Native Korean goats have been raised in the peninsula over the last 2,000 years (Kim et al., 2014). Because native Korean goats produce low volumes of milk and meat, researchers opted to cross them with Saanen goats that are believed to produce greater volumes of both milk and meat. As Saanens are the largest goat breed among the dairy types and are also consistently good milk producers, they have been used most often to form the crossbred goat group. In addition, the Boer goat, which is developed for meat production, has also been used for crossbreeding. These crossbred black goats in Korea not only have a higher milk yield but also a higher growth rate than the native goats. In addition, when fully grown, the crossbred goats are bigger than the native Korean goats (Son, 1999).
Detecting genetic variants related to phenotypic traits is an important issue in livestock genomic research. Due to their large differences in body size, weight, muscle mass, milk production, and coat color (
Whole-blood samples were collected from 11 crossbred goats and 15 native Korean goats in Korea. Blood samples from the native Korean goats were collected from the Animal Genetic Resources Station, National Institute of Animal Science, Rural Development Administration in Korea. For the crossbred goats, we obtained the samples from a Korean small black goat farm. Blood (10 ml) was drawn from the carotid artery and treated with heparin to prevent clotting. DNA was isolated from the whole blood solution using a G-DEXTMIIb Genome DNA Extraction Kit (iNtRoN Biotechnology, Korea) according to the manufacturer’s protocol. We randomly sheared 3 μg of genomic DNA using the Covaris System to generate inserts of about 300 bp. The fragments of sheared DNA were end-repaired, A-tailed, adaptor-ligated, and amplified using a TruSeq DNA Sample Prep Kit (Illumina, USA). Paired-end sequencing of the goat genomes to about ten-fold coverage was conducted at NICEM (National Instrumentation Center for Environment Management, Seoul, Korea) using the Illumina HiSeq2000 platform with the TruSeq SBS Kit vs-HS (Illumina). All of the short-read data have been deposited at the Short Read Archive (SRA) under accession lot SRA160379.
Approximately 220,000,000–230,000,000 paired-end reads were mapped to the reference goat genome (Dong et al., 2013) from the Goat Genome Database Web site (
To identify and compare highly variable regions, the distribution of variants and nucleotide diversity information was obtained based on the goat reference genome (Dong et al., 2013) from the Goat Genome Database website (
To identify SNPs that contribute to differences in phenotypes between native Korean goats and crossbred goats, VCF format files of candidate SNPs were separately concatenated using VCFtools (Danecek et al., 2011). Variant regions were then annotated using SNPEff (Cingolani et al., 2012) with a GFF format file (Dong et al., 2013) from the Goat Genome Database website (
We performed principal component analysis (PCA) (Jackson, 2005) to examine population differentiation between native Korean goats and crossbred goats using genotype data from 15 native Korean goats and 11 crossbred goats. We used GCTAtool (Browning and Browning, 2007), which implements PCA in EIGENSTRAT (Price et al
We used STRUCTURE software (Evanno et al
We used the cross-population extended haplotype homozygosity (XP-EHH) method (Sabeti et al
We generated NGS pair-end reads to about ten-fold coverage for 15 native Korean goats and 11 crossbred goats using Illumina HighSeq2000 to obtain goat re-sequencing data. For each individual goat, over 92% of all the reads [excluding possible polymerase chain reaction (PCR) duplicates] were successfully aligned to the reference goat genome [domestic goat,
In total, 22,759,033 SNVs and 2,450,921 INDELs were identified, and 26.6% of these SNVs and 26.8% of the INDELs were located in genic regions (
We examined the nucleotide diversity of native Korean goats and crossbred goats using VCFtools (Danecek et al., 2011). We also counted the number of SNVs and integrated them for each 1-Mb bin region of the genome using VCF format files, which contained variant information on 26 goats based on the reference goat genome (Dong et al., 2013). The overall distributions of native Korean goats and crossbred goats in terms of nucleotide diversity differed. Native Korean goats generally showed lower nucleotide diversity (total average nucleotide diversity = 0.0007) than the crossbred goats (total average nucleotide diversity = 0.0010) and the SNV density of the native Korean goats (total average SNV density = 6662.83) was also lower than the crossbred goats (total average SNV density = 7701.61) in the same genomic regions (
According to the PCA results (Jackson, 2005), the native Korean goats were clearly distinct from the crossbred goats (Fig. 2). We then examined the genetic structures of native Korean and crossbred goat populations through admixture analysis using STRUCTURE software (Evanno et al., 2005). We observed that the native Korean and crossbred goats shared a majority of their ancestral states and that the crossbred goats originated from the crossbreeding of native Korean native goats with other breeds (Fig. 2).
We took the top 5% of the highly variable regions among all the chromosomal regions in each population as significant. We referenced the Goat Genome Database (Dong et al., 2013) and the Ensembl Genome Database (Hubbard et al., 2002) to identify gene locations and annotation information (
Among 16,570,906 SNV sites, we identified 76 significant non-synonymous SNP sites of native Korean goats using generated re-sequenced data and SNPEff Software. We identified the chymosin (
We estimated the XP-EHH values (Sabeti et al., 2007) to detect selective sweep regions and performed a pairwise test of the native Korean and crossbred goat populations (Fig. 3). The genome was split into non-overlapping segments of 50 kb and we computed the maximum XP-EHH score in each segment. To define the empirical P-value, the genomic windows were binned in increments of 500 SNPs according to methods used elsewhere (Lee et al
In addition, we used an XP-CLR (Chen et al., 2010) to detect selective sweep regions between the two populations (Fig. 3). Using the top 100 XP-CLR score regions, 161 significant genes were identified in the native Korean population. Based on the 161 genes, we performed GO-term analysis using the DAVID analysis tool (Dennis Jr et al., 2003). GO-term cell adhesion (FDR < 0.05) was among the most enriched functional categories that might be related to lumbar paralysis. In addition, neuron development was enriched in the GO-term (FDR < 0.05) (
Native Korean goat populations and some outbred goat lines were used to form crossbred goat populations; this population was based on native Korean goats but was meant to improve on the inferior traits of these same goats. From whole-genome sequencing data, we observed a reduction in nucleotide diversity in native Korean goats compared with the crossbred goats; this might be an indication of inbreeding in native Korean goats (
The results of our admixture analysis using STRUCTURE (Evanno et al., 2005) are presented in Fig. 2. The proportion of imported alleles increased during crossbreeding, while a significant portion of the alleles became indigenous allele majorities in the native Korean goat population. In the crossbred goat population, nucleotide diversity in the genome may have increased due to the consistent introduction of new alleles, unlike in the native Korean goat population.
Recombination rates are known to affect nucleotide diversity (Nachman, 2001) and the terminal regions of chromosomes are known to be prone to recombination events. Based on the distribution of variation in the goat genome (Fig. 1), the terminal regions of chromosomes showed more variation than the other regions, and the top 5% of highly variable regions were enriched with genes involved in olfactory sensors and neurological systems. From the results of the non-synonymous SNP analysis in our study, we identified the enrichment of amino acid substitution in genes that were related to olfactory sensors.
Genes involved in olfactory systems may have had more opportunities to gain variants in nucleotide sequences. Olfactory receptors interact with odorant molecules in the nose to initiate a neuronal response that triggers the perception of a smell. Odor molecules in the environment are detected by olfactory receptors. For animals, olfactory receptors are essential to finding nutritious food and in their ability to avoid eating toxic substances, avoid predators, and identify suitable mating partners and their offspring (Mombaerts, 2004; Niimura, 2009). Native Korean goats were developed under feed shortage conditions and were as a result forced to graze in fields, freely or confined, and tend to overgraze shrubs and the bark of trees. The digestibility of these plants is low. The positive selection of olfactory genes might therefore have arisen as an adaptation to these conditions. Saanen goats, on the other hand, represent commercial breeds that have been artificially selected for intensive production (Choi et al
Native Korean goat populations are also distinct from crossbred goat populations based on non-synonymous SNP patterns of the
Native Korean goats are resistant to lumbar paralysis, a condition that has a severe effect on goats of exotic origin introduced into the peninsula (Son, 1999). Lumbar paralysis is a common disease in ungulate mammals such as goats, sheep, or cattle, and it is transmitted through mosquitos that carry filarial parasites called
Major candidate genes obtained from XP-CLR and XP-EHH analysis
|Candidate genes||Chromosome||Start||End||Max XP-CLR||XP-CLR ||Max XP-EHH||XP-EHH ||Description|
|22||52849231||52882844||23.73496||0.005||-||-||Receptor for a C-C type chemokine|
|13||59025564||59064557||21.42064||0.01||-||-||Minor histocompatibility antigen H13|
|1||114342294||114369614||18.15857||0.01||-||-||Immunoglobulin superfamily member 10|
|1||24953553||26069449||24.10493||0.01||-||-||Roundabout homolog 1|
|1||22148573||22808722||19.23946||0.05||-||-||Roundabout homolog 2|
|6||102650000||102700000||-||-||3.61921||0.0074||Cytokine-dependent hematopoietic cell linker|
|4||54037221||54172686||15.234912||-||-||-||Myosin, Heavy Polypeptide Kinase|
CCR3 region sequence information of the 11 loci
CHR1 = Chromosome Number; BP2 = SNP physical location; Ref 3 = Reference allele code; Alt4 = Alternate allele code; Native5 = Korean native goats alternative allele frequency; Cross6= Cross breed goats alternative allele frequency; CHISQ7= chi-square test statistic (1df); P8 = p-value; OR9 = Odds ratio