Background: Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RTqPCR) is a technique used to measure mRNA species copy number as a way to determine key genes involved in different biological processes. However, the expression level of these key genes may vary among tissues or cells not only as a consequence of differential expression but also due to different factors, including choice of reference genes to normalize the expression levels of the target genes; thus the selection of reference genes is critical for expression studies. For this purpose, ten candidate reference genes were investigated in bovine muscular tissue.

The results of an exhaustive data collection from a bovine population with a low level of exchangeability, the Lidia breed, are presented. A total of 1683 individuals from 79 herds were sampled and genetic diversity within and among lineages was assessed using 24 microsatellite loci on 22 different chromosomes. Expected heterozygosity ranged between 0.46 and 0.68 per lineage and there was significant inbreeding in the lineages, which included several farms [mean F(IS) = 0.11, bootstrap 95% confidence interval (0.09, 0.14)], mainly because of the high genetic divergence between herds within those lineages. High genetic differentiation between lineages was also found with a mean F(ST) of 0.18 [bootstrap 95% confidence interval (0.17, 0.19)], and all pairwise values, which ranged from 0.07 to 0.35, were highly significant. The relationships among lineages showed weak statistical support. Nonetheless, lineages were highly discrete when analysed using correspondence analysis and a great proportion of the individuals were correctly assigned to their own lineage when performing standard assignment procedures.

The distribution of genetic diversity and the genetic relationships among western Mediterranean horse breeds were investigated using microsatellite markers. The examined sample included seven Spanish and three Italian local horse breeds and populations, plus a Spanish Thoroughbred outgroup. The total number of animals examined was 682 (on average 62 animals per breed; range 20-122). The microsatellite marker set analysed provided 128 alleles (10.7 alleles per locus). Within-breed genetic diversity was always high (>0.70), with breeds contributing about 8% of the total genetic variability. The mean molecular coancestry of the entire population examined was 0.205, Losino being the breed that contributed most. In addition to Nei's standard and Reynolds' genetic distances, pair-wise kinship distance and molecular coancestry were estimated. Remarkably similar breed rankings were obtained with all methods. Clustering analysis provided an accurate representation of the current genetic relationships among the breeds. Determining coancestry is useful for analysing genetic diversity distribution between and within breeds and provides a good framework for jointly analysing molecular markers and pedigree information. An integrated analysis was undertaken to obtain information on the population dynamics in western Mediterranean native horse populations, and to better determine conservation priorities.

To clarify the genetic ancestry and the mitochondrial DNA (mtDNA) diversity of the Lidia cattle breed, a 521-bp D-loop fragment was sequenced in 527 animals belonging to 70 herds distributed across 29 lineages. The mtDNA diversity recorded was similar to that seen for Middle Eastern breeds and greater than that recorded for the majority of European breeds. Haplotype T3 was the most common (81%), followed by the African T1 haplotype (17%); very low frequencies were recorded for haplotypes T and T2. The results agree with there being two major ancestral lines for the Lidia breed, European and African, similar to that seen for other Mediterranean breeds. A wide range of variation in haplotype frequencies was seen between the examined lineages. Haplotype T3 was present in all those analysed; in five it was the only one present, and in only one lineage (Miura) was its frequency lower than that of T1. T1*, a haplotype reported in Criollo breeds and to date in only a single European breed (the Retinta breed from Spain), was found in a single animal belonging to the Concha y Sierra lineage. Network analysis of the Lidia breed revealed the presence of two major haplotypes: T3 and T1. The Lidia breed appears to be more closely related to prehistoric Iberian and Italian than to British aurochs.

To assess the paternal gene pool in the Lidia bovine breed (or fighting bull), a total of 603 animals belonging to 81 herds classified in 33 lineages were genotyped for six Y chromosome microsatellites, one single nucleotide polymorphism and one indel. A total of 10 haplotypes were determined with a high level of frequency variation between them, ranging from 0.2 to 74%. All the haplotypes identified belong to two previously defined major haplogroups (Y1 and Y2). Two major paternal influences were identified, corresponding to the two most common haplotypes (H1Y1 and H3Y2) with frequencies of 74 and 18%, respectively. The detection of the INRA189-104 allele evidenced an African influence in the Lidia bovine breed. Low levels of haplotype diversity have been achieved and only eight lineages showed more than one haplotype. Analysis of molecular variance showed a high level of interlineage variance (F(ST) = 86%). Network results evidenced two main clusters made for those haplotypes belonging to Y1 and Y2 haplogroups, respectively. The findings support a high level of genetic structure together with a low level of genetic diversity in the Lidia bovine breed.