beijing, China (http://www.kangpusen.com/), and manufactured by Illumina (San Diego, CA, USA). Using PLINK v1.90 [23], SNPs were filtered with call rate ≥95% and minimum allele frequency (MAF) ≥0.05. The SNP posi tions within a chromosome were based on the current pig genome assembly (Sscrofa11.1).

Genetic diversity
The following parameters of genetic diversity were calculat ed for each SNP and each breed using PLINK v1.90 [23]:

Runs of homozygosity
The ROHs were identified using autosomal SNPs passing the quality control and PLINK v1.09 [23]. We referred to the previous studies listed in the review [15] and the following criteria were employed in the analysis. The ROHs were de fined by a minimum of 40 homozygous SNPs, length of 1,000 kb, minimum SNP density of one SNP/1,000 kb, and the largest possible gap between SNPs of 1 Mb. One heterozy gous SNP and a maximum of five missing markers per ROH were permitted in the analysis. The number of ROH seg ments and the total length of ROHs in each individual were calculated and their mean estimated for each breed. Further more, the genomic inbreeding coefficient based on ROH (F ROH ) was calculated for each individual as described by McQuillan et al [24], i.e. F ROH = ∑L ROH /L AUTO , where ∑L ROH refers to the total length of ROH and L AUTO is the length of the autosomal genome covered by SNPs.

Linkage disequilibrium analysis and effective population size
The LD was determined using the squared correlation coef ficient (r 2 ) between two SNPs for all marker pairs less than 5 Mb for each breed independently using PLINK v1.09 [23]. To evaluate the LD pattern along chromosomes, the data were sorted into groups based on pairwise marker distances, defined every 0.01 Mbp until 0.05 Mbp and every 0.05 Mbp until 1 Mbp, and r 2 was then averaged for each group. Aver age r 2 values for each breed were plotted against physical distance using the R program.
Ne was calculated for each breed using the following equa tion: Ne = (1/4c)×(1/r 2 -1) [25], where c is the genetic distance between two SNPs expressed in Morgans and r 2 is the LD of different distances. The average highdensity recombination rate reported by Tortereau et al [26] was considered to lead more correct estimates of Ne. The current Ne was calculated using the mean value of r 2 for all 1 Mb bins across the entire genome as described by HerreroMedrano [18], whereas the past Ne at generation T, where T = 1/2c, was estimated using the above equation [27].

Population structure and relationship analyses
Principal component analysis (PCA) was performed using PLINK v1.09 [23], and the scatterplot of the first and second PCs was constructed to visualise clusters formed by indi viduals belonging to the same population. The program admixture version 1.3.0 [28] was used to examine historical admixture patterns of the analysed breeds. Crossvalidation (CV) errors were calculated for each K value to identify the K value with the best predictive accuracy. F statistics (F ST ) values were estimated using PLINK v1.90 [23] based on Weir and Cockerham's formula [29]. Additionally, pairwise evolutionary distances among the breeds were calculated using the following options, variance estimation method: none, substitution model: TajimaNei model [30], rates among sites: uniform rates, pattern among lineages: homo geneous, gaps/missing data treatment: pairwise deletion. Evolutionary distances were visualised by constructing the neighbourjoining (NJ) phylogenetic tree [31] using MEGA v7.0 [32].  ROHs for the analysed populations were estimated to de termine the inbreeding levels. As shown in Table 2 The LD along physical distance between markers was also plotted to visualise the LD along chromosomes, and the plot is presented in Figure 1. All breeds showed the similar trend of the average r 2 with distance, i.e., it decreased rapidly over the first 0.2 Mb distance, and then decreased gradually over the remaining 0. The present Ne was estimated for each breed based on the estimated r 2 ( Table 1). The present Ne of the 12 breeds analysed ranged from 69 to 129, with an average of 96.59. Generally, Shandong indigenous breeds had larger Ne than

Population structure analysis
Firstly, PCA was performed to explore the clustering of indi viduals of different breeds (Figure 2). The results indicated that 89.59% of the total variance was explained by the first three PCs (53.06% by PC1, 20.30% by PC2, and 16.23% by PC3). As shown in Figure 2, visibly separated clusters were observed for the commercial breeds, Berkshire, Duroc, and Pietrain, whereas separated but slightly overlapped clusters were observed for Landrace and Yorkshire. This suggested that Western commercial breeds had distinct population structures. In contrast, a single large cluster was formed by the seven Shandong indigenous breeds. Laiwu and Dapulian slightly overlapped at the left end of the large cluster, and Yimeng and Yantai were located at the right end, close to the clusters of Western commercial breeds, particularly Duroc and Landrace. Licha, Heigai, and Wulian completely over lapped in the middle, indicating the close relationship among them. The areas of the seven pig breeds distributed are not far away and have had frequent economic and social ex changes over the history. Thus, intensive gene flow may have occurred among them. Then, historical admixture patterns of the analysed breeds were assessed using K values from 2 up to 12 with Admix ture. The CV errors (Supplementary Table S5) decreased as the K value increased, with a rapid decrease when K ranged from 2 to 8. The population admixture patterns with K = 2 and K ranging from 8 to 12 are shown in Figure 3. The Western commercial breeds formed one large cluster (marked in light blue) when K = 2, while Laiwu and Dapulian formed another one (indicated in dark blue). Laiwu, Dapulian, and five Western breeds appeared as differentiated clusters when K = 8, while the other five Shandong breeds shared the same cluster. Yimeng, Heigai, and Wulian were separated as dis tinct clusters at K = 9, 10, and 11, respectively, with an increase in K value. However, no noticeable differentiation between Yantai and Licha appeared with K values up to 12, suggesting that considerable admixture existed between them.

Population relationship analysis
Pairwise F ST values were further estimated to determine the extent of population genetic differentiation, and the results are shown in the lower diagonal of Table 3 Finally, pairwise evolutionary distances among the anal ysed breeds were calculated (the upper diagonal of Table 3) and visualised using an NJ phylogenetic tree (Figure 4). Gen erally, the evolutionary distances corresponded with the F ST values. The Western commercial breeds clustered together at the top of the tree, while the seven Shandong breeds clus tered together at its bottom. However, the clades formed by Shandong indigenous and Western commercial breeds were not completely separated. Yimeng and Yantai clustered first with Duroc before clustering with the other Shandong in digenous breeds, indicating the close relationship between the two breeds with Western commercial breeds, especially with Duroc.

DISCUSSION
The progressive population decline of Shandong indigenous pig breeds has called for attention towards their conserva tion. Here, we conducted an analysis of the conservation genetics for the Shandong indigenous pig breeds based on HDSNP BeadChip, Porcine SNP55K, to provide essential information for the sustainable protection and use of these genetic resources.
Porcine SNP55K BeadChip used in the present study was designed for genetic research of Chinese local breeds, and SNPs found in the Chinese local breeds were considered in it to improve the coverage of chromosomes and MAF of Chinese local breeds. The total SNP of the chip was lower compared to widely used Illumina 60K BeadChips [33] and its improved versions. However, all the SNPs could be un ambiguously mapped to the current pig genome (Sscrofa11.1) with an even distribution. The results of our study demon strated that the informative SNP rates with MAF >0.05 were more consistent among breeds, with an average of 0.87 and 0.80 for Shandong and Western breeds, respectively. In a previous study conducted in 304 Chinese and Western pigs using Illumina Porcine SNP60 BeadChip, severe SNP bias was observed between these pigs for a common subset of 15,911 SNPs with MAF >0.2, with 0.87 to 0.95 and 0.67 to 0.98 polymorphic SNPs for Western and Chinese breeds, re spectively. However, no informative SNP rates with MAF >0.05 were displayed [19]. Thus, the Porcine SNP55K Bead Chip SNPs are more suitable for genetic studies of Chinese  indigenous breeds than those of Illumina Porcine SNP60 BeadChip.
Our results indicated genetic diversity varied largely among Shandong indigenous breeds. Laiwu and Dapulian were smaller than the Western commercial breeds, while the other five breeds were larger than the Western commercial breeds. Our results are consistent wholly or partly with the previous studies based on microsatellite [13], and SLAFseq [21], but contrary to those based on mtDNA sequence [14,34]. Wang et al [14] sequenced a control region sequence and observed that genetic diversity of Laiwu and Dapulian were comparable with that of Western commercial breeds. Quan et al [34] analysed the mtDNA hypervariable regions of 70 Chinese native pig breeds and showed that most na tive pig breeds have low genetic diversity, especially for those distributed in the Sichuan and Shandong Provinces. The inconsistencies between the genome sequence and mtDNA sequence results could be attributed to mtDNA being mater nally inherited in sexually reproducing organisms, including pigs [35,36]. Also, a small segment sequence was analysed in the mtDNA based studies. These results were indicative of the influence sample size and representation has on re search results.
Western commercial pigs have undergone intensive selec tion over decades, which has resulted in their reduced genetic diversity, high LD, and inbreeding [37,38]. Unlike Western commercial pig breeds, Chinese indigenous pigs have not undergone the intensive selection, and they are assumed to sustain higher levels of genetic diversity. The inconsistencies in our study could mainly be attributed to the small popula tion size of the two breeds; only a few founders were left at the beginning of the preservation programs. Currently, sev eral Western commercial breeds have been dominantly used to produce pork in the Chinese pig industry, and only a limited number of pigs from each breed are raised for conservation or highquality product production. Small population sizes have probably led to some genetic variability loss.
Inbreeding levels and Ne are two essential factors in pop ulation protection. Traditionally, they have been evaluated based on pedigree information, which is however incom plete or completely unavailable for most indigenous breeds. Several methods, independent of the pedigree, have been developed to estimate inbreeding and Ne based on genomic data. F ROH , defined as the percentage of the genome covered by ROH, is regarded as an indicator reflecting the recent in breeding history of a population and has also shown a good correlation with pedigree inbreeding coefficients [15,39]. Ne is estimated based on LD patterns and has been proven to be similar to that estimated based on the pedigree data [27,38]. Our results showed that breeds with high genetic diversity displayed low F ROH and high Ne values. These congruous re sults demonstrated that the reliability in using F ROH to indicate inbreeding and LD to estimate Ne.
Of the Shandong indigenous breeds, Laiwu presented the highest F ROH (0.2263) value, even higher than most Western commercial breeds. The F ROH values of the other Shandong breeds were low to moderate, with Ne above 100 except Yimeng (Ne = 83). Nevertheless, there was significantly large withinbreed variation in F ROH values for these breeds, indi cating the presence of individuals with high inbreeding levels. Such individuals should be specifically considered when planning matings. Furthermore, an effective population size of 100 should be considered to maintain a population's ge netic diversity [40]. Some of Shandong indigenous breeds have Ne values slightly higher than the recommended number, while some are close to the recommended number. Thus, appropriate methods should be applied to control the inbreed ing levels and improve Ne to protect these genetic resources.
All population structure and relationship analyses per formed in the present study demonstrated that Shandong indigenous pig breeds were genetically distant from the Western ones. Particularly, F ST values, which are based on allele frequency differences among populations [41], quan titatively indicated the large genetic differentiation among these breeds. Shandong indigenous and Western commer cial breeds had an average F ST value of 0.21, ranging from 0.1043 to 0.3479. Based on the degree of genetic differenti ation using F ST thresholds (0.05, 0.15 and 0.25) specified by Hartl and Clark [42], moderate to high degrees of differen tiation existed between them. These results are consistent with previous studies based on microsatellite and SLAF seq [13,21].
Of the seven Shandong indigenous breeds, Laiwu and Dapulian had markedly high F ST values with the Western commercial breeds, with averages of 0.3226 and 0.2666, re spectively. Furthermore, Laiwu and Dapulian located at the distal end of the large cluster with slight overlap with other indigenous pigs (Figure 2), differentiated from Western com mercial breeds when K = 2, and were firstly grouped together in the NJ trees ( Figure 4). Collectively, these results indicated that Laiwu and Dapulian were less influenced by Western commercial breeds, being two indigenous pig breeds with unique genetic characteristics. Laiwu and Dapulian are dis tributed mainly in the middle of Shandong province, where traffic and economic development are relatively underdevel oped. In addition, their conservation farms were constructed earlier and in a better management compared to other Shan dong indigenous breeds. Thus, they are better protected and less influenced by Western commercial breeds. These results are consistent with the results of previous studies based on mtDNA sequences [14], microsatellite markers [13] and SLAF seq [21].
In contrast, the other five Shandong indigenous pig breeds had relatively small genetic differentiation with the Western breeds (F ST values from 0.1043 to 0.2536). They were located close to Western breeds clusters ( Figure 2) and had some ex tent of mixture pattern with the Western commercial breeds ( Figure 3). All these results support that they have been in fluenced to some extent by the Western commercial breeds, especially Yimeng and Yantai. The five indigenous breeds are in the northeastern Shandong peninsula, where the traffic and economic development are relatively developed. Histor ically, western pig breeds were introduced to these districts in the early of 20th century and were used to improve the growth performance of the local breeds [3,8]. Additionally, there are relatively low pairwise F ST values (0.0441 to 0.1489), overlapped in the cluster of PCA (Figure 2), and separated only when the K value was high (Figure 3). This suggested that extensive gene flow ever progressed among these breeds. Notably, the five breeds presented high genetic diversity, which may be attributed to gene flow among them as well as hybrid ization with the Western commercial breeds.

CONFLICT OF INTEREST
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu script.