Identification of KLF3 gene polymorphism in Indonesian Friesian Holstein cattle

The KLF3 gene was a potential genetic marker candidate due to its involvement in many biological processes such as cell proliferation, cell differentiation, and apoptosis. This study was conducted to identify the KLF3 gene polymorphism in the Indonesian FH cattle. A total of 302 individual cattle blood samples from West Java (n=138), Central Java (n=34), and East Java (n=130) provincies were used to obtain DNA samples. The DNA sequencing was performed using 9 samples (each location of sampling represented by 3 samples) for initial identification of the KLF3 gene polymorphism and followed by PCR-RFLP analysis using all DNA samples. There were two SNPs identified in the intron region of the KLF3 gene i.e. SNP g.59607486delC and SNP g.59607554A>G. The SNP g.59607554A>G could be detected using PCR-RFLP method with HpyCH4IV restriction enzyme and resulted three genotypes of the KLF3 gene (AA, AG, and GG) but its heterozygosity value was low in the Indonesian FH cattle. The AA genotype has the highest frequency (0.73), while the AG and GG genotypes frequency were 0.24 and 0.03, respectively. Information about the KLF3 gene polymorphism in this study can be considered for further study to analyse its association with milk and protein yield traits.


INTRODUCTION
The Friesian Holstein (FH) cattle breed is the most famous cattle breed in dairy industry.There were 0.5 million heads of FH cattle that spread over in Indonesia and it was concentrated in Java Island especially in West Java, Central Java, and East Java Provinces (Ministry of Agriculture of the Republic of Indonesia, 2017).Milk and protein yield were substantial traits in dairy cattle industry.Based on several studies, the milk and protein yield of the FH cattle in Indonesia still low-medium levels (Lestari et al., 2015;Martono et al., 2016;Widyobroto et al., 2018) and lead the high demmand of the imported milk.The FH cattle that have good quality in milk production trait can be generated through the breeding program that assisted by molecular technology due to the molecular information of specific genes which highly associated with economic traits can be identified and used as genetic markers in cattle breeding programs (Ribeca et al., 2014).
KLF3 gene is a member of the Kruppel-Like Factors (KLFs) family of zinc finger proteins which are involved in many biological processes such as cell proliferation, differentiation, apoptosis, and neoplastic transformation (Eaton et al., 2008).In addition, KLF3 gene was important component of transcriptional control of adipogenesis (Sue et al., 2008;Bell-Anderson et al., 2013) and regulated muscle specific gene expression and synergizes with serum response factor on KLF binding sites (Himeda et al., 2010).In dairy cattle breeding, KLF3 gene could be used for marker assisted selection program as a genetic marker for milk and protein yields (Yin et al., 2010).
The KLF3 gene polymorphism information in Indonesian dairy cattle was unknown.In other hand, information about polymorphism in certain genetic marker candidates was required to design a strategy of cattle breeding program.This study was conducted to identify the KLF3 gene polymorphism in the Indonesian FH cattle using polymerase chain reaction-sequencing-restriction fragment length polymorphism analysis.

Blood Samples and DNA Extraction
A total of 302 heads of FH cattle consist of 138 cattle from West Java, 34 cattle from Central Java, and 130 cattle from East Java were included in this study to obtain blood samples.Blood samples (3-5 mL) were taken from coccygeal vein using Venoject and collected in Vaccutainer tubes containing an anticoagulant.DNA was extracted from blood samples using the Genomic DNA Mini kit (Geneaid Biotech Ltd., Taiwan) following the manufacturer's protocol.

Amplification and Sequencing
The PCR reaction was performed in a Mastercycler® gradient (Eppendorf, Hamburg, Germany) with a pair of primer to amplify the KLF3 gene that recommended by Yin et al. (2010) i.e. forward primer 5'-gccagtgttgttaatgctt-3' and reverse primer 5'-tggtaactttcaagatgcct-3'.The expected size of the KLF3 gene in this study was 329 bp.The PCR reagents composed of: 6.25 µL KAPA2G Fast Ready Mix PCR Kit 1X (Kapa Biosystems, Cape Town, South Africa), 0.5 µL forward and reverse primers (0.2 μM final concentration each), 1 µL DNA samples (5-50 ng>μL), and ddH 2 O up to 12.5 μL final volume.The optimum condition for PCR programme was as follows: initial denaturation at 94°C for 5 minutes; followed by 35 cycles of denaturation at 94°C for 30 seconds, annealing 55°C for 45 seconds, extension at 72°C for 45 seconds; and terminated by a final extension at 72°C for 5 minutes.The PCR products were visualized by electrophoresis process (1% agarose gel, SyBr® staining, and captured in GBOX documentation System (Syngene, UK)).
The DNA sequencing was performed using 9 PCR products (3 samples represented each location of sampling) for initial identification of the KLF3 gene polymorphism.The PCR products from individual cattle were sequenced using the ABI Prims 3100-Avant Genetic Analyzer in the 1st BASE Laboratory, Malaysia.The FH cattle KLF3 gene sequences were aligned and compared with the Bos taurus KLF3 gene sequence available in the GenBank (NCBI) database with accession number AC_000163 using MEGA ver 6.0 programs (Tamura et al. 2013).The position of the sequence in this study was relative to Bos taurus KLF3 gene sequence (AC_000163).

RFLP and Data Analysis
Identification of the KLF3 gene polymorphism in the Indonesian FH cattle was performed by RFLP method using HpyCH4IV restriction enzyme (Biolabs Inc., New England) with restriction site of 5'...A||CGT... where: χ ii = frequency of A i A i genotype (homozygote); χ ij = frequency of A i A j genotype (heterozygote); χ i = frequency of i th allele; n ii = number of individuals with A i A i genotype; n ij = number of individuals with A i A j genotype; H o = observed heterozygosity; H e = expected heterozygosity; N = number of samples.The polymorphism information content (PIC) was calculated based on Botstein et al. (1980): where: p i = allele frequency of A i ; p j =allele frequency of A j .

RESULTS AND DISSCUSION
The KLF3 gene in the Indonesian FH cattle was successfully amplified using a pair of primer designed by Yin et al. (2010).The results indicated that amplification fragment had good specificity (Figure 1), which could proceed directly to sequencing process.The PCR products of the KLF3 gene in this study indicated the same size as reported by Yin et al. (2010) i.e. 329 base pair (bp) approximately.
The DNA sequence alignment analysis revealed that there are two SNPs found in the Indonesian FH cattle KLF3 gene (Figure 2), i.e.SNP g.59607486delC and SNP g.59607554A>G.
Based on the KLF3 gene sequence available in the GenBank (AC_000163), two SNPs that occurred in the Indonesian FH cattle were located in the intron region.In consequence, the KLF3 gene polymorphism in this study was intron region polymorphism.The SNP g.59607554A>G in the Indonesian FH cattle population can be detected by using HpyCH4IV restriction enzyme in the PCR-RFLP analysis.In previous study, Yin et al. (2010) have identified one single nucleotide polymorphism (SNP) in the fourth exon of the KLF3 gene i.e.SNP g.59607644G>C that was associated to milk production traits in Chinese Holstein, but this was not found in the Indonesian FH cattle.
The result of PCR-RFLP analysis revealed that the KLF3 gene in the Indonesian FH cattle were polymorphic with three genotypes (AA, AG, and GG).Each genotype was identified based on the differences in size and the number of bands (RFLP product) that appear in the visualization process (Figure 3).On the A allele, the base A at the position of 59607554 were not changed so that the HpyCH4IV enzyme could not recognize the restriction site and could not cuts the DNA fragments.While on the G allele, the HpyCH4IV enzyme recognize the restriction site at the position of 59607554 due to base substitution (A to G) and cuts the DNA fragments into two fragments (60 and 290 bp).
The allele and genotype frequencies were calculated (Table 1) and found that the highest KLF3 Gene Polymorphism in Indonesian Dairy Cattle (P.P. Agung and F. Laoditta) 197 genotype frequency in the Indonesian FH cattle was AA (0.73) while the lowest was GG (0.03).In general, the H o value of the KLF3 gene was lower than the H e value (0.24<0.25), indicating that the genetic diversity in the Indonesian FH cattle KLF3 gene is quite low and the population was not in Hardy-Weinberg equilibrium.Based on PIC value, diversity level of the KLF3 gene in this study was also in low level (PIC value=0.22).The low level of diversity of the KLF3 gene in this study can be caused by several factors, including selection and assortative mating (Cervini et al., 2006), limitation in the number of sires that used in the farms (Agung et al., 2017), and also inbreeding factor (Loeschche et al., 1994).In addition, the low frequency of GG genotypes and also G allele in this study can be an indication there has been a selection process in the Indonesian FH cattle population.However, the H o value was higher than H e value in West Java subpopulation.This condition can be an indication that random mating still happened in the FH cattle population in West Java.
The utilization of genetic markers in order to achieve breeding objectives was highly dependent on its polymorphism and its association with dairy cattle productivity parameters (e.g.milk yield, protein, fat, total solid, total solid non-fat, and  lactose percentage).Therefore, validating the genetic markers of milk production traits is the initial step to establish the marker assisted selection (MAS) programme in the Indonesian FH cattle.In order to ensure the KLF3 gene utilization in the future, the genotypes in this study can be used for conducting the association study in larger cattle populations.

CONCLUSION
It can be concluded that the KLF3 gene was polymorphic with two SNPs were found in the intron region (SNP g.59607486delC and SNP g.59607554A>G).The genotypes of KLF3 gene based on SNP g.59607554A>G can be detected using RFLP method with HpyCH4IV restriction enzyme and resulted three genotypes (AA, AG, and GG) but its heterozygosity value was low in the Indonesian FH cattle.Information about the KLF3 gene polymorphism in this study can be considered for further study to analyse its association with milk and protein yield traits.
3' (temperature and incubation time following the producer's guidelines).The PCR and RFLP products were visualized were visualized by electrophoresis process.Individual cattle genotype in this study was determined based on the differences in the number and size of the visualized bands.The genotype frequency (equation a and b), allele frequency (equation c), observed heterozygosity (equation d), and expected heterozygosity (equation e) were calculated based on Nei and Kumar (2000): a. χ ii = (n ii /N); b. χ ij = (n ij /N); c. χ i = (2n ii + Σn ij )/(2N); d.H o = n ij /N;

Figure 2 .
Figure 2. The Nucleotide Differences Based on the 2 SNPs between the Indonesian FH Cattle KLF3 Gene and Bos taurus (Hereford) KLF3 Gene Sequence in the GenBank with Accession Number AC_000163; [.] Symbol in the Indonesian FH Cattle Sequence Represent the Same Nucleotide with GenBank sequence; [*] Symbol Represent the Insertion or Deletion

Table 1 .
The allele and Genotype Frequencies of the KLF3 Gene in the Indonesian FH Cattle = West Java; CJ = Central Java; EJ = East Java; n = individuals genotyped; H e = expected heterozygosity; H o = observed heterozygosity; PIC = polymorphism information content; n = number of samples WJ