Abstract
The objective of this study was the optimization and implementation of a reliable and economical molecular screening method for the detection of the mutant allele of CVM (complex vertebral malformation, c.559G>T, SLC35A3 ) by HRM analysis, as well as analyzing its existence in a representative sample of Holstein cows from the Milk Genomic DNA Bank of Uruguay. The optimization of the HRM methodology in the RotorGene ™ 6000 equipment ( Corbett Life Science , Australia) by amplification of the 79 bp PCR products clearly differentiated the two genotypes: homozygous, wild type: GG; and heterozygous, carrier for the mutation CVM: GT (c.559G>T; SLC35A3 ). In the analyzed sample, the frequency of the mutant allele (T) for CVM was high ( q = 0.032), with a prevalence of carrier cows of 6.45%. It is concluded that the PCR-HRM analysis is a fast, easily interpretable, low cost, and highly accurate technique for the detection of this mutation in Holstein cattle, which may be implemented in genetic selection programs.
Highlights
Complex vertebral malformation (CVM, OMIA 001340-9913) is a recessive autosomal disease that causes miscarriages and perinatal problems
Among the bulls used for artificial insemination, CVM carrier animals were identified with a very high prevalence (10-30 %) in several countries (Kearney et al, 2005; Citek et al, 2006; Thomsen et al, 2006)
The disease and several CVM carriers have been identified in other countries (Revell, 2001; Duncan et al, 2001; Nagahata et al, 2002; Konersmann et al, 2003; Berglund et al, 2004; Rusc and Kaminski, 2007; Chu et al, 2008)
Summary
Complex vertebral malformation (CVM, OMIA 001340-9913) is a recessive autosomal disease that causes miscarriages and perinatal problems. The single site mutation causing this disease is a substitution of Guanine (G) by Thymine (T) in the position 559 of exon 4 of gene SCL35A3 (c.559G>T; Thomsen et al, 2006), which plays an essential role in the development of the axial skeleton. This gene codes the UDP-Nacetylglucosamine transporter, and the mutation replaces a valine with a phenylalanine (V180F) at position 180 (Rusc and Kaminski, 2007). The disease and several CVM carriers have been identified in other countries (Revell, 2001; Duncan et al, 2001; Nagahata et al, 2002; Konersmann et al, 2003; Berglund et al, 2004; Rusc and Kaminski, 2007; Chu et al, 2008)
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