Genotyping of crossbred cattle for SNP G22231T and SNP T25025A of BRCA1 gene and its association with somatic cell count

Introduction

Livestock sector in India is very important as it contributes to about 20.5 million of people for their livelihood. It provides an income source to about two-thirds of the rural community. According to the 20th Livestock Census, this sector employs 8.8% of the country's total population and accounts for 25.6% of India's agricultural GDP and 4.11% of the country's overall GDP. Indigenous cattle breeds are known for their resistance to tropical diseases compared to exotic and crossbred cattle, and comparatively lower milk production. Crossbred cattle have a significant role in increased milk production due to their high milk production efficiency. However, they are more susceptible to mastitis than the low producers. Mastitis is a global problem which causes huge economic losses by decreasing milk production. In India, the projected annual losses attributed to mastitis were approximately Rs.7165.51 crore per annum (Bansal et al., 2009) [1] and Rs. 16,702 million per annum (Deb et al., 2014) [4,5] . Mastitis is a multi-etiological disease, but genetic causes are very common. There are many genes responsible for mastitis, but BRCA1 is one of the important genes. The length of the Bovine BRCA1 gene is 68.69 kb and is located on the 19 th chromosome. It encodes 1849 amino acids, consisting of 22 exons and 22 introns. A mutation in BRCA1 itself and errors that occur in the repair of BRCA1 may cause damage to DNA, which eventually increases the risk of cancer. Somatic Cell Count below 1xl0 5 /ml is considered normal milk, and above 1x10 6 /ml is considered infected milk. With advances in molecular genetics techniques, various molecular markers have been identified that can help target candidate genes responsible for production-related diseases such as mastitis. Selection through the markers in breeding will eventually increase production. The genotypes linked with mastitis resistance can be incorporated in selection. Identifying SNPs linked to mastitis and using them for selection offers hope of producing mastitis-resistant animals with improved production in the near future. Considering the abovediscussed facts, the present study is focused on analysing the BRCA1 gene and its association with SCC in the milk of crossbred cattle.

Materials and Methods

The present study comprised 60 unrelated crossbred cows (HF x Gir and Jersey x Gir) reared at Bombay Gorakshak Mandali, Kandivali, Mumbai. All the cows were apparently healthy and between 3 rd to 5 th lactation, tested for CMT positive (Ganguly et al., 2008) [7] . Milk samples were collected for estimation of somatic cell count by DeLaval Cell counter and further converted into somatic cell score by using formula SCS = log2 [SCC/100] + 3 given by Rupp and Boichard (1999) [15] . The blood samples were also collected for isolation of genomic DNA using traditional Phenol: Chloroform: Isoamyl alcohol (P: C: I) method as described by Sambrook and Russell (2006) [16] . Purity and integrity of the isolated genomic DNA samples were checked on 0.8 percent agarose gel at 90 V for 60 minutes.

The DNA fragments were amplified with 25 µl of PCR reaction mixture; nuclease free water-10.5 μl, PCR Master Mix-12.5 μl, forward primer-0.5 μl (10 pmole), reverse primer-0.5 μl (10 pmole) and template DNA-1.0 μl (50~100 ng/μl) for SNP G22231T and SNP T25025A of BRCA1 gene, respectively using primer (Table .1) sequences by Deb et al. (2014) [4,5] . Cycling conditions for PCR amplification of SNP G22231T and SNP T25025A of BRCA1 were standardized as: initial denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 sec, annealing temperature at 55 °C (SNP 1)/60 °C (SNP 2) for 30 sec, Synthesis at 72 °C for 30 sec, and final Extension was done at 72 °C for 10 min. The restriction enzymes used in the present study were HhaI for intron-6 (SNP G22231T) and Mael/(BfaI) for exon-9 (SNP T25025A). The PCR products were digested with restriction enzymes in the final reaction volume of 15 μl (Table 2). The reaction mixture was centrifuged for a few seconds for uniform mixing and incubated at 37 °C for 3 hours in the water bath, and then electrophoresed on 2.5 percent agarose gel for both intron-6 and exon-9. According to the expected fragment sizes, electrophoresis was performed at a constant voltage of 90V for 60 minutes, and a 100 bp ladder was used; bands were visualised under UV light and documented by a gel documentation system. RFLP analysis of SNP G22231T and SNP T25025A of BRCA1 gene revealed three genotypes of each. The somatic cell score of each genotype was estimated, and association studies were established between genotypes and corresponding somatic cell scores. Further, breed-wise and combined genotypes of SNP G22231T and SNP T25025A association were established. Single-factor one-way ANOVA was applied for further statistical analysis of the data for the association study. [4,5] 2 Exon 9 F:5'-GCAGTGGAAATTCCAAATAAACT-3' R:5'-GAATTAGATCTTCAGCTATGTGGC-3' 209 bp Deb et al. (2014) [4,5]

Results and Discussion

This study has been planned in crossbred cows (HF × Gir and J × Gir) to study polymorphism of SNP G22231T and SNP T25025A of BRCA1 gene and its association with somatic cell count (SCC). PCR amplification of SNP G22231T and SNP T25025A of BRCA1 gene fragments of 321 bp (Fig. 1) and 209 bp (Fig. 2) were successfully obtained at an annealing temperature of 55 °C and 60 °C for 30 seconds, respectively which coincided with Deb et al.

(2014) [4,5] and Yuan et al. (2012) [21,22] used 54.5 °C for intron-6 while for exon-9 it was 60 °C. HhaI/PCR-RFLP of SNP G22231T analysis revealed two-band patterns as three bands of size 321 bp, 272 bp, 49 bp and two bands 272, 49 bp (Fig. 3). Based on RFLP band pattern observed, crossbred cows were genotyped as AB (321 bp, 272 bp, 49 bp) and AA (272 bp and 49 bp) as reported by Yuan et al.

(2012 b) [21,22] . The allelic frequencies of SNP G22231T for A and B were 0.769 and 0.230, and only two genotypes were observed, i.e., AB and AA; genotype BB was absent. [11] revealed three genotypes GG, GT and TT for SNP G43737229T with genotypic frequencies 0.255, 0.333 and 0.412 and three genotypes GG, AG and AA for SNP G43761121A with genotypic frequencies 0.392, 0.402 and 0.206. Sonwane et al. (2023) [19] reported two genotypes, BB and AB, for exon 3 of TLR4 gene in crossbred cattle, respectively, with genotypic frequencies of 0.775 and 0.225, while allelic frequencies for allele B and A were 0.887 and 0.112. Deb et al. in 2014 [4,5] found only two genotypes in Frieswal cattle for SNP G22231T, and genotypic frequencies were AA: 0.22 and AB: 0.78, and the gene frequencies were 0.61 and 0.39. PCR-RFLP pattern of 209 bp fragment of SNP T25025A revealed three genotypes (Fig. 4) as DD (209 bp), CD (209, 185, 24 bp) and CC (185, 24 bp). The genotypic frequencies for SNP T25025A recorded were CC, CD and DD, which were 0.04, 0.48 and 0.48. Gene frequency for HF crossbred cows was 0.288 and 0.711 for C and D. For Jersey crossbred, gene frequencies were 0.28 and 0.72 for C and D, and the genotype frequencies were DD, CD and CC, which were 0.485, 0.371 and 0.142. The association studies were established between the two genotypes of SNP G22231T (Intron-6) and somatic cell score of 60 crossbred cows. One-way ANOVA showed non-significant effect , 2012 [21,22] , who observed a non-significant association between genotypes of SNP G22231T, i.e. AB, BB and AA and SCS. They reported highest SCS for genotype AB and lowest for AA and further justified that the (G22231T) introns are not the coding sequence, but previous research on introns has enlightened the role of introns in mRNA splicing, gene expression, etc. Sonwane et al. (2023) [19] reported nonsignificant association between mean somatic cell score and genotypes BB and AB in exon 3 of TLR4 gene in crossbred cattle. For the confirmatory role of SNP G22231T locus of BRCA1 gene in Bovine mastitis, more work is suggested to be undertaken. For the SNP T25025A (Exon-9) non-significant (p = 0.30, α = 0.05) association was observed between the genotypes DD, CD and CC and somatic cell score. The mean value for SCS of CC genotype was found to be higher, i.e. 5.42 + 0.86 followed by DD and CD with SCS values 4.78 + 0.33 and 4.32 + 0.30, respectively. Similar results have been noted by Yuan et al. (2012) [21,22] , who found a non-significant association of SNP T25025A locus of BRCA1 gene and the somatic cell count for the CC genotype compared to the CD and DD genotypes. However, they observed a lower somatic cell count compared to the CC genotype (3.68), and it was found close to a significant level (p < 0.05), and the highest SCS was for genotype DD (4.47). They also studied the locus C28300A of BRCA1 gene and found a significant association with SCS, in which animals belonging to EE (3.22) genotypes had significantly lower somatic cell count than genotypes EF (4.94) and FF (5.53) in Holstein, Sanhe and Simmental cattle breeds. One-way ANOVA revealed no significant association between combined genotypes and corresponding somatic cell score. However, lowest SCS (4.00 + 0.30) and highest SCS (5.32) were observed in ABCD and AACC genotypes, respectively. Whereas, Yuan et al. (2012 b) [21,22] observed a significant association between combined genotypes (24) of three SNPs genotypes and somatic cell score in Chinese commercial cattle. Yuan et al. (2012a) [21,22] studied SNPs of BRCA1 gene and their relation with mastitis in Chinese commercial cattle (Total-404 animals, Holstein-107, Sanhe-201, Simmental-96) and reported three SNPs (c.26198 C>T, c.5682 G>C, c.46126 G>T) in exon-9, in intron-2 and exon-

Acknowledgement

AcknowledgementsThe authors are thankful to Associate Dean, Mumbai Veterinary College, for providing the necessary facilities required to complete this experiment. We express our gratitude to Bombay Gorakshak Mandli, Kandivali Farm, Mumbai, for their cooperation in providing blood and milk samples, along with relevant data of cows. We duly acknowledge the Department of Physiology, Mumbai Veterinary College, Mumbai for providing facility for the estimation of somatic cell count from the milk samples.

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