Polymorphisms of the Leptin gene in Jabres cattle

Many genes, including the leptin gene, control growth performance. Polymorphism or SNPs variant within the gene could change its expression in phenotypes. This study aimed to identify the SNP and haplotype variation of the leptin gene in Jabres cattle (n = 47 head). The SNPs variant was detected using the BioEdit version 7.0 program by sequence alignment. The HaploView program analyzed the haplotype pattern created from the SNP variants. As a result, 20 SNPs were found within the partial sequence of the leptin gene. Only 3 SNPs are located in the coding sequence (CDS) region, SNP g. 12215T>C, g.12237C>T, and g.12238G>A. For the haplotype analysis, we used only SNPs with HW p-value cutoff and minimum minor allele frequency (MAF) higher than 0.05 (Jabres = 12 SNPs). The result showed a distinctive haplotype pattern of SNPs. All the blocks of LD plot in Jabres cattle showed a high linked disequilibrium (LD) (R2 > 0.33, LOD > 2) except for the block containing SNP g.12238G>A (R2 < 0.33, LOD < 2). In conclusion, the polymorphism and haplotype pattern found in this study could be used for further association analysis to the phenotypes, and its utilization could be used as an effective marker selection tool.


Introduction
Leptin (LEP), a polypeptide having a molecular weight of 16 kDa, is crafted from a sequence of 167 amino-acid units and originates from adipocytes [1].This unique molecule governs not only the storage of body fat but also influences feeding habits, weight gain, linear growth, constitutional maturation, and functions related to the immune and reproductive systems [2,3].A significant role played by leptin involves regulating the amount of adipose tissue, its energy usage, and feed intake [4].Leptin's primary release source is adipose tissue, and its role in controlling feeding, maintaining energy equilibrium, and managing reproduction are carried out through its interaction with the Leptin receptor (LEPR) in the hypothalamus [2].The bovine LEP gene, located on chromosome 4, consists of three coding regions (exons) and two non-coding regions (introns) [5].
Single nucleotide polymorphisms (SNPs) are the most common genetic variation investigated in association research.However, it's essential to recognize that the intricate process of genetic inheritance is guided by connected sequences of closely linked nucleotides [6].This gap is where phased multimarker structures, known as haplotypes, come into play.Haplotypes offer the potential to enhance the current understanding of various complex aspects, such as domestication, population diversity, inbreeding effects, the merging of genetic lineages, shifts in demographics, identification of specific genetic regions linked to economically important traits, and the more accurate prediction of breeding values [7].A single SNP association analysis with phenotypic traits in breeding programs is probably quicker and less expensive.However, haplotype analysis aids in assessing how genes affect phenotypic features.Alleles at several loci are not randomly distributed in a condition known as linkage disequilibrium (LD) [8].This LD sheds light on the intricate relationships that define genetic interactions.
The Haploview software is designed to provide a comprehensive range of features, including the generation of quality data about genetic markers, insights into linkage disequilibrium (LD), the identification of segments of shared DNA sequence known as haplotype blocks, calculation of the frequencies of these blocks within populations, and the computation of statistical measurements relevant to the association of individual genetic markers [9].This collection of tools proves particularly beneficial for conducting in-depth analyses centered on haplotypes-patterns of genetic variation-within DNA sequences.These analyses serve as a means to investigate the complex interplay between specific DNA variations [10].
The polymorphisms of the LEP gene in cattle have been reported in Nellore cattle [11], Holstein cattle [12], Pasundan cattle [13], Ongole Grade cattle [14], Madura [15], and Bali cattle [16], except in Jabres cattle.The LD analysis of the LEP gene in Indonesian cattle also lacks reported.Hence, this study aims to identify the polymorphism (SNP) of the LEP gene and LD analysis (haplotype variation), specifically in Jabres cattle.

Ethics statement
The animal use and care in this research follow the guidelines from The Institutional Animal Care and Use Committee (IACUC) of the Indonesian Agency for Agricultural Research and Development.This research was approved with the permit number: Balitbangtan/Lolitsapi/Rm/16/2021.

Blood samples collection and analysis
Using a venoject and an EDTA vacutainer tube, three milliliters of blood samples were drawn from the jugular vein of Jabres (n = 47) cattle.The gSYNC DNA extraction kit (Geneaid, Taiwan) was then used to extract the DNA from the blood samples.A 25 µl volume containing 2 µl of genomic DNA, 0.5 µl of forward and reverse primers, 9.5 µl of ddH2O, and 12.5 µl of MyTaq HS Red Mix (Bioline, UK) were used to amplify the LEP gene.The amplification was performed in Thermocycler (Sensoquest Labcycler, Germany).The PCR procedure consisted of 94 C for 5 min pre-denaturation, followed by 35 cycles of 94 C for 45 sec (denaturation), 60 C for 45 sec (annealing), and 72 C for 1 min (extension) with a final extension at 72 C for 5 min.The first BASE Laboratory then received all PCR products for the sequencing process.

Data analysis
The software BioEdit was used to alter the raw sequencing data.Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used to construct sequence alignments to genotype the samples and find FSHR gene single nucleotide polymorphisms (SNPs).The polymorphic location discovered by sequence comparison was verified manually by looking at electropherograms.Using the HaploView program, the SNPs were examined for LD, and a haplotype block analysis was carried out.The haplotype block was also formed, and the r-square (r 2 ), D', LOD (log of odd), and haplotype IOP Publishing doi:10.1088/1755-1315/1341/1/0120083 frequency were all calculated.The software uses selected markers to visually display connections between these genetic variations by grouping and setting these markers.This grouping may be customized through manual selection and clarified using a variety of color schemes, providing a userfriendly and educational way to investigate these crucial genetic relationships.

Results
In this study, the nucleotide variants were confirmed by sequence alignment and direct examination of the chromatogram of each sample.Based on that procedure, 20 single nucleotide polymorphisms (SNP) were within the studied partial LEP gene (Figure 1).Only 3 SNPs are located in the coding sequence (CDS) region, SNP g. 12215T>C, g.12237C>T, and g.12238G>A.Meanwhile, the other 17 SNPs were located in intron 1 (g.11996G>A, g.12012C>T, g.12035C>T, g.12046G>A, g.12058G>C, g.12075T>C, g.12101C>T, and g.12103A>G) and intron 2 (g.12351G>A, g.12356G>A, g.12368C>T, g.12427C>T, g.12428A>G, g.12468C>T, g.12470A>C, g.12501T>C, and g.12576C>G).All SNPs are named by their position within the sequence of the Bos indicus LEP gene (GenBank accession number NC_032653.1:95655848…95672652).Only SNPs with a minimum minor allele frequency (MAF) higher than 0.05 and an HW p-value cutoff of 0.05 (within equilibrium) were considered for the haplotype analysis (Jabres = 12 SNPs).The outcome revealed a unique haplotype pattern of SNPs.Except for the block containing the SNP g.12238G>A (r 2 0.33, LOD 2), every block in the LD plot in Jabres cattle displayed high linked disequilibrium (LD) (r 2 > 0.33, LOD > 2).Visualization of haplotype built from the twelve SNPs is shown in Figure 2.

Discussion
In this study, there were twenty SNPs found in partial LEP gene sequences.Eighty-five percent of the SNPs are located in a non-coding region of the LEP gene (intron one and intron 2).SNPs occurring within exons have the potential induce amino acid substitutions, resulting in alterations to the primary structure of the protein.Such changes can influence protein folding, stability, and interactions, affecting biological activities.Conversely, introns are non-coding segments of DNA that do not contribute directly to protein synthesis.SNPs residing in intron regions are considered less likely to cause direct protein alterations.However, they can exert regulatory effects on gene expression through alternative splicing and enhancer modulation.Nakaya et al. [17] stated that the non-coding region RNAs (introns), which regulate transcriptional and post-transcriptional gene expression, may still impact the phenotype even when the SNPs are placed there.SNP g. 12215T>C, g.12237C>T, and g.12238G>A were located in exon 2 of LEP gene.Previously, the polymorphism within the exon two regions of the LEP gene also has been reported, especially the SNP 1180C>T.This SNP is polymorphic in Korean cattle [18], Holstein cattle [19], and crossbred cattle [20].In Indonesian cattle, the SNP 1180C>T was reported in Kebumen Ongole cattle [21].The SNP 1180C>T was found to be associated with milk content, meat quality, and body measurement.The amino acid Arginine is changed to Cysteine via the base mutation of Cytosine to Tymin at position 1180.It was previously believed that this amino acid shift is the causal mutation that alters the physiological function of the hormone leptin in energy metabolism [22].
The LD analysis based on 12 SNPs showed a unique pattern.Almost all blocks have a high LD (r 2 > 0.33, LOD > 2) except the block containing the SNP g.12238G>A.A haplotype block pertains to a cluster of closely associated genetic markers on a chromosome that displays notable linkage disequilibrium (LD) and is inherited collectively.This approach effectively reduces the quantity of SNPs necessitating assessment within association studies, as highlighted in the work by Qanbari [23].
The inherent linkage between r 2 and attributes of the population, such as adequate population size and the pattern of recombination landscape, signifies that population-specific factors influence the estimated LD value.This relationship between reasonable size estimations or recombination maps and anticipated r 2 values is intrinsically influenced by the frequency distribution of alleles, an observation underscored by Ober et al. [24].Consequently, this frequency-dependent nature of LD estimations highlights the intricate interplay between genetic parameters and the outcomes of population genetics investigations.

Conclusion
Twenty SNPs were revealed in a partial sequence of the LEP gene in Jabres cattle.12 SNPs were in Hardy-Weinberg equilibrium.The LD analysis showed that all blocks have high LD except blocks containing SNP g.12238G>A.Hence, this haplotype pattern could be recommended for further association analysis to the phenotypes, and its utilization could be used as an effective marker selection tool.

Figure 2 .
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