Genetic Characterization of Thyroglobulin and Leptin Genes in Pasundan Cattle at West Java

The Thyroglobulin (TG) and Leptin (LEP) genes are two candidate genes that widely used for molecular selection to improve carcass traits in beef cattle. This research was carried out to identify the genetic characterization of TG and LEP genes from 47 heads of Pasundan cows at West Java using PCR-RFLP method. Research shows that TG gene of Pasundan cattle is monomorphic with C allele as the dominant allele (1.00). However, LEP gene of Pasundan cattle is polymorphic with C allele as the dominant allele (0.98) and T as the rare allele (0.02). The polymorphic informative content (PIC) and number of effective allele (ne) values in the LEP gene in the animal studied were 0.04 and 1.04 respectively. It was concluded that TG/BstYI and LEP/Sau3AI gene in the present study can not be used as molecular selection in Pasundan cattle. These results are important as the basic information for preparing the molecular selection program in the future.


Introduction
Pasundan cattle is one of Indonesian native cattle from West Java province and was kept by smallholds as the beef cattle.This cattle was declared as Indonesian native cattle trough decision of Ministry of Agriculture of Republic Indonesia No: 1051/Kpts/SR.120/10/2014(Anonymous, 2014).Recently, the population of Pasundan cattle is low because of reducing pasture area in West Java (Arifin et al., 2015).In addition, low calving rate in the Pasundan herds was affected to the low number of Pasundan population (Said et al., 2017).The genetic improvement program for Pasundan cattle is important to increase the productivity.Despite, Pasundan cattle with desirable traits can be increased the sell price in the market.This condition can affect for livestock demand and increasing to the number of Pasundan population.Genetic improvement in the livestock can be obtained through molecular selection in the some candidate genes that affecting productivity such as Thyroglobulin (TG) and Leptin (LEP) genes (Carvalho et al., 2012).
The study of molecular genetic in Pasundan cattle are limited, particulary no study has been made to assess the genetic diversity of TG and LEP genes in this breed.The objectives of the present study were to identify the typical allele and genotype of TG and LEP genes through PCR-RFLP method.The results of this study can be used as basic information for arranging the molecular selection program of Pasundan cattle in the future.

Materials and Methods
A total of 47 heads of Pasundan cattle from the breeding station (BPPIBT-SP Ciamis, West Java) were used for blood sampling purpose.Blood samples (3-5 mL) were taken from cocygeal vein using venoject and collected in vaccutainer tubes containing anticoagulant (EDTA).The bloods sample were used for DNA extraction process using the Genomic DNA Mini Kit (Geneaid Biotech Ltd., Taiwan) following the manufacturer's protocol.Amplification of TG and LEP genes were performed in a Mastercycler ® gradient (Eppendorf, Germany) with two different of PCR reagent composition volume.Total of 10 μL of PCR product per sample was used for TG gene amplification containing 1 μL of DNA template, 4 μL of PCR mix KAPA2G Fast ReadyMix (Kapa Biosystems Inc., USA), 4 μL of DDH2O and 0.5 μL of each primer.Thus, total of 10 μL of PCR product per sample was used for LEP gene amplification containing of 4 μL of DNA template, 5 μL of PCR mix MyTag TM Hot Start Red Mix (Bioline, USA), 0.6 μL DDH2O and 0.2 μL of each primer.The PCR programs for TG and LEP genes amplification were showed in Table 1.
The RFLP reagent of TG gene was performed in 10 μL per sample containing 5 μL of PCR product, 3.7 μL of DDH2O, 1 μL of 10 x buffer and 0.3 μL of BstYI restriction enzyme.Thus, the RFLP reagent of LEP gene was performed in 7 μL per sample containing 5 μL of PCR product, 1 μL of DDH2O, 0.7 μL of 10 x buffer and 0.3 μL of Sau3AI restriction enzyme.The RFLP reagents of TG and LEP genes were digested at 60°C and 37°C respectively in the water bath along 1 h.Therefore, the PCR and RFLP products were visualized using 1% and 2% of agarose gels respectively and captured in GBOX Documentation System (Syngene, UK) with GelRed TM staining (Biotium, USA).The genotip of TG/BstYI gene purposed be consisted of CC (295 bp, 178 bp and 72 bp), TT (473 bp and  72 bp) and CT (473 bp, 295 bp, 178 bp and 72  bp).The genotype of LEP/Sau3AI gene purposed be consisted of CC (389 bp and 32 bp), TT (304 bp, 85 bp and 32 bp) dan CT (389 bp, 304 bp, 85 bp and 32 bp).
The statistical analysis for the TG and LEP genes were consisted of allele frequency, expected heterozygosity (He), observed heterozygote (Ho), number of effective allele (ne), polymorphic informative content (PIC) and Chisquare (χ 2 ) based on Nei and Kumar (2000).

Result and Discussion
The TG and LEP genes of Pasundan cattle were successfully to amplify along 545 bp and 421 bp respectively (Figure 1).The results indicated that amplification fragment had good specificity, which could proceed directly to RFLP analysis.The RFLP analysis of TG/BstYI gene in Pasundan cattle was monomorphic with CC genotype shows in all animal studied (Figure 2).Previous study reported that TG/BstYI gene in Bali (Bos javanicus) and Nellore/Ongole (Bos indicus) cattle were monomorphic with C allele as the common allele (Table 3).It can be concluded that absence of T allele in Pasundan cattle reveals that this cattle had genetic material Bos indicus and Bos javanicus breeds.
According to Table 4, the C allele was more frequent in Bos taurus and Bos indicus cattle.Shin and Chung (2007) reported that homozygote TT animals in the TG/BstYI gene of Hanwoo population had lowest of marbling score value (P<0.05)than other genotypes animals.In    addition, the C allele had trend to increase marbling score trait.Anton et al. (2012) reported that homozygote CC animals in the TG/BstYI gene had the highest of milk yield than other genotype animals in Jersey and Hungarian Simmental populations (P<0.05).Bonilla et al. (2010) reported that homozygote CC animals in the TG/BstYI gene of Mexican beef population had lowest of intramuscular fat content (IMF) than heterozygote animals (P<0.05).
The LEP/Sau3AI gene of Pasundan cattle in the present study was polymorphic with two genotype of CC and CT (Figure 4).According to Figure 2, two DNA fragments of 85 bp and 32 bp were not showed clearly in 2% agarose gel.The polymorphism of LEP/Sau3AI gene in the present study had low PIC value (0.04) and can not be used as molecular selection (Table 2).
The PIC value is one of parameter for measuring the level polymorphism in genetic marker.According to Nei and Kumar (2000), PIC value was consisted of three category of low (PIC<0.25),moderate (0.25<PIC<0.50)and high (PIC>0.50).High PIC value indicated that the genetic marker had multiple allele and can be used for molecular selection (Chesnokov and Artemyeva, 2015).Therefore, the Ho and He values in the present study are similar and indicated that the crossing in Pasundan population was occured.Meanwhile, inbreeding in the population was detected when Ho < He and random mating in the population was detected when Ho > He (Chesnokov and Artemyeva, 2015).The LEP/Sau3AI gene in the population sample is under genetic equilibrium (χ 2 =0.02) and can be caused by random mating and no selection program (Falconer and Mackay, 1996).The ne value of LEP/Sau3AI in Pasundan cattle was 1.04 and explains that only one common allele that identify in LEP/Sau3AI gene.
Previous study reported that the frequency of C allele in Bos indicus breeds was 0.53 to 1.00 (Table 5).According to the Table 5, most of Bos indicus and Bos taurus cattle had T allele with lower frequency than C allele.Trakovicka et al. (2013) reported that CC genotype in the LEP/Sau3AI gene had highest of milk yield, protein yield and fat yield than other genotypes in mix population between Pinzgau and Slovak Spotted cows (P<0.05).In addition, heterozygote animals had lowest of age at first calving (P<0.05).
The TG and LEP genes can not be used as molecular selection for carcass traits of Pasundan cattle.The further research to identify SNP in other regions of TG and LEP genes are important in order to obtain marker assisted selection (MAS) for carcass traits of Pasundan cattle in the future.

Conclusions
The study showed TG/BstYI and LEP/Sau3AI genes can not be used for molecular selection in Pasundan cattle.The 5'UTR of TG gene in Pasundan cattle was monomorphic with C allele as the common allele.The genetic diversity in the intron 2 of LEP/Sau3AI gene in Pasundan cattle was low (PIC<0.25)with C allele as the dominant allele.bloods sample collection support and BP2D of West Java for the financial support.

Figure 1 .
Figure 1.The amplification of TG and LEP genes in Pasundan cattle were separated in 1% agarose gel along 545 bp (left) and 421 bp (right) respectively.M: DNA ladder 100 bp; line 1-12: number of sample.

Figure 2 .
Figure 2. The RFLP analysis for TG and LEP genes of Pasundan cattle in 2% agarose gel.The TG/BstYI gene is monomorphic with one genotype of CC (295 bp; 178 bp dan 72 bp) in all animal studied.The LEP/Sau3AI gene is polymorphic with two genotype of CC (389 bp) and CT (389 bp and 304 bp) in the animal studied.M: DNA ladder 100 bp.

Table 1 .
Primer and PCR program in the TG and LEP genes

Table 2 .
Statistical analysis in the TG and LEP genes of Pasundan cattle at West Java * N: number of sample; Ho: observe heterozigosity; He: expected heterozigosity; ne: number of effective allele; PIC: polymorphic informative content; X 2 : Chi-square value; * genetic equilibrium (X 2 <5.99).

Table 3 .
Allelic and genotypic frequency of TG/BstYI gene in several breeds cattle

Table 4 .
Allelic and genotypic frequency of LEP/Sau3AI gene in several breeds cattle