Genome-wide association study for backfat thickness in Canchim beef cattle using Random Forest approach

  • Fabiana Barichello Mokry1Email author,

    Affiliated with

    • Roberto Hiroshi Higa2,

      Affiliated with

      • Maurício de Alvarenga Mudadu3,

        Affiliated with

        • Andressa Oliveira de Lima1,

          Affiliated with

          • Sarah Laguna Conceição Meirelles4,

            Affiliated with

            • Marcos Vinicius Gualberto Barbosa da Silva5,

              Affiliated with

              • Fernando Flores Cardoso6,

                Affiliated with

                • Maurício Morgado de Oliveira6,

                  Affiliated with

                  • Ismael Urbinati7,

                    Affiliated with

                    • Simone Cristina Méo Niciura3,

                      Affiliated with

                      • Rymer Ramiz Tullio3,

                        Affiliated with

                        • Maurício Mello de Alencar3 and

                          Affiliated with

                          • Luciana Correia de Almeida Regitano3

                            Affiliated with

                            BMC Genetics201314:47

                            DOI: 10.1186/1471-2156-14-47

                            Received: 18 February 2013

                            Accepted: 23 May 2013

                            Published: 5 June 2013

                            Abstract

                            Background

                            Meat quality involves many traits, such as marbling, tenderness, juiciness, and backfat thickness, all of which require attention from livestock producers. Backfat thickness improvement by means of traditional selection techniques in Canchim beef cattle has been challenging due to its low heritability, and it is measured late in an animal’s life. Therefore, the implementation of new methodologies for identification of single nucleotide polymorphisms (SNPs) linked to backfat thickness are an important strategy for genetic improvement of carcass and meat quality.

                            Results

                            The set of SNPs identified by the random forest approach explained as much as 50% of the deregressed estimated breeding value (dEBV) variance associated with backfat thickness, and a small set of 5 SNPs were able to explain 34% of the dEBV for backfat thickness. Several quantitative trait loci (QTL) for fat-related traits were found in the surrounding areas of the SNPs, as well as many genes with roles in lipid metabolism.

                            Conclusions

                            These results provided a better understanding of the backfat deposition and regulation pathways, and can be considered a starting point for future implementation of a genomic selection program for backfat thickness in Canchim beef cattle.

                            Keywords

                            Bovine Lipid metabolism Machine learning Single nucleotide polymorphism (SNP) Subcutaneous fat Tropical composite cattle

                            Background

                            Beef cattle production in Brazil is based on several breeds, depending on the geography and climate of a given area. Breeds based on Bos taurus are commonly raised as livestock for beef in the South of Brazil, but in most parts of the country, beef cattle production is based on Bos indicus (zebu) breeds raised on natural pastures. A good description of Brazilian beef cattle production was recently published [1]. Zebu breeds are considered highly adapted to the tropical environment in Brazil [25], but they are known for their lower meat quality in certain aspects, such as tenderness, palatability, and marbling [610], and for their lower reproduction efficiency [11, 12] when compared to Bos taurus. The Canchim (3/8 zebu + 5/8 Charolais) breed was developed in the early 1960’s in Brazil [13] with the intention of combining fitness traits from zebu to the higher reproduction efficiency and meat quality from the Charolais breed.

                            Although the Canchim breed has fared well when raised on natural pastures in Brazil, some carcass traits have still remained inferior when compared to Bos taurus. One such trait is backfat thickness, which has been a concern for Canchim producers, and for the beef cattle industry in general, due to its low fat deposition in animals raised on pasture (1.90mm ± 0.77) [14]. Improvement of this trait in Canchim beef cattle using traditional selection techniques has had limited success because of its relatively low heritability (0.23) [14], and because it is measured late in an animal’s life. Most studies available in the literature regarding backfat thickness have been conducted on animals raised in feedlot systems, which permits earlier ultrasound measurements, and has also shown moderate to high heritabilities [1519], thereby allowing traditional selection techniques under these conditions to be more successful than compared to the Canchim breed.

                            In attempts to improve meat quality, previous studies have focused on the identification of candidate markers associated with meat quality traits, as well as backfat thickness, in Canchim and other Bos indicus × Bos taurus crosses in Brazil [2024]. However, these have had limited success, particularly in response to markers on the DDEF1 and LEP genes [20, 23]. Therefore, the identification of genetic markers linked to backfat thickness by novel methodologies is an important strategy for genetic improvement of carcass and meat qualities. One recently developed approach relies on examining how SNPs (single nucleotide polymorphisms) are associated with these qualitative traits [25]. More specifically, this method has been used successfully in studies that examined fat-related traits, such as intramuscular fat percentage, marbling, rib fat, backfat thickness and rump fat depth [2635]. By the use of high-density SNP panel assays for different breeds and crosses, these studies have collectively found such traits associated with regions on nine bovine chromosomes (6, 15, 17, 20, 21, 24, 25, 26, and 28) [27, 28, 32, 35]. However, another study suggested that some of the effects attributed to each SNP can show variation based on the breed’s origin, resulting from variation in indicine and taurine-indicine composite cattle [35], thereby justifying the investigation of SNPs based on the breed of interest.

                            A previous study using high-density SNP panel has associated 100 SNPs to backfat thickness in a Canchim population using an approach that selected animals with extreme phenotypes for genotyping [33]. Those SNPs were located on several bovine autosomes, and from them, the authors further investigated and validated two regions on chromosome (chr) 14 associated with backfat thickness, where the haplotypes were responsible for 0.24% to 1.1% of the phenotypic variance for this trait.

                            Although these results are useful, it is well known that quantitative traits are polygenic as each SNP may account for only a small part of the phenotypic variance, therefore joint analysis of many SNPs has become a more interesting strategy [36, 37]. This, however, exacerbates the ‘large p, small n’ problem faced by genome-wide studies, which means that there is a small number of phenotypes (n) to predict a large number of SNP (p) effects [38].

                            One solution to this problem is through the use of Random Forest, a machine learning algorithm capable of dealing with certain datasets for building model independent classification and/or regression problem predictors [39]. Specifically, it embeds a procedure of accounting for predictor variable importance, which results in a score that can be used for prioritizing variables (SNPs), similar to p-values from statistical tests [4042]. Because of these features, the variable importance of the random forest method has been recognized as an useful methodology for genome-wide association studies [43].

                            Considering all of the above, the objectives of this study were to identify SNPs associated with backfat thickness in Canchim beef cattle using the random forest approach for genome-wide association studies, to shed insight on potential genes associated with this trait, and to discover potential SNPs for future implementation of genomic selection (GS). The set of SNPs identified by this methodology explains as much as 50% of the deregressed estimated breeding value variance associated with the observed phenotype. These results intend to provide a better understanding of the backfat deposition and regulatory pathways, and to enable the use of the identified SNPs in validation studies for genomic selection.

                            Methods

                            Animals and phenotypes

                            Animals used in this study were part of the Canchim Breeding Association from seven herds located in two Brazilian states (São Paulo and Goiás). This research is in agreement with the ethical principles of animal experimentation of Embrapa Southeast Livestock Ethical Committee of Animal Use (CEUA-CPPSE), and has been performed with the approval of CEUA-CPPSE under protocol number 02/2009. An initial sample of 987 animals (males and females) was evaluated for backfat thickness by ultrasound in vivo over the 12th rib around the age of 18 months. All animals evaluated were born between 2003 and 2005 and raised on natural pastures.

                            These 987 animals had the estimated breeding value (EBV) predicted by restricted maximum likelihood using the MTDFREML software [44]. The animal model included fixed effects of contemporary group (sex, year, herd, and genetic group) and age at measurement as a linear covariate, the additive genetic effect and error were included as random effects. From these animals, a sample of 400 was selected considering: EBV, accuracy, family size, and proportion between males (196) and females (204). These 400 animals were offspring of 50 different sires (with 1 to 30 offspring per sire).

                            Genotyping and SNPs quality control

                            The selected 400 animals were genotyped using the BovineHD BeadChip (Illumina Inc., San Diego, CA). The quality control filters included call rate (< 0.90) for samples and SNPs, minor allele frequency (MAF < 0.01), and heterozygosity (< 3 standard deviations). After quality control processing, 396 animals and 708,641 SNPs with an average call rate higher than 0.99 remained in the study.

                            Genome-wide association analysis

                            Genome-wide association (GWA) analysis was performed on deregressed EBVs (dEBV) [45], which takes into account the pedigree matrix, estimated heritability (0.16, data not shown), EBVs, and EBV's accuracies obtained by the same animal model described above. For the estimation of dEBVs the data set was enhanced with data collected from animals born between 2005 and 2008 totaling 1,648 individuals with phenotypes for backfat thickness, with 6,801 animals in the pedigree matrix.

                            Association of SNPs to dEBVs was undertaken by a random forest package [46] available in the R-project software [47]. The association analysis was composed of a two-step procedure. In the first step, the SNPs with the highest 1% importance score by chromosome were selected, and in the second step, the outcome set of SNPs from the first step was re-analyzed disregarding the chromosome classification, and the SNPs with the highest 1% importance score were selected. For the association analysis, the missing genotypes were imputed by the näive method provided in the random forest package (which imputes column median values for missing genotypes), the number of trees to grow and the number of randomly selected candidate SNPs at each split were set to 5,000 and 10% from the SNPs being evaluated, respectively. This procedure was done using the 396 samples available.

                            Taking into account the unbalanced offspring range among sires, 10 subsamples consisting of 198 animals each were also analyzed in the same two-step process as previously described. The 10 subsamples were selected as follows: i) The first animal was chosen at random from the 396 genotyped animals; ii) The next animal was selected based on the lowest relationship with the previous selected animal, but most representative from the rest of the genotyped animals; and iii) Step ii was repeated until 198 animals were selected.

                            Two approaches were considered for further SNP investigation among the results obtained by the random forest analysis. One approach selected the SNPs in common among the analysis with the 396 animals and the 10 subsamples, called the Common SNPs strategy. Another approach selected only the top 1% (importance score) from the analysis with 396 animals, called the Highest 1% SNPs.

                            Finally, after both sets of SNPs (Common SNPs and Highest 1% SNPs) had been selected, each set of selected SNPs were fitted into a final stepwise regression model using SAS/STAT software [48] to estimate the amount of variance explained by the selected SNPs in the data set (final model R2 values correspond to the dEBV variance explained by the model, which are reported in Table 1). For doing so, the SNPs were coded as 0, 1, and 2 for the AA, AB, and BB genotypes, respectively. In order to evaluate the significance of the results, a permutation test was conducted to estimate the bias associated with the R2 obtained from the stepwise regression analysis. In the permutation test, the dEBV values were shuffled and then regressed to the same SNPs previously selected. The permutation test was repeated 1,000 times.
                            Table 1

                            Number of candidate and final SNPs selected through the Common SNP and Highest 1% SNP strategies

                             

                            Candidate SNPs

                            Final Model SNPs

                            % dEBV variance(1)

                            Permutation R2(2)

                            Common SNP

                            162

                            19

                            50.59%

                            0.00 ± 0.02

                            Highest 1% SNP

                            70

                            21

                            53.27%

                            0.00 ± 0.02

                            1 dEBV deregressed estimated breeding value variance explained by the final fitting of SNPs. The % dEBV variance is the model R2 from the final analysis which fits all SNPs as fixed effects into a regression analysis.

                            2 Permutation R2: average values and standard deviations for R2 from 1,000 permutation tests.

                            Candidate genes and pathways

                            A pathway analysis was conducted to characterize the genomic regions identified by the set of SNPs previously selected and to identify candidate genes influencing biological functions and pathways related to backfat thickness and fat-related traits.

                            The software fastPHASE version 1.4.0 [49] was used for reconstructing the haplotypes for each chromosome. Afterwards, the reconstructed haplotypes were analyzed by the software Haploview [50] (using default parameters) for estimating haplotype blocks and linkage disequilibrium (LD), which was calculated based on the squared correlation coefficient between SNP pairs (r2). Considering the extent of LD based on the overall average r2 (average r2 = 0.12 at a distance of 250Kb, data not shown), a window of 500Kb (SNP position ± 250Kb) surrounding each SNP previously selected by the stepwise regression was considered to define the region used for candidate gene discovery and pathway annotation.

                            The Cattle Genome Browser through the UMD 3.1 Cattle genome assembly [51], was used for visualization of the selected SNPs and surrounding areas for localization and identification of QTLs, genes, and other interesting genomic landmarks. Other databases, such as the NCBI BioSystems database [52], and Kyoto Encyclopedia of Genes and Genomes (KEGG) [53, 54] were also used for pathway annotation to gain insight into the biological processes involved in backfat thickness deposition.

                            Results

                            We performed regression analysis for both strategies (Common SNP and Highest 1% SNP), and the results were very similar in the final number of SNPs selected, and the percentages of dEBV variance explained by the final set of SNPs (Table 1) enabling the discussion to be focused on the set of 21 SNPs selected from the Highest 1% SNP strategy due to its higher % of dEBV variance explained. Also, the first five SNPs (rs133046994, rs137294146, rs109349988, rs136717249, rs134790147) in the regression model were the same and in the same order for both strategies. These first five SNPs were responsible for 34.13% of dEBV variance for backfat thickness.

                            As a precaution against spurious artifacts that can result from splitting small samples into training and validation datasets, this was not performed here. An alternative option is to use a permutation test, which calculates the probability of obtaining a value more extreme than or equal to the observed value of a test statistic by shuffling the data and recalculating the test statistic. The proper test statistic for multiple regression is the coefficient of multiple determination, R2[55].

                            A permutation test was carried out to evaluate the probability of bias associated with the R2 from the stepwise regression analysis (Table 1). The average R2 from 1,000 permutation tests was 0.00 ± 0.02 for the Highest 1% SNP strategy, showing that there is a small bias associated with the R2 from the stepwise regression analysis. However, this is very small when compared to the 53.27% obtained from the Highest 1% SNP strategy, and therefore reinforces the significance of the results presented in Table 1.

                            Table 2 shows the 21 SNPs selected by the stepwise regression, their chromosome, position, % of dEBV variation explained by the SNP, genes annotated within ± 250Kb, fat-related QTLs described in the current literature, and references. Table 3 shows a summary of pathway annotation using the genes within ± 250Kb from the 21 selected SNPs using the KEGG [53, 54] pathway database.
                            Table 2

                            Summary of information available for the Highest 1% SNPs selected by the stepwise regression

                            dbSNP1

                            Chr2

                            Position

                            % dEBV3

                            Genes4in ± 250Kb

                            Fat-related QTL5

                            QTL reference

                            rs133046994

                            10

                            18129602

                            11.12

                            THSD4, LRRC49

                            SF, MS

                            [65, 66]

                            rs137294146

                            1

                            132385787

                            9.41

                            SOX14, CLDN18, DZP1L

                            FT12R, IF

                            [29, 71]

                            rs109349988

                            3

                            15814096

                            5.21

                            KCNN3, EFNA3, EFNA4, DCST2, LOC100294774, PMVK, ADAR, CHRNB2, ADAM15, ZBTB7B, LOC100294857, DCST1, FLAD1, PYGO2, CKS1B, PBXIP1, SHC1, LOC100294894

                            FT12R, MS

                            [66]

                            rs136717249

                            19

                            37969870

                            4.88

                            B4GALNT2, GNGT2, ABI3, PHOSPHO1, ZNF652, NGFR, PHB, IGF2BP1, GIP

                            OAC, PAC

                            [84, 85]

                            rs134790147

                            13

                            20780821

                            3.51

                            CCDC7, ARL5B, MGC152301, LOC100848675, LOC100847992

                            FT12R

                            [29]

                            rs136287610

                            25

                            42678992

                            2.89

                            FAM20C, LOC783396, LOC100300875, LOC100337322, PRKAR1B, PDGFA, PTCHD3, LOC783852, LOC783961

                            FT12R

                            [29]

                            rs136393667

                            11

                            65619399

                            2.51

                            LOC786621, LOC100139826, ETAA1

                            FT12R, MS

                            [29]

                            rs41790889

                            16

                            990255

                            2.07

                            OPTC, PRELP, FMOD, BTG2, LOC789413, LOC789394, CHI3L1, MYBPH, ADORA1, LOC100847554

                            FT12R, MS

                            [29]

                            rs42126516

                            4

                            52535108

                            1.99

                            TFEC, LOC100296613, TES

                            MS

                            [29, 97]

                            rs42021729

                            3

                            64737352

                            1.46

                             

                            MS

                            [98]

                            rs137001098

                            8

                            95507919

                            1.46

                            SMC2, LOC100337180

                            MS

                            [29]

                            rs43341824

                            1

                            50110036

                            1.23

                            LOC785980, ALCAM

                            OAC, FT12R

                            [85, 99]

                            rs41683753

                            13

                            33219105

                            1.04

                            CACNB2, NSUN6, EPC1, LOC10084770

                            PAC, MS

                            [29, 85]

                            rs136348926

                            12

                            10043410

                            0.90

                            LOC786945

                              

                            rs109869647

                            3

                            13195543

                            0.72

                            LOC100849046, LOC100848852, LOC784007, LOC783963

                            MS

                            [66]

                            rs110833507

                            11

                            42856561

                            0.69

                            LOC100296234, LOC100296682, BCL11A

                              

                            rs42923911

                            2

                            12761205

                            0.57

                            LOC787311, LOC100848878

                              

                            rs135638125

                            10

                            18147174

                            0.55

                            THSD4, LRRC49

                            MS, SF

                            [65, 66]

                            rs110607520

                            9

                            96622647

                            0.66

                            SYTL3, TULP4, TMEM181, EZR, LOC781263, DYNLT1, RSPH3, LOC782714, TAGAP, LOC782637

                            MS

                            [29]

                            rs110025080

                            9

                            11710300

                            0.52

                            RIMS1

                              

                            rs109697559

                            2

                            61906393

                            0.58

                            LOC100847709, LOC100297008, LCT, UBXN4, MCM6, DARS, R3HDM1, MIR128-1

                            MS

                            [29]

                            1 Reference SNP cluster report.

                            2 Chromosome in B. taurus.

                            3 % dEBV variance is the model R2 for each of the SNPs in the final analysis which fits all SNPs as fixed effects into a regression analysis.

                            4 Gene symbol.

                            5 SF subcutaneous fat, FT12R fat thickness at the 12th rib, IF intramuscular fat, FT fat thickness, MS marbling score, OAC oleic acid content, PAC palmitoleic acid content.

                            Table 3

                            Summary of pathway description from the KEGG Pathway Database

                            Global Pathway

                            Subpathway

                            Pathway

                            Gene

                            SNP

                            Metabolism

                            Carbohydrate Metabolism

                            Galactose metabolism

                            LCT

                            rs109697559

                              

                            Amino sugar and nucleotide sugar metabolism

                            CHI3L1

                            rs41790889

                             

                            Lipid Metabolism

                            Glycerophospholipid metabolism

                            PHOSPHO1

                            rs136717249

                             

                            Metabolism of Terpenoids and Polyketides

                            Terpenoid backbone biosynthesis

                            PMVK

                            rs109349988

                             

                            Metabolism of Cofactors and Vitamins

                            Riboflavin metabolism

                            FLAD1

                            rs109349988

                            Genetic Information Processing

                            Replication and Repair

                            DNA replication

                            MCM6

                            rs109697559

                             

                            Folding, Sorting and Degradation

                            RNA degradation

                            BTG2

                            rs41790889

                             

                            Translation

                            Aminoacyl-tRNA biosynthesis

                            DARS

                            rs109697559

                            Environmental Information Processing

                            Signal Transduction

                            MAPK signaling pathway

                            CACNB2, PDGFA

                            rs41683753, rs136287610

                              

                            ErbB signaling pathway

                            SHC1

                            rs109349988

                             

                            Signaling Molecules and Interaction

                            Cell adhesion molecules (CAMs)

                            CLDN18, ALCAM

                            rs137294146, rs43341824

                              

                            Neuroactive ligand-receptor interaction

                            CHRNB2, GIP, ADORA1

                            rs109349988, rs136717249, rs41790889

                              

                            Cytokine-cytokine receptor interaction

                            NGFR, PDGFA

                            rs136717249, rs136287610

                            Cellular Processes

                            Cell Motility

                            Regulation of actin cytoskeleton

                            EZR, PDGFA

                            rs110607520, rs136287610

                             

                            Cell Growth and Death

                            Cell cycle

                            MCM6

                            rs109697559

                              

                            Apoptosis

                            PRKAR1B

                            rs136287610

                             

                            Cell Communication

                            Tight junction

                            CLDN18

                            rs137294146

                              

                            Focal adhesion

                            SHC1, PDGFA

                            rs109349988, rs136287610

                              

                            Gap junction

                            PDGFA

                            rs136287610

                             

                            Transport and Catabolism

                            Peroxisome

                            PMVK

                            rs109349988

                            Organismal Systems

                            Circulatory System

                            Cardiac muscle contraction

                            CACNB2

                            rs41683753

                             

                            Immune System

                            Leukocyte transendothelial migration

                            EZR, CLDN18

                            rs110607520, rs137294146

                              

                            Chemokine signaling pathway

                            GNGT2, SHC1

                            rs136717249, rs109349988

                              

                            Cytosolic DNA-sensing pathway

                            ADAR

                            rs109349988

                              

                            Natural killer cell mediated cytotoxicity

                            SHC1

                             
                             

                            Digestive System

                            Gastric acid secretion

                            EZR

                            rs110607520

                              

                            Carbohydrate digestion and absorption

                            LCT

                            rs109697559

                             

                            Nervous System

                            Glutamatergic synapse

                            GNGT2

                            rs136717249

                              

                            GABAergic synapse

                            GNGT2

                            rs136717249

                              

                            Cholinergic synapse

                            GNGT2, CHRNB2

                            rs136717249, rs109349988

                              

                            Dopaminergic synapse

                            GNGT2

                            rs136717249

                              

                            Serotonergic synapse

                            GNGT2

                            rs136717249

                              

                            Retrograde endocannabinoid signaling

                            GNGT2, RIMS1

                            rs136717249, rs110025080

                              

                            Synaptic vesicle cycle

                            RIMS1

                            rs110025080

                              

                            Neurotrophin signaling pathway

                            SHC1, NGFR

                            rs109349988, rs136717249

                             

                            Development

                            Axon guidance

                            EFNA3, EFNA4

                            rs109349988

                             

                            Endocrine System

                            Insulin signaling pathway

                            SHC1, PRKAR1B

                            rs109349988, rs136287610

                            Human Diseases

                            Cardiovascular Diseases

                            Hypertrophic cardiomyopathy (HCM)

                            CACNB2

                            rs41683753

                              

                            Arrhythmogenic right ventricular cardiomyopathy (ARVC)

                            CACNB2

                            rs41683753

                              

                            Dilated cardiomyopathy (DCM)

                            CACNB2

                            rs41683753

                             

                            Infectious Diseases

                            Pathogenic Escherichia coli infection

                            EZR

                            rs110607520

                              

                            Hepatitis C

                            CLDN18

                            rs137294146

                              

                            Measles

                            ADAR

                            rs109349988

                              

                            Influenza A

                            ADAR

                            rs109349988

                              

                            Bacterial invasion of epithelial cells

                            SHC1

                            rs109349988

                              

                            HTLV-I infection

                            PDGFA

                            rs136287610

                             

                            Substance Dependence

                            Morphine addiction

                            GNGT2, ADORA1

                            rs136717249, rs41790889

                              

                            Nicotine addiction

                            CHRNB2

                            rs109349988

                              

                            Alcoholism

                            GNGT2, SHC1

                            rs136717249, rs109349988

                             

                            Cancers

                            Pathways in cancer

                            CKS1B, PDGFA

                            rs109349988, rs136287610

                              

                            Small cell lung cancer

                            CKS1B

                            rs109349988

                              

                            Glioma

                            SHC1, PDGFA

                            rs109349988, rs136287610

                              

                            Chronic myeloid leukemia

                            SHC1

                            rs109349988

                              

                            Transcriptional misregulation in cancers

                            NGFR, PDGFA

                            rs136717249, rs136287610

                              

                            Melanoma

                            PDGFA

                            rs136287610

                              

                            Prostate cancer

                            PDGFA

                            rs136287610

                            Discussion

                            The use of the random forest approach as a first step, to filter candidate SNPs without taking into consideration a statistical model specification, is advantageous in genome-wide association studies, as long as little is known about candidate areas and the genetic architecture of the specific trait. Furthermore, the fact that results were obtained using two different strategies (Common SNPs and Highest 1% SNPs) and are very similar, provides reliability to the random forest methodology as can be seen in the previous study [43].

                            With the exception of four selected SNPs in the Highest 1% SNPs strategy (chr 12: rs136348926; chr 11: rs110833507; chr 2: rs42923911; chr 9: rs110025080), all other SNPs presented a fat-related QTL described in their chromosome region. Also, only one SNP on chr 3 (rs42021729) is not close to any described gene in the surrounding area (± 250kb) (Table 2).

                            In a previous genome-wide association study in Canchim, 100 SNPs on several chromosomes were considered the optimal set of SNPs to differentiate the 30 individuals with extreme phenotypes for backfat thickness. Among these SNPs, two haplotypes on chr 14 were genotyped and their association to the phenotype was validated in the whole population [33]. In the current study, even though SNPs from chr 14 were associated with backfat thickness by the random forest approach (in the Common SNP and Highest 1% SNP strategies, data not show), these SNPs were not selected in the stepwise regression model. Conflicting results and/or studies that cannot be replicated in the post-genomic area are not so uncommon [5659], and these differences can be attributed to partially insufficient power, false-positive results, bias, sample size, and to differences in populations, controls, and methodologies [5658], or true heterogeneity associations [56]. In these two GWA studies with Canchim, the base population is very similar, but the sample size and methodologies are not, which could explain the difference in the findings. A future option to help clarify the inconsistency in these findings would be to perform a meta-analysis, which combines data together to increase sample size and power, while reducing error risks [58, 60].

                            Another outcome from this study and the previous one [33] is the possibility of including these SNPs in the development of a low density SNP (LD-SNP) panel for implementation of genomic selection in Canchim beef cattle. The most widespread strategy for developing small panels is by applying methods of variable selection to identify a diminutive set of SNPs that have good predictive power for the trait or breeding value [61]. The increase in accuracy of genomic breeding values obtained by using LD-SNP panels can be highly similar (around 90%) compared to the accuracies obtained by high density panels [62, 63], at a more cost-effective price. Therefore, it is more likely to be adopted by farmers and the beef industry [64]. Furthermore, LD-SNP panels developed with SNPs selected on the basis of their effects perform better than LD-SNP panels with SNPs evenly spaced [62, 63]. Importantly, SNPs identified in these studies need to undergo a prior validation in a population of animals which are not included in the population used for the SNP discovery (training population), enabling confidence in genomic predictions for future populations.

                            From the SNPs identified in this study, there were two on chr 10 (rs133046994, rs135638125) associated with backfat thickness, which together accounted for almost 12% of the dEBV variation (Table 2). These two SNPs are in the same chromosomal region as fat-related QTLs identified in previous studies [65, 66], and they map to the same genes (THSD4 - thrombospondin, type I, domain containing 4, and LRRC49 - leucine-rich repeat-containing protein 49) thereby indicating THSD4, LRRC49 and the surrounding areas as strong candidates for further investigations and validation. The LRRC49 gene has been linked to breast cancer in humans, but very little is known about the biological function of the protein encoded by this gene [67].

                            The THSD4 gene in Bos taurus and in Homo sapiens has a provisional status from RefSeq [68], which, by definition, supports that this gene is both transcribed and expressed. Further evidence for the annotation of this gene is given by its sequence identity in the UniGene database [52] when compared to orthologous sequences from M. musculus (95.1%), which has a validated status in RefSeq, and to H. sapiens (93.1%), suggesting a well-conserved homology of the THSD4 gene in these species.

                            The THSD4 gene encodes a protein with conserved disintegrin and metalloprotease domains, which it shares with the ADAM-TS1 protein family, and plays an import role in adipogenesis [69]. Previous studies have shown that this protein family interferes with the availability of differentiation-inducing or differentiation-inhibiting growth factors, either by modifying the extracellular matrix, affecting cell migration and adhesion, or by activating other pathways, which are key for regulating the differentiation of adipocytes, allowing their growth and expansion during adipogenesis [70].

                            The subcutaneous fat percentage QTL reported on chr 10 (Table 2) is from a Charolais × Holstein crossbred cattle population, and is described as highly significant with additive effects estimated to be 0.5 phenotypic standard deviation units [65]. The study also reveals that the Charolais allele was associated with higher fat levels.

                            The SNP on chr 1 (rs137294146) associated with backfat thickness is responsible for approximately 9.4% of the dEBV variation (Table 2). There is also a reported QTL for fat thickness over the 12th rib [29] and another for intramuscular fat percentage [71], indicating that there should be one or more genes in this area affecting fat metabolism. In the 500Kb window surrounding this SNP, three genes are annotated, SOX14 (sex determining region Y – box 14), CLDN18 (claudin 18), and DZIP1L (DAZ interacting protein 1-like). The SOX14 gene seems to be involved in the regulation of embryonic development, whereas CLDN18 belongs to a multigene family that encodes a tetraspanning membrane protein acting on components at tight junctions, but its regulatory mechanisms, and roles in physiology and pathology are still under investigation [72]. The DZIP1L gene encodes a zinc finger protein, but how it affects either adipogenesis or lipid metabolism has not been depicted from the current literature. Nonetheless, the functions of these gene products are still being elucidated.

                            The 500Kb window around the SNP on chr 3 (rs109349988) reveals many annotated genes, of which some have been reported as participating in lipid metabolism. For example, PMVK (phosphomevalonate kinase) catalyzes the conversion of mevalonate 5-phosphate with ATP to form mevalonate 5-diphosphate and ADP, which is one of the initial reactions involved in the cholesterol biosynthetic pathway [73]. Other proteins in this region include ADAR (adenosine deaminase, RNA-specific), which encodes an RNA-editing enzyme by site-specific deamination of adenosines, resulting in changes in protein function or gene expression. A study in humans was conducted that found ADAR enzymes were associated with serum triglyceride and adiponectin levels, abdominal circumference, and body mass index [74]. Interestingly, this region also contains SHC1 (Src homology 2 domain containing – transforming protein 1) which has been reported as having a role in human obesity [75], and as being one of the mediators for regulating the insulin-like growth factor 1 (IGF-1) pathway, which plays a key role in regulating cell proliferation, differentiation and apoptosis [76]. Lastly, this region contains ADAM15 (ADAM metallopeptidase domain 15), which belongs to the ADAM protein family previously discussed. These studies corroborate our findings and require further investigation to elucidate how these genes are affecting the deposition of subcutaneous fat in bovines.

                            The SNP associated with backfat thickness on chr 19 (rs136717249) is responsible for approximately 4.88% of the dEBV variance. This region contains the PHOSPHO1 (phosphatase, orphan 1) gene, which encodes a phosphatase enzyme that has been implicated in the mineralization of the extracellular matrix, a key process for skeletal development [77]. The PHOSPHO1 gene product has high activities toward phosphoethanolamine (PEA) and phosphocholine (PCho) [78], which are the main metabolites involved in the pathway for the formation of phosphatidylcholine and phosphatidylethanolamine [79]. These compounds are implicated in the metabolism of complex glycerolipids, prostaglandins, leukotrienes, glycosylphosphatidylinositol-anchors, and some amino acids, such as glycine, serine and threonine. Also included in this region is the PHB gene (prohibitin), which is thought to be involved in regulating cell proliferation, gene transcription, and apoptosis. In recent studies, deficient PHB activity in the liver has been associated with non-alcoholic steatohepatitis and obesity, although the mechanism remains unknown [80, 81]. Other examples include the IGF2BP1 (insulin-like growth factor 2 mRNA binding protein 1) gene, which encodes a protein that binds to the mRNAs of certain genes and regulates their translation. Lastly, the GIP (gastric inhibitory polypeptide, also known as the glucose-dependent insulinotropic polypeptide) gene has a known effect on stimulating the release of insulin from pancreatic β cells, but also has an insulin-like effect on adipocytes, suggesting that the GIP gene product enhances adipocyte glucose uptake, and that, at least in humans, it has an important role in the development of nutrition-induced obesity [82]. A recent study suggests that the GIP gene product has an effect on reducing free fatty acid release from adipose tissues, either by increasing reesterification or by inhibition of lipolysis [83]. Indeed, QTL studies reveal oleic acid content (OAC) and palmitoleic acid content (PAC) QTLs [84, 85] in close proximity to the GIP gene in the bovine genome, which further suggests an association between this gene and free fatty acid processing.

                            The SNP rs134790147 on chr 13 also was associated with backfat thickness, and it is carrying 3.51% of the dEBV. Within this SNP region, a QTL for fat thickness over the 12th rib was found and described in an Angus population [29]. Also, a set of four genes are localized in the ±250kb window from the SNP position. The CCDC7 gene (coiled-coil domain containing 7) seems to be associated with human cancer [86, 87], and there is no information available for bovines. The ARL5B gene product (ADP-ribosylation factor-like 5B), also known as ARL8, belongs to a family of proteins that show similar structure to ADP-ribosylation factors (ARFs family). ARLs and ARFs belong to the RAS superfamily of small GTPases, which function as modulators of complex and diverse cellular processes [88, 89], of which the most canonical are cell proliferation and differentiation. However, they are also involved in protein trafficking through the trans-Golgi network (TGN). The TGN has a central role in protein sorting and directs the transport of newly synthesized proteins to different transport vesicles [9092], and also receives recycled molecules and extracellular materials by retrograde transport. Recently, it was observed that ARL5B enhances retrograde transport from endosomes to the TGN [93]. The MGC152301 (uncharacterized LOC783682) and the LOC524240 (Alk-like) genes do not have any available information in terms of function of their gene products, but both show the same two conserved domains: cd00112 (LDLa) and cd06263 (MAM) [94]. The LDLa is a low density lipoprotein receptor class A domain, that plays an important role in mammalian cholesterol metabolism, the protein receptor binds LDL and transports it into the cell by endocytosis [95]. The MAM is an extracellular domain that mediates protein-protein interactions, and is found in a variety of proteins, of which many are known to function in cell adhesion [96]. The remaining 16 SNPs, which were not described in detail here, accounted for 19.14% of dEBV variation for backfat thickness and, as seen in Table 2, most of them present some fat-related QTL described within their regions [29, 65, 66, 85, 9799], and are of further interest for future investigations on how these SNPs can be influencing backfat thickness deposition in Canchim beef cattle.

                            Conclusions

                            In this study, we were able to identify a set of SNPs that correlates with approximately 50% of the deregressed estimated breeding value variance for backfat thickness in Canchim beef cattle, which introduces the possibility of including these SNPs in the development of a low density SNP panel for future implementation of genomic selection program in Canchim beef cattle. We also have applied a new methodology using the Random Forest approach to identify novel gene candidates for improving backfat thickness in Canchim beef cattle. In addition, although this study used backfat thickness as a target trait, other analyses of this type have successfully used other traits, thereby supporting the random forest approach as a means of future investigations of livestock production traits. Lastly, some regions identified are not conspicuously associated with any specific genes. This suggests that they may be involved in as of yet unidentified regulatory functions of gene expression or processing. Given the intrinsic complexity of biochemical pathways, these regions and the genes within them merit a great deal of future investigations, specifically to how they correlate with backfat thickness deposition in Canchim beef cattle and to other breeds.

                            Declarations

                            Acknowledgements

                            The authors would like to acknowledge the technicians Wilson Mallagó Jr, Flávia A. Bressani, and Gilberto A. Agostinho involved in this study for providing excellent assistance, and also to the Canchim Breed Association for providing DNA material. We thank Dr. David Z. Mokry for English revisions and corrections. This project was funded by grants from CAPES/PNPD no 02663/09-0 (Coordination for Enhancement of Higher Education Personnel), and Embrapa (Brazilian Agricultural Research Corporation). The funding agencies had no role in study design, data collection and analysis, interpretation, decision to publish, or preparation of the manuscript.

                            Marcos Vinicius Gualberto Barbosa da Silva, Fernando Flores Cardoso, Maurício Mello de Alencar and Luciana Correia de Almeida Regitano are CNPq fellows. Fabiana Barichello Mokry and Andressa Oliveira de Lima are CAPES fellows.

                            Authors’ Affiliations

                            (1)
                            Department of Genetics and Evolution, Federal University of São Carlos
                            (2)
                            Embrapa Agricultural Informatics
                            (3)
                            Embrapa Southeast Livestock
                            (4)
                            Department of Animal Science, Federal University of Lavras
                            (5)
                            Embrapa Dairy Cattle
                            (6)
                            Embrapa Southern Region Animal Husbandry
                            (7)
                            Department of Exact Science, São Paulo State University

                            References

                            1. Ferraz JBS, Felício PE: Production systems–an example from Brazil. Meat Sci 2010, 84:238–243.PubMedView Article
                            2. Carvalho FA, Lammoglia MA, Simoes MJ, Randel RD: Breed affects thermoregulation and epithelial morphology in imported and native cattle subjected to heat stress. J Anim Sci 1995, 73:3570–3573.PubMed
                            3. Alencar MM, Fraga AB, Silva AM: Adaptação de genótipos a ambientes tropicais: resistência à mosca-dos-chifres (Haematobia irritans, linnaeus) e ao carrapato (Boophilus microplus, CANESTRINI). Agrociencia 2005, IX:579–585.
                            4. Silva AM, Alencar MM, Regitano LCA, Oliveira MCS: Infestação natural de fêmeas bovinas de corte por ectoparasitas na Região Sudeste do Brasil. Revista Brasileira de Zootecnia 2010, 39:1477–1482.View Article
                            5. Turner JW: Genetic and biological aspects of Zebu adaptability. J Anim Sci 1980, 50:1201–1205.PubMed
                            6. Bianchini W, Silveira AC, Jorge AM, Arrigoni MDB, Martins CL, Rodrigues É, Hadlich JC, Andrighetto C: Efeito do grupo genético sobre as características de carcaça e maciez da carne fresca e maturada de bovinos superprecoces. Revista Brasileira de Zootecnia 2007, 36:2109–2117.View Article
                            7. Connor SFO, Tatum JD, Wulf DM, Green RD, Smith GC: Genetic effects on beef tenderness in Bos indicus composite and Bos taurus cattle. J Anim Sci 1997, 75:1822–1830.
                            8. Whipple G, Koohmaraie M, Dikeman ME, Crouse JD, Hunt MC, Klemm RD: Evaluation of attributes that affect longissimus muscle tenderness in Bos taurus and Bos indicus cattle. J Anim Sci 1990, 68:2716–2728.PubMed
                            9. Highfill CM, Esquivel-Font O, Dikeman ME, Kropf DH: Tenderness profiles of ten muscles from F1 Bos indicus x Bos taurus and Bos taurus cattle cooked as steaks and roasts. Meat Sci 2012, 90:881–886.PubMedView Article
                            10. Thrift FA, Sanders JO, Brown MA, Brown AH, Herring AD, Riley DG, DeRouen SM, Holloway JW, Wyatt WE, Vann RC, Chase CC, Franke DE, Cundiff LV, Baker JF: Review: Preweaning, Postweaning, and Carcass Trait Comparisons for Progeny Sired by Subtropically Adapted Beef Sire Breeds at Various US Locations. Profl Anim S 2010, 26:451–473.
                            11. Chase CC, Chenoweth PJ, Larsen RE, Olson TA, Hammond AC, Menchaca MA, Randel RD: Growth and reproductive development from weaning through 20 months of age among breeds of bulls in subtropical Florida. Theriogenology 1997, 47:723–745.PubMedView Article
                            12. Nogueira GP: Puberty in South American Bos indicus (Zebu) cattle. Anim Reprod Sci 2004, 82–83:361–372.PubMedView Article
                            13. Vianna AT: Formação do gado Canchim pelo cruzamento charoles-zebu. Rio de Janeiro: Ministério da Agricultura; 1960:48.
                            14. Meirelles SL, Alencar MM, Oliveira HN, Regitano LCA: Efeitos de ambiente e estimativas de parâmetros genéticos para características de carcaça em bovinos da raça Canchim criados em pastagem. Revista Brasileira de Zootecnia 2010, 39:1437–1442.View Article
                            15. Rhoades R, Ponce C, Smith S, Herring A, TEDESCHI L, Lunt DK, Dean D, Ribeiro F, Choi C, Riley D, Sawyer J: Evaluation of Growth-Based Predictions of Carcass Fat and Marbling at Slaughter Using Ultrasound Measurements. Profl Anim S 2009, 25:434–442.
                            16. Utrera AR, Van Vleck LD: Heritability estimates for carcass traits of cattle: a review. Genet Mol Res: GMR 2004, 3:380–394.PubMed
                            17. Lopes JS, Rorato PRN, Weber T, Rodrigues RD, Comin JG, Dornelles M d A: Metanálise para características de carcaça de bovinos de diferentes grupos genéticos. Ciência Rural 2008, 38:2278–2284.View Article
                            18. Kemp DJ, Herring WO, Kaiser CJ: Genetic and environmental parameters for steer ultrasound and carcass traits. J Anim Sci 2002, 80:1489–1496.PubMed
                            19. Crews DH, Shannon NH, Crews RE, Kemp RA: Weaning, yearling, and preharvest ultrasound measures of fat and muscle area in steers, bulls, and heifers. J Anim Sci 2002, 80:2817–2824.PubMed
                            20. Veneroni GB, Meirelles SL, Grossi DA, Gasparin G, Ibelli AMG, Tizioto PC, Oliveira HN, Alencar MM, Regitano LCA: Prospecting candidate SNPs for backfat in Canchim beef cattle. Genet Mol Res: GMR 2010, 9:1997–2003.PubMedView Article
                            21. Fortes MRS, Curi RA, Chardulo LAL, Silveira AC, Assumpção MEOD, Visintin JA, Oliveira HN: Bovine gene polymorphisms related to fat deposition and meat tenderness. Genet Mol Biol 2009, 32:75–82.PubMed
                            22. Curi RA, Krauskopf MM, Hadlich JC, Fortes MRS, Vankan DM, Silva JAIV, de Oliveira HN, da Mota MDS: Candidate SNPs for carcass and meat traits in Nelore animals and in their crosses with Bos taurus. Pesquisa Agropecuária Brasileira 2012, 47:294–301.View Article
                            23. De Carvalho TD TD, Siqueira F, de Torres Júnior RAA, de Medeiros SR, Feijó GLD, de Souza Junior MD, Blecha IMZ, Soares CO: Association of polymorphisms in the leptin and thyroglobulin genes with meat quality and carcass traits in beef cattle. Revista Brasileira de Zootecnia 2012, 41:2162–2168.View Article
                            24. Veneroni GB, Meirelles SL, De Oliveira HN, De Alencar MM MM, Gasparin G: Association of CSSM066 and ILSTS011 microsatellite markers and thyroglobulin gene SNP with backfat in Canchim cattle. Sci Agric 2012, 69:1–5.
                            25. Schenkel FS, Miller SP, Ye X, Moore SS, Nkrumah JD, Li C, Yu J, Mandell IB, Wilton JW, Williams JL: Association of single nucleotide polymorphisms in the leptin gene with carcass and meat quality traits of beef cattle. J Anim Sci 2009, 83:2009–2020.
                            26. Barendse W: Haplotype analysis improved evidence for candidate genes for intramuscular fat percentage from a genome wide association study of cattle. PLoS ONE 2011, 6:e29601.PubMedView Article
                            27. Bolormaa S, Porto Neto LR, Zhang YD, Bunch RJ, Harrison BE, Goddard ME, Barendse W: A genome-wide association study of meat and carcass traits in Australian cattle. J Anim Sci 2011, 89:2297–2309.PubMedView Article
                            28. Kim Y, Ryu J, Woo J, Kim JB, Kim CY, Lee C: Genome-wide association study reveals five nucleotide sequence variants for carcass traits in beef cattle. Anim Genet 2011, 42:361–365.PubMedView Article
                            29. McClure MC, Morsci NS, Schnabel RD, Kim JW, Yao P, Rolf MM, McKay SD, Gregg SJ, Chapple RH, Northcutt SL, Taylor JF: A genome scan for quantitative trait loci influencing carcass, post-natal growth and reproductive traits in commercial Angus cattle. Anim Genet 2010, 41:597–607.PubMedView Article
                            30. Lindholm-Perry AK, Sexten AK, Kuehn LA, Smith TPL, King DA, Shackelford SD, Wheeler TL, Ferrell CL, Jenkins TG, Snelling WM, Freetly HC: Association, effects and validation of polymorphisms within the NCAPG - LCORL locus located on BTA6 with feed intake, gain, meat and carcass traits in beef cattle. BMC Genet 2011, 12:103.PubMedView Article
                            31. Soma Y, Uemoto Y, Sato S, Shibata T, Kadowaki H, Kobayashi E, Suzuki K: Genome-wide mapping and identification of new quantitative trait loci affecting meat production, meat quality, and carcass traits within a Duroc purebred population. J Anim Sci 2011, 89:601–608.PubMedView Article
                            32. Peters SO, Kizilkaya K, Garrick DJ, Fernando RL, Reecy JM, Weaber RL, Silver GA, Thomas MG: Bayesian genome wide association analyses of growth and yearling ultrasound measures of carcass traits in Brangus heifers. J Anim Sci 2012, 90:3398–3409.PubMed
                            33. Veneroni-Gouveia G, Meirelles SL, Grossi DA, Santiago AC, Sonstegard TS, Yamagishi MEB, Matukumalli LK, Coutinho LL, Alencar MM, Oliveira HN, Regitano LCA: Whole-genome analysis for backfat thickness in a tropically adapted, composite cattle breed from Brazil. Anim Genet 2012, 43:518–524.PubMedView Article
                            34. Gill JL, Bishop SC, McCorquodale C, Williams JL, Wiener P: Association of selected SNP with carcass and taste panel assessed meat quality traits in a commercial population of Aberdeen Angus-sired beef cattle. Genet, Sel, Evol: GSE 2009, 41:36.PubMedView Article
                            35. Porto Neto LR, Bunch RJ, Harrison BE, Barendse W: Variation in the XKR4 gene was significantly associated with subcutaneous rump fat thickness in indicine and composite cattle. Anim Genet 2012, 43:785–789.PubMedView Article
                            36. Hoh J, Ott J: Mathematical multi-locus approaches to localizing complex human trait genes. Nat Rev Genet 2003, 4:701–709.PubMedView Article
                            37. Balding DJ: A tutorial on statistical methods for population association studies. Nat Rev Genet 2006, 7:781–791.PubMedView Article
                            38. Long N, Gianola D, Rosa GJM, Weigel KA, Avendaño S: Machine learning classification procedure for selecting SNPs in genomic selection: application to early mortality in broilers. J Anim Breed Genet 2007, 124:377–389.PubMedView Article
                            39. Breiman L: Random Forests. Mach Learn 2001, 45:5–32.View Article
                            40. Breiman L: Classification and regression trees. Belmont, Calif: Wadsworth International Group; 1984:358.
                            41. Breiman L: Bagging Predictors. Mach Learn 1996, 140:123–140.
                            42. Ho TK: The random subspace method for constructing decision forests. IEEE Trans Pattern Anal Mach Intell 1998, 20:832–844.View Article
                            43. Goldstein BA, Hubbard AE, Cutler A, Barcellos LF: An application of Random Forests to a genome-wide association dataset: methodological considerations & new findings. BMC Genet 2010, 11:49.PubMedView Article
                            44. Boldman KG, Kriese LA, Vleck V, Tassell V, Kachman SD: A Manual for Use of MTDFREML. A Set of Programs to Obtain Estimates of Variances and Covariances. [Draft]. Washington. DC: U.S. Department of Agriculture, Agricultural Research Service; 1995.
                            45. Garrick DJ, Taylor JF, Fernando RL: Deregressing estimated breeding values and weighting information for genomic regression analyses. Genet, Sel, Evol: GSE 2009, 41:55.PubMedView Article
                            46. Liaw A, Wiener M: Classification and Regression by randomForest. R News 2002, 2:18–22.
                            47. R Development Core Team: R: A Language and Environment for Statistical Computing. R Found Stat Comput 2011, 1:409.
                            48. SAS Institute Inc: SAS/STAT Software. SAS Institute Inc: Version 9.3. Cary NC; 2011.
                            49. Scheet P, Stephens M: A fast and flexible statistical model for large-scale population genotype data: applications to inferring missing genotypes and haplotypic phase. Am J Human Genet 2006, 78:629–644.View Article
                            50. Barrett JC, Fry B, Maller J, Daly MJ: Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics (Oxford, England) 2005, 21:263–265.View Article
                            51. Cattle Genome UMD3.1 http://​www.​animalgenome.​org/​cgi-bin/​gbrowse/​bovine/​
                            52. Geer LY, Marchler-Bauer A, Geer RC, Han L, He J, He S, Liu C, Shi W, Bryant SH: The NCBI BioSystems database. Nucleic Acids Res 2010, 38:D492-D496.PubMedView Article
                            53. Kanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 2000, 28:27–30.PubMedView Article
                            54. Kanehisa M, Goto S, Sato Y, Furumichi M, Tanabe M: KEGG for integration and interpretation of large-scale molecular data sets. Nucleic Acids Res 2012, 40:D109-D114.PubMedView Article
                            55. Anderson MJ: Permutation tests for univariate or multivariate analysis of variance and regression. Can J Fish Aquatic Sci 2001, 58:626–639.View Article
                            56. Colhoun H, Mckeigue P, Smith G: Problems of reporting genetic associations with complex outcomes. Lancet 2003, 361:865–872.PubMedView Article
                            57. Sun Q, Song K, Shen X, Cai Y: The association between KCNQ1 gene polymorphism and type 2 diabetes risk: a meta-analysis. PLoS ONE 2012, 7:e48578.PubMedView Article
                            58. Song Q, Zhu B, Hu W, Cheng L, Gong H, Xu B, Zheng X, Zou L, Zhong R, Duan S, Chen W, Rui R, Wu J, Miao X: A common SMAD7 variant is associated with risk of colorectal cancer: evidence from a case–control study and a meta-analysis. PLoS ONE 2012, 7:e33318.PubMedView Article
                            59. Luo Y, Jin C, Ling Z, Mou X, Zhang Q, Xiang C: Association study of IL28B: rs12979860 and rs8099917 polymorphisms with SVR in patients infected with chronic HCV genotype 1 to PEG-INF/RBV therapy using systematic meta-analysis. Gene 2013, 513:292–296.PubMedView Article
                            60. Pabalan NA: Meta-analysis in cancer genetics. Asian Pac J Cancer Prev: APJCP 2010, 11:33–38.PubMed
                            61. Habier D, Fernando RL, Dekkers JCM: Genomic selection using low-density marker panels. Genetics 2009, 182:343–353.PubMedView Article
                            62. Moser G, Khatkar MS, Hayes BJ, Raadsma HW: Accuracy of direct genomic values in Holstein bulls and cows using subsets of SNP markers. Gen, Sel, Evol: GSE 2010, 42:37.View Article
                            63. Zhang Z, Ding X, Liu J, Zhang Q, De Koning D-J: Accuracy of genomic prediction using low-density marker panels. J Dairy Sci 2011, 94:3642–3650.PubMedView Article
                            64. Garrick DJ: The nature, scope and impact of genomic prediction in beef cattle in the United States. Gen, Sel, Evol: GSE 2011, 43:17.View Article
                            65. Gutiérrez-Gil B, Williams JL, Homer D, Burton D, Haley CS, Wiener P: Search for quantitative trait loci affecting growth and carcass traits in a cross population of beef and dairy cattle. J Anim Sci 2009, 87:24–36.PubMedView Article
                            66. Casas E, Shackelford SD, Keele JW, Koohmaraie M, Smith TPL, Stone RT: Detection of quantitative trait loci for growth and carcass composition in cattle. J Anim Sci 2003, 81:2976–2983.PubMed
                            67. De Souza Santos E, De Bessa SA, Netto MM, Nagai MA: Silencing of LRRC49 and THAP10 genes by bidirectional promoter hypermethylation is a frequent event in breast cancer. Int J Oncol 2008, 33:25–31.PubMed
                            68. Pruitt KD, Tatusova T, Brown GR, Maglott DR: NCBI Reference Sequences (RefSeq ): current status, new features and genome annotation policy. Nucleic Acids Res 2012, 40:130–135.View Article
                            69. McDaniel AH, Li X, Tordoff MG, Bachmanov AA, Reed DR: A locus on mouse Chromosome 9 (Adip5) affects the relative weight of the gonadal but not retroperitoneal adipose depot. Mamm Genome 2006, 17:1078–1092.PubMedView Article
                            70. Lilla J, Stickens D, Werb Z: Metalloproteases and adipogenesis: a weighty subject. Am J Pathol 2002, 160:1551–1554.PubMedView Article
                            71. Gutiérrez-Gil B, Wiener P, Nute GR, Burton D, Gill JL, Wood JD, Williams JL: Detection of quantitative trait loci for meat quality traits in cattle. Anim Genet 2008, 39:51–61.PubMedView Article
                            72. Lal-Nag M, Morin PJ: The claudins. Genome Biol 2009, 10:235.PubMedView Article
                            73. Herdendorf TJ, Miziorko HM: Functional evaluation of conserved basic residues in human phosphomevalonate kinase. Biochemistry 2007, 46:11780–11788.PubMedView Article
                            74. Oguro R, Kamide K, Katsuya T, Akasaka H, Sugimoto K, Congrains A, Arai Y, Hirose N, Saitoh S, Ohishi M, Miura T, Rakugi H: A single nucleotide polymorphism of the adenosine deaminase, RNA-specific gene is associated with the serum triglyceride level, abdominal circumference, and serum adiponectin concentration. Exp Gerontol 2012, 47:183–187.PubMedView Article
                            75. Feigelson HS, Teras LR, Diver WR, Tang W, Patel AV, Stevens VL, Calle EE, Thun MJ, Bouzyk M: Genetic variation in candidate obesity genes ADRB2, ADRB3, GHRL, HSD11B1, IRS1, IRS2, and SHC1 and risk for breast cancer in the Cancer Prevention Study II. Breast Cancer Res: BCR 2008, 10:R57.PubMedView Article
                            76. Wagner K, Hemminki K, Grzybowska E, Klaes R, Butkiewicz D, Pamula J, Pekala W, Zientek H, Mielzynska D, Siwinska E, Försti A: The insulin-like growth factor-1 pathway mediator genes: SHC1 Met300Val shows a protective effect in breast cancer. Carcinogenesis 2004, 25:2473–2478.PubMedView Article
                            77. Huesa C, Yadav MC, Finnilä MAJ, Goodyear SR, Robins SP, Tanner KE, Aspden RM, Millán JL, Farquharson C: PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures. Bone 2011, 48:1066–1074.PubMedView Article
                            78. Roberts SJ, Stewart AJ, Sadler PJ, Farquharson C: Human PHOSPHO1 exhibits high specific phosphoethanolamine and phosphocholine phosphatase activities. Biochem J 2004, 382:59–65.PubMedView Article
                            79. Walkey CJ, Donohue LR, Bronson R, Agellon LB, Vance DE: Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase. Proc Nat Acad Sci U S Am 1997, 94:12880–12885.View Article
                            80. Theiss AL, Sitaraman SV: The role and therapeutic potential of prohibitin in disease. Biochimica et Biophysica Acta 1813, 2011:1137–1143.
                            81. Sánchez-Quiles V, Santamaría E, Segura V, Sesma L, Prieto J, Corrales FJ: Prohibitin deficiency blocks proliferation and induces apoptosis in human hepatoma cells: molecular mechanisms and functional implications. Proteomics 2010, 10:1609–1620.PubMedView Article
                            82. Song DH, Getty-Kaushik L, Tseng E, Simon J, Corkey BE, Wolfe MM: Glucose-dependent insulinotropic polypeptide enhances adipocyte development and glucose uptake in part through Akt activation. Gastroenterology 2007, 133:1796–1805.PubMedView Article
                            83. Gögebakan Ö, Andres J, Biedasek K, Mai K, Kühnen P, Krude H, Isken F, Rudovich N, Osterhoff MA, Kintscher U, Nauck M, Pfeiffer AFH, Spranger J: Glucose-dependent insulinotropic polypeptide reduces fat-specific expression and activity of 11β-hydroxysteroid dehydrogenase type 1 and inhibits release of free fatty acids. Diabetes 2012, 61:292–300.PubMedView Article
                            84. Morris CA, Cullen NG, Glass BC, Hyndman DL, Manley TR, Hickey SM, McEwan JC, Pitchford WS, Bottema CDK, Lee MAH: Fatty acid synthase effects on bovine adipose fat and milk fat. Mamm Genome 2007, 18:64–74.PubMedView Article
                            85. Morris CA, Bottema CDK, Cullen NG, Hickey SM, Esmailizadeh AK, Siebert BD, Pitchford WS: Quantitative trait loci for organ weights and adipose fat composition in Jersey and Limousin back-cross cattle finished on pasture or feedlot. Anim Genetics 2010, 41:589–596.View Article
                            86. Shen Y-M, Arbman G, Sandström P, Gullstrand P, Wei Y-Q, Zhang H, Rosell J, Olsson B, Peng F, Yang H-S, Wang C-T, Sun X-F: Novel gene hBiot2 is an independent prognostic factor in colorectal cancer patients. Oncol Rep 2012, 27:376–382.PubMed
                            87. Shen YM, He X, Deng HX, Xie YP, Wang CT, Wei YQ, Zhao X: Overexpression of the hBiot2 gene is associated with development of human cervical cancer. Oncol Rep 2011, 25:75–80.PubMed
                            88. Macara IG, Lounsbury KM, Richards SA, McKiernan C, Bar-Sagi D: The Ras superfamily of GTPases. FASEB J 1996, 10:625–630.PubMed
                            89. Wennerberg K, Rossman KL, Der CJ: The Ras superfamily at a glance. J Cell Sci 2005, 118:843–846.PubMedView Article
                            90. Griffiths G, Simons K: The trans Golgi network: sorting at the exit site of the Golgi complex. Science (New York, N.Y.) 1986, 234:438–443.View Article
                            91. Gu F, Crump CM, Thomas G: Trans-Golgi network sorting. Cell Mol Life Sci 2001, 58:1067–1084.PubMedView Article
                            92. Bonifacino JS, Rojas R: Retrograde transport from endosomes to the trans-Golgi network. Nature reviews. Mol Cell Biol 2006, 7:568–579.
                            93. Houghton FJ, Bellingham SA, Hill AF, Bourges D, Ang DKY, Gemetzis T, Gasnereau I, Gleeson PA: Arl5b is a Golgi-localised small G protein involved in the regulation of retrograde transport. Exp Cell Res 2012, 318:464–477.PubMedView Article
                            94. Marchler-Bauer A, Zheng C, Chitsaz F, Derbyshire MK, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Lu S, Marchler GH, Song JS, Thanki N, Yamashita RA, Zhang D, Bryant SH: CDD: conserved domains and protein three-dimensional structure. Nucleic Acids Res 2013, 41:D348-D352.PubMedView Article
                            95. Huang W, Dolmer K, Gettins PG: NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor-related protein. J Biol Chem 1999, 274:14130–14136.PubMedView Article
                            96. Aricescu AR, Hon W-C, Siebold C, Lu W, Van der Merwe PA, Jones EY: Molecular analysis of receptor protein tyrosine phosphatase mu-mediated cell adhesion. EMBO J 2006, 25:701–712.PubMedView Article
                            97. Yokouchi K, Mizoguchi Y, Watanabe T, Iwamoto E, Sugimoto Y, Takasuga A: Identification of a 3.7-Mb region for a marbling QTL on bovine chromosome 4 by identical-by-descent and association analysis. Anim Genetics 2009, 40:945–951.View Article
                            98. Casas E, Stone RT, Keele JW, Shackelford SD, Kappes SM, Koohmaraie M: A comprehensive search for quantitative trait loci affecting growth and carcass composition of cattle segregating alternative forms of the myostatin gene. J Anim Sci 2001, 79:854–860.PubMed
                            99. Imumorin IG, Kim E-H, Lee Y-M, De Koning D-J, Van Arendonk JA, De Donato M, Taylor JF, Kim J-J: Genome scan for parent-of-origin QTL effects on bovine growth and carcass traits. Front Genetics 2011, 2:44.

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                            This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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