Polymorphisms in A-FABP gene associated with meat quality traits of beef cattle in Gansu

A-FABP gene belongs to fatty acid binding proteins (FABPs). The family was discovered in the intestinal mucosa by Ockner et al., (1972). His research concerned the regulation of the intestinal fatty acid absorption in rats in the 1970 s at University of California. So far, experts have discovered that there are at least nine different types about FABPs structure. FABPs genes of various types consisting of four exons and three introns can be expressed in different tissues and cells. FABP gene’s expression, which was very rich in mammal cells, specifically combining with fatty acid, so it was considered as important protein that participates in fatty acid transport in cells (Yan et al., 2008) A-FABP gene was considered to be important candidate genes that affected intramuscular fat content of animals (Wang et al., 2008). The research of polymorphisms showed that A-FABP gene polymorphisms in connection with intramuscular fat deposition in fowls, ducks (Zhao et al., 2009), pigs, cattle and could be used as meat quality’s candidate genes for beef marbling score and IMF content. The mutation of A-FABP gene had influence on marbling score, hypodermal fat thickness and back fat thickness in Japanese black × Limousin F2 crossbreed cattle (Cho et al., 2008) and on marbling score in Korean cattle (Lee et al., 2010). In view of the important role of A-FABP gene in process of fat substances, five cattle groups were conducted as research objects by using PCR-SSCP technology to detect of polymorphism for A-FABP gene exon3 and analysis associated with polymorphic websites and meat quality, in order to find the genetic markers associated with production characters to provide genetic theory basis for the breeding of excellent beef. 

Cattle ear tissue samples were placed in 5ml EP tubes containing 75% alcohol and were stored in -20°C. Cattle neck intravenous blood sample collected about 6- 8ml and 1ml ACD anticoagulant was add to it, it was stored at -20°C. cDNA was extracted from ear tissue and blood through phenol chloroform method, it was dissolved by TE. DNA purity and concentration were tested by agarose electrophoresis and ultraviolet spectroscopy, next samples were diluted to 100 ng.ml^-1 (Table1).



The five groups were randomly selected for testing meat quality traits (pressing loss, marbling score, meat color, shear force, cooking loss, pH value). The tested cattle were 22 months old bulls in good health without blood relationships on same management conditions. The assay methods of beef character referred to Cattle Science (Wang, 2000). A pair of primers was designed by Primer 5.0 software according to A-FABP gene sequence published by Gene Bank (NC_007312). The primer sequences: F: 5'- AATAGTAAGCCTACCCTGA - 3', R: 5'- CATTTCCTTACC CACTTC - 3'. It could be used in SSCP analysis. Synthesis of primers was performed by Shanghai Sang on biological engineering Technology Co., LTD. Expected length of amplification fragment was 219bp. PCR reaction system was 25ul: 12.5ul (1.25U) Premix Taq, 1ul (100ng.ul^-1) DNA template, 0.5ul (10pmol.ul^-1) F/R primer, respectively, 10.5ul ddH2O. Reaction condition: pre degeneration time was 3min at 94°C, degeneration time was 30s at 94°C, annealing time was 30s at 59°C, extension time was 30s at 72°C, cycle numbers were 35, extension time was 10min at 72°C, then it was stored at 40°C.

PCR products were tested by employment of 1% agarose electrophoresis. PCR products were taken about 2ul, added 8ul denatured loading buffer (Formamide Deionized (98%), EDTA (10mmol.l^-1, pH8.0), xylenecyanol (0.025%) and bromophenol blue (0.025%) to it. Degeneration time was 10 min at 98°C, ice-bath time was 10 min. The products were detected by 14% acrylamide gel electrophoresis, next, were silver staining after electrophoresis that cost 10h on 180V. According to the result of SSCP analysis, the PCR products of different genotypes were purified and sequenced by Shanghai biological Engineering Co., LTD. The sequence obtained from the research was compared in Gen Bank (NM-174314.2) sequence by MEGA5 software.

It was calculated that genotypic, homozygous (Ho), heterozygous (He), effective number of alleles (Ne) and Hardy-Weinberg equilibrium test of genotypes distribution was conducted in groups by PopGen32 software. PIC was calculated by PIC software, genotypes distribution were analyzed by chi-square test independently in groups and compounded to next model to conduct least square analysis of variance.

Next, characteristics’ differences were compared between different genotypes by SPSS19.0’S GLM (general linear model) procedure.
The model was as follows:
 

Yij= µ+GENi+GROj+(GEN×GRO)ij+eij

 
Where:
Yij: Phenotypic value of individual characteristics
µ: Population mean
GENi: Genotypic response
GROj: Group response
(GEN×GRO) ij: Interaction response of the genotype and the representative of the population
eij: Random error. 

The result of SSCP discovered three types of bands, which means that there were three kinds of genotypes. The sequence obtained from the research was compared to the original sequence (NM-174314.2) published in data bank only to find a mutation site at G408>C of exon3 of A-FABP gene, which was a G’°C base mutation (Fig 1 and 2).



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The analysis discovered that expected Simmental beef crossbreed’s genotype GC frequencies were higher than genotype GG and the other four groups were lower than the genotype GC and genotype GG frequency was higher than genotype CC in the test results based on SSCP. Allele G frequencies were greater than the allele C frequencies. In five beef cattle groups, so it was dominant group.

The chi-square fitting test showed that the c.408G> Cx2 value of site reached a significant level in Qingyang native beef cattle and Qinchuan beef cattle, which indicated that the genotypes distribution in Hardy-Weinberg disequilibrium (P<0.05), while the other three groups the genotype distribution was in Hardy-Weinberg equilibrium (P <0.05) (Table 2).

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The chi-square test for independence showed  that genotypes distribution of Pingliang group and Simmental cross breed group were significantly different (P<0.05), among Qingyang native beef cattle group, Qinchuan beef cattle group and South Devon crossbreed beef group’s were highly significant difference (P<0.01), respectively (Table 3).

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It could be learned from table 4 that homozygous (Ho), heterozygous (He), number of effective alleles (Ne) and PIC in C.408G> C site of A-FABP gene in five groups. Qingyang group and Pingliang group were belonged to the low polymorphism due to their PIC was lesser than 0.25. The Other three groups were in moderate polymorphism (0.25<PIC <0.5) (Table 4).

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Six meat quality traits of harvest cattle (included pressing loss, marbling score, meat color, shear force, cooking loss, pH value) were conducted analysis of variance after exon3 of A-FABP gene genotyping, the results showed that genotype GG’s pressing loss was significantly greater than genotype CC’s (P<0.05), genotype GG’s shear force was highly significant greater than the genotype GC’s and genotype CC’s (P<0.01), difference between the genotype GC and genotype CC was not significant, genotype GG’s cooking loss and pH were significantly greater than genotype GC’s (P<0.05), were highly significant greater than genotype CC’s (P<0.01).The difference between meat color and marbling score among the three genotypes was not significant (P>0.05) (Table 5).

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The research of polymorphisms of A-FABP gene was discovered that intron1 and exon4 existed mutation at A-FABP gene of Qinchuan native beef cattle, while exon3 was relatively conservative. The other researches were studied that A-FABP gene’s 5’flanking region, exon2, exon3 and 3’flanking region all existed variations in Qinchuan cattle, Nanyang cattle, Luxi cattle, Xianan cattle and Jiaxian red cattle (Liu et al., 2010), exon2, exon3, exon4 of Korean cattle had mutation sites (Oh et al., 2012). In this study, the polymorphisms were detected at exon3 of A-FABP gene (c.408G>C) in five cattle groups, it showed that Pingliang group was without genotype CC, while three genotypes (GG, GC and CC) were presented in the other groups. In other words, G and C were detected at the A-FABP gene’s coding region and it was similar to mutation of A-FABP gene’s exon3 in Korean cattle (Oh et al., 2012). The results showed that genotype CC was not detected in Pingliang native beef cattle, the difference of genotypes distribution might due to Pingliang group’s genetic characteristics were different from the other four groups, genotype CC was non-dominant genotype, so it was eliminated in the long-term natural selection or artificial selection.

According to the chi-square test, genotypes distribution of Pingliang group and Simmental crossbreed groups were significantly different (P<0.05) and Qingyang group, Qinchuan cattle group, South Deven crossbreed group had highly significant differences(P<0.01), further confirmed there were differences of genetic characteristics among different groups wing to there were significant differences in genetic back ground itself among groups, which led to indirect selection pressure and genetic variation level of different alleles in the breeding process. It was considered as indexes to measured levels of genetic variation among populations that polymorphic information content, the number of alleles and heterozygosity were consistent in size and had a large polymorphism information content, therefore an effective number of alleles and heterozygosity was relatively large. These three parameters were high, groups’ variability was large at this site and there was great select potential, the site could be used to make marker-assisted selection. The analysis results from genetic polymorphisms showed that PIC of Qingyang group and Pingliang group was 0.21, 0.24, respectively, it belonging to a low degree of polymorphism (PIC<0.25). PIC that South Devon crossbreed group, Qinchuan group and Simmental crossbreed group was 0.37,0.28,0.30, respectively, ranged from 0.25 to 0.5, belonging to moderately polymorphic. The phenomenon indicated that the G408C sites of the A-FABP gene coding region could be conducted marker-assisted selection.

The chi-square fitting test showed that Qingyang group and Qinchuan group were in Hardy-Weinberg non-equilibrium state, indicating that natural selection or artificial selection in these groups had great influence to gene distribution and resulted in imbalanced distribution, which might be related to individual selection of breeding. The other three groups belonged to Hardy-Weinberg equilibrium state, it indicated that these three groups had formed genetic characteristics that adapted to several living environment, so the mutation could be stably inherited in the host population. Relationship between gene polymorphisms and meat quality traits.

A-FABP gene was considered important candidate genes that can affect intramuscular fat content in animal. Fat that intramuscular deposition, also known as IMF content has direct relationship with meat tenderness and flavor. In view of an important function of A-FABP gene on meat quality traits, at present, the gene was researched from various cattle populations in China and other foreign countries.

The polymorphic sites were typed that were discovered on intron1 and exon4 of Qinchuan group’s A-FABP gene and analyzed their correlation with meat quality traits by Wang Zhuo (2005). We discovered that the mutation site of intron1 was related to indicators that back fat thickness, tenderness, marbling and so on. The indicators of genotype GG that back fat thickness, marbling and tenderness were significantly greater than genotype CC. Liu et al., (2010) thought the mutation of the 5’flanking has significant effect on meat production traits of beef, SNPs of exon2 were related to slaughter rate, tenderness, carcass length and loin eye area, SNPs of exon3 were related to loin eye area and the SNPs of the 3’ flanking region were associated with beef carcass deep. Cho et al. (2008) discovered that SNPs of exon2 of A-FABP gene associated with cattle’s back fat thickness. Lee et al., (2010) discovered that a synonymous mutation site in exon3 had a significant influence on beef marbling score at Korean cattle’s A-FABP gene. Barend et al., (2009) discovered that there was a splice mutation site between exon3 and intron3 in FABP4 gene, the SNP significantly associated with fat deposition of the Australian cattle.

In this research, the G408C locus of exon3 of the A-FABP gene was consistent with mutation sites that were researched by Barend et al., (2009) and Lee et al., (2010), but the impact of the synonymous mutation site on the marbling score was not reached a significant level, which was not consistent with the reported result, so we could research correlation between SNPs of A-FABP gene and marbling score’s level on the other sites.

The research’s mutation site was a synonymous mutation which did not cause a corresponding change in the amino acid sequences. Once the sequences of the nucleotide were changed, speed of translation of mRNA was affected, the expression quality of protein was altered and even spatial structure of the protein was changed, thereby its function was affected (Barend et al., 2009). In this research, there are some differences of SNPs and A-FABP gene and some differences among the distribution of five groups, it associated with indicators that carcass weight, meat weight, dressing percentage, meat percentage, loin eye area, shear stress, pressure lose, cooking loss and pH value. In this research, the correlation between SNPs of the exon3 region of the A-FABP gene and indicators that carcass weight, dressing percentage, meat percentage, loin eye area, shear force, pressure lose, cooking loss and pH value did not reported in literatures, the results could provide reference data for subsequent testing. The correlation between genetic mutation sites of A-FABP gene and major economical traits of meat quality in the experimental cattle population, indicated that the A-FABP gene site SNPs could be served as molecular marker to enhance breeding and genetic effects of meat quality traits.

In this study, a mutation site was detected in exon3 of A-FABP gene meat quality traits had a big difference in different genotypes. Genotype GG’s pressing loss was significantly greater than genotype CC’s (P<0.05); genotype GG’s shear force was highly significant greater than the genotype GC and genotype CC’s (P<0.01), the difference between the genotype GC and genotype CC was not significant; genotype GG’s cooking loss and pH value were significantly greater than genotype GC’s (P<0.05) and highly significant greater than genotype CC’s (P<0.01).The difference between meat color and marbling score among the three genotypes was not significant. It could be used as genetic marker during the process of cattle’s meat quality traits in breeding.

The work was supported by the National Scientific and Technological Plan in Rural Areas of "Twelfth Five-year"(2015BAD03B04-4), the Agriculture Science and technology innovation of Gansu (GNCX-2014-32) and the Scientific Research Project of Colleges and Universities in Gansu Province (2013A-063).