Resource population and phenotypic data
The Illinois Meat Quality Pedigree (IMQP), generated from a Berkshire × Duroc intercross, has been generally described [1]. This pedigree represents a three-generation resource population in which each of three purebred Berkshire boars was mated to six or seven different purebred Duroc sows (n = 19) to produce seven F1 boars and fifty-six F1 sows. Non-sibling F1 individuals were then intermated. Six of the seven F1 boars were primarily selected for matings such that two paternal half-sib boars sired by each of three founder Berkshire sires were used to generate F2 individuals; these boars were named 108110, 108120, 207050, 207061, 308102 and 309050, where the first number indicates the founder sire of each F1 boar. The seventh boar, 207020, sired only one family of thirteen offspring and was therefore not analyzed individually in this study. Only F2 pigs for which shear force and sensory tenderness data were available were used. These F2 pigs included 801 individuals from eighty-six full-sib families; the number of full-sib families and the total number of offspring sired by each F1 boar are indicated in Table 3. Individual full-sib families represented up to three litters and ranged in size from two to twenty-four pigs, with an average family size of approximately nine pigs.
The phenotypic data used in this study have previously been reported, and the appropriate methods of collection have been described [1]; briefly, shear force was measured using a Universal Testing Machine (Instron) with a Warner-Bratzler shear attachment, and sensory tenderness was scored, using an integer scale of 1 (tough) to 15 (tender), by a trained panel of six independent testers.
RH mapping of microsatellite marker SW1517
Primer sequences for microsatellite marker SW1517 were obtained from the United States Department of Agriculture/Roman L. Hruska Meat Animal Research Center (USDA/MARC) [2]. This marker was then amplified by PCR, using INRA-Minnesota porcine radiation hybrid (IMpRH) panel DNA templates, as described for previously mapped markers [3]. SW1517 vector data was then added to existing SSC2 marker vector data and used to construct a multipoint maximum likelihood RH map as described.
Selection and screening of publicly available markers
Primer sequences for microsatellite markers previously mapped to SSC2q were obtained from the USDA/MARC website [2]. PCR was typically performed in a 10-μl reaction volume containing 20–25 ng of template DNA, 1× PCR buffer (containing 1.5 mM MgCl2; QIAGEN), 200 μM each dNTP (Fermentas), 0.5 μM each primer, and 0.25 U HotStarTaq DNA polymerase (QIAGEN). Typical PCR cycling parameters included an initial denaturation step of 95°C for 15 min followed by 35 cycles of 94°C for 30 s, 55–66°C for 45 s, and 72°C for 45 s, plus a final extension step of 72°C for 5 min. One primer of each pair was radioactively end-labeled, using an appropriate reaction volume containing 1× T4 polynucleotide kinase (PNK) buffer (New England Biolabs), 10 μM primer, 5 U T4 PNK, and 0.3 μCi γ-32P dATP. Labeling was performed for 30 min at 37°C, followed by an enzyme inactivation step of 65°C for 10 min. Radioactively-labeled primers were then used to PCR-amplify each marker from genomic DNA of the six F1 boars. Length polymorphism was assessed by polyacrylamide gel electrophoresis. Markers with at least three alleles and determined to be polymorphic in at least three of six F1 boars were used for genotyping all individuals in the IMQP.
Development of novel informative markers
Construction of standard BAC subclone libraries
Twenty-three anchored BAC clones, as well as any potential CAST-containing clones, were obtained from the appropriate BAC libraries (CHORI-242 or RPCI-44) [21, 22]. BAC clones were individually cultured overnight in 3 ml 2×LB media containing 20 μg/ml chloramphenicol, at 37°C with shaking. Aliquots of these cultures were then used to inoculate one of two 100-ml cultures (2×LB media, 20 μg/ml chloramphenicol). For the pooled BAC library, 100 μl of each appropriate culture (2.3 ml total) was used for inoculation, and for the CAST-containing BAC library, 2.5 ml of the one appropriate culture was used. Both cultures were incubated at 37°C, with shaking, for an additional 6.75 hrs, until an OD600 of ~2.0 was attained. Cultures were then centrifuged for 15 min at 3,000 × g, and BAC DNA was isolated using the NucleoBondR® BAC 100 protocol (Macherey-Nagel). STS content was confirmed by PCR, using purified BAC DNA as template, for all 23 BACs in the BAC pool, as well as for the CAST-containing BAC.
Subclone libraries were generated using the TOPO® Shotgun Subcloning Kit (Invitrogen). For each library, ~5 μg of purified BAC DNA was sheared, by nebulization, to obtain DNA fragments with a median size of ~2 kb (pooled BAC library) or ~900 bp (CAST-containing BAC library). As an optional step in the protocol, sheared DNA was size-fractionated and purified before proceeding; SizeSep™ 400 Spun Columns (Amersham Pharmacia Biotech) were used, according to instructions and using 1× NEBuffer2 (New England Biolabs; 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM Dithiothreitol, pH 7.9) for column equilibration, to remove any small molecules and DNA fragments < 400 bp in size. Subsequent DNA modification and cloning steps were done according to the manufacturer's protocol.
Construction of a microsatellite-enriched BAC subclone library
As described above, ~5 μg of the same purified BAC DNA used to construct the standard pooled BAC subclone library was nebulized to obtain DNA fragments with a median size of ~2 kb. Sheared DNA was then size-fractionated/purified and the DNA ends were blunted, also as described.
DNA linkers were prepared, by mixing equal Molar volumes of two HPLC-purified oligonucleotides, cDNA-1b (5'-GTCACGCAAGCTTCTCACAGG-3') and cDNA-2b (5'phos-CCTGTGAGAAGCTTGCGTGACTT-3'), boiling for 5 min, and slowly cooling to room temperature. Linkers were then ligated to the blunt-ended DNA fragments in a 100-μl reaction volume of 1× T4 DNA ligase buffer (New England Biolabs; 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM Dithiothreitol, 25 μg/ml BSA, pH 7.5) containing approximately a 1:10 ratio of fragment ends:linker (~3 pmol ends:~30 pmol linker) and an excess (2,000 U) of T4 DNA ligase. Ligation was allowed to proceed for 2 hrs at room temperature.
Ten microliters of the ligation reaction was then used as template in a 200-μl PCR reaction containing 1× PCR buffer (containing 1.5 mM MgCl2; QIAGEN), 200 μM each dNTP (Fermentas), 1 μM non-HPLC purified cDNA-1b primer, and 5 U Taq DNA polymerase (QIAGEN). PCR cycling parameters included initial elongation and denaturation steps of 63°C for 10 min and 95°C for 3 min, respectively, followed by 26 cycles of 94°C for 1 min, 64°C for 1 min, and 72°C for 2.5 min, plus a final extension step of 72°C for 5 min. Ten micrograms of library PCR product DNA was then purified using the QIAquick PCR purification kit, according to the protocol, and eluted in 40 μl Buffer EB (10 mM Tris-Cl, pH 8.5).
Purified library DNA was then enriched twice for CAn microsatellites using a 5'-biotinylated CA15 oligonucleotide probe and streptavidin-coated beads (Dynal). For the first enrichment, 0.5 μg of library DNA was hybridized with 10 μM biotinylated CA15 oligonucleotide in a 10-μl reaction volume of 1× hybridization buffer [1.5 M NaCl, 10 mM NaHPO4 (pH 7.2), 10 mM EDTA, 10× Denhardt's Solution (0.2% Ficoll, 0.2% polyvinylpyrolidone, 0.2% BSA), 0.2% SDS]. Hybridization was allowed to proceed overnight, at 72°C, following an initial denaturation step of 95°C for 10 min. The hybridization reaction was then placed on ice for 3 min, before adding 50 μl of Bead Binding Buffer (BBB; 10 mM Tris (pH 7.5), 1 mM EDTA, 1 M NaCl), and transferring the entire volume to a microcentrifuge tube containing 100 μl of pre-washed streptavidin-coated beads in BBB (washed twice with 1 ml BBB plus 1× BSA, then once with 1 ml BBB). Hybridization of DNA to beads was allowed to proceed for 25 min at room temperature, followed by three 15-min washes with 1 ml of pre-warmed wash solution at 72°C (1× SSC, 0.1% SDS). DNA was then eluted from the beads by addition of 50 μl of 50 mM NaOH for 5 min, transferred to a new tube and neutralized with 50 μl 1 M Tris (pH 7.5). DNA fragments were then purified using the QIAquick PCR purification kit and eluted in 50 μl Buffer EB.
Ten microliters of purified, enriched library DNA was then used as template in a 200-μl PCR reaction containing 1× PCR buffer (containing 1.5 mM MgCl2; QIAGEN), 200 μM each dNTP (Fermentas), 1 μM non-HPLC purified cDNA-1b primer, and 5 U HotStarTaq DNA polymerase (QIAGEN). PCR cycling parameters included an initial denaturation step of 95°C for 15 min, followed by 30 cycles of 94°C for 1 min, 64°C for 1 min, and 72°C for 2.5 min, plus a final extension step of 72°C for 5 min. Products were again purified using the QIAquick PCR purification kit and eluted in 40 μl sterile Optima water (Fisher Scientific).
The second enrichment involved an additional hybridization reaction, containing 30 μl of purified, enriched PCR product in 1× hybridization buffer with ~1.6 μM biotinylated CA15 oligonucleotide. Hybridization proceeded for ~3 hrs, following an initial denaturation step of 95°C for 10 min. Hybridization was then stopped, beads were washed, and DNA was eluted as described above. PCR was performed, as above, in a 100-μl reaction volume. One microliter of double-enriched PCR product was then for ligation and cloning, according to the protocol for the TOPO® TA cloning kit for sequencing (Invitrogen, pCR®4-TOPO® cloning vector).
Skim sequencing of subclone libraries
Library transformants were randomly picked and grown overnight, at 37°C with shaking, in 96-well culture plates containing 1.2–1.5 ml 2× LB media plus 100 μg/ml ampicillin per well. Plasmid DNA was isolated using a standard alkaline lysis protocol, and 100–200 ng of each plasmid was used as template for cycle sequencing. Sequencing was performed in an 8-μl reaction volume containing ~1.3 μM primer T3 (5'-ATGACCATGATTACGCCAAGC-3') or T7 (5'-ATACGACTCACTATAGGGCGAA-3'), 0.25 μl Big Dye v3.1, 0.08 μl Big Dye dGTP v3.0, and 3.62 μl dilution buffer (0.16 M Tris base (pH 9.0), 3 mM MgCl2, 4.9% tetramethylene sulfone, 0.0001% Tween-20® surfactant) [23]. Cycle sequencing parameters included an initial denaturation step of 96°C for 1.5 min followed by 45 cycles of 96°C for 15 s, 53°C for 15 s, and 60°C for 3 min, plus a final extension step of 60°C for 10 min. Sequencing products were then purified by size exclusion using Sephadex® G-50 Fine (Amersham Biosciences) and run on an ABI 3730 capillary sequencer (Applied Biosystems). Sequences were analyzed using Phred base-calling and Phrap assembly software [24]; sequences were trimmed of low-quality reads (Phred quality score < 20) as well as vector sequence prior to assembly. Any contaminating E. coli sequence, as identified by BLASTn, was removed, and assembled contigs or individual reads were screened for simple repeats (with a minimum length of 8 per dimer, 6 per trimer, and 6 per tetramer repeat) using the online Simple Sequence Repeat Identification Tool (SSRIT) [25]. Sequence contigs or reads containing SSRs were then masked of additional porcine repetitive elements using RepeatMasker [26] prior to primer design. Primers were designed using available tools including Primer Designer 2 (Scientific and Educational Software) and Primer 3 [27].
Markers
Markers developed in this study were named according to assigned GenBank accession numbers [GenBank:EF444909,EF444910,EF444911,EF444912,EF444913,EF444914,EF444915,EF444916,EF444917,EF444918].
Genotyping and linkage analysis
One primer of each pair per selected marker was labeled with one of four fluorescent dyes (PET™, NED™, VIC®, 6-FAM™; Applied Biosystems), and grouped into multiplex PCR reactions based on color and size combinations. PCR conditions were optimized accordingly, resulting in five multiplexes of two to six markers. IMQP DNA templates (including "no DNA" controls) were prepared in 10.5 96-well PCR plates, and PCR was performed using 0.4–1 μM primer, and either QIAGEN Multiplex PCR Master Mix or HotStarTaq, with appropriate buffers. Multiplex PCR conditions are described in Additional file 2.
PCR products from three and two multiplexes, respectively, were combined and purified using Promega Wizard® SV96 binding plates. Briefly, 5 μl of each multiplex PCR product was combined, with or without water, to a final volume of 15 μl. Combined products were then mixed with 75 μl of isopropanol, and transferred to a binding plate. Following binding for ~1 min, liquid was removed by vacuum filtration. Bound products were then washed three times with 200 μl of 80% ethanol and eluted in 80 μl Optima water. GeneScan™ 500 LIZ® size standard (Applied Biosystems) was added to 10-μl aliquots prior to loading on an ABI 3730 capillary sequencer. Automated allele-calling was performed using either GeneMapper® v4.0 (Applied Biosystems) or GeneMarker® (SoftGenetics, LLC) software. Allele calls were checked manually and edited if necessary.
A multilocus linkage map was constructed using CRI-MAP v2.4 [28]. The TWOPOINT option was used to calculate two-point linkage between marker pairs, and markers displaying a recombination fraction of 0.0 were haplotyped. The BUILD option was then used to map markers, in decreasing order of informativeness, with a LOD score threshold of 3.0. The CHROMPIC option was used to identify and remove potential genotype errors.
QTL analyses
Phenotypic and marker data were analyzed, both across and within families, using the outbred F2 analysis servlet of QTL Express [14, 16]. QTL, additive and dominance effects were estimated every 1 cM for both Instron shear force and taste panel tenderness traits, using a general linear model including an overall trait mean, additive and dominance effects, fixed effects, and a residual error term. Fixed effects included sex (2 levels) and birth month and year (BYM; 14 levels). Chromosome-wise significance thresholds were determined by permutation (n = 5,000), and 95% confidence intervals were obtained by bootstrapping (n = 1,000). Both 1- and 2-QTL models were used; however, chromosome-wise significance thresholds could not be determined for a 2-QTL model using QTL Express.
