Characterization of the 3' untranslated region of bovine parathyroid hormone gene in N'Dama and Boran cattle

We describe a polymorphism in the bovine gene PTHG which can be readily typed by PCR assay. The polymorphism is codominantly inherited and the allele frequencies appear characteristic of Bos indicus and B. taurus cattle.     

PCR-RFLP typing of the bovine myoglobin gene

We have identified substitutions in the 3₁ untranslated region of the bovine myoglobin gene, one of which affects an MboII restriction enzyme site resulting in a bi-allelic restriction fragment length polymorphism. Co-dominant inheritance of the alleles in three reference families was observed using a polymerase chain reaction—restriction fragment length polymorphism assay. The distribution of the alleles seems characteristic of cattle type—one of the alleles was not detected in purely taurine breeds. Furthermore, we mapped, using the polymerase chain reaction on a bovine–rodent somatic cell hybrid panel, the myoglobin gene to bovine chromosome five. It is therefore syntenic with γ-interferon and insulin-like growth factor in which we have not found polymorphism. The myoglobin locus therefore serves as a type one marker on bovine chromosome five.     

A panel of bovine, ovine and caprine polymorphic microsatellites

We report a set of six new bovine microsatellite polymorphisms based on (CA)_n repeats. They are highly polymorphic and thus represent valuable markers for genome mapping. Four of the six are polymorphic in sheep and two are polymorphic in goats. One, which is polymorphic in cattle and sheep and apparently monomorphic in goats, is X-chromosome specific and has potential value in, for example, sex determination and detection of chimaerism.     

Geographic and breed distribution of an Msp I PCR-RFLP in the bovine growth hormone (bGH) gene

Information is presented on the frequency of the Msp I (-) allele in the third intron of the bovine growth hormone gene in a large number of cattle breeds. Consideration of the breed frequencies in relation to their geographic origin shows a low frequency for breeds originating in Northern Europe, moderate frequencies for breeds originating in Eastern Europe or the countries surrounding the Mediterranean basin, and very high frequencies for breeds originating in the Indian subcontinent. Consideration of breed frequencies in relation to breed type, shows low to moderate frequencies for the humpless breeds, high frequencies for the humped breeds. Various explanations for this distribution are discussed, among them the possibility that the Msp I (-) allele originated in the Bos indicus breeds of the Indian subcontinent, from which it diffused through the humpless Bos taurus breeds of Eastern Europe, the Mediterranean basin, eventually reaching Western, Northern Europe, Western Africa in low frequencies.     

A polymorphism in randomly amplified DNA that differentiates the Y chromosomes of Bos indicus and Bos taurus

A small number of west African Bos taurus cattle breeds, including the N'Dama, constitute a valuable genetic resource by virtue of their ability to remain productive under trypanosomiasis challenge. However, introgression of Bos indicus genes into the trypanotolerant breeds, particularly by introduction of zebu bulls, is a threat to this resource. This work describes the characterization and cloning of a bovine randomly amplified polymorphic DNA (RAPD) that is generated in polymerase chain reaction (PCR) with the 10 base primer ILO1065 from Bos indicus male templates, but not from B. taurus male templates or female templates of either type. Male-specific sequences with homology to the RAPD also occur in B. taurus breeds. This suggests that the polymorphism may be due to base substitution(s) in an ILO1065 priming site, or insertion/deletion events either affecting priming sites or occurring between sites on the cattle Y chromosome. We have shown that cattle, whether of B. indicus or B. taurus phenotype, which possess a typically B. indicus metaphase Y chromosome on the basis of QFQ banding, have a B. indicus ILO1065-generated genotype. The ILO1065-primed RAPD can be used in a simple dot blot assay as a probe of RAPD-PCR products, to provide a convenient, reliable and effective means of detecting introgression of zebu genes in B. taurus cattle populations.     

Cattle breed-variation in infestation by the horn fly Haematobia irritans

A study was carried out to assess the resistance of pure and cross-bred groups of cattle to the horn fly Haematobia irritans (Linnaeus) (Diptera: Muscidae) in northern Argentina. Pure-bred cattle were Criolla, Iberian Bos taurus Linnaeus (Artiodactyla: Bovidae) and Nellore, Bos indicus Linnaeus (Artiodactyla: Bovidae). Cross-bred cattle were Hereford, British B. taurus (34%) X Nellore (66%) and Hereford (66%) X Nellore (34%). All were heifers and animals were maintained in two groups, each containing a mixture of pure and cross-breeds. The lowest fly numbers were found on Criolla heifers and the highest on Hereford X Nellore cross-breeds. However, it could not be determined from this study whether this was a consequence of breed and/or size, as Criolla heifers were lighter than the corresponding Hereford X Nellore heifers. Fly numbers on the heifers followed an approximately negative binomial distribution. However, the ranking of individual animals in their level of infestation within subgroups was not consistent. Hence, culling the most infested heifers on any given date would at best give only a small improvement in H. irritans control.     

Estimate of the population of preantral follicles in the ovaries of Bos taurus indicus and Bos taurus taurus cattle

The number of oocytes recovered from Bos taurus indicus females subjected to ovum pick-up averaged two to four times greater compared to Bos taurus taurus females. The objective of the present study was to test the hypothesis that this difference in oocyte yield was due to more preantral follicles in the ovaries of Bos indicus females. Ovaries (n = 64) from Nelore (Bos indicus) fetuses (n = 10), heifers (n = 12), and cows (n = 10), and Aberdeen Angus (Bos taurus) fetuses (n = 10), heifers (n = 12), and cows (n = 10) were cut longitudinally into halves, fixed, and processed for histological evaluation. The number of preantral follicles was estimated by counting them in each histological section, using the oocyte nucleus as a marker and employing a correction factor. The average number of preantral follicles in the ovaries of Bos indicus vs Bos taurus was (mean ± SD) 143,929 ± 64,028 vs 285,155 ± 325,195 for fetuses, 76,851 ± 78,605 vs 109,673 ± 86,078 for heifers, and 39,438 ± 31,017 vs 89,577 ± 86,315 for cows (P > 0.05). The number of preantral follicles varied greatly among individual animals within the same category, as well as between breeds. In conclusion, we inferred that the higher oocyte yield from Bos indicus females was not due to a greater ovarian reserve of preantral follicles. Therefore, mechanisms controlling follicle development after the preantral stage likely accounted for differences between Bos indicus and Bos taurus females in number of oocytes retrieved at ovum pick-up.     

Detection of the Bcl I polymorphism of the glucocorticoid receptor gene by single-tube allele-specific polymerase chain reaction

The Bcl I polymorphism of the glucocorticoid receptor gene, recently identified as an intronic C to G change 646 nucleotides downstream of exon 2, has been associated with increased sensitivity to glucocorticoids and its potential relevance in metabolic disturbances and in various disorders has been extensively investigated. In the present study, we designed a single-tube allele-specific polymerase chain reaction for genotyping this polymorphism in peripheral blood DNA samples. When the Bcl I polymorphism was detected with this novel method in a cohort of 247 healthy subjects, the observed genotype distribution matched the Hardy–Weinberg equilibrium (100 subjects homozygous for the wild-type, 124 heterozygous and 23 homozygous for the mutant allele). In 50 randomly selected subjects the Bcl I polymorphism was also determined using a traditional restriction fragment length polymorphism technique and DNA sequencing, and the results showed 100% coincidence with those obtained by our novel method. The method proved to be more rapid and less labour-intensive compared to currently used techniques, and it avoided the use of extensive instrumentals. We assume that this novel method may have a broad utility in clinical and molecular epidemiological studies aimed to elucidate the impact of the Bcl I polymorphism of the glucocorticoid receptor gene either on metabolic disturbances, or various disorders, including cancer treatment and hormone substitution therapies.     

Nucleotide Sequence and Variation of IGF2 Gene Exon 6 in Bos Taurus and Bos Indicus Cattle

The major assumption of this study is that polymorphism of a gene could be used to investigate its allele-specific expression as well as its methylation and imprinting status. Therefore, the aim of this study was to analyze the polymorphism of the coding region of the bovine IGF2 gene and to determine the sequence of its gene exon 6 in Bos taurus and Bos indicus cattle. A single nucleotide “C” deletion/insertion polymorphism was found in both cattle subspecies and a G/T transversion (RFLP-MboII) in the Bos indicus IGF2 gene. A 407-bp fragment of bovine IGF2 exon 6 was sequenced and the sequences (including variable nucleotides) were deposited in the GenBank database. A comparative analysis was performed for this fragment from different species; 99.5% identity was found between Bos taurus and Bos indicus cattle.     

Detection of selection signatures within candidate regions underlying trypanotolerance in outbred cattle populations

Breeding indigenous African taurine cattle tolerant to trypanosomosis is a straightforward approach to control costs generated by this disease. A recent study identified quantitative trait loci (QTL) underlying trypanotolerance traits in experimental crosses between tolerant N'Dama and susceptible Boran zebu cattle. As trypanotolerance is thought to result from local adaptation of indigenous cattle breeds, we propose an alternative and complementary approach to study the genetic architecture of this trait, based on the identification of selection signatures within QTL or candidate genes. A panel of 92 microsatellite markers was genotyped on 509 cattle belonging to four West African trypanotolerant taurine breeds and 10 trypanosusceptible European or African cattle breeds. Some of these markers were located within previously identified QTL regions or candidate genes, while others were chosen in regions assumed to be neutral. A detailed analysis of the genetic structure of these different breeds was carried out to confirm a priori grouping of populations based on previous data. Tests based on the comparison of the observed heterozygosities and variances in microsatellite allelic size among trypanotolerant and trypanosusceptible breeds led to the identification of two significantly less variable microsatellite markers. BM4440, one of these two outlier loci, is located within the confidence interval of a previously described QTL underlying a trypanotolerance-related trait.
Detection of selection signatures appears to be a straightforward approach for unravelling the molecular determinism of trypanosomosis pathogenesis. We expect that a whole genome approach will help confirm these results and achieve a higher resolving power.     

Restriction fragment length polymorphism in amplification products of the bovine butyrophilin gene: assignment of bovine butyronhilin to bovine chromosome 23

A polymorphism was identified in the bovine butyrophilin (BTN) gene by digesting poly-merase chain reaction products with the restriction enzyme Hae III. This polymorphism was segregating in a Holstein-Friesian sire selected as part of an ongoing study directed towards the identification of quantitative trait loci affecting milk composition. Screening of a half-sib family established for the heterozygous sire allowed the localization of BTN to bovine chromosome 23 (BTA23).     

A simple method for genotyping the bovine growth hormone gene

An allele-specific polymerase chain reaction (PCR) amplification method was developed to determine the genotypes at the bovine growth hormone locus that result from two nucleotide substitutions in exon 5 of the gene. This method was a multiplex PCR (ASM–PCR) employing a common primer pair and two allele-specific reverse primers. The common primer pair was designed to amplify a target region containing two substitution points from the three variants of the bovine growth hormone gene. The allele-specific primers were designed to be mismatched with other genotypes at the 3' end of oligonucleotides. When the common and allele-specific reverse primers competed with each other, the shorter allele-specific fragments were amplified preferentially. Consequently, the PCR products of the variant-specific fragments were 347, 483 and 656 bp for alleles A, B and C, respectively, of the bovine growth hormone gene. Genotypes of the bovine growth hormone gene were easily identified by agarose gel electrophoresis of PCR products. The results suggested that this multiplex PCR method would be useful for identification of genetic variants caused by point mutations.     

Sequence comparison of mitochondrial tRNA genes and origin of light strand replication in Bos taurus and Nellore (Bos indicus) breeds

Although the complete bovine mitochondrial DNA molecule has been previously sequencedand sequence comparisons of the mitochondrial displacement loop have been performed, detailed sequence information is limited on coding regions of mitochondrial DNA within and among breeds of Bos taurus and Bos indicus. This study analysed polymorphism of the mitochondrial DNA transfer RNA genes for trypto-phan, alanine, asparagine, cysteine, tyrosine and the origin of light strand replication among Ayrshire, Canadian, Belgium Blue, Brown Swiss, Hereford, Jersey, Limousine, Piedmon-taise, Red Angus, Simmental (Bos taurus) and a Nellore (Bos indicus). Nucleotide sequence analysis of a 420-bp fragment of mitochondrial DNA comprising the five transfer RNA genes showed 100% homology among single individuals of the Bos taurus breeds. The Nellore breed showed guanine to adenine substitutions in the DHU arm of asparagine tRNA and in the origin of light-strand replication. This equates to a 0.5% sequence difference between the Nellore andBos taurus breeds and may reflect an independent evolutionary origin of the species.     

Refinement of quantitative trait loci on bovine chromosome 18 affecting health and reproduction in US Holsteins

Selection for increased milk yield is associated with decreased fertility in US Holsteins. Previously, a putative quantitative trait locus (QTL) on chromosome 18 affecting daughter pregnancy rate (DPR) was identified in one family. Our aim was to determine the validity of the QTL using additional markers and an extended pedigree. Twelve markers were genotyped in 940 descendants of the original sire in which the QTL was identified. Analysis of the extended pedigree detected significant and suggestive QTL for DPR, productive life and somatic cell score. Further analysis is underway to refine the QTL region so that positional candidate genes can be identified.     

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.     

Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA

Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67 bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.     

Restriction fragment length polymorphism in amplification products of the bovine PIT1 gene and assignment of PIT1 to bovine chromosome 1

A polymorphism was identified in the bovine PIT1 gene by digesting polymerase chain reaction (PCR) products with the restriction enzyme Hinfl . This polymorphism was segregating in five diverse breeds of cattle. PIT1 was sublocalized to the centromeric region of bovine chromosome 1 by linkage analysis.     

Organization and polymorphism of bovine major histocompatibility complex class II genes as revealed by genomic hybridizations with bovine probes

Previous studies on restriction fragment length polymorphism of bovine major histocompatibility complex class II genes have primarily been based on the use of human probes. In the present study bovine probes for DQA, DQB, DRB and DYA were used for RFLP analysis of cattle genomic DNA digested with PvuII and TaqI. There was an excellent agreement between the RFLP results obtained with homologous and heterologous probes. Although a few 'new' restriction fragments were revealed with the bovine probes there was no discrepancy with regard to the classification of allelic types with the two types of probes. The major advantages of using bovine probes were a better hybridization signal and reduced cross-hybridization between loci. Hybridization experiments with DQA probes for the first domain exon from two different genomic clones revealed the presence of two distinct types of bovine DQA genes. Surprisingly, these probes did not cross-hybridize at high stringency, indicating that the two genes are quite divergent. Hybridization with a recently described genomic clone for a novel bovine alpha-chain gene confirmed that it corresponds to the DYA gene which had previously been identified by cross-hybridization to a human DQA probe.     

Target region amplification polymorphism: A novel marker technique for plant genotyping

The advent of large-scale DNA sequencing technology has generated a tremendous amount of sequence information for many important organisms. We have developed a rapid and efficient PCR-based technique, which uses bioinformatics tools and expressed sequence tag (EST) database information to generate polymorphic markers around targeted candidate gene sequences. This target region amplification polymorphism (TRAP) technique uses 2 primers of 18 nucleotides to generate markers. One of the primers, the fixed primer, is designed from the targeted EST sequence in the database; the second primer, the arbitrary primer, is an arbitrary sequence with either an AT-or GC-rich core to anneal with an intron or exon, respectively. PCR amplification is run for the first 5 cycles with an annealing temperature of 35°C, followed by 35 cycles with an annealing temperature of 50°C. For different plant species, each PCR reaction can generate as many as 50 scorable fragments with sizes ranging from 50–900 bp when separated on a 6.5% polyacrylamide sequencing gel. The TRAP technique should be useful in genotyping germplasm collections and in tagging genes governing desirable agronomic traits of crop plants.