Xinyang Brown layers (Kang et al., 2012) is a hybrid line of brown shell layer, which was bred from Rhode Island Red and Rhode Island White with continuous selection by Shanghai Poultry Breeding Co. Ltd. The complete production population consists of the grandparent, the parent and the commercial layer chickens. About 330 Xinyang Brown Layers were randomly selected from the population of commercial generation layers in the same farm and used in the association study. The laying pattern of each hen was recorded daily. Egg production traits including age at first egg (AFE) and total number of eggs at 28 weeks of age (E28) were used for polymorphism–trait association analysis. A Chinese indigenous breed (Wenchang) and a hybrid line (Hy-line Brown) were also included in the genotyping analysis. Genomic DNA from birds was extracted from blood samples using the TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China). The hens were handled and treated according to the Animal Care and Use Committee of Shandong Agricultural University.

Primer pair was designed to amplify a fragment of the promoter region according to chicken MMP9 gene sequence (GenBank accession NC_006107.3). The primer sequences were as follows: forward 5′-TTGGCGGCCAGTGGGCCC -3′ and reverse 5′-ACACAGCTGGCTCCAGGGTG -3′. Polymerase chain reaction (PCR) was performed by mixing 1 μL (50 to 100 ng) of genomic DNA, 2 μL of dNTPs (2.5 mM each), 2 μL of MgCl₂ (25 mM), 1 μL of each primer (25 μM), 0.2 μL of Taq polymerase (5 U/μL, TaKaRa Bio, Dalian, China), and 2.5 μL of 10×Taq buffer in a 25 μL volume, and running according to the following program: 95°C for 4 min, 35 cycles of 95°C for 40 s, 58°C for 40 s, and 72°C for 40 s and final extension at 72°C for 5 min. The PCR products were electrophoresed on 1% agarose gel, purified with AxyPrep DNA Gel Extraction Kit (Axygen Biotechnology, Hangzhou, China) and sequenced using the reverse primer. Sequences were edited and aligned using DNAMAN program.

Based on the sequencing analysis, three genotypes (C⁺C⁺, C⁺C⁻, C⁻C⁻) at the −1,954 of MMP9 promoter region were discovered. Using the following primers: forward 5′-TTTCTCGTGCCGCATTGTTCCT-3′ and reverse 5′-CAGAGGAGGGTGAAGCTGTGCC-3, the 5′-regulatory region from −3,842 to −14 bp of the chicken MMP9 gene, where +1 is the transcription initiation site, was cloned. The PCR fragments contain each of the alleles were cloned into the pGL-3 basic luciferase reporter vector (Promega, Madison, WI, USA).

The granulosa cell isolation and transfection procedures were according to previous protocol (Zhu et al., 2012). The hen ovaries were isolated and placed in phosphate buffered saline (HyClone, Logan, UT, USA). The granulosa layer from F2 to F4 follicles were separated from the follicle and the granulosa cells were dispersed by treatment with 0.3% collagenase at 37°C for 10 min with gentle agitation in a flask. After centrifugation, the cells were suspended in culture medium (M199 with 2.5% fetal bovine serum and 1% penicillin/streptomycin) and subsequently seeded in 24-well culture plates at a density of 2×10⁵/well. The number of viable cells (>90%) was estimated using Trypan blue. Cells were cultured at 39°C in a water-saturated atmosphere of 95% air and 5% CO₂. The cells were transfected with the luciferase reporter plasmids (800 ng/well) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Transfection efficiency was normalized by co-transfection of 30 ng of the Renilla luciferase reporter plasmid (pRL-CMV vector, Promega, Madison, WI, USA). At 48 h after transfection, the cells were lysed and assayed for luciferase activity using the dual-luciferase reporter assay system (Promega, USA). The enzymatic activity of luciferase was measured with a Modulus Luminometer (Turner Biosystems, Sunnyvale, CA, USA).

Hardy-Weinberg equilibrium was tested employing the exact probability test in POPGENE version 1.32. The associations of MMP9 promoter genotypes with egg production traits including AFE and E28 were analyzed in Xinyang Brown chicken populations using the general linear model of SAS (SAS Institute Inc). The estimated genotype values were compared by Duncan’s Multiple Range Test of SAS. The statistical model was shown as following:

where Y_ij was phenotypic value of traits, μ was the population mean, G_i was the fixed effect of genotypes (include additive effect and dominance effect), T_j was the fixed effect of tier floor of the farm house, and e_ij was the random residual error. The difference between genotypic effects were considered as significant when p<0.05. The additive effects and dominance effects of the MMP9 gene promoter polymorphism were estimated using the REG procedure of the SAS version 8.0 (Liu, 1998). In short, Additive = (AA − BB)/2, Dominance = AB − (AA+BB)/2, where AA, AB, and BB are the least squares means of genotype C⁺C⁺, C⁺C⁻, and C⁻C⁻ groups, respectively. One-way analysis of variance was used to examine the difference of the luciferase activity of different genotypes C⁺C⁺ and C⁻C⁻ of MMP9 promoter-reporters and when p<0.05 the difference was considered as significant.