INTRODUCTION
The genetic diversity of a commercial pig breed must be monitored to ensure the sustainable use of genetic resources and continuous genetic improvements in the future [
1]. The importance of proper management of inbreeding through systemic breeding programs should be emphasized [
2]. Traditionally, inbreeding has been estimated from the pedigree information [
3], and the inbreeding rate can be converted to an effective population size (Ne), which is considered a general indicator of the risk of genetic erosion [
4]. However, pedigree-based estimates of Ne depend on the completeness of the available pedigree. Genomic data are widely used improve the accuracy of Ne estimation without pedigree data, as these data allow estimations of current and previous Ne. This estimation is based on linkage disequilibrium (LD) patterns [
5–
7]. Genome-wide single nucleotide polymorphism (SNP) genotyping of in pigs has been possible since 2009 [
8]. In animal breeding, recent genomic methods such as genome-wide association studies and SNP-based genomic selection depend on the extent of LD and the association between the rate of LD decline with the distance between loci within a population. Researchers or animal breeders can apply mass SNP data, made available by SNP Genotyping Beadchips, to several genomic analyzes of domestic animals, including pigs. Researchers have already applied SNP chips to genome-wide association studies [
9–
11] and genomic selection of pigs [
12–
15]. This new SNP technology provides useful tools for studying the genetic diversity of pig populations and enables more detailed comparisons of populations than did earlier pedigree-based approaches.
In finite populations, several meaningful evolutionary processes involving artificial or natural selection can induce LD, or the nonrandom association of alleles in two different loci [
16]. In particular, most traits of interest in animal breeding are complex traits, and genomic selection techniques are more successful than genome-wide association, which is used to investigate some significant genomic regions closely linked to each trait [
17]. In animal breeding, these genomic methods strongly depend on the extent of LD and the sample size. Therefore, the characterization of LD is essential when planning future genomic technique-based animal breeding studies of complex traits. LD between loci can provide insights into the evolutionary history of each population through Ne, the number of individuals in an idealized population that would give rise to the degree of inbreeding in the current population [
18]. If we accurately estimate Ne, we can use this value to investigate genetic diversity in each domesticated pig population and explain the observed extents and patterns of genetic variation. Using Ne, we can also prospectively predict the loss of genetic variation and infer the accuracy of genomic selection before applying genomic selection at an industrial level for a particular domesticated animal. Additionally, we can infer the ancestral Ne from the strength of LD at different genetic distances between markers. Knowledge of the historical Ne pattern in each pig population could increase our understanding of the effects of recent animal breeding strategies.
Although the Korea pig industry includes several breeds, this study focuses on the Landrace population. Although this is not a domestic breed in Korea but rather has been imported from several countries, Landrace is considered a representative maternal pig breed in Korea, and Korean grand-grandparent (GGP) Farms maintain a considerable number of Landrace animals as breeding stock. For the Korean Landrace population, a good growth rate, efficient feed conversion rate, and increased piglet number are considered the main selection targets. The patterns of LD in Landrace populations from other countries have already been characterized, and estimated Ne values have been predicted using SNP chip data. Badke [
19] characterized the extent of LD in four US pig breeds, including Landrace. Lei Wang [
20] characterized the extent of LD in three Danish pig breeds, including Landrace. Veroneze [
21] characterized the extent of LD of six commercial pig breeds (including Landrace) in the Netherlands. This study identified that the LD declined as a function of distance (using 37,326 SNPs with an average minor allelic frequency of 0.283 in the Illumina PorcineSNP60 chip) and all pig lines had an average r
2 above 0.3 for markers 100 to 150 apart. Uimari [
22] characterized the extent of LD and estimated the LD-based actual and ancestral Ne values using 86 Finnish Landrace boars. This study reported average LD (r
2) between adjacent SNP in the Illumina PorcineSNP60 BeadChip was 0.43 (57% of the adjacent SNP pairs had r
2 >0.2) for Finnish Landrace and Ne estimates based on the decay of r
2 with distance were similar to those based on the pedigree data: 80 for Finnish Landrace.
The objective of the present study was to characterize LD within the Korean Landrace population, using data from the Illumina PorcineSNP60 BeadChip and to estimate the current and ancestral Ne values and thus dissect the genetic characteristics of Korean Landrace population. The estimated Ne (±standard deviation [SD]) of the Korean Landrace population was 92.27 [79.46; 105.07] individuals. Additionally, ancestral Ne was estimated in previous generations. Compared with other studies, our results are considered in the context of current knowledge regarding the establishment of genomic methods for the Korean Landrace population.
DISCUSSION
In this study, we used whole genome SNP data to investigate the extent of LD, as well as the current and previous Ne of the Korean Landrace population. Here, the observed LD (r
2) extended for long distances when the adjacent 100 SNPs of each SNP in the genome were used. Although a previous study used both mass pedigree and small genomic data [
20], we used large-scale genomic data from GGP farms to characterize LD and estimate Ne with the aim of obtaining an unbiased picture of LD in the Korean Landrace population. Because domesticated pig breeds such as Landrace were strongly and artificially selected for a long period of time, the observed LD is higher at short distances and more extensive than that observed in human populations. The pattern of LD decline in the Korean Landrace population was consistent with those reported by previous studies of domesticated pig breeds [
20,
28] and other domesticated animals [
24,
25].
We estimated the Ne of Korean Landrace population using a formula published by Sved [
5], in which a non-linear regression model was used to describe the relationship between genetic distance and LD. However, this method of estimating Ne is associated with difficulties when addressing values within the limits of the parameter space (i.e., if r
2 = 0.0, estimated Ne is infinite and if r
2 = 1.0, estimated Ne is zero). Uimari [
22] noted this limitation of the method devised by Sved [
5]. In this study, we calculated r
2 between one SNP with its adjacent 100 SNPs to reduce bias of r
2 estimation. If we used r
2 between one SNP with its adjacent few SNPs in this estimation, we could not take enough information about relationship between r
2 and distance because two SNPs interval could be short or long. So we used adjacent 100 SNPs per SNPs in r
2 eatimation and this was why the results could yielded accurate Ne. Another concern associated with the relationship between the estimated LD and the distance between SNPs involves the accuracy of the porcine reference genome (Build 9) used in this study. In future studies, updates to the porcine reference genome will refine the order and distances between SNPs on the commercial Illumina PorcineSNP60 version2 BeadChip. However, we considered that bias resulting from incorrect ordering of or distances between SNPs would be diluted by the large number of SNP pairs used in this study; therefore, slight overestimation and/or underestimation of LD would not be an issue. Moreover, the relationships between genetic and physical distances are known to vary across chromosomes and chromosomal regions. Therefore, we inferred the cM/Mb ratio per chromosome using position data from a physical map of the porcine reference genome and a genetic map generated using the USDA pedigree (derived from a population composed of ¼ Duroc, ¼ Large White, ¼ maternal Landrace, and ¼ high growth Landrace) in a previous study [
26]. We further used genetic distances based on physical distances to estimate Ne. Accordingly, we were able to estimate Ne more reliably from these detailed estimates of genetic distances between SNPs, compared with other studies [
24,
25]. Finally, another study reported that a limited sample size could bias the estimates of r
2 and recommended correcting these estimates for a sample size n (r
2 – 1/2n) before using the Sved [
5] equation. However, given our large sample size, we did not need to correct the r
2 estimates or use corrected r
2 values. When estimating the Korean Landrace population Ne, we used an alternative version of the Sved [
5] equation derived by Tenesa [
26], which incorporated a new parameter a (equal to 2) to account for mutations. Using this formula [
26], the initial value of parameter a was 2 when parameters were estimated using the non-linear regression model of R. As a result, the estimated parameter a per chromosome ranged from 2.45 to 3.35 (
Figure 3), and the estimated parameter b per chromosome ranged from 39.12 to 139.77. Regarding heterogeneity in the variance of the observed r
2 per chromosome that declined as the distances between SNPs increased (
Supplementary Figure S2), this might have affected our estimation of parameter b in
Equation (2). In one study, a significant negative relationship was observed between the chromosome length and parameter b estimates from the non-linear model [
24], whereas other studies of domestic livestock species reported a positive relationship [
29] and still others did not investigate either type of relationship [
25]. We therefore considered that the relationship between the chromosomal length and the estimated parameter b differed for each population, as did the evolution histories of each species and breed. In this study, all marker pairs were calculated only in each bin so that r
2 would not be affected by the chromosome length. These results were consistent with the Yorkshire LD characterization results reported by Uimari [
22] and the Korean Yorkshire Ne study (in review) [
14]. Furthermore, we did not observe a relationship between the chromosome length and the estimated b values.
Our estimated b value represents an estimated Ne with an assumed constant present population size because we used genetic data of a Korean Landrace population that comprised candidate replacement breeder pigs from each major GGP or GP farms during the period of 2015 through 2016. When calculating Ne, parameter b in
Equation (2) represents a conceptual average Ne over the period inferred from the range of SNP pair distances per chromosome [
30]; we regarded the combined parameter b from a meta-analysis as the current Ne of the Korean Landrace population. Therefore, we inferred that the current Ne (±SD) of the Korean Landrace population is approximately 92.27 [79.46; 105.07]. This Ne was less than the effective Korean Yorkshire population size (122.87, [106.90; 138.84]) (in review). Because Landrace and Yorkshire are both major maternal breeds in the Korean pig industry, this difference in Ne values was interesting. In Korea, genetic diversity within each breed population is affected by two main factors: the breeding pig selection system used at GGP farms and the addition of breeding pigs imported from other countries. First, we thought that the genetic diversity in the Landrace population might have been less than that in the Yorkshire population because a higher number of Yorkshire individuals had been included in pig trait tests at GGP or GP farms (
Supplementary Figure S5). Second, relative to the Yorkshire population, fewer Landrace individuals were imported during the past 20 years (
Supplementary Figure S6). The Ne of the Korean Landrace population might have been less than that of the Yorkshire population because of the importance of imported individuals with regard to increased genetic diversity.
As shown in
Supplementary Table S3, LD over short distances reflects the Ne of many generations ago, whereas LD over long distances reflects the recent population history [
6,
7].
Supplementary Figures S1 and
S6 show that the historical Ne assumed a linear population in accordance with Hayes [
7]. The most interesting aspect of
Supplementary Figure S1 was the rapid decrease in Ne from 20 to 10 generations ago. This pattern was also observed in estimations of historical Ne in the Korean Yorkshire population (in review). We presume that an important event must have affected multiple pig populations, including Landrace and Yorkshire. The most likely event was an outbreak of contagious disease, such as foot-and-mouth disease (FMD). We note that the Republic of Korea had been free of FMD between 1934 and a recent outbreak in 2000. Since this outbreak, however, Korea has not remained free from FMD, and during outbreaks, huge numbers of living domestic animals, including pigs and cattle, were buried to prevent the spread of disease. We considered that repeating cycles of contagious disease spread and recovery might have affected population sizes of the main pig breeds. As shown in
Supplementary Figure S1, the observed pattern exhibited a steady decrease in Ne from 100 generations ago to the current population. Furthermore, the Ne of Korean Landrace population had decreased by 99.6% from 10,000 generations ago to the present in this study as Yorkshire population. Several factors could explain this pattern, including bottlenecks associated with domestication, selection, and breed administration. Therefore, it would useful to consider our results in the context of the demographic history of the Korean Landrace population. The reliability of this method, however, depends on both the technical implementation and data from previous studies [
24,
25].
This study aimed to characterize LD and estimate the effective size of the Korean Landrace population, using genomic data from thousands of individuals. In agreement with previous studies, the observed LD pattern of our own study of the Korean Landrace population was similar to the average value presented by Du [
28] for Landrace, the findings of a 2001 study of Finnish Landrace pig breeds by Uimari [
20,
28]. However, the overall LD in the Finnish Landrace population appeared to be slightly stronger than that in the Korean Landrace population. We thought that because Korea Landrace populations included breeding pigs imported from several countries, the genetic diversity of Korean population was larger than the single Finnish breed population.
Although the UN Food and Agriculture Organization (FAO [
4]) recommends a minimum of 50 breeding animals, Meuwissen [
31] considered this recommendation to be the lower limit for a critical population size, and proposed that the actual critical size should be range between 50 and 100. However, the current Ne of the Korean Landrace population is 92.27, which is not sufficient to maintain genetic diversity. Therefore, we suggest that the Korean Landrace population contains insufficient genetic variation and has an acceptable rate of inbreeding, including compromising genetic gains in commercially important traits. The importance of the Landrace breed to the Korean pig industry suggests that this population requires a higher level of genetic diversity. Sufficient genetic diversity is also needed when applying selection methods that maximize selection responses at a fixed inbreeding rate [
31] or methods that optimize the use of genetic resources from the parental generation .
Currently, the Ne of the Korean Landrace population would remain very small or continue to decrease if we were to apply an effective new method for estimating breeding values (e.g., genomic selection) [
17]. Therefore, we must emphasize breed management and the avoidance of inbreeding, using measurements of genetic diversity. Although this might affect short-term genetic gains, it is essential for maintaining the long-term genetic variability of the Korean Landrace population. Continuous monitoring and long-term efforts to maintain genetic diversity are also needed to control the pig population and avoid an unintended reduction of the Ne. The maintenance of a sufficient Ne within a production population is the best way to maintain a sustainable pig population. Therefore, efficient monitoring and management, as described in this report, are essential.