All practices and procedures in this study strictly followed “The Guide for Care and Use of Laboratory Animals” published by the Institutional Animal Care and Use Committee of the National Institute of Animal Science (NIAS) (2012-C-037) in Korea.

The chicken population described in previous studies was used [10–12]. It consisted of five F₁ lines of KNCs: Black line (88 chickens), Grey line (111 chickens), Red line (135 chickens), White line (121 chickens), and Yellow line (130 chickens), and the parents (F₀, 88 chickens). All the birds were nurtured under the standard feeding and environmental conditions of the National Institute of Animal Science (NIAS) Korea. Phenotypes were measured during the experiment period as part of the breeding program. The body weight of each individual was measured at two-week intervals from hatching to 20-week-old and used to calculate the weight gain in two-week intervals. The longitudinal body weight data were fitted to a logistic growth model (W_(t) = α/[1+βe^−γt]) to obtain three growth curve parameters: α = asymptotic body weight; β = the log-function for the proportion of the asymptotic mature weight to be gain after birth (week); γ = scale proportional to the overall growth rate. For the molecular analysis, blood samples were collected in ethylene diamine tetra acetic acid containing tubes and were provided by NIAS Korea. Genomic DNAs were isolated according to the published method [13]. The purity and concentration of the DNAs were assessed by spectrophotometry method (NANODrop 2000, Thermo Scientific, Wilmington, DE, USA). Stock DNAs were diluted with ultrapure water to produce a working concentration of 25 ng/μL and stored at −20°C until use.

Three SNP polymorphisms of chicken POU1F1 gene were genotype by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method. SNPs were named as M_1 to M_3. The M_1 SNP is a mutation at g. 6758 T>C in exon 5 of POU1F1 and not reported previously. The SNP M_2 (g. 9432T>C) and M_3 (g.11041T>C) were referred to gene bank accession number: rs13687127, rs13687128, respectively and were previously reported in chicken [9] (Table 1). All the Three SNPs were first screened to confirm by using the next generation sequence data of KNCs parents’ population. Three pairs of PCR primers were designed based upon the chicken POU1F1 gene sequences using Primer 3.0 software (Table 1). PCR was performed to amplify sequence fragments of the markers (Thermal cycler T100, Bio-Rad, Hercules, CA, USA) in exons 5, 6, and intron 5 of POU1F1 gene. The PCR mixture (20 μL) contained 50 ng/μL of chicken genomic DNA, Prime PCR buffer (Genetbio Inc., Daejeon, Korea), 1.6 μL dNTPs, 0.2 U Taq DNA polymerase (Genetbio Inc., Korea), 0.8 pmol of each forward and reverse primer (M_1 to M_3), and distilled water. PCR was performed using the following PCR thermal cycler conditions. Initial denaturation at 94°C for 10 min; 34 to 35 cycles of 94°C for 30 s, specific annealing temperature for each marker (60°C to 67°C) for 30 s, and 72°C for 30 s; and final extension at 72°C for 10 min. The PCR products were digested at 37°C for 6 to 12 h with HhaI, EcoRI, or BspHI (Table 1). The digestion mixture (20 μL) contained 15 μL PCR product, 1× digestion buffer, two units of each enzyme and distilled water. Genotypes of M_1 to M_3 were determined using an ultraviolet trans illuminator after 3.0% agarose gel electrophoresis of the digestion mixture at 120 V for 30 minutes.

Genotype and allele frequencies were calculated by the direct counting method [14]. Hardy–Weinberg equilibriums for KNC population was analysed using Chi Square (x²) tests with a significance level of p<0.05, using the ‘Hardy-Weinberg’ package in R version 3.2.5 [15]. Based on the allele distribution results, all the lines were pooled. Single SNP marker association analyses were performed with the general linear model procedure of analysis of variance in Minitab version 15 [16]. Haplotype inferences and haplotype frequencies were obtained using Phase 2.1 software [17] and the Haploview program [18]. Associations between haplogroups and traits were calculated using same statistical model by replacing Genotype_i to Haplogroup_i term in model.

Linear mixed model analysis was performed using following mathematical model;

Where, Y_ijklmnn is the response of each growth trait, μ is the overall mean, Sex_j is the fixed effect of the i^th sex (i = male, female), Batch_k is the fixed effect of the k_th batch (k = 1, 2), Line_l is the fixed effect of the l^th line (l = 1, 2, 3, 4, 5), Sire_m(line) is the effect of the m^th sire nested in the l^th line, Dam_n(lineSire) is the effect of the n^th dam nested in the l^th line and m^th sire, and e_ijklmno is the random residual effect. The level of significance for an association was taken and pairwise comparisons between genotypes were obtained using Tukey’s test at p value<0.05. Regression analyses of traits and SNPs were performed to obtain regression coefficients for dominance and additive effects [14]. To calculate the dominance and additive effects of each SNP, “genotype effect” in the model was replaced by dominance effect (X_dom), (genotypes were coded as 0–1-0 for CC, CT, TT, respectively) and additive effect (X_add) (coded as 0, 1, for two homozygous and 2 for heterozygous based on the minor allele frequency) [19].

In this study, we describe three SNPs (M_1 to M_3) in the KNC (Figure 1); two of these SNPs have previously been identified in the reciprocal crosses of white recessive rock male and Chinese Xinghua female chicken breed [9]. Digestion of PCR fragment of the M_1 SNP in exon 5 with HhaI, produced fragments lengths of 626 bp for the TT genotype, and 417 bp and 209 bp for the CC genotype and 626,417,209 for CT genotype. The CC, CT, and TT genotype frequencies of M_1 were 0.86, 0.10, and 0.04, respectively. The corresponding C and T allele frequencies were 0.91 and 0.09, respectively. The M_2 SNP in intron 5 (genebank accession number rs13687127) PCR product was digested with EcoR1, produced a 442 bp fragment for the CC homozygote, 246 and 196 bp fragments were in the TT homozygote and 442, 246, and 196 bp in heterozygous CT genotype. The frequency of the CC genotype was 0.87 while the frequency of the CT and TT genotypes were 0.11 and 0.022, respectively. PCR product of M_3 SNP (genebank accession number rs13687128) produced fragments of 283, 251, and 216 bp for CC the genotype and 467 bp and 283 bp for the TT genotype and 467,283,251, and 216 bp for heterozygous CT genotype after digest with BspH1. In M_3 SNP, CC genotype was most frequent (0.83) and the TT genotype was least frequent (0.03); the CT genotype also occurred at a low frequency value of 0.14.

Genotypes and allele frequencies for all markers (M_1 to M_3) and Chi Square (x²) tests for Hardy-Weinberg equilibrium were calculated and are summarized in Table 2. The CC genotype was more frequently observed in KNC than in other genotypes, and the C allele frequency was higher than that of the T allele in all SNPs. None of the SNPs was in Hardy-Weinberg equilibrium in KNC population.

Four haplotypes were reported in this study. Among these, CCC (H1) was the major haplotype (88.01%, 1,029/1168), three minor haplotypes were also found at frequencies of lower than 1%: CTT, TCC, TTT (H4 (32/1,168), (H5 49/1,168), and H6 (58/1,168), respectively).

Log of the likelihood odds ratio scores (LOD), a pairwise measure of linkage disequilibrium D′ = |D|/D_max [20] between the three SNP loci were estimated. Higher D′ indicates a low recombination rate between the loci. The D′, LOD, and regression coefficient (r²) values between M_1 and M_2 were 0.596, 36.83%, and 0.294, respectively; between M_1 and M_3, they were 0.563, 32.63%, and 0.314, respectively. The highest D′ and r² values were found between M_2 and M_3 and were 0.999, and 0.836, respectively (Figure 2).

A single marker trait analysis showed that M_1 SNP was not significantly associated with any of the growth traits tested in KNC. Whereas, M_2 SNP was significantly associated with body weight at two weeks (BW02), weight gain from hatch to two weeks (GR0–2), weight gain from 16 to 18 weeks (GR16–18) (p<0.05), and with asymptotic body weight (α) (p<0.1) (Table 3). The highest BW02 and GR0–2 were found in the TT type animals, while the lowest was observed in the CT type. However, for weight gain, GR16–18, the CC animals had a higher weight gain than the TT animals, while the CT type showed moderate weight gain. The M_3 SNP was significantly associated with weight gain from 14 to 16 weeks (GR14–16), weight gain from 16 to 18 weeks (GR16–18), and with asymptotic body weight (α) (p<0.05). The CC type had the highest weight gain and asymptotic body weight (α), while TT type had the lowest values. Overall, GR14–16, GR16–18, and α trait in the CC animals were significantly different from those in the TT animals by 19%, 25%, and 1.1%, respectively (Table 3).

We screened the haplotypes of the 584 individuals in this study and found four haplotypes and eight diploids: H1H1 (482), H1H2 (14), HIH3 (14), H1H4 (37), H2H2 (5), H2H4 (8), H3H3 (11), H3H4 (13). The haplogroup H1H3 was associated with GR14–16 and had higher weight gain than those of the H1H4 and H2H2 (Supplementary Table S3).