Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Association study of resistance to Soilborne wheat mosaic virus in U.S. winter wheat

Soilborne wheat mosaic virus (SBWMV) is one of the most important winter wheat pathogens worldwide. To identify genes for resistance to the virus in U.S. winter wheat, association study was conducted using a selected panel of 205 elite experimental lines and cultivars from U.S. hard and soft winter wheat breeding programs. Virus symptoms were evaluated twice in virus-infected fields for the panel at Manhattan, KS in spring 2010 and 2011 and for a subpanel of 137 hard winter wheat accessions at Stillwater, OK in spring 2008. At the two locations, 69.8 and 79.5% of cultivars were resistant or moderately resistant to the disease, respectively. After 282 simple-sequence repeat markers covering all wheat chromosome arms were scanned for association in the panel, marker Xgwm469 on the long arm of chromosome 5D (5DL) showed a significant association with the disease rating. Three alleles (Xgwm469-165bp, -167bp, and -169bp) were associated with resistance and the null allele was associated with susceptibility. Correlations between the marker and the disease rating were highly significant (0.80 in Manhattan at P < 0.0001 and 0.63 in Stillwater at P < 0.0001). The alleles Xgwm469-165bp and Xgwm469-169bp were present mainly in the hard winter wheat group, whereas allele Xgwm469-167bp was predominant in the soft winter wheat. The 169 bp allele can be traced back to 'Newton', and the 165 bp allele to Aegilops tauschii. In addition, a novel locus on the short arm of chromosome 4D (4DS) was also identified to associate with the disease rating. Marker Xgwm469-5DL is closely linked to SBWMV resistance and highly polymorphic across the winter wheat accessions sampled in the study and, thus, should be useful in marker-assisted selection in U.S. winter wheat.

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.

An unusual splice defect in the mitofusin 2 gene (MFN2) is associated with degenerative axonopathy in Tyrolean Grey cattle

Tyrolean Grey cattle represent a local breed with a population size of approximately 5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.

Arachnomelia in Brown Swiss cattle maps to chromosome 5

Arachnomelia in Brown Swiss cattle is a monogenic autosomal recessive inherited congenital disorder of the skeletal system giving affected calves a spidery look (OMIA ID 000059). Over a period of 20 years 15 cases were sampled in the Swiss and Italian Brown cattle population. Pedigree data revealed that all affected individuals trace back to a single acknowledged carrier founder sire. A genome scan using 240 microsatellites spanning the 29 bovine autosomes showed homozygosity at three adjacent microsatellite markers on bovine Chr 5 in all cases. Linkage analysis confirmed the localization of the arachnomelia mutation in the region of the marker ETH10. Fine-mapping and haplotype analysis using a total of 34 markers in this region refined the critical region of the arachnomelia locus to a 7.19-Mb interval on bovine Chr 5. The disease-associated IBD haplotype was shared by 36 proven carrier animals and allows marker-assisted selection. As the corresponding human and mouse chromosome segments do not contain any clear functional candidate genes for this disorder, the mutation causing arachnomelia in the Brown Swiss cattle might help to identify an unknown gene in bone development.

Genome and transcriptome sequencing identifies breeding targets in the orphan crop tef (Eragrostis tef)

Background: Tef (Eragrostis tef), an indigenous cereal critical to food security in the Horn of Africa, is rich in minerals and protein, resistant to many biotic and abiotic stresses and safe for diabetics as well as sufferers of immune reactions to wheat gluten. We present the genome of tef, the first species in the grass subfamily Chloridoideae and the first allotetraploid assembled de novo. We sequenced the tef genome for marker-assisted breeding, to shed light on the molecular mechanisms conferring tef's desirable nutritional and agronomic properties, and to make its genome publicly available as a community resource. Results: The draft genome contains 672 Mbp representing 87% of the genome size estimated from flow cytometry. We also sequenced two transcriptomes, one from a normalized RNA library and another from unnormalized RNASeq data. The normalized RNA library revealed around 38000 transcripts that were then annotated by the SwissProt group. The CoGe comparative genomics platform was used to compare the tef genome to other genomes, notably sorghum. Scaffolds comprising approximately half of the genome size were ordered by syntenic alignment to sorghum producing tef pseudo-chromosomes, which were sorted into A and B genomes as well as compared to the genetic map of tef. The draft genome was used to identify novel SSR markers, investigate target genes for abiotic stress resistance studies, and understand the evolution of the prolamin family of proteins that are responsible for the immune response to gluten. Conclusions: It is highly plausible that breeding targets previously identified in other cereal crops will also be valuable breeding targets in tef. The draft genome and transcriptome will be of great use for identifying these targets for genetic improvement of this orphan crop that is vital for feeding 50 million people in the Horn of Africa.

Application of TILLING for Orphan Crop Improvement

People in developing countries mostly depend for their diet on special staple crops, so called orphan crops. These crops play a key role in food security since they are grown by many resource-poor farmers and consumed locally. Despite their huge importance in the economy and livelihood of the developing world, orphan crops have received little attention in terms of scientific improvement. Although conventional breeding is widely implemented to improve crop plants, alternative methods such as marker-assisted breeding and reverse genetics approaches have proved to be efficient in developing crop cultivars. In this review, we present detailed description of a non-transgenic and reverse genetics technique called TILLING (Targeting Induced Local Lesion IN Genomes). The method was originally optimized in the model plant Arabidposis thaliana and subsequently applied to crops such as maize, wheat, and rice. We also present detailed procedures for several TILLING strategies and discuss their benefits and drawbacks. The application of the technique for orphan crop improvement is also discussed based on several TILLING platforms currently carried-out on these understudied crops of the world.

Robust expression of CCR3 as a single basophil selection marker in flow cytometry

Basophil activation tests (BAT) rely on different combinations of basophil selection and activation markers. Whereas activation markers, especially CD63, are widely validated, the most suitable and robust marker for basophil selection is still a matter of debate.

Adiponectin as a marker of success in intracytoplasmic sperm injection/embryo transfer cycles

Adiponectin (Acrp30) is an adipose tissue-derived protein whose serum concentrations, in contrast to leptin, are reported to be negatively correlated to body mass. In spite of the comparatively high circulating adiponectin concentrations, this protein has not been studied in the context of assisted reproduction to date. The aim of this preliminary project was thus to examine the potential of adiponectin to serve as a marker for fertility. We compared adiponectin levels in serum before and after controlled ovarian hyperstimulation, as well as in follicular fluid (FF), between two groups: those with successful outcome (clinical pregnancies) and those with implantation failure. In the former, adiponectin concentrations were higher than in the negative outcome group; this difference was statistically significant (p < 0.05) in serum on the day of oocyte pick-up (OPU) as well as two or three days before OPU, but not in FF or in serum at the beginning of the stimulation phase. This finding adds a new perspective to the suggested but still controversial reduction in FF leptin concentrations in the positive outcome group, and may become a useful tool for early prediction of success of in vitro fertilization treatment for a given patient.

Systematic Review and Cumulative Analysis of Oncologic and Functional Outcomes After Robot-assisted Radical Cystectomy

CONTEXT Although open radical cystectomy (ORC) is still the standard approach, laparoscopic radical cystectomy (LRC) and robot-assisted radical cystectomy (RARC) are increasingly performed. OBJECTIVE To report on a systematic literature review and cumulative analysis of pathologic, oncologic, and functional outcomes of RARC in comparison with ORC and LRC. EVIDENCE ACQUISITION Medline, Scopus, and Web of Science databases were searched using a free-text protocol including the terms robot-assisted radical cystectomy or da Vinci radical cystectomy or robot* radical cystectomy. RARC case series and studies comparing RARC with either ORC or LRC were collected. A cumulative analysis was conducted. EVIDENCE SYNTHESIS The searches retrieved 105 papers, 87 of which reported on pathologic, oncologic, or functional outcomes. Most series were retrospective and had small case numbers, short follow-up, and potential patient selection bias. The lymph node yield during lymph node dissection was 19 (range: 3-55), with half of the series following an extended template (yield range: 11-55). The lymph node-positive rate was 22%. The performance of lymphadenectomy was correlated with surgeon and institutional volume. Cumulative analyses showed no significant difference in lymph node yield between RARC and ORC. Positive surgical margin (PSM) rates were 5.6% (1-1.5% in pT2 disease and 0-25% in pT3 and higher disease). PSM rates did not appear to decrease with sequential case numbers. Cumulative analyses showed no significant difference in rates of surgical margins between RARC and ORC or RARC and LRC. Neoadjuvant chemotherapy use ranged from 0% to 31%, with adjuvant chemotherapy used in 4-29% of patients. Only six series reported a mean follow-up of >36 mo. Three-year disease-free survival (DFS), cancer-specific survival (CSS), and overall survival (OS) rates were 67-76%, 68-83%, and 61-80%, respectively. The 5-yr DFS, CSS, and OS rates were 53-74%, 66-80%, and 39-66%, respectively. Similar to ORC, disease of higher pathologic stage or evidence of lymph node involvement was associated with worse survival. Very limited data were available with respect to functional outcomes. The 12-mo continence rates with continent diversion were 83-100% in men for daytime continence and 66-76% for nighttime continence. In one series, potency was recovered in 63% of patients who were evaluable at 12 mo. CONCLUSIONS Oncologic and functional data from RARC remain immature, and longer-term prospective studies are needed. Cumulative analyses demonstrated that lymph node yields and PSM rates were similar between RARC and ORC. Conclusive long-term survival outcomes for RARC were limited, although oncologic outcomes up to 5 yr were similar to those reported for ORC. PATIENT SUMMARY Although open radical cystectomy (RC) is still regarded as the standard treatment for muscle-invasive bladder cancer, laparoscopic and robot-assisted RCs are becoming more popular. Templates of lymph node dissection, lymph node yields, and positive surgical margin rates are acceptable with robot-assisted RC. Although definitive comparisons with open RC with respect to oncologic or functional outcomes are lacking, early results appear comparable.

Continental-Scale Footprint of Balancing and Positive Selection in a Small Rodent (Microtus arvalis)

Genetic adaptation to different environmental conditions is expected to lead to large differences between populations at selected loci, thus providing a signature of positive selection. Whereas balancing selection can maintain polymorphisms over long evolutionary periods and even geographic scale, thus leads to low levels of divergence between populations at selected loci. However, little is known about the relative importance of these two selective forces in shaping genomic diversity, partly due to difficulties in recognizing balancing selection in species showing low levels of differentiation. Here we address this problem by studying genomic diversity in the European common vole (Microtus arvalis) presenting high levels of differentiation between populations (average FST = 0.31). We studied 3,839 Amplified Fragment Length Polymorphism (AFLP) markers genotyped in 444 individuals from 21 populations distributed across the European continent and hence over different environmental conditions. Our statistical approach to detect markers under selection is based on a Bayesian method specifically developed for AFLP markers, which treats AFLPs as a nearly codominant marker system, and therefore has increased power to detect selection. The high number of screened populations allowed us to detect the signature of balancing selection across a large geographic area. We detected 33 markers potentially under balancing selection, hence strong evidence of stabilizing selection in 21 populations across Europe. However, our analyses identified four-times more markers (138) being under positive selection, and geographical patterns suggest that some of these markers are probably associated with alpine regions, which seem to have environmental conditions that favour adaptation. We conclude that despite favourable conditions in this study for the detection of balancing selection, this evolutionary force seems to play a relatively minor role in shaping the genomic diversity of the common vole, which is more influenced by positive selection and neutral processes like drift and demographic history.