Re-sequencing of DNA from a diverse panel of cattle reveals a high level of polymorphism in both intron and exon

Abstract

In order to assess the extent of DNA sequence variation in cattle, introns and exons from both the leptin and Amyloid Precursor Protein (APP) genes have been sequenced in a panel of DNAs derived from 22 diverse animals. Direct DNA sequencing of PCR products was used; thus, 44 chromosomes were studied. Polymorphisms were identified by manual scanning of sequence chromatograms and computerized sequence analysis. Twenty Single Nucleotide Polymorphisms (SNPs) were detected in 1788 bp sequenced from the leptin gene, giving a frequency of 1 SNP per 89 bp. Twenty-four SNPs were detected in a 458-bp fragment of the APP gene; 23 of the polymorphisms were contained in a 302-bp intron 16 fragment. This equates to an SNP frequency of 1 per 13 bp for the intron. We can thus conclude that this portion of the bovine APP gene constitutes a hypermutable region. Nucleotide sequence diversity values of 0.019 and 0.0026 were obtained for APP and leptin respectively.

Association of a missense mutation in the bovine leptin gene with carcass fat content and leptin mRNA levels

“…A microsatellite has been located in the 5 UTR of the leptin gene [32] and nine SNPs have been discovered in intron 2 of the bovine leptin gene [20,23]. The polymorphisms reported in this study that change the amino acid in exons 2 (Arg/Cys) and 3 (Ala/Val) have been confirmed by Konfortov et al [19], who also report several other intronic polymorphisms in a diverse panel of cattle breeds. Haegeman et al [12] also report the alanine to valine substitution as well as an amino acid change from Glu to Arg in exon 3.…”

DNA polymorphism of leptin gene in Bos indicus and Bos taurus cattle

“…The T allele frequency was comparatively low in crossbred population as compared to the two pure breeds of taurine cattle, possibly due to the absence of this mutation in Hariana cattle which contributed only the C allele to the crossbred population. Our findings for taurine cattle are similar to those of Buchanan et al (2002) who reported T allele frequencies of 0.58 in Angus cattle, 0.34 in Charolais cattle, 0.55 in Hereford cattle and 0.32 in Simmental cattle (all taurine cattle) and Konfortov et al (1999) who found the T allele frequency to be 0.41 in taurine cattle. In all the four breeds of indicine cattle, the C allele frequency was 1.0, possibly due to the absence of the V allele (valine) in these populations.…”

“…Our results agree with those of Lien et al (1997) who for the first time described a guanine (G) to adenine (A) substitution (G ® A substitution) in intron 2 of the leptin gene of Norwegian cattle. Although Konfortov et al (1999) have reported that some of the B. indicus crossbred cattle have leptin gene polymorphisms that are not found in B. taurus breeds, our findings are the first report of leptin gene polymorphisms in purebred B. indicus cattle. Taken together, the present results suggest that leptin gene mutation might have occurred far back in evolution before the divergence of cattle into taurine (B. taurus) and indicine (B. indicus) cattle.…”

“…In our work involving the 94 bp leptin fragment we identified three Kpn2I digestion patterns in the Bos taurus and crossbreds indicating three genotypes: an intact 94 bp fragment as TT genotype; 75 and 19 bp fragments as CC genotype; and 94, 75 and 19 bp fragments as CT genotype. Konfortov et al (1999) also reported this mutation in both B. taurus (Limousin, Charolais, Charolais x Friesian, Simmental x Friesian, Limousin x Friesian, Charolais x Hereford/Friesian, Jersey, Simmental breeds) and its crossbreds with Bos indicus. In our study, the Hariana, Sahiwal, Gir and Nimari breeds of B. indicus cattle showed only the 75 and 19 bp (CC genotype) restriction pattern, indicating absence of the C ® T mutation.…”

“…Leptin is a protein involved intricately in the growth and metabolism of animals and which plays an important role in the regulation of feed intake, energy metabolism, growth and reproduction of cattle (Ramsay and Cranwell, 1999) and thus the leptin gene is a potential candidate gene for QTL studies. Although many polymorphic studies on the bovine leptin gene have been reported (Pomp et al, 1997;Lien et al, 1997;Wilkins and Davey, 1997;Konfortov et al 1999;Haegeman et al, 2000;Buchanan et al, 2002), to our knowledge there has been no reported polymorphic study involving cattle originating in the Indian subcontinent (indicine cattle, Bos indicus). From each animal, about 15 mL of venous blood was collected from the jugular vein and placed into a sterile 15 mL polypropylene centrifuge tube containing 0.5 mL of 2.7% EDTA solution as an anticoagulant and transported in an icebox to the laboratory and stored at 4°C until needed for DNA extraction.…”