Mapping and association of GAD2 and GIP gene variants with feed intake and carcass traits in beef cattle (short communication)

Glutamate decarboxylase 2 (GAD2) is a major autoantigen in insulin-dependent diabetes, while glucosedependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone. Both genes are involved in insulin secretion and play a role in the regulation of feed intake and metabolic rate in animals. In the present study, we assigned the bovine GAD2 and GIP by RH mapping to BTA 13 and BTA 19, respectively. We discovered SNPs from these genes by a PCR-sequencing approach. We identified one SNP (T/C transition) from the 14 intron of the bovine GAD2 gene and another SNP (A/C transversion) from the 4 intron of the bovine GIP gene. Genotyping over 300 animals from five different beef populations revealed that the average allelic frequency for the GAD2 allele A was 0.48 and allele B 0.52, while for the GIP allele A 0.80 and allele B 0.20, respectively. There were significant associations observed between the GAD2 and GIP gene variants and Average Daily Feed Intake (ADFI) (p<0.05) in beef cattle. In addition, the GAD2 SNP was also associated with Meat Percent (MP) (p<0.05), whereas the GIP SNP was also significantly associated with Backfat Thickness (BF) (p<0.05) and Ratio of Feed-to-Meat (RFM) (p<0.05). There were no significant effects found for other traits. Although both SNPs could be used as candidate markers for MAS to improve feed intake and carcass traits, further investigations in large populations and in other breeds are recommended in order to understand the effect of the GAD2 and GIP polymorphisms on these quantitative traits in beef cattle. Key Word: beef cattle, GAD2, GIP, RH mapping, single nucleotide polymorphism, feed intake, carcass traits


Introduction
The GAD2 gene encodes the glutamic acid decarboxylase enzyme (GAD65), catalyzes the formation of Gamma-aminobutyric acid (GABA) from L-glutamic acid and is expressed in both pancreatic islets and brain (ERDO et al., 1990).GABA is colocalized in neuropeptide Y (NPY) neurons, and is involved in the leptin pathway through the arcuate nucleus in the hypothalamus, suggesting that GABA and NPY interact in the paraventricular nucleus to stimulate food intake (OVESJO et al., 2001).Several lines of investigation support a role for GABA in regulation of feed intake and growth.For example, in turkey, it has been demonstrated that injection of varying doses of muscimol, a potent GABA agonist, caused a dose-dependant increase of food intake (DENBOW, 1991); Transgenic mice over-expressing the GABA transporter exhibited heritable obesity, with increased body weight and fat deposition (MA et al., 2000).A genome-wide scan performed in 158 multiplex French obese Caucasian families (514 individuals) revealed a microsatellite marker (D10S197) on human chromosome (HSA) 10p that is significantly linked to obesity (HAGER et al., 1998).This marker is located within the 7 th intron of the human GAD2 gene (http://www.ncbi.nlm.nih.gov/genome/sts/sts.cgi?uid=8782).BOUTIN et al. (2003) investigated SNPs of the GAD2 gene and performed association studies with obesity, obtaining evidence to implicate GAD2 as a candidate gene for human obesity.The study in cases with extreme forms of obesity have shown mutations in the gene involved in the leptin-melanocortin pathway (CLEMENT et al., 2002), which primarily impairs food intake regulation.Recently, MEYRE et al. found that the GAD2 promoter variation was significantly associated with childhood obesity in the French population and also influenced fetal growth and feeding behavior (MEYRE et al., 2005).Therefore, GAD2 could play a role in regulating growth and development in animals (ERLANDRE et al., 1992).Glucose-dependent insulinotropic polypeptide (GIP, formerly known as gastric inhibitory polypeptide) is a gastrointestinal hormone (BUCHAN et al., 1978).The role of GIP generally has been thought to stimulate insulin secretion from pancreatic betacells.In humans, GIP appears to play a role in lipid physiology and is associated with obesity (MEIER et al., 2003).However, obesity is a result of the interaction of environmental and genetic factors that mediate energy intake and expenditure (SPIEGELMAN et al., 2001).There is experimental evidence indicating that GIP regulates fat metabolism in adipocytes, including enhanced insulin-stimulated incorporation of fatty acids into triglycerides, stimulation of lipoprotein lipase activity, and stimulation of fatty acid synthesis (YIP et al., 2000).In 1999, by investigating the effect of gastric inhibitory polypeptide on bovine fat metabolism, DAWSON et al. found that cattle fed on grass silage diets tend to deposit more fat than animals fed on dried forages.They concluded that, although GIP may regulate nutrient utilization, it is unlikely that the gene plays a major role in promoting fat accretion in cattle (DAWSON et al., 1999).GIP also stimulates the growth, differentiation, proliferation and survival of pancreatic b-cells (POSPISILIK et al., 2003;TRUMPER et al., 2001).In this study, our main goal is to identify polymorphisms from the bovine GAD2 and GIP genes and to determine whether the polymorphisms are associated with quantitative traits, such as feed intake, growth and meat quality.This is the first step towards understanding whether bovine GAD2 and GIP gene variants can serve as genetic markers for marker assisted selection (MAS) to improve feed intake, growth and carcass traits in cattle.

Animals
A total of 320 animals including Simmental (n = 25), Angus (n = 22), Hereford (n = 21), Sanhe (n = 201) and Simmental crossbred steers (Simmental crossed with indigenous female yellow cattle in China) (n = 51) were randomly selected from commercial populations and used to analyze the GAD2 allelic frequencies.A total of 331 animals including Simmental (n = 28), Angus (n = 23), Hereford (n = 28), Sanhe (n = 201) and Simmental crossbred (n = 51) were used to analyze GIP allelic frequencies.Only the 51 Simmental crossbreed steers were used for the association study.The crossbreed steers (405 ± 50.5 kg; 30 ± 2 months of age) were housed in a concrete-floored cowshed (in a single pan for each animal).The pre-trial period (for adaptation to treatment) was 15 days, and the test period was 180 days.Steers were fed according to the NY/T 815-2004 feeding standard of beef cattle (Agricultural Department of People's Republic of China 2004).The composition of the diet fed is 60% maize, 20% artifactitious soybean, 5% artifactitious cotton seed, 10% bran and 5% specific mixture feed (including vitamins, microelements and mineral elements), which contained a total of energy 45.98 MJ, 18% CP, 69% TDN, 2.3% Ca, and 1.0% P. Animals had free access to water and feed during the entire 195 days of the experimental period.The average dry matter intake (DMI) is 7.46 kg/d for each cow during the test period.All animals were weighed on days 1, 75 and 195 at 8:00 AM, with prior removal of feed and water (12 hours).Feed not consumed was weighed and recorded at 10:00 PM each day.Immediately after the trial, steers were harvested at a processing facility (KeErQing Beef cattle Co., Ltd.P. R. China).Carcass traits were measured according to the criterion GB/T 17238-1998 Cutting Standard of Fresh and Chilled Beef in China (China Standard Publishing House).The following traits, Dressing Percent (DP), Backfat Thickness (BF), Carcass Weight (CW), Meat Percent (MP), Average Daily Gain (ADG), Average Daily Feed Intake (ADFI), Ratio of Feedto-Meat (RFM), Beginning Average Daily Gain (BADG; an average daily gain in the test period from day 1 to day 75), Finishing Average Daily Gain (FADG; an average daily gain in the test period from day 75 to day 180), Stomach Weight (SW) and Intestine Weight (IW) were measured or calculated.

DNA preparation, SNP detection and genotyping
Blood samples were collected from all 331 animals studied.The routine phenol chloroform extraction method was used to isolate the genomic DNA, which was diluted to 50 ng/µl for PCR.The mRNA sequence for the human GAD2 (NM_000818) and GIP (NM_004123) genes were obtained from NCBI and used to search for bovine expressed sequence tags (ESTs) from EST databases using a standard BLAST (http://www.ncbi.nlm.nih.gov/blast/)search (WANG et al., 2003;YANG et al., 2003;ZHU et al., 2005).The bovine ESTs that are ~ 500 bp in length and have more than 80% similarity with the corresponding human mRNA were identified as candidates.
These candidate ESTs were then assembled into contigs.Finally, a contig of bovine ESTs (GenBank acc.nos.CK846169, DY453982, BE899934) was used for GAD2 PCR primer design, and an EST (GenBank acc.no.CK833953) was used for GIP primer design.Primer pairs (Table 1) were designed using Primer 5.0 (PREMIER Biosoft International, Palo Alto, CA, USA) and synthesized by Shanghai Bioasia Biotechnology Co. Ltd. in China.PCR was performed in a PTC-200 Peltier Thermal Cycler (MJ Research Inc, Hercules, CA, USA).Amplified products were obtained for GAD2 (primers: F5-R5) and for GIP (primers: F1-R1) in a 20 μl reaction volume consisting of 50 ng of bovine genomic DNA, 1× PCR buffer, 0.3 μM of each primer, 75 μM of each dNTP, 1.5 mM MgCl 2 (2 mM MgCl 2 for GIP) and 1U Taq DNA polymerase (Promega, Madison, WI, USA).PCR conditions were: 5 min at 95 ˚C followed by 30 s at 94 ˚C, 30 s at the annealing temperature (Table 1) and 30 s at 72 ˚C for 34 cycles and a final extension at 72 ˚C for 10 min.PCR products were analyzed on a 1.5% agarose gel stained with 0.5 µg/mL ethidium bromide.To verify their identity, the products were purified using a Wizard Prep PCR Purification Kit (Shanghai Bioasia Biotechnology Co., Ltd.P. R. China), cloned into the pGEM-T Easy Vector Kit (Promega, Madison, WI, USA), and sequenced (Applied Biosystems 3730xl DNA Analyzer, Foster City, CA, USA).Sequences of the fragments were used in BLASTn searches at NCBI to ensure that they were the expected bovine sequences.After alignment of the amplified sequences from different individuals, several putative single polymorphism sites were identified.PCR-RFLP was used to genotype the SNPs in the bovine GAD2 and GIP genes.The PCR products for GAD2 and GIP were digested with Hae III and Msp I at 37 ˚C for 4 hrs.respectively.Restriction fragments were scored on 1.5% agarose gels.A total of 320 cattles from five breeds (Simmental, Angus, Hereford, Sanhe, crossbreed) were genotyped and allele frequencies were determined (Table 2).

Statistical analyses
The collected data were analyzed using SAS (SAS Institute Inc. Cary, NC, USA) according to the following model: Where Y ij stands for observed value; µ: population mean; G i : ith genotype; b ij : regression coefficient; w ij : weight; ε ij : random error.a Primer pair F5-R5 was used to isolate the 630 bp genomic DNA fragment of GAD2 and primer pair F6-R5 was used for RH mapping.b Primer pair F1-R1 was used to isolate the 902 bp genomic DNA fragment of GIP and primer pair F2-R1 was used for RH mapping.

RH mapping
A 7000-rad whole-genome cattle-hamster RH panel (SUNbRH), consisting of 92 hybrids, was used to map the GAD2 gene with the primer pair F6-R5 and the GIP gene with the primer pair F2-R1 (Table 1).PCR typing, vector scoring and RH mapping were conducted following the methods described by GUO et al. (2005) andLIU et al. (2005).

SNP identification
Using the primer pair F5-R5 that flanks the 14 th intron of the GAD2 gene (Table 1), we amplified a fragment of 630 bp (GenBank acc.no.DQ139317) by PCR from 10 individuals, which represent four breeds (Simmental, Angus, Hereford, and Sanhe).Sequencing of the fragment confirmed the origin of the bovine GAD2 gene.By careful comparison of the sequences from the 10 individuals, we found a SNP (T/C transition) at position nt 185 in the fragment located within the 14 th intron of the gene.Digestion of the PCR fragments with Hae III revealed two alleles: allele A (T-base) -one fragment of 630 bp, and allele B (C-base) -two fragments of 445 bp and 185 bp (Table 2).Similarly, using the primer pair F1-R1 (Table 1) that flanks the 4 th intron of GIP, a fragment of 902 bp was amplified (GenBank acc.no.DQ160296).Sequence comparison among the 10 individuals revealed a SNP (A/C transversion) at position nt 758 of the PCR fragment.Digestion of the 902 bp PCR product with Msp I revealed two alleles: allele A (A-base) -two bands with sizes of 740 bp and 162 bp, and allele B (C-base) -three bands with sizes of 596 bp, 162 bp and 144 bp, which were observed on agarose gels.Allele frequency for both the GAD2 and GIP genes was investigated in five different beef populations (Table 2).The frequency of the GAD2 allele A ranged from 0.43 to 0.55.The frequency of the GIP allele A ranged from 0.58 to 0.98) (Table 2).We found that the allelic frequency varied among the five cattle populations studied.For the GAD2 gene, the Chinese breed Sanhe cattle and the crossbreed that contains 50% of Chinese Yellow Cattle blood had frequency of allele A (≥ 0.51), in contrast to the Western beef breeds (Simmental, Angus and Hereford) (≤ 0.48); whereas for the GIP gene, the Chinese breed and the crossbreed had a frequency of ≤ 0.60 for allele A, which was significantly lower than that of ≥ 0.89 in the Western beef breeds (Table 2).The X 2 test of allelic frequency of both the GAD2 and GIP genes was performed among 5 breeds of cattle.For the GAD2 gene, there was not a significant difference in the allelic frequency among breeds (P>0.05).For the GIP gene, there was a significant difference in allelic frequency between the Chinese native breed (and the crossbreed) and the Western beef breeds (Simmental, Hereford and Angus) (p≤0.001).

RH mapping of the bovine GAD2 and GIP genes
By typing on the 7000-rad SUNbRH panel and analyzing the vector together with 3,216 framework markers (http://www.animalgenome.org/cattle/maps)(ITOH et al. 2005), the GAD2 gene was assigned, at a 2-point (DIK5201) LOD score of 32, to the bovine chromosome (BTA) 13 (Table 3), mapping between DIK2709 and DIK5201 with a distance of ~ 8.08 cR to DIK2709 and ~ 4.48 cR to DIK5201.Comparative mapping has demonstrated that human chromosome (HSA) 10, where the human GAD2 gene is located, is conserved with homologous segments to BTA13, BTA26 and BTA28 (EVERTS- VAN DER WIND et al., 2004;ITOH et al., 2005), which supports the mapping of the bovine GAD2 gene to BTA13 in this work.GIP was assigned, at a 2-point (BM17132) LOD score of 38, to BTA19 between BM17132 and DIK5224 with a small interval of ~ 0.6 cR to BM17132 and ~ 0.61 cR to DIK5224.The human GIP gene maps to HSA17, which is completely conserved with BTA19 (EVERTS- VAN DER WIND et al., 2004), supporting the present assignment of GIP to BTA19.Association analysis of the GAD2 185-SNP and GIP 758-SNP It is interesting the bovine GAD2 and GIP genes are located into several QTL regions in bovine chromosome 13 and 19, which have been indicated to affect body growth and carcass traits (http://bovineqtlv2.tamu.edu/home.php).A preliminary association study was performed to test the candidacy of GAD2 185-SNP and GIP 758-SNP as QTL contributors.Three genotypes, namely AA, AB and BB, were observed for both the GAD2 and GIP genes among the animals investigated.The genotypes of 51 crossbreed steers were compared with their phenotypic data for 11 traits.Effects of different genotypes on these traits are given in Table 4.It is interesting to see that both the GAD2 and GIP genes were significantly associated with ADFI (p<0.05).Animals with the AA and AB genotype of GAD2 had a higher ADFI and BB had a lower ADFI (Table 4).For the GIP gene, it was the BB genotype that had a higher ADFI, while AA and AB had a lower ADFI.Besides feed intake, the GAD2 185-SNP was also significantly associated with Meat Percent (MP) (p<0.05),whereas the GIP SNP was also significantly associated with Backfat Thickness (BF) (p<0.05) and Ratio of Feed-to-Meat (RFM) (p<0.05)(Table 4).Although we noticed differences among different genotypes of both the GAD2 and GIP genes for the rest of the traits (DP, CW, ADG, BADG, FADG, SW, and IW), these differences were not statistically significant (p≥0.05).Based on the data in Therefore, we propose that both the GAD2 and GIP SNPs are markers for feed intake and could also be used as markers for carcass traits such as MP, BF and RFM.Usually animals with higher feed intake have either a higher growth rate or a higher requirement for maintenance.The latter may be true for the Chinese cattle, as they, in general, have a lower growth rate in comparison to the Western beef breeds.
Although both SNPs could be used as candidate markers for MAS to improve feed intake and carcass traits, precautions must be taken when applying the markers, as the animal population used in this study is relatively small.In order to validate the findings present in this paper and to understand the effects of the GAD2 and GIP polymorphisms on these quantitative traits and the efficiency of using these markers for MAS, further investigations in large populations and in other beef breeds are recommended.

Table 1 Bovine
GAD2 and GIP gene primers used for SNP analysis and RH mapping (Nutzung boviner GAD2 und GIP Genprimer für die SNP Analyse sowie RH Kartierung)

Table 2
Allelic frequencies of the bovine GAD2 and GIP genes in different cattle breeds (Allelfrequenzen boviner GAD2 und GIP Gene verschiedener Rassen) aWithin columns means with different superscripts indicate significant differences at P < 0.001

Table 3
Chromosomal assignment of bovine GAD2 and GIP genes (Chromosomale Zuordnung boviner GAD2 und GIP Table 4, selection of animals with the BB genotype of GAD2 would have ~ 7.8 % improvement in ADFI over the AA and AB genotypes.It also leads to ~ 4.6 % increase in MP.Selection of animals with the AB genotype of GIP would have ~ 18.8 % improvement in ADFI over the BB genotype.At the same time, it would also result in a ~ 35.5 % increase in RFM and ~ 15.8 % decrease in BF.

Table 4
Effects of GAD2 and GIP genotypes on phenotypic traits in beef cattle (Zusammenhänge zwischen GAD2 und GIP Genotypen und phänotypischen Merkmale der Fleischrinder) Average Daily Gain (ADG), Carcass Weight (CW), Dressing Percent (DP), Meat Percent (MP), Backfat Thickness (BF), Beginning Average Daily Gain (BADG; average daily gain in the test period from day 1 to day 75), Finishing Average Daily Gain (FADG; average daily gain in the test period from day 75 to day 180), Ratio of Feed-to-Meat (RFM), Average Daily Feed Intake (ADFI), Stomach Weight (SW), and Intestine Weight (IW). 2 Within columns means with different superscripts indicate significant differences at P < 0.05. 1