Inferring the population structure of the Maghreb sheep breeds using a medium-density SNP chip.

North Africa has a great diversity of indigenous sheep breeds whose origin is linked to its environmental characteristics and to certain historical events that took place in the region. To date, few genome-wide studies have been conducted to investigate the population structure of North African indigenous sheep. The objective of the present study was to provide a detailed assessment of the genetic structure and admixture patterns of six Maghreb sheep populations using the Illumina 50K Ovine BeadChip and comparisons with 22 global populations of sheep and mouflon. Regardless of the method of analysis used, patterns of multiple hybridization events were observed within all North African populations, leading to a heterogeneous genetic architecture that varies according to the breed. The Barbarine population showed the lowest genetic heterogeneity and major southwest Asian ancestry, providing additional support to the Asian origin of the North African fat-tailed sheep. All other breeds presented substantial Merino introgression ranging from 15% for D'man to 31% for Black Thibar. We highlighted several signals of ancestral introgression between North African and southern European sheep. In addition, we identified two opposite gradients of ancestry, southwest Asian and central European, occurring between North Africa and central Europe. Our results provide further evidence of the weak global population structure of sheep resulting from high levels of gene flow among breeds occurring worldwide. At the regional level, signs of recent admixture among North African populations, resulting in a change of the original genomic architecture of minority breeds, were also detected.

Identification of major milk fat globule membrane proteins from pony mare milk highlights the molecular diversity of lactadherin across species.

Although several studies have been devoted to the colloidal and soluble protein fractions of mare milk (caseins and whey proteins), to date little is known about the milk fat globule membrane (MFGM) protein fraction from mare milk. The objective of this study was thus to describe MFGM proteins from Equidae milk and to compare those proteins to already described MFGM proteins from cow and goat milk. Major MFGM proteins (namely, xanthine oxidase, butyrophilin, lactadherin, and adipophilin) already described in cow or goat milk were identified in mare milk using mass spectrometry. However, species-specific peculiarities were observed for 2 MFGM proteins: butyrophilin and lactadherin. A highly glycosylated 70-kDa protein was characterized for equine butyrophilin, whereas proteins of 64 and 67 kDa were characterized for cow and goat butyrophilin, respectively. Prominent differences across species were highlighted for lactadherin. Indeed, whereas 1 or 2 polypeptide chains were identified, respectively, by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight analysis for caprine and bovine lactadherin, 4 isoforms (60, 57, 48, and 45 kDa) for lactadherin from mare milk were identified by 10% sodium dodecyl sulfate-PAGE. Polymerase chain reaction experiments on lactadherin transcripts isolated from milk fat globules revealed the existence of 2 distinct lactadherin transcripts in the horse mammary gland. Cloning and sequencing of both transcripts encoding lactadherin showed an alternative use of a cryptic splice site located at the end of intron 5 of the equine lactadherin-encoding gene. This event results in the occurrence of an additional alanine (A) residue in the protein that disrupts a putative atypical N-glycosylation site (VNGC/VNAGC) described in human lactadherin. Liquid chromatography coupled with tandem mass spectrometry analyses confirmed the existence of both lactadherin variants in mare MFGM. We show here that lactadherin from Equidae milk is much more complex than that from Bovidae milk (i.e., cow and goat milk), therefore raising questions regarding the precise function of these different isoforms, if any, in the equine mammary gland.

Validation of RNA isolated from milk fat globules to profile mammary epithelial cell expression during lactation and transcriptional response to a bacterial infection.

Mastitis, an inflammation of the mammary gland, is the most costly infectious disease of dairy ruminants worldwide. Although it receives considerable attention, the early steps of the host response remain poorly defined. Here, we report a noninvasive method using milk fat globules (MFG) as a source of mammary RNA to follow the dynamics of the global transcriptional response of mammary epithelial cells (MEC) during the course of a bacterial infection. We first assessed that RNA isolated from MFG were representative of MEC RNA; we then evaluated whether MFG RNA could be used to monitor the MEC response to infection. Sufficiently high yields of good-quality RNA (RNA integrity numbers ranging between 6.7 and 8.7) were obtained from goat MFG for subsequent analyses. Contamination of MFG by macrophages and neutrophils, which can be trapped during creaming, was assessed and when using quantitative real-time PCR for cell-type specific markers, was shown to be weak enough (<8%) to affect MFG gene expression profiling. Using microarrays, we showed that RNA extracted from MFG and from mammary alveolar parenchyma shared approximately 90% of the highlighted probes corresponding in particular to genes encoding milk proteins (CSN, BLG, LALBA) and enzymes involved in milk fat synthesis and secretion (FASN, XDH, ADRP, SCD, and DGAT1). In addition, a gene involved in the acute-phase reaction, coding for the serum amyloid A3 (SAA3) protein, was found within the first 50 most highly expressed genes in a noninfectious context in both mammary alveolar parenchyma and MFG, strongly suggesting that SAA3 is expressed in MEC. We took advantage of this noninvasive RNA sampling to follow the early proinflammatory response of MEC during the course of a bacterial infection and showed that the levels of mRNA encoding SAA3 sharply increased at 24h postinfection. Taken together, our results demonstrate that MFG represent a unique source of MEC RNA to noninvasively sample sufficient amounts of high-quality RNA to assess the dynamics of MEC gene expression in vivo, especially during the first steps of infection, thereby paving the way for the discovery of early biomarkers for the control of intramammary infections. Furthermore, this noninvasive technique could be used to provide mammary transcriptomic data on a large scale, thus filling the gap between genomic and phenotypic data.

Terminal differentiation of goat mammary tissue during pregnancy requires the expression of genes involved in immune functions.

Terminal differentiation of mammary tissue into a functional epithelium that synthesizes and secretes milk occurs during pregnancy. The molecular mechanisms underlying this complex process are poorly understood, especially in ruminants. To obtain an overview of the ruminant mammary gland's final differentiation process, we conducted time-course gene expression analysis of five physiological stages: four during pregnancy (P46, P70, P90, and P110) and one after 40 days of lactation (L40). An appropriate loop experimental design was used to follow gene expression profiles. Using three nulliparous (pregnancy) or primiparous (lactation) goats per stage, we performed a comparison starting from nine dye-swaps and using a 22K bovine oligoarray. Statistical analysis revealed that the expression of 1,696 genes varied significantly at least once in the study. These genes fell into 19 clusters based on their expression profiles. Identification of biological functions with Ingenuity Pathway Analysis software revealed several similarities, in keeping with physiological stages described in mice. As in mice, expression of milk protein genes began at midpregnancy, and genes regulating lipid biosynthesis were induced at the onset of lactation. During the first half of pregnancy, the molecular signature of goat mammary tissue was characterized by the expression of genes associated with tissue remodeling and differentiation, while the second half was mainly characterized by the presence of messengers encoding genes involved in cell proliferation. A large number of immune-related genes were also induced, supporting recent speculation that mammary tissue has an original immune function, and the recruitment of migrating hematopoietic cells possibly involved in the branching morphogenesis of the mammary gland. These data hint that the induction of differentiation occurs early in pregnancy, very likely before P46. This period is therefore crucial for obtaining a healthy and productive mammary gland.