Landscape Genetics of Raccoons (Procyon lotor) Associated with Ridges and Valleys of Pennsylvania: Implications for Oral Rabies Vaccination Programs

Raccoons are the reservoir for the raccoon rabies virus variant in the United States. To combat this threat, oral rabies vaccination (ORV) programs are conducted in many eastern states. To aid in these efforts, the genetic structure of raccoons (Procyon lotor) was assessed in southwestern Pennsylvania to determine if select geographic features (i.e., ridges and valleys) serve as corridors or hindrances to raccoon gene flow (e.g., movement) and, therefore, rabies virus trafficking in this physiographic region. Raccoon DNA samples (n = 185) were collected from one ridge site and two adjacent valleys in southwestern Pennsylvania (Westmoreland, Cambria, Fayette, and Somerset counties). Raccoon genetic structure within and among these study sites was characterized at nine microsatellite loci. Results indicated that there was little population subdivision among any sites sampled. Furthermore, analyses using a model-based clustering approach indicated one essentially panmictic population was present among all the raccoons sampled over a reasonably broad geographic area (e.g., sites up to 36 km apart). However, a signature of isolation by distance was detected, suggesting that widths of ORV zones are critical for success. Combined, these data indicate that geographic features within this landscape influence raccoon gene flow only to a limited extent, suggesting that ridges of this physiographic system will not provide substantial long-term natural barriers to rabies virus trafficking. These results may be of value for future ORV efforts in Pennsylvania and other eastern states with similar landscapes.

Using Genetics to Assess Differentiation Among Raccoons in an Area with Variable Rabies Status in Alabama

The western spread of raccoon rabies in Alabama has been slow and even appears to regress eastward periodically. While the disease has been present in the state for over 30 years, areas in northwest Alabama are devoid of raccoon rabies. This variation resulting in an enzootic area of raccoon rabies primarily in southeastern Alabama may be due to landscape features that hinder the movement of raccoons (i.e., gene flow) among different locations. We used 11 raccoon-specific microsatellite markers to obtain individual genotypes to examine gene flow among areas that were rabies free, enzootic with rabies, or had only sporadic reports of the disease. Samples from 70 individuals were collected from 5 sampling localities in 3 counties. The landscape feature data were collected from geographic information system (GIS) data. We inferred gene flow by estimating FST and by using Bayesian tests to identify genetic clusters. Estimates of pairwise FST indicated genetic differentiation and restricted gene flow between some sites, and an uneven distribution of genetic clusters was observed. Of the landscape features examined (i.e., land cover, elevation, slope, roads, and hydrology), only land cover had an association with genetic differentiation, suggesting this landscape variable may affect gene flow among raccoon populations and thus the spread of raccoon variant of rabies in Alabama.

Contribution and perspectives of quantitative genetics to plant breeding in Brazil

The purpose of this article is to show how quantitative genetics has contributed to the huge genetic progress obtained in plant breeding in Brazil in the last forty years. The information obtained through quantitative genetics has given Brazilian breeders the possibility of responding to innumerable questions in their work in a much more informative way, such as the use or not of hybrid cultivars, which segregating population to use, which breeding method to employ, alternatives for improving the efficiency of selection programs, and how to handle the data of progeny and/or cultivars evaluations to identify the most stable ones and thus improve recommendations.

Is missing maxillary lateral incisor in complete cleft lip and palate a product of genetics or local environment?

Objective: To test the null hypothesis: Subjects with isolated complete unilateral cleft lip and palate (UCLP) show no differences in overall frequency of tooth agenesis (hypodontia), comparing a subsample with cleft-side maxillary lateral incisor (MxI2) agenesis to a subsample without cleftside MxI2 agenesis. Findings could clarify the origins of cleft-side MxI2 agenesis. Materials and Methods: Tooth agenesis was identified from dental radiographs of 141 subjects with UCLP. The UCLP cohort was segregated into four categories according to the status and location of MxI2 in the region of the unilateral cleft: group M: subjects with one tooth, located on the mesial side of the alveolar cleft; group D: subjects with one tooth, located on the distal side of the alveolar cleft; group MD: subjects with two teeth present, one mesial and one distal to the cleft; and group ABS: subjects with lateral incisor absent (agenesis) in the cleft area. Results: The null hypothesis was rejected. Among UCLP subjects, there was a twofold increase (P < .0008) in overall frequency of tooth agenesis outside the cleft region in a subsample with cleftside MxI2 agenesis (ABS), compared to a subsample presenting with no agenesis of the cleft-side MxI2 (M+D+MD). Conclusions: Cleft-side MxI2 agenesis in CLP subjects appears to be largely a genetically controlled anomaly associated with cleft development, rather than a collateral environmental consequence of the adjacent cleft defect, since increased hypodontia involving multiple missing teeth observed remote from a cleft clearly has a significant genetic basis. (Angle Orthod. 2012;82:959-963.)

Mercury-induced autoimmunity : Genetics and immunoregulation

The existence of immune self-tolerance allows the immune system to mount responses against infectious agents, but not against self-molecular constitutes. Although self-tolerance is a robust phenomenon, in some individuals as well as in experimental models, the self-tolerance breaks down and as a result, a self-destructive autoimmune disease emerges. The underlying mechanisms for the development of autoimmune diseases are not known, but genetic, environmental and immunological factors are suggested to be involved. In this thesis, we used murine mercury-induced autoimmunity to test this suggestion. In susceptible mice mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of IgG1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. In contrast, in resistant DBA/2 (H-2d) mice, none of these characteristics develop after exposure to mercury. By crossing and backcrossing mercury-resistant DBA/2 mice to mercury susceptible strains, we found that the resistance was inherited as a dominant trait in F1 hybrids and that one gene or a cluster of genes located in the H-2 loci determined the resistance to ANolA production, whereas resistance to the other characteristics was found to be controlled by two or three non-H-2 genes. We further put forward the “cryptic peptide hypothesis” to investigate whether mercury and another xenobiotic metal use similar pathway(s) to induce the H-2 linked production of ANolA. We found that while mercury stimulated ANolA synthesis in all H-2 susceptible (H-2s, H-2q and H-2f) mouse strains, silver induced only ANolA responses in H-2s and H-2q mice, but not in H-2f mice. Further studies showed that the resistance to silver-induced ANolA production in H-2f mice was inherited as a dominant trait. We next tested the proposition that mercury induces more adverse immunological effects in mouse strains, which are genetically prone to develop autoimmune diseases, using tight-skin 1 mice, an animal model for human Scleroderma. It was found that in this strain, mercury induced a strong immune activation with autoimmune characteristics, but did not accelerate the development of dermal fibrosis, a characteristic in Tsk/1 mice. Finally we addressed the Th1/Th2 cross-regulation paradigm by examining if a Th1-type of response could interact with a Th2-type of response if simultaneous induced in susceptible mice. Our findings demonstrated that mercury-induced autoimmunity (Th2-type) and collagen-induced arthritis (CIA) (Th1-type) can interact in a synergistic, antagonistic or additive fashion, depending on at which stage of CIA mercury is administered.

The Integrated European Project "GEHA - GEnetics of Healthy Aging": recruitment, health status assessment and survival of the Italian 90+ sibpairs

The present study is part of the EU Integrated Project “GEHA – Genetics of Healthy Aging” (Franceschi C et al., Ann N Y Acad Sci. 1100: 21-45, 2007), whose aim is to identify genes involved in healthy aging and longevity, which allow individuals to survive to advanced age in good cognitive and physical function and in the absence of major age-related diseases. Aims The major aims of this thesis were the following: 1. to outline the recruitment procedure of 90+ Italian siblings performed by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The procedures related to the following items necessary to perform the study were described and commented: identification of the eligible area for recruitment, demographic aspects related to the need of getting census lists of 90+siblings, mail and phone contact with 90+ subjects and their families, bioethics aspects of the whole procedure, standardization of the recruitment methodology and set-up of a detailed flow chart to be followed by the European recruitment centres (obtainment of the informed consent form, anonimization of data by using a special code, how to perform the interview, how to collect the blood, how to enter data in the GEHA Phenotypic Data Base hosted at Odense). 2. to provide an overview of the phenotypic characteristics of 90+ Italian siblings recruited by the recruiting units of the University of Bologna (UNIBO) and Rome (ISS). The following items were addressed: socio-demographic characteristics, health status, cognitive assessment, physical conditions (handgrip strength test, chair-stand test, physical ability including ADL, vision and hearing ability, movement ability and doing light housework), life-style information (smoking and drinking habits) and subjective well-being (attitude towards life). Moreover, haematological parameters collected in the 90+ sibpairs as optional parameters by the Bologna and Rome recruiting units were used for a more comprehensive evaluation of the results obtained using the above mentioned phenotypic characteristics reported in the GEHA questionnaire. 3. to assess 90+ Italian siblings as far as their health/functional status is concerned on the basis of three classification methods proposed in previous studies on centenarians, which are based on: • actual functional capabilities (ADL, SMMSE, visual and hearing abilities) (Gondo et al., J Gerontol. 61A (3): 305-310, 2006); • actual functional capabilities and morbidity (ADL, ability to walk, SMMSE, presence of cancer, ictus, renal failure, anaemia, and liver diseases) (Franceschi et al., Aging Clin Exp Res, 12:77-84, 2000); • retrospectively collected data about past history of morbidity and age of disease onset (hypertension, heart disease, diabetes, stroke, cancer, osteopororis, neurological diseases, chronic obstructive pulmonary disease and ocular diseases) (Evert et al., J Gerontol A Biol Sci Med Sci. 58A (3): 232-237, 2003). Firstly these available models to define the health status of long-living subjects were applied to the sample and, since the classifications by Gondo and Franceschi are both based on the present functional status, they were compared in order to better recognize the healthy aging phenotype and to identify the best group of 90+ subjects out of the entire studied population. 4. to investigate the concordance of health and functional status among 90+ siblings in order to divide sibpairs in three categories: the best (both sibs are in good shape), the worst (both sibs are in bad shape) and an intermediate group (one sib is in good shape and the other is in bad shape). Moreover, the evaluation wanted to discover which variables are concordant among siblings; thus, concordant variables could be considered as familiar variables (determined by the environment or by genetics). 5. to perform a survival analysis by using mortality data at 1st January 2009 from the follow-up as the main outcome and selected functional and clinical parameters as explanatory variables. Methods A total of 765 90+ Italian subjects recruited by UNIBO (549 90+ siblings, belonging to 258 families) and ISS (216 90+ siblings, belonging to 106 families) recruiting units are included in the analysis. Each subject was interviewed according to a standardized questionnaire, comprising extensively utilized questions that have been validated in previous European studies on elderly subjects and covering demographic information, life style, living conditions, cognitive status (SMMSE), mood, health status and anthropometric measurements. Moreover, subjects were asked to perform some physical tests (Hand Grip Strength test and Chair Standing test) and a sample of about 24 mL of blood was collected and then processed according to a common protocol for the preparation and storage of DNA aliquots. Results From the analysis the main findings are the following: - a standardized protocol to assess cognitive status, physical performances and health status of European nonagenarian subjects was set up, in respect to ethical requirements, and it is available as a reference for other studies in this field; - GEHA families are enriched in long-living members and extreme survival, and represent an appropriate model for the identification of genes involved in healthy aging and longevity; - two simplified sets of criteria to classify 90+ sibling according to their health status were proposed, as operational tools for distinguishing healthy from non healthy subjects; - cognitive and functional parameters have a major role in categorizing 90+ siblings for the health status; - parameters such as education and good physical abilities (500 metres walking ability, going up and down the stairs ability, high scores at hand grip and chair stand tests) are associated with a good health status (defined as “cognitive unimpairment and absence of disability”); - male nonagenarians show a more homogeneous phenotype than females, and, though far fewer in number, tend to be healthier than females; - in males the good health status is not protective for survival, confirming the male-female health survival paradox; - survival after age 90 was dependent mainly on intact cognitive status and absence of functional disabilities; - haemoglobin and creatinine levels are both associated with longevity; - the most concordant items among 90+ siblings are related to the functional status, indicating that they contain a familiar component. It is still to be investigated at what level this familiar component is determined by genetics or by environment or by the interaction between genetics, environment and chance (and at what level). Conclusions In conclusion, we could state that this study, in accordance with the main objectives of the whole GEHA project, represents one of the first attempt to identify the biological and non biological determinants of successful/unsuccessful aging and longevity. Here, the analysis was performed on 90+ siblings recruited in Northern and Central Italy and it could be used as a reference for others studies in this field on Italian population. Moreover, it contributed to the definition of “successful” and “unsuccessful” aging and categorising a very large cohort of our most elderly subjects into “successful” and “unsuccessful” groups provided an unrivalled opportunity to detect some of the basic genetic/molecular mechanisms which underpin good health as opposed to chronic disability. Discoveries in the topic of the biological determinants of healthy aging represent a real possibility to identify new markers to be utilized for the identification of subgroups of old European citizens having a higher risk to develop age-related diseases and disabilities and to direct major preventive medicine strategies for the new epidemic of chronic disease in the 21st century.

Ecological genetics and conservation genomics of wolf (Canis lupus)

In the present work, we apply both traditional and Next Generation Sequencing (NGS) tools to investigate some of the most important adaptive traits of wolves (Canis lupus). In the first part, we analyze the variability of three Major Histocompatibility Complex (MHC) class II genes in the Italian wolf population, also studying their possible role in mating choice and their influence on fitness traits. In the second section, as part of a larger canid genome project, we will exploit NGS data to investigate the transcript-level differences between the wolf and the dog genome that can be correlated to domestication.

Novel tools for conservation genetics in marine fish: population structure and evolution of the European hake (Merluccius merluccius) inferred by SNP variation and applications to traceability

The research presented in my PhD thesis is part of a wider European project, FishPopTrace, focused on traceability of fish populations and products. My work was aimed at developing and analyzing novel genetic tools for a widely distributed marine fish species, the European hake (Merluccius merluccius), in order to investigate population genetic structure and explore potential applications to traceability scenarios. A total of 395 SNPs (Single Nucleotide Polymorphisms) were discovered from a massive collection of Expressed Sequence Tags, obtained by high-throughput sequencing, and validated on 19 geographic samples from Atlantic and Mediterranean. Genome-scan approaches were applied to identify polymorphisms on genes potentially under divergent selection (outlier SNPs), showing higher genetic differentiation among populations respect to the average observed across loci. Comparative analysis on population structure were carried out on putative neutral and outlier loci at wide (Atlantic and Mediterranean samples) and regional (samples within each basin) spatial scales, to disentangle the effects of demographic and adaptive evolutionary forces on European hake populations genetic structure. Results demonstrated the potential of outlier loci to unveil fine scale genetic structure, possibly identifying locally adapted populations, despite the weak signal showed from putative neutral SNPs. The application of outlier SNPs within the framework of fishery resources management was also explored. A minimum panel of SNP markers showing maximum discriminatory power was selected and applied to a traceability scenario aiming at identifying the basin (and hence the stock) of origin, Atlantic or Mediterranean, of individual fish. This case study illustrates how molecular analytical technologies have operational potential in real-world contexts, and more specifically, potential to support fisheries control and enforcement and fish and fish product traceability.

Epidemiology and population genetics of Podosphaera fusca and Golovinomyces orontii, causal agents of cucurbit powdery mildew

In 2010, 2011 and 2012 growing seasons, the occurrence of the ascomycetes Podosphaera fusca and Golovinomyces orontii, causal agents of powdery mildew disease, was monitored on cultivated cucurbits located in Bologna and Mantua provinces to determine the epidemiology of the species. To identify the pathogens, both morphological and molecular identifications were performed on infected leaf samples and a Multiplex-PCR was performed to identify the mating type genes of P. fusca isolates. The investigations indicated a temporal succession of the two species with the earlier infections caused by G. orontii, that seems to be the predominant species till the middle of July when it progressively disappears and P. fusca becomes the main species infecting cucurbits till the end of October. The temporal variation is likely due to the different overwintering strategies of the two species instead of climatic conditions. Only chasmothecia of P. fusca were recorded and mating type alleles ratio tended to be 1:1. Considering that only chasmothecia of P. fusca were found, molecular-genetic analysis were carried out to find some evidence of recombination within this species by MLST and AFLP methods. Surprisingly, no variations were observed within isolates for the 8 MLST markers used. According to this result, AFLP analysis showed a high similarity within isolates, with SM similarity coefficient ranging between 0.91-1.00 and also, sequencing of 12 polymorphic bands revealed identity to some gene involved in mutation and selection. The results suggest that populations of P. fusca are likely to be a clonal population with some differences among isolates probably due to agricultural practices such as fungicides treatments and cultivated hosts. Therefore, asexual reproduction, producing a lot of fungal biomass that can be easily transported by wind, is the most common and useful way to the spread and colonization of the pathogen.