Resolving cryptic species complexes of major tephritid pests

An FAO/IAEA Co-ordinated Research Project (CRP) on “Resolution of Cryptic Species Complexes of Tephritid Pests to Overcome Constraints to SIT Application and International Trade” was conducted from 2010 to 2015. As captured in the CRP title, the objective was to undertake targeted research into the systematics and diagnostics of taxonomically challenging fruit fly groups of economic importance. The scientific output was the accurate alignment of biological species with taxonomic names; which led to the applied outcome of assisting FAO and IAEA Member States in overcoming technical constraints to the application of the Sterile Insect Technique (SIT) against pest fruit flies and the facilitation of international agricultural trade. Close to 50 researchers from over 20 countries participated in the CRP, using coordinated, multidisciplinary research to address, within an integrative taxonomic framework, cryptic species complexes of major tephritid pests. The following progress was made for the four complexes selected and studied: Anastrepha fraterculus complex – Eight morphotypes and their geographic and ecological distributions in Latin America were defined. The morphotypes can be considered as distinct biological species on the basis of differences in karyotype, sexual incompatibility, post-mating isolation, cuticular hydrocarbon, pheromone, and molecular analyses. Discriminative taxonomic tools using linear and geometric morphometrics of both adult and larval morphology were developed for this complex. Bactrocera dorsalis complex – Based on genetic, cytogenetic, pheromonal, morphometric, and behavioural data, which showed no or only minor variation between the Asian/African pest fruit flies Bactrocera dorsalis, B. papayae, B. philippinensis and B. invadens, the latter three species were synonymized with B. dorsalis. Of the five target pest taxa studied, only B. dorsalis and B. carambolae remain as scientifically valid names. Molecular and pheromone markers are now available to distinguish B. dorsalis from B. carambolae. Ceratitis FAR Complex (C. fasciventris, C. anonae, C. rosa) – Morphology, morphometry, genetic, genomic, pheromone, cuticular hydrocarbon, ecology, behaviour, and developmental physiology data provide evidence for the existence of five different entities within this fruit fly complex from the African region. These are currently recognised as Ceratitis anonae, C. fasciventris (F1 and F2), C. rosa and a new species related to C. rosa (R2). The biological limits within C. fasciventris (i.e. F1 and F2) are not fully resolved. Microsatellites markers and morphological identification tools for the adult males of the five different FAR entities were developed based on male leg structures. Zeugodacus cucurbitae (formerly Bactrocera (Zeugodacus) cucurbitae) – Genetic variability was studied among melon fly populations throughout its geographic range in Africa and the Asia/Pacific region and found to be limited. Cross-mating studies indicated no incompatibility or sexual isolation. Host preference and genetic studies showed no evidence for the existence of host races. It was concluded that the melon fly does not represent a cryptic species complex, neither with regard to geographic distribution nor to host range. Nevertheless, the higher taxonomic classification under which this species had been placed, by the time the CRP was started, was found to be paraphyletic; as a result the subgenus Zeugodacus was elevated to genus level.

Effects Of 5' Flanking Sequences And Changes In The 5' Internal Control Region On The Transcription Of Rice Transfer-Rna Gcc Cly Gene

A stretch of 71 nucleotides in a 1.2 kilobase pair Pst I fragment of rice DNA was identified as tRNA~ gene by hybridization and nucleotide sequence analyses. The hybridization of genomic DNA with the tRNA gene showed that there are about 10 glycine tRNA genes per diploid rice genome. The 3' and 5' internal control regions, where RNA polymerase III and transcription factors bind, were found to be present in the coding sequence. The gene was transcribed into a 4S product in an yeast cell-free extract. The substitution of 5' internal control region with analogous sequences from either M13mpl9 or M13mpl8 DNA did not affect the transcription of the gene in vitro. The changes in three highly conserved nucleotides in the consensus 5' internal control region (RGYNNARYGG; R = purine, Y = pyrimidine, N = any nucleotide) did not affect transcription showing that these nucleotides are not essential for promotion of transcription. There were two 16 base pair repeats, 'TGTTTGTTTCAGCTTA' at - 130 and - 375 positions upstream from the start of the gene. Deletion of 5' flanking sequences including the 16 base pair repeat at - 375 showed increased transcription indicating that these sequences negatively modulate the expression of the gene.

A consensus genetic map of sorghum that integrates multiple component maps and high-throughput Diversity Array Technology (DArT) markers

Background: Sorghum genome mapping based on DNA markers began in the early 1990s and numerous genetic linkage maps of sorghum have been published in the last decade, based initially on RFLP markers with more recent maps including AFLPs and SSRs and very recently, Diversity Array Technology (DArT) markers. It is essential to integrate the rapidly growing body of genetic linkage data produced through DArT with the multiple genetic linkage maps for sorghum generated through other marker technologies. Here, we report on the colinearity of six independent sorghum component maps and on the integration of these component maps into a single reference resource that contains commonly utilized SSRs, AFLPs, and high-throughput DArT markers. Results: The six component maps were constructed using the MultiPoint software. The lengths of the resulting maps varied between 910 and 1528 cM. The order of the 498 markers that segregated in more than one population was highly consistent between the six individual mapping data sets. The framework consensus map was constructed using a "Neighbours" approach and contained 251 integrated bridge markers on the 10 sorghum chromosomes spanning 1355.4 cM with an average density of one marker every 5.4 cM, and were used for the projection of the remaining markers. In total, the sorghum consensus map consisted of a total of 1997 markers mapped to 2029 unique loci ( 1190 DArT loci and 839 other loci) spanning 1603.5 cM and with an average marker density of 1 marker/0.79 cM. In addition, 35 multicopy markers were identified. On average, each chromosome on the consensus map contained 203 markers of which 58.6% were DArT markers. Non-random patterns of DNA marker distribution were observed, with some clear marker-dense regions and some marker-rare regions. Conclusion: The final consensus map has allowed us to map a larger number of markers than possible in any individual map, to obtain a more complete coverage of the sorghum genome and to fill a number of gaps on individual maps. In addition to overall general consistency of marker order across individual component maps, good agreement in overall distances between common marker pairs across the component maps used in this study was determined, using a difference ratio calculation. The obtained consensus map can be used as a reference resource for genetic studies in different genetic backgrounds, in addition to providing a framework for transferring genetic information between different marker technologies and for integrating DArT markers with other genomic resources. DArT markers represent an affordable, high throughput marker system with great utility in molecular breeding programs, especially in crops such as sorghum where SNP arrays are not publicly available.

p53 and DNA damage checkpoint responses in the human prostate

Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer mortality in men. In 2004, 5237 new cases were diagnosed and altogether 25 664 men suffered from prostate cancer in Finland (Suomen Syöpärekisteri). Although extensively investigated, we still have a very rudimentary understanding of the molecular mechanisms leading to the frequent transformation of the prostate epithelium. Prostate cancer is characterized by several unique features including the multifocal origin of tumors and extreme resistance to chemotherapy, and new treatment options are therefore urgently needed. The integrity of genomic DNA is constantly challenged by genotoxic insults. Cellular responses to DNA damage involve elegant checkpoint cascades enforcing cell cycle arrest, thus facilitating damage repair, apoptosis or cellular senescence. Cellular DNA damage triggers the activation of tumor suppressor protein p53 and Wee1 kinase which act as executors of the cellular checkpoint responses. These are essential for genomic integrity, and are activated in early stages of tumorigenesis in order to function as barriers against tumor formation. Our work establishes that the primary human prostatic epithelial cells and prostatic epithelium have unexpectedly indulgent checkpoint surveillance. This is evidenced by the absence of inhibitory Tyr15 phosphorylation on Cdk2, lack of p53 response, radioresistant DNA synthesis, lack of G1/S and G2/M phase arrest, and presence of persistent gammaH2AX damage foci. We ascribe the absence of inhibitory Tyr15 phosphorylation to low levels of Wee1A, a tyrosine kinase and negative regulator of cell cycle progression. Ectopic Wee1A kinase restored Cdk2-Tyr15 phosphorylation and efficiently rescued the ionizing radiation-induced checkpoints in the human prostatic epithelial cells. As variability in the DNA damage responses has been shown to underlie susceptibility to cancer, our results imply that a suboptimal checkpoint arrest may greatly increase the accumulation of genetic lesions in the prostate epithelia. We also show that small molecules can restore p53 function in prostatic epithelial cells and may serve as a paradigm for the development of future therapeutic agents for the treatment of prostate cancer We hypothesize that the prostate has evolved to activate the damage surveillance pathways and molecules involved in these pathways only to certain stresses in extreme circumstances. In doing so, this organ inadvertently made itself vulnerable to genotoxic stress, which may have implications in malignant transformation. Recognition of the limited activity of p53 and Wee1 in the prostate could drive mechanism-based discovery of preventative and therapeutic agents.

Genetic analysis for a shared biological basis between migraine and coronary artery disease

Objective: To apply genetic analysis of genome-wide association data to study the extent and nature of a shared biological basis between migraine and coronary artery disease (CAD). Methods: Four separate methods for cross-phenotype genetic analysis were applied on data from 2 large-scale genome-wide association studies of migraine (19,981 cases, 56,667 controls) and CAD (21,076 cases, 63,014 controls). The first 2 methods quantified the extent of overlapping risk variants and assessed the load of CAD risk loci in migraineurs. Genomic regions of shared risk were then identified by analysis of covariance patterns between the 2 phenotypes and by querying known genome-wide significant loci. Results: We found a significant overlap of genetic risk loci for migraine and CAD. When stratified by migraine subtype, this was limited to migraine without aura, and the overlap was protective in that patients with migraine had a lower load of CAD risk alleles than controls. Genes indicated by 16 shared risk loci point to mechanisms with potential roles in migraine pathogenesis and CAD, including endothelial dysfunction (PHACTR1) and insulin homeostasis (GIP). Conclusions: The results suggest that shared biological processes contribute to risk of migraine and CAD, but surprisingly this commonality is restricted to migraine without aura and the impact is in opposite directions. Understanding the mechanisms underlying these processes and their opposite relationship to migraine and CAD may improve our understanding of both disorders.

Shared genetic basis for migraine and ischemic stroke: A genome-wide analysis of common variants

OBJECTIVE To quantify genetic overlap between migraine and ischemic stroke (IS) with respect to common genetic variation. METHODS We applied 4 different approaches to large-scale meta-analyses of genome-wide data on migraine (23,285 cases and 95,425 controls) and IS (12,389 cases and 62,004 controls). First, we queried known genome-wide significant loci for both disorders, looking for potential overlap of signals. We then analyzed the overall shared genetic load using polygenic scores and estimated the genetic correlation between disease subtypes using data derived from these models. We further interrogated genomic regions of shared risk using analysis of covariance patterns between the 2 phenotypes using cross-phenotype spatial mapping. RESULTS We found substantial genetic overlap between migraine and IS using all 4 approaches. Migraine without aura (MO) showed much stronger overlap with IS and its subtypes than migraine with aura (MA). The strongest overlap existed between MO and large artery stroke (LAS; p = 6.4 x 10(-28) for the LAS polygenic score in MO) and between MO and cardioembolic stroke (CE; p = 2.7 x 10(-20) for the CE score in MO). CONCLUSIONS Our findings indicate shared genetic susceptibility to migraine and IS, with a particularly strong overlap between MO and both LAS and CE pointing towards shared mechanisms. Our observations on MA are consistent with a limited role of common genetic variants in this subtype.

Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine

Migraine is a debilitating neurological disorder affecting around 1 in 7 people worldwide, but its molecular mechanisms remain poorly understood. Some debate exists over whether migraine is a disease of vascular dysfunction, or a result of neuronal dysfunction with secondary vascular changes. Genome-wide association (GWA) studies have thus far identified 13 independent loci associated with migraine. To identify new susceptibility loci, we performed the largest genetic study of migraine to date, comprising 59,674 cases and 316,078 controls from 22 GWA studies. We identified 45 independent single nucleotide polymorphisms (SNPs) significantly associated with migraine risk (P < 5 x 10-8) that map to 38 distinct genomic loci, including 28 loci not previously reported and the first locus identified on chromosome X. Furthermore, a subset analysis for migraine without aura (MO) identified seven of the same loci as from the full sample, whereas no loci reached genome-wide significance in the migraine with aura (MA) subset. In subsequent computational analyzes, the identified loci showed enrichment for genes expressed in vascular and smooth muscle tissues, consistent with a predominant theory of migraine that highlights vascular etiologies.

Inference of the genetic architecture underlying BMI and height with the use of 20,240 sibling pairs

Evidence that complex traits are highly polygenic has been presented by population-based genome-wide association studies (GWASs) through the identification of many significant variants, as well as by family-based de novo sequencing studies indicating that several traits have a large mutational target size. Here, using a third study design, we show results consistent with extreme polygenicity for body mass index (BMI) and height. On a sample of 20,240 siblings (from 9,570 nuclear families), we used a within-family method to obtain narrow-sense heritability estimates of 0.42 (SE = 0.17, p = 0.01) and 0.69 (SE = 0.14, p = 6 x 10(-)(7)) for BMI and height, respectively, after adjusting for covariates. The genomic inflation factors from locus-specific linkage analysis were 1.69 (SE = 0.21, p = 0.04) for BMI and 2.18 (SE = 0.21, p = 2 x 10(-10)) for height. This inflation is free of confounding and congruent with polygenicity, consistent with observations of ever-increasing genomic-inflation factors from GWASs with large sample sizes, implying that those signals are due to true genetic signals across the genome rather than population stratification. We also demonstrate that the distribution of the observed test statistics is consistent with both rare and common variants underlying a polygenic architecture and that previous reports of linkage signals in complex traits are probably a consequence of polygenic architecture rather than the segregation of variants with large effects. The convergent empirical evidence from GWASs, de novo studies, and within-family segregation implies that family-based sequencing studies for complex traits require very large sample sizes because the effects of causal variants are small on average.

Androgenetic alopecia: Identification of four genetic risk loci and evidence for the contribution of WNT signaling to its etiology

The pathogenesis of androgenetic alopecia (AGA, male-pattern baldness) is driven by androgens, and genetic predisposition is the major prerequisite. Candidate gene and genome-wide association studies have reported that single-nucleotide polymorphisms (SNPs) at eight different genomic loci are associated with AGA development. However, a significant fraction of the overall heritable risk still awaits identification. Furthermore, the understanding of the pathophysiology of AGA is incomplete, and each newly associated locus may provide novel insights into contributing biological pathways. The aim of this study was to identify unknown AGA risk loci by replicating SNPs at the 12 genomic loci that showed suggestive association (5 x 10(-8)

Pharmacogenetic Variation at CYP2D6, CYP2C9, and CYP2C19: Population Genetic and Forensic Aspects

Pharmacogenetics deals with genetically determined variation in drug response. In this context, three phase I drug-metabolizing enzymes, CYP2D6, CYP2C9, and CYP2C19, have a central role, affecting the metabolism of about 20-30% of clinically used drugs. Since genes coding for these enzymes in human populations exhibit high genetic polymorphism, they are of major pharmacogenetic importance. The aims of this study were to develop new genotyping methods for CYP2D6, CYP2C9, and CYP2C19 that would cover the most important genetic variants altering the enzyme activity, and, for the first time, to describe the distribution of genetic variation at these loci on global and microgeographic scales. In addition, pharmacogenetics was applied to a postmortem forensic setting to elucidate the role of genetic variation in drug intoxications, focusing mainly on cases related to tricyclic antidepressants, which are commonly involved in fatal drug poisonings in Finland. Genetic variability data were obtained by genotyping new population samples by the methods developed based on PCR and multiplex single-nucleotide primer extension reaction, as well as by collecting data from the literature. Data consisted of 138, 129, and 146 population samples for CYP2D6, CYP2C9, and CYP2C19, respectively. In addition, over 200 postmortem forensic cases were examined with respect to drug and metabolite concentrations and genotypic variation at CYP2D6 and CYP2C19. The distribution of genetic variation within and among human populations was analyzed by descriptive statistics and variance analysis and by correlating the genetic and geographic distances using Mantel tests and spatial autocorrelation. The correlation between phenotypic and genotypic variation in drug metabolism observed in postmortem cases was also analyzed statistically. The genotyping methods developed proved to be informative, technically feasible, and cost-effective. Detailed molecular analysis of CYP2D6 genetic variation in a global survey of human populations revealed that the pattern of variation was similar to those of neutral genomic markers. Most of the CYP2D6 diversity was observed within populations, and the spatial pattern of variation was best described as clinal. On the other hand, genetic variants of CYP2D6, CYP2C9, and CYP2C19 associated with altered enzymatic activity could reach extremely high frequencies in certain geographic regions. Pharmacogenetic variation may also be significantly affected by population-specific demographic histories, as seen within the Finnish population. When pharmacogenetics was applied to a postmortem forensic setting, a correlation between amitriptyline metabolic ratios and genetic variation at CYP2D6 and CYP2C19 was observed in the sample material, even in the presence of confounding factors typical for these cases. In addition, a case of doxepin-related fatal poisoning was shown to be associated with a genetic defect at CYP2D6. Each of the genes studied showed a distinct variation pattern in human populations and high frequencies of altered activity variants, which may reflect the neutral evolution and/or selective pressures caused by dietary or environmental exposure. The results are relevant also from the clinical point of view since the genetic variation at CYP2D6, CYP2C9, and CYP2C19 already has a range of clinical applications, e.g. in cancer treatment and oral anticoagulation therapy. This study revealed that pharmacogenetics may also contribute valuable information to the medicolegal investigation of sudden, unexpected deaths.

Occurrence, divergence and evolution of intrinsic terminators across Eubacteria

In Escherichia coli, the canonical intrinsic terminator of transcription includes a palindrome followed by a U-trail on the transcript. The apparent underrepresentation of such terminators in eubacterial genomes led us to develop a rapid and accurate algorithm, GeSTer, to predict putative intrinsic terminators. Now, we have analyzed 378 genome sequences with an improved version of GeSTer. Our results indicate that the canonical E. coli type terminators are not overwhelmingly abundant in eubacteria. The atypical structures, having stem-loop structures but lacking ‘U’ trail, occur downstream of genes in all the analyzed genomes but different phyla show conserved preference for different types of terminators. This propensity correlates with genomic GC content and presence of the factor, Rho. 60–70% of identified terminators in all the genomes show “optimized” stem-length and ΔG. These results provide evidence that eubacteria extensively rely on the mechanism of intrinsic termination, with a considerable divergence in their structure, positioning and prevalence. The software and detailed results for individual genomes are freely available on request

The application of DArTseq technology to pineapple

DArTseq technology is potentially the most appropriate system to discover hundreds of polymorphic genomic loci, scoring thousands of unique genomic-wide DNA fragments in one single experiment, without requiring existing DNA sequence information. The DArT complexity reduction approach in combination with Illumina short read sequencing (Hiseq2000) was applied. To test the application of DArTseq technology in pineapple, a reference population of 13 Ananas genotypes from primitive wild accessions to modern cultivars was used. In a comparison of 3 systems, the combination of restriction enzymes PstI and MseI performed the best producing 18,900 DArT markers and close to 20,000 SNPs. Based on these markers genetic relationships between the samples were identified and a dendrogram was generated. The topography of the tree corresponds with our understanding of the genetic relationships between the genotypes. Importantly, the replicated samples of all genotypes have a dissimilarity of close to 0.0 and occupy the same positions on the tree, confirming high reproducibility of the markers detected. Eventually it is planned that molecular markers will be identified that are associated with resistance to Phytophthora cinnamomi (Pc), the most economically important pathogen of pineapple in Australia, as genetic resistance is known to exist within the Ananas. Marker assisted selection can then be utilized in a pineapple breeding program to develop cultivars resistant to Pc.

CRC50150 Targeting mechanisms of phosphine resistance in stored grain pests

The objectives of this projects are: 1)To ensure the identification of genomic DNA markers for phosphine resistance in Rhyzopertha dominica and Tribolium castaneum; 2) To determine gene function of identified phosphine resistance genes in Rhyzopertha dominica and Tribolium castaneum; and 3) Predict future problems by characterising international resistances using our genes as a starting point to determine strong resistance can get by determining similarities with Australia.

Examination of Genetic Components Affecting Human Obesity-Related Quantitative Traits

Obesity increases the risk for several conditions, including type 2 diabetes mellitus, cardiovascular disease, hypertension, osteoarthirits and certain types of cancer. Twin- and family studies have shown that there is a major genetic component in the determination of body mass. In recent years several technological and scientific advance have been made in obesity research. For instance, novel replicated loci have been revealed by a number of genome wide association studies. This thesis aimed to investigate the association of genetic factors and obesity-related quantitative traits. The first study investigated the role of the lactase gene in anthropometric traits. We genetically defined lactose persistence by genotyping 31 720 individuals of European descent. We found that lactase persistence was significantly correlated with weight and body mass index but not with height. In the second study we performed the largest whole genome linkage scan for body mass index to date. The sample consisted of 4401 twin families and 10 535 individuals from six European countries. We found supporting evidence for two loci (3q29 and 7q36). We observed that the heritability estimate increased substantially when additional family members were removed from the analyses, which suggests reduced environmental variance in the twin sample. In the third study we assessed metabonomic, transcriptomic and genomic variation in a Finnish population cohort of 518 individuals. We formed gene expression networks to portray pathways and showed that a set of highly correlated genes of an inflammatory pathway associated with 80 serum metabolites (of 134 quantified measures). Strong association was found, for example, with several lipoprotein subclasses. We inferred causality by using genetic variation as anchors. The expression of the network genes was found to be dependent on the circulatory metabolite concentrations.

Molecular mechanisms of cancer predisposition in HNPCC/Lynch syndrome

Hereditary non-polyposis colorectal carcinoma (HNPCC; Lynch syndrome) is among the most common hereditary cancers in man and a model of cancers arising through deficient DNA mismatch repair (MMR). It is inherited in a dominant manner with predisposing germline mutations in the MMR genes, mainly MLH1, MSH2, MSH6 and PMS2. Both copies of the MMR gene need to be inactivated for cancer development. Since Lynch syndrome family members are born with one defective copy of one of the MMR genes in their germline, they only need to acquire a so called second hit to inactivate the MMR gene. Hence, they usually develop cancer at an early age. MMR gene inactivation leads to accumulation of mutations particularly in short repeat tracts, known as microsatellites, causing microsatellite instability (MSI). MSI is the hallmark of Lynch syndrome tumors, but is present in approximately 15% of sporadic tumors as well. There are several possible mechanisms of somatic inactivation (i.e. the second hit ) of MMR genes, for instance deletion of the wild-type copy, leading to loss of heterozygosity (LOH), methylation of promoter regions necessary for gene transcription, or mitotic recombination or gene conversion. In the Lynch syndrome tumors carrying germline mutations in the MMR gene, LOH was found to be the most frequent mechanism of somatic inactivation in the present study. We also studied MLH1/MSH2 deletion carriers and found that somatic mutations identical to the ones in the germline occurred frequently in colorectal cancers and were also present in extracolonic Lynch syndrome-associated tumors. Chromosome-specific marker analysis implied that gene conversion, rather than mitotic recombination or deletion of the respective gene locus accounted for wild-type inactivation. Lynch syndrome patients are predisposed to certain types of cancers, the most common ones being colorectal, endometrial and gastric cancer. Gastric cancer and uroepithelial tumors of bladder and ureter were observed to be true Lynch syndrome tumors with MMR deficiency as the driving force of tumorigenesis. Brain tumors and kidney carcinoma, on the other hand, were mostly MSS, implying the possibility of alternative routes of tumor development. These results present possible implications in clinical cancer surveillance. In about one-third of families suspected of Lynch syndrome, mutations in MMR genes are not found, and we therefore looked for alternative mechanisms of predisposition. According to our results, large genomic deletions, mainly in MSH2, and germline epimutations in MLH1, together explain a significant fraction of point mutation-negative families suspected of Lynch syndrome and are associated with characteristic clinical and family features. Our findings have important implications in the diagnosis and management of Lynch syndrome families.