Evaluating genetic resources and analyzing molecular markers related to egg yield traits in lien minh chicken breed

n enzyme (1 µl), the incubation time is 16 hours, the incubation temperature is corresponding to each enzyme cut (Table 2.4). Table 2.4. Information for polymorphisms and restriction enzyme (RE) Locus Polymophism RE GenBank accession number PCR-RFLP size [bp] Ta [oC] Manufacturer PRL C-2402T (PRL5) AluI AB011438 439/304/160/144/81/54 37 Themo, America – 358/ indel 24 bp (PRL24) - AF288765 154/130 37 Themo, America C2161G Csp6I AB011438 439/405/34 37 Themo, America VIP C+338T HinfI NC_006090 520/480/40 37 Themo, America G5138982T ApoI NC_006090 520/486/34 37 Themo, America VIPR1 C1715301T TaqI NC_006089 486/310/176 65 Themo, America C1704887T HhaI NC_006089 434/253/181 37 Themo, America NPY Indel 4 bp tại 3139135 DraI M87298 248(252) 167/81 37 Themo, America C31394761T KpnI GI:35797382 9 324/200/124 200/124 37 Themo, America GH C-2983T SacI JN675393 1164/1020/ 570/450/144 37 Themo, America GHR C571T (intron 5) NspI NC_006127 750/550/200 37 Themo, America GHR A294-541G (intron 2) HinIII NC_006127 718/428/290 258/170/290 37 Themo, America 2.1.2. The chemicals Chemicals used in molecular biology research of Sigma (Germany), Merck (Germany), and Thermo (USA). 2.1.3. Machinery used PCR machine eppendorf AQ mastercycle 5332YN056927 (Germany), horizontal electrophoresis machine FCIE-PLAS-HU1030012479 (UK), water bath (JSR- JSWB22T-Korea), storage refrigerator 40C (Panasonic - Japan), gel scanner (UVP-M- 7 26V / P / N95-0458-02, USA), centrifuge (HETTICH-UNI version 320R, Germany), egg pressure gauge, Nano Drop Spectrophotometer. 2.2. RESEARCH CONTENT - Content 1: Assessing genetic diversity of Lien Minh chicken based on mitochondrial DNA - Content 2: Analyzing the relationship between genetic markers and egg production traits in Lien Minh chicken + Experiment 1: Identifying indicators related to egg production traits in 90 Lien Minh hens; + Experiment 2: Determining genotypic and allelic frequency in candidate genes (PRL, VIP, VIPR1, NPY, GH and GHR) related to egg yield traits; Identifing the relationship between candidate genes and egg yield traits in Lien Minh chickens. 2.3. METHODS 2.3.1. Blood sampling The blood samples from Lien Minh chickens were taken with a 5 ml syringe from the vein of the wing vein, then quickly transferred to a blood tube containing EDTA-K anticoagulant. Blood tube was stored at 4ºC, and -20ºC in the lab. 2.3.2. DNA extraction Genomic DNA was extracted by a standard procedure using Proteinase K digestion followed by phenol-chloroform extraction and precipitation with ethanol (Ausubel et al, 1995). A nanodrop spectrometer and agarose gel electrophoresis were used to estimate DNA concentration and purity. DNA samples were stored at -20oC until analysis 2.3.3. PCR amplification The PCR primers were designed based on the chicken candidate gene sequences using Primer 3.0 software. PCR was performed in a 25 µl reaction containing 1x PCR Buffer, 1.5 mM MgCl2, 1.25 mM each dNTPs, 5 pM primer, 1U Taq-polymerase (Fermentas), and 100 ng genomic DNA. In PCR amplification, an initial denaturation at 94oC for three minutes followed by 35 cycles of denaturation at 94oC for 45 seconds, annealing for 45 seconds and extension at 72oC for 90 seconds, and an additional extension of 72oC for seven minutes was set. 2.3.4. PCR purification and gene sequencing Purification of DNA fragments for sequencing: The purification process was used with the QIAquick PCR Purification Kit (QIAGEN). The purified product was 8 sequenced on the ABI-3100 Avant Gentic Analyzer from Macrogen Company (Korea). 2.3.5. Restriction Fragment Length Polymorphism Genetic length polymorphism analysis used PCR-RFLP (Restriction Fragment Length Polymorphism (RFLP) technique with appropriate restriction fragment enzymes. PCR products were then digested with restriction enzymes overnight. The restriction fragments were separated on 2% agarose gel stained with ethidium bromide. 2.3.6. Evaluate indicators related to reproductive traits in chickens Based on the description of Bui Huu Doan et al (2011), the indicatorsto produce eggs include age at first egg laying (days), weight of first egg (gram), energy egg yield (total number of eggs/hens/20 weeks) egg weight (grams) and egg shape index (large diameter size/small diameter D/R). 2.3.7. Body weight The body weight of Lien Minh chickens was weighed every Friday at 19 o'clock (start from 01 day to 22 weeks), when chickens go to barns, using a scale of 5 g accuracy. 2.3.8. Statistical analyses Nucleotide sequences were identified with the Basic Local Alignment Search Tool (BLAST) (Altschul et al, 1990). Multiple nucleotide alignments were carried out with BioEdit (Hall, 1999). The diversity parameters, including the nucleotide diversity haplotypes D-loop of all sequences were estimated using DnaSP v.5 software (Librado and Rozas, 2009). Neighbor Joining (NJ) phylogenetic tree was conducted using MEGA version 6.1 (Tamura et al, 2007). Phylogenetic tree analysis was constructed based on classification of Oka et al, (2007) with reference mtDNA sequences from the GenBank database. , The gene frequencies were calculated by counting method as: p= 2(AA) + (AB)/2N and q= 2(BB) + (AB)/2N where p = the gene frequency of allele A, q = the gene frequency of allele B and N = the total number of chickens tested. The Hardy- Weinberg Equilibrium (HWE) was estimated using the method of Rodriguez et al, 2009. The association between genotype and egg production was analyzed based on General Linear Model of Minitab software version 16.0: Yij= µ + Gi + ξij (where Yij: traits observed; μ: general mean, Gi: influence of genotype; ξij: random error). 9 Chapter III. RESULTS AND DISCUSSION 3.1. GENETIC DIVERSITY ANALYSIS IN INDIGENOUS LIEN MINH CHICKEN 3.1.1. DNA extraction In order to carry out an evaluation of the Lien Minh chicken genetic diversity based on the nucleotide sequence of D-loop region of mitochondrial DNA, it is necessary to obtain pure and intact genomic DNA. Table 3.1. Results of genomic Spectrophotometer DNA samples OD260nm/280nm Concentration (ng/µl) GLM01 → GLM24 1,82 → 1,99 100,56 → 694,80 G9C01 → G9C06 1,81 → 1,90 257,50 → 428,96 GDT01 → GDT18 1,85 → 1,97 225,60 → 515,37 Table 3.1 shows that the OD260/280 ratio of DNA samples are in the range of 1.8 - 2.0, which proves the extracted DNA is purified, the DNA concentration is from 100.56 to 694.80 ng/µl. Therefore, the quality of genomic DNA samples is assurance to subsequent experiments. 3.1.2. Amplification of the mitochondrial DNA D-loop region As shown in Figure 3.2, DNA sample is a bright band and has a molecular expected size of about 1300 bp (the electrophoresis band is located between two standard DNA bands of 1000 bp and 1500 bp). Figure 3.2. Eectrophoresis of PCR products on 1% agarose gel Wells 1 → 9: PCR products of Lien Minh chickens, M: Ladder DNA1kb (Merck) 3.1.3. Nucleotide sequence in D-loop region The result of the nucleotide sequence in D-loop region of Lien Minh chicken were registered on GenBank with the codes: from KY172116 to KY172121 and from 10 MH425591 to MH425608. 3.1.4. Analysis of nucleotide polymorphism in D-loop region of Lien Minh chickens and other native chickens with code on Genbank 3.1.4.1. Diversity analysis in D-loop region DnaSP was used to analyze the nucleotide polymorphism in mtDNA D-loop region of Lien Minh chicken . The analytical parameters include: Number of polymorphic sites (S); number of haplotype (h); haplotype diversity (Hd); nucleotide diversity (Pi); Tajima test (D). The analysis results of the nucleotide polymorphic parameters (1050 bp) in Lien Minh chickens, other native chickens in Vietnam were shown in Table 3.2. Table 3.2: Polymorphic sites, haplotype and nucleotide diversity of chicken breeds Breeds N S h Hd Pi NC D Lien Minh 24 23 12 0.913 0.007 4 0.187* Dong Tao 18 11 7 0.856 0.004 1 1.721* Nhieu Ngon 6 4 3 0.867 0.002 1 0.562* N: number of samples, S: number of polymorphic sites (excluding sites with gaps), h: Number of haplotypes, Hd: Haplotype diversity, Pi: nucleotide diversity, NC: number of clades, D:Tajima’s test. 3.1.4.2. Polymorphic nucleotide sequence in D-loop region a. Analysis of D-loop nucleotide polymorphisms between Lien Minh chickens and native Vietnamese chicken breeds (455 bp) The analysis results indicate, there were 21 nucleotide substitution sites and no nucleotide insert/deletion polymorphism when compared to a native Vietnamese chicken coded GU564373 (Dong Tao chicken). Figure 3.4 showed that among 21 nucleotide substitution sites there are 11 T→C substitutive positions, four A→G substitutive positions, five substitutive positions C→T, but only one G→A substitutive nucleotide sites. Especially, T→C substitution at position 241 appeared in all Lien Minh chickens, in addition this substitution at position 206 appeared in 17 of 24 studied samples. b. Analysis of D-loop nucleotide polymorphisms between Lien Minh chickens and native breeds in the world (in Genbank) The results showed that from 24 nucleotide sequences were identified by 23 nucleotide polymorphic positions between Lien Minh chicken and the branch E breed with code AB268540 (Gallus gallus domesticus), including 22 nucleotide substitution sites and a nucleotide insert site. Special nucleotide G insertion at position 860 appears in all Lien Minh chickens. Besides, nucleotide substitution T→C appears in 10 positions (167, 199, 217, 243, 246, 256, 270, 315, 367, 888) and substitutions C→T appears only in position. 225, 261, 310 and 446. 11 3.1.4.3. Nucleotide identity of D-loop region a. Analysis nucleotide identity of D-loop region between Lien Minh chickens and native Vietnamese chicken breeds (455 bp) Mostly, genetic diversity of indigenous chicken was analyzed by using 455 bp D- loop region (Le Tien et al, 2009; Cuc et al, 2011). Therefore, 455 bp D-loop region was used to compare the level of nucleotide diversity of D-loop between Lien Minh chicken and other Vietnam native chickens. The analytical results indicated that the nucleotide identity among Lien Minh chickens reached 97.5% - 100%, in which 14 Lien Minh individuals of Group I have the nucleotide identity of 100% (GLM 01, GLM02, GLM03, GLM06, GLM 07, GLM 08, GLM 09, GLM 11, GLM12, GLM13, GLM14, GLM15, GLM16 và GLM18) have Nucleotide identity index that reaches. 100%. The above Lien Minh chickens have a nucleotide identity to the Lien Minh chickens group II (GLM10 and GLM17) reaching 97.5%. As demonstrated from the pairwise comparison that the Lien Minh chickens shared high nucleotide identity with their native counterparts in Vietnam, ranging from 96.8 to 99.7%. Particularly, the percent nucleotide identity of group I Lien Minh chicken (n=14) compared to branch B native chicken ranged from 99.3 to 99.7%. Also, Lien Minh chickens in this group have a high nucleotide identity coefficient when compared to Dong Tao and Multi-Finger chickens (99.1-99.7). On the contrary, when compared to Vietnamese native chicken breeds in branches C, D, E, G, I, the percent nucleotide identity of Lien Minh chickens in group I reaching only from 97,3 % to 97,9%. Lien Minh chickens of group II (GLM10 and GLM17) shared a high percent nucleotide identity compared to native chickens in branch B8 (99.1%), while the nucleotide identity of Lien Minh chickens in group III (GLM04, GLM19, GLM23), GLM24) reached 99,1% of nucleotide identity whenc compared with the Vietnamese native chickens in D1 branch. Lien Minh chickens in group IV (GLM20) and group V (GLM05) had high nucleotide identity with Vietnamese native chickens of branch C1 (99.5-99.7%). Lien Minh chickens of groups VI (GLM21) and VII (GLM22) had high percent nucleotide identity to those of E1 branched chickens (99.3-99.5%). b. Analysis of D-loop nucleotide identity between Lien Minh chickens and domestic chickens in the world (1050 bp) 1050 nucleotides in the D-loop region of Lien minh chicken were used to compare with the native chickens of the world (Oka et al, 2007). Result analysis illustrated that the nucleotide identity coefficient among Lien Minh chickens reached 12 98.7% - 100%. In particular, Lien Minh chickens in groups I (GLM 01, GLM 07, GLM 08, GLM 09, GLM 11), group II (GLM02, GLM18), group III (GLM12, GLM13, GLM14, GLM15), group IV ( GLM06) and group V (GLM03, GLM16) shared a fairly high nucleotide identity from 99.7% to 99.9%, Lien Minh chickens in the same group have a nucleotide identity of 100%. The nucleotide identity of Lien Minh chickens and domestic chickens according to research results of Oka et al (2007) reached 98.2% - 100%. Lien Minh chickens of groups I, II, III, IV and V shared nucleotide identity with chickens of branch E1, E2, E3, E4 (Oka et al, 2007) reaching 99.6% - 100.0%. Lien Minh chickens GLM10, GLM17, GLM21, GLM22 shared a high nucleotide identity with chicken in branch A (99.6% - 100.0%),Lien Minh chickens of group VIII (GLM04, GLM19, GLM23, GLM24) shared a high nucleotide identity with domestic chickens in branch C, reaching 99.5%. The nucleotide identity between GLM05, GLM20 with branch D chicken is 99.8-99.9%. On the other hand, the nucleotide identity is lower when compared Lien Minh chicken breed to the domestic chicken in branches G, F, merely ranged 98.2 to 99.2%. 3.1.4.4. Distribution Haplotype Lien Minh chickens Using DnaSP software version 5, determine haplotype on 1050 bp and 455 bp Dloop region. A total of 12 haplotypes in 24 Lien Minh chickens based on 1050 bp of mtDNA D-loop sequence, with in are focused most on three haplotypes (1, 3 and 8). Beside this, a total of 7 haplotypes in 24 Lien Minh chickens based on 455 bp of mtDNA D-loop sequence, with in are focused on haplotype 1 (14 chickens). 3.1.4.5. The Phylogenetic analysis of the Lien Minh chicken is based on the D- loop nucleotide sequence a. Phylogenetic analysis between Lien Minh chicken and native Vietnamese chicken breeds (455 bp) Phylogenetic tree was constructed based on 48 D-loop sequences (24 GLM sequences, six G9C sequences and 18 GDT sequences) along with 11 reference sequences that corresponded to the nine clades defined by Liu et al, (2006) and 37 sequences of indigenous Vietnamese chickens distributed seven branches (Cuc et al, 2011). Using 455 bp D-loop mitochondrial DNA to determine the genetic relationship between Lien Minh chickens and native Vietnamese chicken breeds (Figure 3.10). 13 Figure 3.10. The phylogenetic tree in Lien Minh chicken with native Vietnamese chickens The phylogenetic tree was generated by the neighbor‐joining method using MEGA6.1 with bootstrap values for interior clades after 1,000 replications. Different clades and haplotypes are indicated. GLM, GNN, and GDT breeds are marked (circle●, triangular▲, and square■, respectively). Numbers in bracket are number of samples that appeared at identical haplotype. 14 As can be seen from the Phylogenetic tree which was constructed based on D- loop, Lien Minh chickens were distributed on seven haplotypes of 5 clades (A, B, C, D, E). In general, 58,3% of the Lien Minh chickens were presented in clade B that has also known as the dominant clade of indigenous Vietnamese chickens and Southeast Asia. In this study, no samples of Lien Minh, Dong Tao, Many Finger chicken breeds were distributed in clades G, H, I and F. This result was consistent with the study of Cuc et al (2011). b. Phylogenetic analysis between Lien Minh, Dong Tao, Many Fingers chicken breeds and the world native chicken breeds (455 bp) Figure 3.11. Phylogenetic tree in Lien Minh chicken with native chickens in the world The phylogenetic tree was generated by the neighbor‐joining method using MEGA6.1 with bootstrap values for interior clades after 1,000 replications. Different clade and haplotype are indicated. GLM, GNN and GDT breeds are marked (circle●, triangular▲ and square■, respectively). Numbers in bracket are number of samples appeared at identical haplotype. Phylogenetic tree was constructed based on 48 D-loop sequences (24 GLM sequences, six G9C sequences and 18 GDT sequences) along with 11 reference 15 sequences that corresponded to the seven clades defined by Oka et al (2007). Using 1050 bp of D-loop region and using NJ method to build Phylogenetic tree for Lien Minh chicken, Multi Finger chicken, Dong Tao chicken and domestic chicken in the world. Results showed that, 24 GLM samples were observed in five clade A, B, C, D and E. Specifically, 14 samples (58.3%) of Lien Minh chickens belonging to haplotype 1, 2, 3 and 4 were distributed in clade E. Previous studies have shown that chickens of clade E were observed in indigenous chickens in Asia, China, Laos, Japan and India (Liu et al, 2006, Oka et al, 2007, Cuc et al, 2011, Kawabe et al, 2014) and were not observed in native African chickens (Miao et al, 2013). 3.2. ANALYSIS POLYMORPHISM IN CANDIDATE GENES RELATED WITH EGG PRODUCTION TRAITS IN LIEN MINH CHICKEN 3.2.1. Track five egg production traits related to egg yield traits of 90 Lien Minh chickens Experiment was conducted on 90 hens which were kept in separate cages for the period of egg laying (25-44 weeks old). All hens were fed with same diet and veterinary indicators during the experiment aiming to evaluate the correlation between candidate genes and egg yield traits in Lien Minh chickens. During the 20-week spawning period, each Lien minh chicken individual was evaluated on a daily basis against five egg production traits which are as follows age at first egg, first egg’s weight, number of eggs, eggs’ weight, and egg’s shape index (D/d). 3.2.2. The frequencies of the alleles and genotypes of polymorphisms of PRL, PRLR, VIP, VIPR1, NPY, GH and GHR; analyze the relationship with egg yield traits in Lien Minh chickens 3.2.2.1. DNA extraction 3.2.2.2. The frequencies of the alleles and genotypes of polymorphisms of PRL and its association with egg yield traits a. PCR amplification of gen PRL b. Polymorphic analysis of PRL gene by restriction enzyme c. Identification of PRL polymorphism by nucleotide sequencing d. Analysis of the frequencies of the alleles and genotypes of polymorphisms of PRL Table 3.6 illustrates polymorphisms of PRL24, PRL/2161genes were not significantly different from the distribution expected under the assumption of Hardy Weinberg equilibrium (P>0.05). The PRL/2402 polymorphism did not follow the Hardy-Weinberg equilibrium (P<0.05) e. Association of the PRL polymorphisms on egg production traits in Lien Minh chicken 16 Table 3.9. Association between SNP PRL24 and egg production traits in Lien Minh chicken Egg production traits Genotype (N) P ID (24) DD (66) Age at first egg (days) 185.33±8.21 187.12±8.25 0.365 Number of eggs in 20 weeks (egg) 45.29±5.77 43.14±4.73 0.075 Mean eggs weight in 20 weeks (g) 47.57±3.11a 45.05±4.33b 0.011 Mean eggs weight in 36-44 weeks (g) 49.13±3.27a 46.58±4.68b 0.016 First egg’s weight (g) 42.16±4.57 39.95±4.98 0.062 Eggs’ shape index (D/d) 1.28±0.03 1.28±0.03 0.632 a, or b, values with no common superscripts within a column for each site differ significantly (P<0.05) Analysis of the relationship between polymorphism of PRL24 and egg yield traits revealed that the average egg weight of chickens with genotype ID (47.57±3.11 g) was greater than that of chickens with DD (45.05±4.33 g) ( P<0.05) (Table 3.9). For polymorphism of PRL/C2402T, the average egg weight in Lien Minh chickens with genotype CT (46.91±4.29 g) was higher than genotype TT (44.89±3.93 g) (P<0,05) (Table 3.10). Table 3.10. Association between SNP PRL/C2402T and egg production traits in Lien Minh chicken Egg production traits Genotype (N) P CT (37) TT (53) Age at first egg (days) 185.84±7.99 187.21±8.42 0.440 Number of eggs in 20 weeks (egg) 44.95±5.63 42.85±4.53 0.054 Mean eggs weight in 20 weeks (g) 46.91±4.29a 44.89±3.93b 0.023 Mean eggs weight in 36-44 weeks (g) 48.27±4.52 46.56±4.36 0.073 First egg’s weight (g) 41.17±4.44 40.11±5.27 0.319 Eggs’ shape index (D/d) 1.28±0.03 1.28±0.03 0.585 . a, or b, values with no common superscripts within a column for each site differ significantly (P<0.05) For polymorphism of PRL/C2161G, there was no statistically significant association between the SNP PRL/C2161G and egg production traits (Table 11) Table 3.11. Association between SNP PRL/C2161G and egg production traits in Lien Minh chicken Egg production traits Genotype (N) P CC (4) CG (26) GG (60) Age at first egg (days) 187.00±2.94 186.42±8.55 186.72±8.41 0.985 Number of eggs in 20 weeks (egg) 48.50±3.70* 44.00±4.98* 43.27±5.10* - Mean eggs weight in 20 weeks (g) 48.99±3.25 45.10±3.58 45.77±4.41 0.221 Mean eggs weight in 36-44 weeks 51.14±3.37 46.94±3.74 47.14±4.76 0.206 First egg’s weight (g) 41.48±4.45 39.69±5.89 40.85±4.55 0.565 Eggs’ shape index (D/d) 1.27±0.02 1.27±0.02 1.29±0.03 0.107 . * : Data were not subjected for statistical analysis. 17 f. Effect of haplotype PRL gene at positions: -385; 2402; and 2161 to egg yield traits in Lien Minh chickens The analysis of the combination of two polymorphisms PRL24 and PRL/2402 (Table 3.12) showed that the average egg weight of haplotype IDTT (48.17±1.83) was higher than that of haplotype IDCT (47.20±3.68), DDCT (46.71±4.73) and DDTT (44.22±3.91) (P<0.01). Table 3.12. Effect of nucleotide polymorphism of PRL24 and PRL/2402 on egg production traits in Lien Minh chicken Egg production traits Haplotype (N) P DDCT (22) DDTT (44) IDCT (15) IDTT (9) AFE (days) 188.05±7.64 186.66±8.59 182.60±7.61 189.89±7.42 0.128 FEW (g) 40.69±4.76 39.59±5.10 41.88±3.96 42.62±5.67 0.230 EN(eggs) 44.64±4.96 42.39±4.48 45.40±6.64 45.11±4.29 0.107 MEW (g) 46.71±4.73ab 44.22±3.91b 47.20±3.68ab 48.17±1.83a 0.006 MEW 36-44(g) 47.99±5.08 45.88±4.36 48.68±3.66 49.87±2.51 0.022 ESI (D/d) 1.28±0.03* 1.28±0.03* 1.28±0.03* 1.28±0.03* - AFE: age at first egg, FEW: first egg’s weight, EN: number of eggs in 20 weeks, MEW: mean eggs weight in 20 weeks, MEW 36-44: mean eggs weight in 36-44 weeks and ESI: eggs’ shape index. a,b or ab, values with no common superscripts within a column for each site differ significantly (P<0.05); *: Data were not subjected for statistical analysis. Analysis of the combination of two polymorphs PRL24 and PRL/2161 also showed that chickens with haplotype IDCC showed average egg weight and egg yield, higher than chickens with the remaining haplotypes. Table 3.13. Effect of nucleotide polymorphism of PRL24 and PRL/2161 on egg production traits in Lien Minh chicken Haplotype (N) AFE (days) MEW 36- 44(g) EN(eggs) MEW (g) ESI (D/d) DDCC (3) 187.33±3.51 52.34±2.89 47.67±4.04 49.41±3.85 1.27±0.02 DDCG (16) 185.00±8.81 45.70±3.96 42.69±3.98 43.83±3.77 1.28±0.02 DDGG (47) 187.83±8.26 46.52±4.81 43.00±4.93 45.18±4.41 1.29±0.03 IDCC (1) 186.00±0.00* 47.52±0.00* 51.00±0.00* 47.75±0.00* 1.29±0.00* IDCG (10) 188.70±8.03 48.91±2.40 46.10±5.88 47.14±2.09 1.27±0.02 IDGG (13) 182.69±7.99 49.41±3.98 44.23±5.78 47.88±3.87 1.29±0.04 P 0.391 0.038 0.167 0.041 0.372 AFE: age at first egg, FEW: first egg’s weight, EN: number of eggs in 20 weeks, MEW: mean eggs weight in 20 weeks, MEW 36-44: mean eggs weight in 36-44 weeks and ESI: eggs’ shape index (N): Number of chickens Particularly, the average egg weight of chicken with haplotype DDCC (49.41±3.85) was higher than that of chicken with haplotype DDCG (43.83±3.77) with 5.58 grams (P <0.05). In addition, chickens with haplotype DDCC showed high egg yield of 47.76 18 eggs/hen/20 weeks higher than chickens with haplotype DDCG of 4.98 eggs (P = 0.051). It was found from the results that there was a significant interrelation between ID and CC genes and egg weight, egg yield in Lien Minh chickens. Table 3.14. Effect of nucleotide polymorphism of PRL/2402 and PRL/2161 on egg production traits in Lien Minh chicken Haplotype(N) AFE (days) MEW 36- 44(g) EN(eggs) MEW (g) ESI (D/d) CTCC (3) 188.00±2.65 51.84±3.75 50.00±2.65 50.19±2.70 1.28±0.02 CTCG (10) 185.30±9.42 46.63±3.07 46.60±5.46 45.03±2.78 1.27±0.02 CTGG (24) 185.79±8.00 48.51±4.91 43.63±5.56 47.29±4.69 1.28±0.03 TTCC (1) 184.00±0.00* 49.01±0.00* 44.00±0.00* 45.41±0.00* 1.26±0.00* TTCG (16) 187.13±8.20 47.13±4.19 42.38±4.02 45.15±4.09 1.27±0.02 TTGG (36) 187.33±8.73 46.23±4.50