The Sumba Ongole (SO) cattle is included of Bos indicus breed that capable to adapt well in Sumba Island of Indonesia. This cattle was decided as one of Indonesian native cattle through decision of Agriculture Ministry of Indonesia No: 427/Kpts/SR.120/3/2014. This cattle was imported from India since 1900 by Dutch colonial government for the drought animals (Hardjosubroto, 1994). Recently, SO cattle was kept by most farmers as the beef cattle or meat production. Previous studies reported that the average of body weight in SO bulls (±2.5 years age) was 353.86-474.08 kg (Said et al. 2016a). In addition, the average of dress percentage in SO bulls were 51.42-56.34% (Agung et al. 2015). Some of genetic parameters of repeatability, heritability and genetic correlation values of growth trais in SO cattle were high and reveal that growth traits in this cattle can be increased through conventional selection (Said et al. 2016b; Putra et al. 2018). Despite, growth traits in SO cattle can be performed through molecular selection. Up to present, information about genetic markers in the SO cattle used for molecular selection is limited. Agung et al. (2017) reported that polymorphism in the growth hormone (GH) gene in SO cattle had not association with any growth traits. Therefore, information about genetic marker candidates need to be explorated. One of the candidate gene that widely use for the molecular selection is leptin (LEP) gene (Putra and Indriastuti, 2017). The LEP gene was located at fourth chromosome (BTA4q32) with length 16.735 bp and consisted of two introns and three exons (Pfister-Ganskow et al. 1996). Corva et al. (2009) reported that bovine LEP gene (GenBank: U50365) consisted of 34 bp of exon 1, 465 bp of exon 2 (from nuleotide: 877-1342) and 495 bp of exon 3 (from nucleotide: 2961-3456). The LEP is a protein that consisted of 167 amino acids with molecule weight of 16 kDa (Taniguchi et al. 2002). In cattle, LEP was synthesized by adipose tissue and involved in regulation of feed intake, energy balance, fertility and immune functions (Fruhbeck et al. 1998), milk performance (Liefers et al. 2002; Madeja et al. 2004) and reproductive traits (Almeida et al. 2003; Moussavi et al. 2006). Previous studies reported that one single nucleotide polymorphism (SNP) in the intron 2 of bovine LEP gene (GenBank: U50365) were occured at nucleotide position g.2270A/G (Pomp et al. 1997; Rasor et al. 2002; Oner et al. 2017). Morever, this SNP had association with productivity traits of cattle (Oprzadek et al. 2003). Despite, previous studies reported that SNP in the exon 3 of bovine LEP gene (GenBank: U50365) were occured at g.2961A/G; g.3100C/T; g.3260T/C; g.3257C/T; g.3272T/C and g.3356 C/T (Shin and Chung, 2007; Jhala et al. 2009; Orru et al. 2011; Kawaguchi et al. 2016). Putra et al. (2017) reported that 17 SNPs were confirmed in the 3'flanking region of LEP gene in SO cattle but the effect of these SNPs to productivity traits is not reported. There are no studies that reported the SNPs along intron 2 and exon 3 sequences of LEP gene in SO cattle. This research was carried out to detect the SNPs in intron 2 and exon 3 of LEP gene in population and selectied SO cattle through sequencing method. This research results is important as the early information to obtain the genetic marker for productivity traits in SO cattle.
MATERIALS AND METHODS
Animals and DNA extraction
A total of 31 DNA samples of SO female cattle from Sumba Island, West Nusa Tenggara Province of Indonesia were used for analysis in the present study. The animals were kept at the breeding station (PT. Karya Anugerah Rumpin) that located at Rumpin District, Bogor Regency, West Java Province of Indonesia. Amount of 3-5 mL of bloods samples were taken from coccygeal vein using venoject and collected in the vaccutainer tubes containing anticoagulant (EDTA). The DNA extraction was conducted from bloods sample with Genomic DNA Mini Kit (Geneaid Biotech Ltd., Taiwan) following the producers instruction.
In the breeding station, total of 11 SO cows were randomly selected to maintain in the performance test from 20/05/2013 to 24/05/2014 (369 days of test). Unfortunately, only three confirmed cows (ID: 1937, ID: 2097 and ID: 2099) that had LEP gene sequences information in the tested animals. Thus, all cows were ranked based on the final weight (FW) at end of the test. The performance in the three confirmed cows compare to unconfirmed cows were performed in the present study as the early information to obtain genetics marker in SO cattle.
Amplification of LEP gene and DNA sequencing
The amplification of LEP genes were performed in Mastercycler® gradient machine (Eppendorf, Germany) with two pairs of primers (Table 1). The primers were designed to amplify two regions of intron 2 and exon 3 based on GenBank: U50365.
Table 1 The primer pair, amplicon and PCR program for amplification of leptin (LEP) gene in Sumba Ongole cattle
1 Based on GenBank: U50365.
Figure 1 The amplicons of leptin 3 and leptin 4 genes in Sumba Ongole cattle
M: DNA ladder 100 bp and line 1-6: number of sample
Total of 24 μL of PCR reagents containing of 9 μL of KAPA2G Robust HotStart Ready Mix (Kapa Biosystems, South Africa); 1.8 μL of primer forward and reverse; 9.4 μL of ddH2O and 2 μL of DNA template. The polymerase chain reaction (PCR) program in the LEP gene of SO cattle were presented in Tabel 1. The visualization of amplification product were performed in 1% of Agarose gel (Vivantis, Malaysia) and stained with SyBr®. The DNA sequencing analysis was performed for all PCR products and managed by commercial laboratory service (1st BASE Laboratory, Malaysia) using the sequencing machine of ABI Prisms 3100-Avant Genetic Analyzer. Furthermore, DNA sequence of LEP 3 and LEP 4 genes were aligned and compared to the sequence reference (GenBank: U50365) using MEGA 6.0 (Tamura et al. 2013) and BioEdit (Hall, 1999) programs.
The statistical analysis in the LEP gene sequences were consisted of genotype frequency, allele frequency, observed heterozigosity (Ho), expected heterozigosity (He), number of effective allele (ne), polymorphic informative content (PIC) and Chi-square value (χ2) according to Nei and Kumar (2000). Therefore, the genetic diversity data (Ho; He,; ne; PIC and χ2) were calculated with CONVERT (Glaubitz, 2004), CERVUS (Kalinowski et al. 2007) and POPGENE (Yeh and Boyle, 1997) programs.
RESULTS AND DISCUSSION
Two PCR product of bovine LEP gene along 1002 bp (LEP 3 gene) and 1023 bp (LEP 4 gene) were successfully to amplified for the sequencing analysis (Figure 1). Total of 16 SNPs in the LEP gene were confirmed in the present study and consisted of 10 SNPs in LEP 3 gene and 6 SNPs in LEP 4 gene (Table 2). According to the Table 2, the PIC values were ranged from 0.06 (low) to 0.37 (moderate). Seven SNPs in the intron 2 (g.2325G/T; g.2423A/C; g.2448C/T; g.2456C/G; g.2466C/T; g.2778T/A; g.2857G/A) had moderate PIC value (0.25<PIC<0.50). Meanwhile, moderate PIC value in the exon 3 were occured in all SNPs (g.3260T/C; g.3272T/C; g.3356C/T; g.3468G/A). Previous studies reported that SNP g.2270A/G in the inron 2 was occured in many cattle breeds i.e. Angus × Nellore (Almeida et al. 2003), Friesian Holstein (Oprzadek et al. 2003). However, this SNP was not detected in the animals studied and showed AA genotype in all samples (monomorphic). The A allele in the SNP g.2270A/G is considered as the favourable allele for weight at first calving trait in crossbred cattle (Almeida et al. 2003). Despite, the SNP g.2270A/G was not associated with service per conception in Friesian Holstein heifers (Oner et al. 2017), but had significant association with some productive traits in Black-and-White cattle (Oprzadek et al. 2003). The monomorphism in SNP g.2270A/G. in SO cattle can be caused by selection, migration and close breeding system (Falconer and Mackay, 1996). Orru et al. (2011) reported that six SNPs were detected in the exon 3 of bovine LEP gene i.e. g.3100C/T; g.3157A/G; g.3257C/T; g.3260T/C; g.3272T/C and g.3356C/T. Corva et al. (2009) reported that six SNPs in the exon 3 of LEP gene had amino acid changed of alanine/valine (g.3100C/T); asparagine/Serine (g.3157A/G); glycine/glycine (g.3257C/T); valine/valine (g.3260T/C); alanine/alanine (g.3272T/C) and proline/proline (g.3356C/T). The SNP g.3100C/T and g.3157A/G were widely used as molecular because this SNP had amino acid changes (Matteis et al. 2012).
Figure 2 Novel SNP g.3468G/A in the exon 3 of bovine leptin gene in Sumba Ongole cattle was caused amino acid change from glycine (GGG) to arginine (AGG)
Table 2 Detection SNPs in the leptin (LEP) gene of Sumba Ongole cattle based on GenBank: U503651
SNP: single nucleotide polymorphism; N: number of observation; Ho: observed heterozigosity; He: expected heterozigosity; ne: number of effective allele; PIC: polymorphic informative content and χ2: Chi-square value.
* Under Hardy-Weinberg equilibrium (χ2<5.991).
Previous study reported that SNP g.3100C/T was not associated with live weight, carcass weight, dressing percentage, backfat thickness and marbling score in Hanwoo cattle (Shin and Chung, 2007) and reproductive traits in Czech Fleckvieh cattle (Jecminkova et al. 2018). Orru et al. (2011) reported that C allele in the SNP g.3257C/T can be reduced the total lipids contain in Simmental bulls. Moreover, C allele in the SNP g.3100C/T can be increasing the milk protein concentration and milk fat yield in Friesian Holstein cattle (Giblin et al. 2010). Research showed that SNP of g.3100C/T and g.3257C/T were not occured in the LEP gene of SO cattle. New mutation site in the exon 3 of LEP gene of SO was occured in SNP g.3468G/A (Figure 2). This SNP had moderate PIC value (0.25) and caused the amino acid change from glycine (GGG) to arginine (AGG). A SNP with moderate PIC value can be used for molecular selection (Bourdon, 2000). In this study, the effect of SNP g.3468G/A to the productive traits of SO cattle can not observed because of no performance recorded. According to the FW in the performance test, each confirmed cow were reached 2nd rank (cow ID: 1937), 8th rank (cow ID: 2097) and 10th rank (cow ID: 2099) as presented in Table 3. The phenotypic characteristic of second best SO cows (ID: 1937) was presented in Figure 3. According to the confirmed cows LEP gene sequences (Table 4), the second best cows (cow ID: 1937) had three different genotype compare to the other confirmed cows based on SNP g.2325G/T; SNP g.2362A/T and SNP g.2466C/T (intron 2). However, these SNP is important to investigate through depth study with large number of sample and performance records data.
Table 3 The rank of Sumba Ongole cows based on final weight at the end of performance test1
*Animal with the LEP gene sequences information (confirmed cow).
BW: body weight; WW: weaning weight at 205 days of age; YW: yearling weight at 365 days of age and FW: corrected final weight at the end of test (source: PT. Karya Anugerah Rumpin).
Figure 3 The second best of Sumba Ongole cows (confirmed) based on performance test results
Table 4 Profile of leptin (LEP) gene sequences in the three confirmed cows in the performance test
Research showed that the intron 2 and exon 3 regions of LEP gene in SO cattle are polymorphic. However, the association between SNPs with growth traits of SO cattle is important to analysis through depth research. The genetic diversity of LEP gene in the tested cows can be used as an early information to obtain genetics marker in the future. One SNP in intron 2 (g.2270A/G) and two SNPs in exon 3 (g.3100C/T; and g.3157A/G) of LEP gene were widely used as molecular selection in cattle but both SNPs were not detected in SO cattle. One novel SNP of g.3468G/A was occured in the exon 3 of LEP gene in SO cattle. This SNP was changed the amino acid from glycine (Gly) to arginine (Arg).
This research was funded by Research Center for Biotechnology, Indonesian Institute of Sciences (LIPI) through DIPA UNGGULAN LIPI 2016 scheme. We would like to thank all the breeding staff in the PT. KAR for technical assistance in the farm.