Identification of SNPs in the MAP4K4 Gene Coding Sequence and Their Association with Mastitis Susceptibility/Resistance in Nili Ravi Buffaloes

Nili-Ravi water buffalo (Bubalus bubalis) has long been an important part of Asian livestock production and agriculture, contributing to agro-socio-economic growth through milk, meat, hide and draught power. The Nili Ravi buffalo breed is found in Punjab and is extensively spread along the Ravi River, with a concentration in Amritsar, Tarn Taran and Ferozepur districts. Nili Ravi accounts for 6.94 per cent of Punjab’s total buffalo population (NDDB, 2014).

Mastitis is one of the multiple horrible diseases that strike milking cows and impacts both the amount and quality of their production. It is an inflammation of the mammary glands, which may or may not be contagious (Firyal et al., 2017). It’s a bacterial infection that causes physical and chemical alterations in milk, as well as pathological changes in the glandular tissue (DAHD, 2019). The severity of mastitis is reflected in the milk protein profile and biochemical indicators linked with oxidative stress in blood and milk. However, the greatest loss is linked to decreased milk supply in affected cows (Akers and Nickerson, 2011). Mastitis decreases the quantity as well as the quality of milk and milk-based products. Lactose, fat, solids-without-fat and casein levels are all decreased during and after mastitis (Graves and Fetrow, 1993). The concentrations of whey protein, chloride, salt, pH, free fatty acid concentrations and milk somatic cell count (SCC) all increase. Casein levels are dropping, whereas albumin and immunoglobulin levels are increasing (Zhang et al., 2023).

Mitogen-Activated Protein Kinase 4 (MAP4K4) is an important gene that regulates inflammation and its role in mastitis resistance. The MAP4K4 gene is in control of key steps in the innate response’s start (Dror et al., 2007; Bhattarai et al., 2017). The finding of SNPs in bovine MAP4K4 could provide useful information (Eckersall et al., 2006; Schulman et al., 2009) and we hypothesized that the MAP4K4 gene could have an important role in immunity and could be associated with mastitis resistance/ susceptibility in Nili Ravi buffaloes. An association study was carried out to investigate the connection between the MAP4K4 polymorphism with mastitis to test this hypothesis.

Animals and sampling
 
Aseptic sample collection was performed at Guru Angad Dev Veterinary and Animal Sciences University (GADVASU), specifically at the Directorate of Livestock Farms (DLF). A total of 20 mastitis-positive and 20 healthy (normal) Nili Ravi animals contributed to the sample pool. Using aseptic techniques, approximately 10 ml of peripheral blood was meticulously drawn from the jugular veins of the buffaloes. This blood was promptly transported to the laboratory within a thermally insulated container equipped with a gel-ice pack. The blood was transferred to sterile 15 ml tubes containing 0.5 ml of EDTA (0.5 M) as an anticoagulant. During the period preceding RNA isolation, the blood samples were stored at a temperature of 4°C.
 
Molecular analysis
 
RNA extraction and quality checking
 
RNA isolation was accomplished from collected blood samples of Nili Ravi buffalo utilizing the kit extraction methodology. As a means of quality assurance, the RNA samples underwent dilution and subsequent analysis via agarose gel electrophoresis using a 1 per cent Agarose solution in 1X TAE buffer at a pH of 8.0. To initiate the electrophoresis, 5 µl of RNA, combined with 1 µl of 6X gel loading dye, was meticulously loaded into the gel wells utilizing a micropipette. Electrophoresis itself was conducted at a voltage of 97V, spanning a duration of 40 to 45 minutes. Subsequent to the electrophoresis process, the gel was subjected to visualization through employment of a gel documentation system, thereby facilitating an assessment of the RNA’s quality.
 
Quantification of RNA using nanodrop spectrophotometer
 
After being stored at -20°C, the RNA samples were carefully thawed at room temperature. The upper and lower optical surfaces of the microvolume spectrophotometer sample retention system were cleaned by pipetting 1 µl of clean nucleus-free water onto the lower optical surface before attempting to quantify recovered RNA. The lever arm was closed to guarantee that the upper pedestal was in touch with the nuclease-free water and then the arm was elevated to clean, dry and lint-free both optical surfaces. After completing the blank measurement, the lever arm was lowered and both optical surfaces were wiped clean using a dry, lint-free lab wipe. The necessary amount of sample was put onto the lower optical surface and the lever was lowered to detect the RNA concentration and purity measurement. Using the 260/280 nm ratio, total RNA concentration and purity were measured spectrophotometrically.
 
Synthesis of complementary DNA (cDNA) by reverse transcription
 
The RNA template standard concentration was 1000ng/µl in 1 µl volume and the cDNA was synthesized using a kit procedure. The numerous components utilized in cDNA synthesis as priming at 25°C for 5 mintes, reverse transcription at 46°C for 20 minutes, RT inactivation 95°C at 1 minute. For complementary DNA synthesis, we employed the RNA template indicated above, as well as a mixture of 4 µl iScript Reaction Mix (5X), 1 µl Script Reverse Transcriptase (1X) and Nuclease-free water adjusted to the RNA template volume. The measured cDNA was diluted based on its concentration for the subsequent qPCR reaction.
 
PCR amplification and SNP identification
 
PCR was carried out in a final reaction volume of 25µl. The GoTaq® Master-Mix reaction was utilized for amplification. In 0.2 mL thin-walled PCR tubes, this master mix was added. The mixture was put into PCR tubes that were firmly capped and spun for a few seconds. The tubes were placed in a thermal cycler and PCR was performed using the primer 1 and primer 2 conditions stated in Table 1 and 2 respectively. PCR products from all primers were purified from the gel using a gel purification kit and 20 representative samples from each group were sent for DNA sequencing (Livak and Schmittgen, 2001). The purified PCR products were sequenced on both strands (forward and reverse) at AgriGenome Labs Pvt Ltd, India. The sequences were analyzed using multiple sequence alignment (MSA) and other bioinformatic tools such as Finch-TV program, Clustal Omega.



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The RNA was extracted using the kit extraction method from blood samples obtained aseptically from Nili Ravi buffaloes. The RNA samples were utilized to synthesize cDNA, while the rest of the samples were stored at -80°C. A 1 percent Agarose gel was electrophoresed at 80 V for 1 hour to determine the quality of extracted genomic cDNA (Fig 1) followed by visual inspection on a Gel documentation system. For additional PCR procedures, only samples with sharp and high molecular weight bands were chosen. The concentration of RNA was determined using a NanoDrop spectrophotometer. The ratio of OD values obtained at 260 nm and 280 nm was between 1.9 and 2.0, indicating that the RNA was of acceptable quality and devoid of contamination. The measured cDNA was diluted based on its concentration for the subsequent PCR reaction.



Amplification of coding region of the MAP4K4 gene for SNP detection
 
Thermocycler was used to amplify a cDNA fragment of 400 bp and 409 bp of the MAP4K4 gene. The primer 1 of 400 bp was used for the first amplification, with (5' GTGTGCTGTGTCAATGCGG -3') as a forward template and (5' - CTGCTCCTGGCCTTTACA -3') as a reverse template. In the second amplification, primer 2 of 409bp was used, with (5'-AGCACAGCATAGCACATACAAAC-3') as the forward primer and (5' CCACAGCCTCCTAGAAAC-3') as the reverse primer, as shown in Table 2 and 3. To check for contamination, the negative control (NTC) was used. For all samples, the 100 bp marker ladder was used to confirm single, intense, compact bands of 400 bp and 409 bp on a 2 per cent agarose gel (90V for 50 minutes).

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Sanger’s dideoxy chain termination approach was used to genotype a PCR amplicon of the MAP4K4 gene sent to Agrigenome for sequencing (Sanger et al., 1977). BLAST analysis validated the sequencing results and some other buffalo MAP4K4 gene sequences were acquired from the GenBank database for comparison investigations based on the BLAST bit score. Multiple alignments of the sequences were performed using the Clustal Omega. SNPs in the MAP4K4 gene have been discovered in various places. The derived sequences were divided into three groups based on their base pair size i.e; 400 bp, 300 bp and 200 bp. The alignment of group 1 and the other two groups are aligned in the same way (Fig 2).


 
SNP identification in the coding region of the MAP4K4 gene in nili ravi buffalo
 
The FASTA sequences were matched to the reference sequences at NCBI using BLAST and the sequencing results of chromatogram files were then inspected by the Finch-TV program for SNP detection, BLAST (Basic local alignment search tool).

Fig 3, 4 and 5 shows the presence of SNPs at 173, 400 and 459 positions, likewise same the other four SNPs were discovered. In short, a total of seven SNPs at 173, 226, 252, 329, 400, 410 and 459 were identified in both mastitis and non-mastitis samples using multiple sequence alignment (MSA) and Clustal omega. Associations between these SNPs with mastitis and non-mastitis animals are shown in Table 3 and 4. The chi-square test was used to check the association of identified SNPs. NA values were obtained as a chi-square test of SNP 2 for amplicon 1 (7), SNP 1 for amplicon 2 (6) and SNP 3 for amplicon 3 (18). Hence result was found to be indefinite as values were more than 0.5. On the other hand, SNP 1 for amplicon 1 (7), SNP 1 for amplicon 3 (18) and SNP 2 for amplicon 3 (18) were found to be 0.03, 0.023 and 0.004 in Table 5 respectively which means the result was statistically significant.





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In this study, we have highlighted seven SNPs in MAP4K4 that could be regarded as novel candidate genetic molecular markers for mastitis resistance/susceptibility in Nili Ravi buffaloes. This should be based on only Bo-MAP4K4 alleles. Conclusively, the SNPs identified through this investigation hold promise for forthcoming research pertaining to mastitis occurrence in Nili Ravi buffaloes. These SNP associations warrant subsequent validation efforts. Naturally, the findings of this study should be corroborated through independent sample analyses, thereby enhancing the potential for more refined genomic selection strategies within buffalo breeds.
 
Likewise, In dairy sheep, SNP markers that exhibited noteworthy correlations with mastitis attributes were validated for mastitis resistance on chromosomes 2, 3, 5, 16, and 19 (Banos et al., 2017; Guntur et al., 2010). A total of seven SNPs identified on mastitis incidence in Murrah buffalo found significantly associated (p<0.001) NCBP1, FOXN3, TPK1, XYLT2, CPXM2, HERC1 and OPCML. The majority of them were having tumor suppressing action, related to immunogenetics or glycolytic and energy production. Thus, results confirmed the presence of animal genetic variability in mastitis and identified genomic regions associated with SNPs in the Nili Ravi buffaloes. The preserved genetic framework related to mastitis across different buffalo breeds implies the viability of implementing selection programs that span multiple breeds.

Our findings confirm that the MAP4K4 could be regarded as a novel candidate gene against mastitis and, the identified polymorphisms might potentially be strong genetic molecular markers for mastitis resistance/ susceptibility in Nili Ravi buffaloes. SNPs within genes of the local mammary innate immunity response are attracting particular importance as potential DNA markers for mastitis susceptibility. The possible association of such polymorphism with mastitis susceptibility and the occurrence of preferable haplotypes within the MAP4K4 are interesting areas for the future. Thus, the genetic markers associated with mastitis outcomes the genetic improvement through the implementation of marker-assisted selection (MAS).

The work has been supported by the Department of Animal Genetics and Breeding, College of Veterinary Science, College of Animal Biotechnology, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India (141004). The authors thankfully acknowledge the team for their support. All authors read and approved the manuscript.

Sehajpal Singh Dhillon: Lab work, preparation of the manuscript and data analysis, Dr. Simarjeet Kaur: Planning of work, Manuscript editing and proofreading of the manuscript, Shakti Kant Dash: Data analysis, Dr. CS Mukhopadhyay: Data analysis.

There is no conflict of interest among the authors.