Animals and phenotypic data
A total of 1,065 Chinese Holstein cows from44 sire families was used in this study which were detailed described by Shi et al. [40]. We collected milk samples of these 1,065 cows to measure milk fatty acids in the laboratory of Beijing Dairy Cattle Center (www.bdcc.com.cn). By gas chromatograph, the contents of 16 kinds of main milk fatty acids were directly detected, including SFA: C6:0, C8:0, C10:0, C11:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0, C20:0; and UFA: C14:1, C16:1, C17:1 and C18:1cis-9. Based on these phenotype values, we obtained five index traits (C14index, C16index, C17index, C18index, and total index) calculated with the formulas:
and summarized the SFA, UFA, SFA/UFA.
SNP identification and genotyping
We extracted genomic DNA from the blood samples of 1,065 cows and the semen samples of 44 sires using TIANamp Blood DNA Kit (Tiangen, Beijing, China) and salt-out procedure, respectively. DNA quantity and quality were measured by NanoDropTM ND-2000 Spectrophotometer (Thermo Scientific, Hudson, DE, USA) and 2.0% agarose gel electrophoresis.
A total of 15 pairs of primers (Additional file 4: Table S4) were designed using the Primer 3.0 (http://primer3.wi.mit.edu/) based on the sequences of all the exons, and 3,000 bp of 5' and 3' flanking regions of the bovine HTR1B gene (Gene ID: 317707), and were synthesized in the Beijing Genomics Institute (Beijing, China). Two DNA pools’ construction and polymerase chain reaction (PCR) procedure used the same method as those in our previous study [40]. By sequencing PCR products, we identified potential polymorphic sites and further genotyped identified SNPs in 1,065 cows by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS, Sequenom MassARRAY, Bioyong Technologies Inc. HK).
Statistical analysis
First, we used the Haploview 4.1 software (Broad Institute of MIT and Harvard, Cambridge, MA, USA) to identify the LD extent among the identified SNPs of the HTR1B gene.
Subsequently, we performed single SNP-based and haplotype-based association analysis. We traced the pedigrees of the 1,065 Chinese Holstein cows back to three generations, as a result, a total of 3,335 individuals were included for association analysis, which kinship matrix (A-matrix) were constructed with SAS9.2 (SAS Institute, Cary, NC, USA). Then, associations between the identified SNPs and haplotype blocks with 24 milk fatty acid traits were performed by SAS9.2 on the basis of the following mixed animal model:
Prediction of the secondary structure and function changes of the HTR1B protein
We used the NPSA SOPMA SERVER program (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html) to predict whether the identified missense mutation in coding region changed HTR1B protein secondary structure, and set the parameters with similarity threshold (8), and number of states (4-Helix, Sheet, Turn, Coil). Also, we used the SIFT (http://sift.bii.a-star.edu.sg/) and PROVEAN (http://sift.jcvi.org/index.php) to investigate whether the missense mutation altered the protein function. The score thresholds of the SIFT and PROVEAN were 0.05 [43] and -2.5 [44], respectively. When the score is below the threshold, the protein function is considered changed.
Prediction of the changes of transcription factor binding sites (TFBSs)
We predicted whether the SNPs in 5′ UTR and flanking region of the HTR1B gene impacted on TFBSs by using the Genomatix suite v3.9 (http://www.genomatix.de/cgi-bin/welcome/welcome.pl?s=d1b5c9a9015b02bb3b1a806f9c03293f).
Construction of recombinant plasmid, cell culture and luciferase assay
We constructed six luciferase reporter gene fragments with Kpn1 and Nhel restriction sites at the 5' to 3' termini (Figure 3), which contained alleles A and T of g.17307103A>T, T and G of g.17305206T>G, and C and T of g.17303761C>T. The six fragments were synthesized in Genewiz (Suzhou, China), and were cloned into the pGL4.14 Luciferase Assay Vector (Promega, Madison, USA). Subsequently, the plasmids were purified by Endo-free Plasmid DNA Mini Kit Ⅱ (OMEGA, omega bio-tek, Norcross, Georgia, USA), and then were sequenced to confirm the integrity of each construct's insertions.
Human Embryonic Kidney (HEK)-293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM) including 10% heat-inactivated fetal bovine serum (FBS; Gibco, Life Technologies) at 5% CO2 and 37℃. We seeded approximately 2×105 cells per well into 24-well plates, and transfected the cells using Lipofectamine 2000 (Invitrogen, CA, USA). For each well, we transfected 500ng of the constructed plasmid DNA along with 10ng of pRL-TK renilla luciferase reporter vector (Promega), and conducted three replicates for each construct. The cells were cultured for about 36-48h after transfection and then were measured the activities of firefly and renilla luciferases using a Dual-Luciferase Reporter Assay System (Promega) with a Modulus microplate multimode reader (Turner Biosystems, CA, USA). Finally, average statistics of three replicates were calculated as the normalized luciferase data (firefly/renilla).