Biodiversity assessment using markers for ecologically important traits

Most studies of genetic variation within species to date are based on random markers. However, how well this correlates with quantitative variation is contentious. Yet, functional, or'ecotypic' variation in quantitative traits determines the ecological niche of a species, its future evolutionary potential, and, for livestock, crops and their wild relatives, their usefulness as a genetic resource for breeding. But nowadays we can also assess genetic diversity using markers directly targeted at specific genes or gene families. Such gene-targeted, multilocus profiles of markers can contribute to ex-situ management of genetic resources, ecological studies of diversity, and conservation of endangered species.     

Patterns of genetic diversity in the Andean gene pool of common bean reveal a candidate domestication gene

Most studies on the genetic diversity of common bean (Phaseolus vulgaris L.) have focussed on accessions from the Mesoamerican gene pool compared to the Andean gene pool. A deeper knowledge of the genetic structure of Argentinian germplasm would enable researchers to determine how the Andean domestication event affected patterns of genetic diversity in domesticated beans and to identify candidates for genes targeted by selection during the evolution of the cultivated common bean. A collection of 116 wild and domesticated accessions representing the diversity of the Andean bean in Argentina was genotyped by means of 114 simple sequence repeat (SSR) markers. Forty-seven Mesoamerican bean accessions and 16 Andean bean accessions representing the diversity of Andean landraces and wild accessions were also included. Using the Bayesian algorithm implemented in the software STRUCTURE we identified five major groups that correspond to Mesoamerican and Argentinian wild accessions and landraces and a group that corresponds to accessions from different Andean and Mesoamerican countries. The neighbour-joining algorithm and principal coordinate clustering analysis confirmed the genetic relationships among accessions observed with the STRUCTURE analysis. Argentinian accessions showed a substantial genetic variation with a considerable number of unique haplotypes and private alleles, suggesting that they may have played an important role in the evolution of the species. The results of statistical analyses aimed at identifying genomic regions with consistent patterns of variation were significant for 35 loci (~20 % of the SSRs used in the Argentinian accessions). One of these loci mapped in or near the genomic region of the glutamate decarboxylase gene. Our data characterize the population structure of the Argentinian germplasm. This information on its diversity will be very valuable for use in introgressing Argentinian genes into commercial varieties because the majority of present-day common bean varieties are of Andean origin.     

VAPSE-based analysis: a two-phased candidate gene approach for elucidating genetic predisposition to complex disorders.

The extraordinary success of linkage analysis in diseases with Mendelian inheritance has not extended readily to the genetics of common complex diseases. VAPSE-based analysis is a type of candidate gene approach that represents an alternative strategy by which genetic mechanisms can be defined despite the presence of substantial genetic heterogeneity. Recent advances in mutation screening and statistical methodology have enhanced substantially the efficiency and power of this approach. The "bread and butter" of VAPSE-based analysis is genotype-to-phenotype searches in large populations with computerized medical records.     

A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli

Bacterial species are internally diverse in genomic and multi-locus gene comparisons. The ecological causes of phenotypic and genotypic diversity within species are far less well understood. Here, we focus on the competitive fitness for growth on nutrients within Escherichia coli, an internally rich species. Competition experiments in nutrient-limited chemostats revealed that members of the ECOR collection exhibited a wide continuum of competitive abilities, with some fitter and some less fit than the lab strain MG1655. We observed an inverse relationship between competitiveness and the resistance of strains to detergent and antibiotic, consistent with the notion that membrane permeability and competitive fitness are linked by a trade-off between self-preservation and nutritional competence (SPANC); high permeability has a postulated cost in antibacterial sensitivity whereas a low permeability has a cost in nutrient affinity. Isolates moved along the markedly nonlinear trade-off curve by mutational adaptation; an ECOR strain sensitive to antibacterials and a good competitor was easily converted by mutation into a mutant with higher resistance but poorer competition in the presence of low antibiotic concentrations. Conversely, a resistant ECOR strain changed into a better competitor after a short period of selection under nutrient limitation. In both directions, mutations can affect porin proteins and outer membrane permeability, as indicated by protein analysis, gene sequencing and an independent assay of outer membrane permeability. The extensive, species-wide diversity of E. coli in ecologically important traits can thus be explained as an evolutionary consequence of a SPANC trade-off driven by antagonistic pleiotropy.     

Molecular analysis of congenital scoliosis: a candidate gene approach

The etiology of congenital scoliosis is largely unknown. The severe vertebral disorder, spondylocostal dysostosis type 1, is associated with a homozygous delta-like 3 (DLL3) mutation. Scoliosis has been observed in a heterozygous DLL3 carrier, raising the possibility of its involvement in congenital scoliosis. We present the first molecular study of congenital scoliosis by analysis of the candidate gene DLL3 and demonstrate one novel missense variant. However, no novel or previously described mutations are present in our cohort, indicating that DLL3 mutations may not be a major cause of congenital scoliosis. Additionally, we have evaluated patients with congenital scoliosis not diagnosed with a known syndrome and identified a significant number of associated renal and cardiac anomalies and familial incidence of idiopathic scoliosis in this group.     

The candidate gene approach in plant genetics: a review

The candidate gene (CG) approach has been applied in plant genetics in the past decade for the characterisation and cloning of Mendelian and quantitative trait loci (QTLs). It constitutes a complementary strategy to map-based cloning and insertional mutagenesis. The goal of this paper is to present an overview of CG analyses in plant genetics. CG analysis is based on the hypothesis that known-function genes (the candidate genes) could correspond to loci controlling traits of interest. CGs refer either to cloned genes presumed to affect a given trait (`functional CGs') or to genes suggested by their close proximity on linkage maps to loci controlling the trait (`positional CGs'). In plant genetics, the most common way to identify a CG is to look for map co-segregation between CGs and loci affecting the trait. Statistical association analyses between molecular polymorphisms of the CG and variation in the trait of interest have also been carried out in a few studies. The final validation of a CG will be provided through physiological analyses, genetic transformation and/or sexual complementation. Theoretical and practical applications of validated CGs in plant genetics and breeding are discussed.     

TargetScreen: An unbiased approach to identify functionally important microRNA targets

We recently identified miR-19 as the critical activity for leukemogenesis within the oncogenic 17~92 cluster of microRNAs.¹ This finding prompted us to test an unbiased method for pinpointing those miR-19 targets may be key to its oncogenic action. Specifically, we used a large-scale short hairpin RNA screen to identify those miR-19 target genes, whose knockdown could reproduce miR-19's effects on lymphocyte transformation. In this way, we found that miR-19 produces a coordinate clampdown on multiple negative regulators of PI3K-related survival signals. These findings have implications for the therapy of miR-19 expressing tumors. They also validate a new strategy for the unbiased identification of functionally important microRNA target genes. Using the example of miR-19 in leukemia, we will discuss some possibilities and limitations of this new approach.     

An extensive candidate gene approach to speciation: diversity,divergence and linkage disequilibrium in candidate pigmentation genes across the European crow hybrid zone

Colouration patterns have an important role in adaptation and speciation. The European crow system, in which all-black carrion crows and grey-coated hooded crows meet in a narrow hybrid zone, is a prominent example. The marked phenotypic difference is maintained by assortative mating in the absence of neutral genetic divergence, suggesting the presence of few pigmentation genes of major effect. We made use of the rich phenotypic and genetic resources in mammals and identified a comprehensive panel of 95 candidate pigmentation genes for birds. Based on functional annotation, we chose a subset of the most promising 37 candidates, for which we developed a marker system that demonstrably works across the avian phylogeny. In total, we sequenced 107 amplicons (∼3 loci per gene, totalling 60 kb) in population samples of crows (n=23 for each taxon). Tajima''s D, Fu''s F_S, DHEW and HKA (Hudson–Kreitman–Aguade) statistics revealed several amplicons that deviated from neutrality; however, none of these showed significantly elevated differentiation between the two taxa. Hence, colour divergence in this system may be mediated by uncharacterized pigmentation genes or regulatory regions outside genes. Alternatively, the observed high population recombination rate (4N_er∼0.03), with overall linkage disequilibrium dropping rapidly within the order of few 100 bp, may compromise the power to detect causal loci with nearby markers. Our results add to the debate as to the utility of candidate gene approaches in relation to genomic features and the genetic architecture of the phenotypic trait in question.     

Characterising genetic diversity and effective population size in one reservoir and two riverine populations of the threatened Macquarie perch

Nine polymorphic microsatellite loci have been used to infer population genetic diversity and structure of the threatened Australian freshwater fish, Macquarie perch, across three tributaries of the Murrumbidgee River in south-eastern Australia. This investigation has revealed a high level of divergence among all three populations, along with contrasting patterns of genetic diversity. The Cotter Reservoir, which is a stronghold population for the species, has typically higher diversity and effective population size than nearby riverine populations. This suggests that the reservoir population is unlikely to have undergone a genetic bottleneck during and following dam construction. Genetic diversity estimates were comparable with one riverine site but were significantly higher than a population sampled from the Queanbeyan River. This comparison revealed significantly less heterozygotes in the Queanbeyan River and lower estimates of effective population size. Options and considerations for stock replenishment of this population are discussed.     

Molecular enumeration of an ecologically important cyanophage in a Laurentian Great Lake

Considerable research has shown that cyanobacteria and the viruses that infect them (cyanophage) are pervasive and diverse in global lake populations. Few studies have seasonally analyzed freshwater systems, and little is known about the bacterial and viral communities that coexist during the harsh winters of the Laurentian Great Lakes. Here, we employed quantitative PCR to estimate the abundance of cyanomyoviruses in this system, using the portal vertex g20 gene as a proxy for cyanophage abundance and to determine the potential ecological relevance of these viruses. Cyanomyoviruses were abundant in both the summer and the winter observations, with up to 3.1 × 10(6) copies of g20 genes ml(-1) found at several stations and depths in both seasons, representing up to 4.6% of the total virus community. Lake Erie was productive during both our observations, with high chlorophyll a concentrations in the summer (up to 10.3 μg liter(-1)) and winter (up to 5.2 μg liter(-1)). Both bacterial and viral abundances were significantly higher during the summer than during the winter (P < 0.05). Summer bacterial abundances ranged from 3.3 × 10(6) to 1.6 × 10(7) ml(-1) while winter abundances ranged between ～3.4 × 10(5) and 1.2 × 10(6) ml(-1). Total virus abundances were high during both months, with summer abundances significantly higher at most stations, ranging from 6.5 × 10(7) to 8.8 × 10(7) ml(-1), and with winter abundances ranging from 3.4 × 10(7) to 6.6 × 10(7) ml(-1). This work confirms that putative cyanomyoviruses are ubiquitous in both summer and winter months in this large freshwater lake system and that they are an abundant component of the virioplankton group.     

Candidate genes and thermal phenotypes: identifying ecologically important genetic variation for thermotolerance in the Australian Drosophila melanogaster cline

Clinal variation in traits often reflects climatic adaptation; in Drosophila melanogaster clinal variation provides an opportunity to link variation in chromosomal inversions, microsatellite loci and various candidate genes to adaptive variation in traits. We undertook association studies with crosses from a single population of D. melanogaster from eastern Australia to investigate the association between genetic markers and traits showing clinal variation. By genotyping parents and phenotyping offspring, we minimized genotyping costs but had the power to detect association between markers and quantitative traits. Consistent with prior studies, we found strong associations between the clinal chromosomal inversion In(3R)Payne and markers within it, as well as among these markers. We also found an association between In(3L)Payne and one marker located within this inversion. Of the five predicted associations between markers and traits, four were detected (increased heat, decreased cold resistance and body size with the heat shock gene hsr-omega S, increased cold resistance with the inversion In(3L)Payne), while one was not detected (heat resistance and the heat shock gene hsp68). In a set of eight exploratory tests, we detected one positive association (between hsp23a and heat resistance) but no associations of heat resistance with alleles at the hsp26, hsp83, Desat 2, alpha-Gpdh, hsp70 loci, while cold resistance was not associated with Frost and Dca loci. These results confirm interactions between hsr-omega and thermal resistance, as well as between In(3L)Payne and cold resistance, but do not provide evidence for associations between thermal responses and alleles at other clinically varying marker genes.     

Patterns of genetic diversity and candidate genes for ecological divergence in a homoploid hybrid sunflower, Helianthus anomalus

Natural hybridization accompanied by a shift in niche preference by hybrid genotypes can lead to hybrid speciation. Natural selection may cause the fixation of advantageous alleles in the ecologically diverged hybrids, and the loci experiencing selection should exhibit a reduction in allelic diversity relative to neutral loci. Here, we analyzed patterns of genetic diversity at 59 microsatellite loci associated with expressed sequence tags (ESTs) in a homoploid hybrid sunflower species, Helianthus anomalus. We used two indices, ln RV and ln RH, to compare variation and heterozygosity (respectively) at each locus between the hybrid species and its two parental species, H. annuus and H. petiolaris. Mean values of ln RV and ln RH were significantly lower than zero, which implies that H. anomalus experienced a population bottleneck during its recent evolutionary history. After correcting for the apparent bottleneck, we found six loci with a significant reduction in variation or with heterozygosity in the hybrid species, compared to one or both of the parental species. These loci should be viewed as a ranked list of candidate loci, pending further sequencing and functional analyses. Sequence data were generated for two of the candidate loci, but population genetics tests failed to detect deviations from neutral evolution at either locus. Nonetheless, a greater than eight-fold excess of nonsynonymous substitutions was found near a putative N-myristoylation motif at the second locus (HT998), and likelihood-based models indicated that the protein has been under selection in H. anomalus in the past and, perhaps, in one or both parental species. Finally, our data suggest that selective sweeps may have united populations of H. anomalus isolated by a mountain range, indicating that even low gene-flow species may be held together by the spread of advantageous alleles.     

Identification of candidate antigen in Whipple's disease using a serological proteomic approach

Whipple's disease (WD) is a chronic multisystemic infection, caused by Tropheryma whipplei, a Gram-positive rod. Recently, a reliable method has been developed for cultivating T. whipplei in vitro. This together with the availability of complete genome sequence of T. whipplei prompted us to initiate proteome analysis of T. whipplei. The objective of the present study was to identify candidate proteins for serological diagnosis of WD. Immunoreactivities of sera collected from 18 patients with WD were compared with those of 24 control subjects who did not have WD. For this, we used 2-DE, immunoblotting, and MS. In total, we identified 23 candidate antigenic proteins. These included a subset of six proteins, each of which was found significantly more frequently in cases as compared to their controls. The remaining 17 proteins were found exclusively in cases. The methods we used in the current study enabled us to identify candidate antigens that, in our view, might be useful for serological diagnosis of WD.     

Characterising a Eucalyptus cladocalyx breeding population using SNP markers

Population structure, family relatedness and inbreeding within a first-generation Eucalyptus cladocalyx breeding population were analysed with single-nucleotide polymorphism markers to underpin quantitative trait analysis and breeding program management. The breeding population, comprising families selected from wild and cultivated stands, was found to be strongly structured ( $ {{\widehat{F}}_{\mathrm{ST}}} $ ?=?18 %), with two geographically defined groups of South Australian wild subpopulations: Kangaroo Island (KI) and South Flinders Ranges (SFR). The selections from cultivated stands were shown to be derived from SFR subpopulations of SFR and had similar levels of diversity, suggesting that they were established from a broad genetic base. Relatedness and inbreeding among families was heterogeneous, ranging from completely outcrossed and predominantly half-sib (HS) to completely selfed. Families from the cultivated stands had minimal inbreeding and were close to HS on average. Among SFR subpopulations, family-average inbreeding was negatively correlated with growth, suggesting inbreeding depression (ID). Inbreeding was high, on average, in the KI subpopulations; however, evidence of ID was absent, with highly inbred families amongst the most vigorous, perhaps indicative of purging of deleterious recessive alleles in a bottleneck event. The marker-based information suggested that modification of the usual assumptions of relatedness made in undertaking quantitative analysis of the first-generation populations would be desirable.     

Habitat islands, genetic diversity, and gene flow in a Patagonian rodent

The effects of terrestrial habitat islands on gene flow and genetic diversity in animal populations have been predicted and discussed in theoretical terms, but empirical data are needed to test these predictions and provide an understanding of the relationships of life-history characteristics to genetics of insular species. We studied saxicolous mice ( Phyllotis xanthopygus ) in Patagonia to explore genetic structure, phylogeography, and gene flow in a species inhabiting natural habitat islands. Phylogeographic analyses based on mtDNA sequences revealed two haplotype clades, which presumably reflect early Pleistocene factors that temporarily separated the mice into two geographically isolated groups. The Río Chubut, which lies within a glacial drainage basin bisecting northern Patagonia, might have affected gene flow in the species. Although we anticipated isolation by distance and founder phenomena associated with habitat islands, in some habitat patches we found evidence of high local genetic diversity. The amount of divergence in the mitochondrial cytochrome b gene (≈ 3.4%) in animals at a single locality could best be explained through a combination of historical factors and metapopulation source–sink theory. Demographic shifts, dispersal, and episodic recolonization are important in the life history and genetic population structure of P. xanthopygus .