A Genomic Scan for Selection Reveals Candidates for Genes Involved in the Evolution of Cultivated Sunflower (Helianthus annuus)

Genomic scans for selection are a useful tool for identifying genes underlying phenotypic transitions. In this article, we describe the results of a genome scan designed to identify candidates for genes targeted by selection during the evolution of cultivated sunflower. This work involved screening 492 loci derived from ESTs on a large panel of wild, primitive (i.e., landrace), and improved sunflower (Helianthus annuus) lines. This sampling strategy allowed us to identify candidates for selectively important genes and investigate the likely timing of selection. Thirty-six genes showed evidence of selection during either domestication or improvement based on multiple criteria, and a sequence-based test of selection on a subset of these loci confirmed this result. In view of what is known about the structure of linkage disequilibrium across the sunflower genome, these genes are themselves likely to have been targeted by selection, rather than being merely linked to the actual targets. While the selection candidates showed a broad range of putative functions, they were enriched for genes involved in amino acid synthesis and protein catabolism. Given that a similar pattern has been detected in maize (Zea mays), this finding suggests that selection on amino acid composition may be a general feature of the evolution of crop plants. In terms of genomic locations, the selection candidates were significantly clustered near quantitative trait loci (QTL) that contribute to phenotypic differences between wild and cultivated sunflower, and specific instances of QTL colocalization provide some clues as to the roles that these genes may have played during sunflower evolution.     

Contributions of flowering time genes to sunflower domestication and improvement

Determining the identity and distribution of molecular changes leading to the evolution of modern crop species provides major insights into the timing and nature of historical forces involved in rapid phenotypic evolution. In this study, we employed an integrated candidate gene strategy to identify loci involved in the evolution of flowering time during early domestication and modern improvement of the sunflower (Helianthus annuus). Sunflower homologs of many genes with known functions in flowering time were isolated and cataloged. Then, colocalization with previously mapped quantitative trait loci (QTLs), expression, or protein sequence differences between wild and domesticated sunflower, and molecular evolutionary signatures of selective sweeps were applied as step-wise criteria for narrowing down an original pool of 30 candidates. This process led to the discovery that five paralogs in the flowering locus T/terminal flower 1 gene family experienced selective sweeps during the evolution of cultivated sunflower and may be the causal loci underlying flowering time QTLs. Our findings suggest that gene duplication fosters evolutionary innovation and that natural variation in both coding and regulatory sequences of these paralogs responded to a complex history of artificial selection on flowering time during the evolution of cultivated sunflower.     

Climatic adaptation and ecological divergence between two closely related pine species in Southeast China

Climate is one of the most important drivers for adaptive evolution in forest trees. Climatic selection contributes greatly to local adaptation and intraspecific differentiation, but this kind of selection could also have promoted interspecific divergence through ecological speciation. To test this hypothesis, we examined intra‐ and interspecific genetic variation at 25 climate‐related candidate genes and 12 reference loci in two closely related pine species, Pinus massoniana Lamb. and Pinus hwangshanensis Hisa, using population genetic and landscape genetic approaches. These two species occur in Southeast China but have contrasting ecological preferences in terms of several environmental variables, notably altitude, although hybrids form where their distributions overlap. One or more robust tests detected signals of recent and/or ancient selection at two‐thirds (17) of the 25 candidate genes, at varying evolutionary timescales, but only three of the 12 reference loci. The signals of recent selection were species specific, but signals of ancient selection were mostly shared by the two species likely because of the shared evolutionary history. F_ST outlier analysis identified six SNPs in five climate‐related candidate genes under divergent selection between the two species. In addition, a total of 24 candidate SNPs representing nine candidate genes showed significant correlation with altitudinal divergence in the two species based on the covariance matrix of population history derived from reference SNPs. Genetic differentiation between these two species was higher at the candidate genes than at the reference loci. Moreover, analysis using the isolation‐with‐migration model indicated that gene flow between the species has been more restricted for climate‐related candidate genes than the reference loci, in both directions. Taken together, our results suggest that species‐specific and divergent climatic selection at the candidate genes might have counteracted interspecific gene flow and played a key role in the ecological divergence of these two closely related pine species.     

Evidence of selection on fatty acid biosynthetic genes during the evolution of cultivated sunflower

The identification of genes underlying the phenotypic transitions that took place during crop evolution, as well as the genomic extent of resultant selective sweeps, is of great interest to both evolutionary biologists and applied plant scientists. In this study, we report the results of a molecular evolutionary analysis of 11 genes that underlie fatty acid biosynthesis and metabolism in wild and cultivated sunflower (Helianthus annuus). Seven of these 11 genes showed evidence of selection at the nucleotide level, with 1 (FAD7) having experienced selection prior to domestication, 2 (FAD2-3 and FAD3) having experienced selection during domestication, and 4 (FAB1, FAD2-1, FAD6, and FATB) having experienced selection during the subsequent period of improvement. Sequencing of a subset of these genes from an extended panel of sunflower cultivars revealed little additional variation, and an analysis of the genomic region surrounding one of these genes (FAD2-1) revealed the occurrence of an extensive selective sweep affecting a region spanning at least ca. 100 kb. Given that previous population genetic analyses have revealed a relatively rapid decay of linkage disequilibrium in sunflower, this finding indicates the occurrence of strong selection and a rapid sweep.     

Molecular ecology and selection in the drought-related Asr gene polymorphisms in wild and cultivated common bean (Phaseolus vulgaris L.)

ABSTRACT: BACKGROUND: The abscisic acid (ABA) pathway plays an important role in the plants' reaction to drought stress and ABA-stress response (Asr) genes are important in controlling this process. In this sense, we accessed nucleotide diversity at two candidate genes for drought tolerance (Asr1 and Asr2), involved in an ABA signaling pathway, in the reference collection of cultivated common bean (Phaseolus vulgaris L.) and a core collection of wild common bean accessions. RESULTS: Our wild population samples covered a range of mesic (semi-arid) to very dry (desert) habitats, while our cultivated samples presented a wide spectrum of drought tolerance. Both genes showed very different patterns of nucleotide variation. Asr1 exhibited very low nucleotide diversity relative to the neutral reference loci that were previously surveyed in these populations. This suggests that strong purifying selection has been acting on this gene. In contrast, Asr2 exhibited higher levels of nucleotide diversity, which is indicative of adaptive selection. These patterns were more notable in wild beans than in cultivated common beans indicting that natural selection has played a role over long time periods compared to farmer selection since domestication. CONCLUSIONS: Together these results suggested the importance of Asr1 in the context of drought tolerance, and constitute the first steps towards an association study between genetic polymorphism of this gene family and variation in drought tolerance traits. Furthermore, one of our major successes was to find that wild common bean is a reservoir of genetic variation and selection signatures at Asr genes, which may be useful for breeding drought tolerance in cultivated common bean.     

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.     

Molecular population genetics and the search for adaptive evolution in plants

The first papers on plant molecular population genetics were published approximately 10 years ago. Since that time, well over 50 additional studies of plant nucleotide polymorphism have been published, and many of these studies focused on detecting the signature of balancing or positive selection at a locus. In this review, we discuss some of the theoretical and statistical issues surrounding the detection of selection, with focus on plant populations, and we also summarize the empirical plant molecular population genetics literature. At face value, the literature suggests that a history of balancing or positive selection in plant genes is rampant. In two well-studied taxa (maize and Arabidopsis) over 20% of studied genes have been interpreted as containing the signature of selection. We argue that this is probably an overstatement of the prevalence of natural selection in plant genomes, for two reasons. First, demographic effects are difficult to incorporate and have generally not been well integrated into the plant population genetics literature. Second, the genes studied to date are not a random sample, so selected genes may be overrepresented. The next generation of studies in plant molecular population genetics requires additional sampling of local populations, explicit comparisons among loci, and improved theoretical methods to control for demography. Eventually, candidate loci should be confirmed by explicit consideration of phenotypic effects.     

Functional and geographical differentiation of candidate balanced polymorphisms in Arabidopsis thaliana

Molecular population genetic analysis of three chromosomal regions in Arabidopsis thaliana suggested that balancing selection might operate to maintain variation at three novel candidate adaptive trait genes, including SOLUBLE STARCH SYNTHASE I (SSI) , PLASTID TRANSCRIPTIONALLY ACTIVE 7(PTAC7) , and BELL-LIKE HOMEODOMAIN 10 (BLH10). If balanced polymorphisms are indeed maintained at these loci, then we would expect to observe functional variation underlying the previously detected signatures of selection. We observe multiple replacement polymorphisms within and in the 32 amino acids just upstream of the protein–protein interacting BELL domain at the BLH10 locus. While no clear protein sequence differences are found between allele types in SSI and PTAC7, these two genes show evidence for allele-specific variation in expression levels. Geographical patterns of allelic differentiation seem consistent with population stratification in this species and a significant longitudinal cline was observed at all three candidate loci. These data support a hypothesis of balancing selection at all three candidate loci and provide a basis for more detailed functional work by identifying possible functional differences that might be selectively maintained.     

Characterising functionally important and ecologically meaningful genetic diversity using a candidate gene approach

Over the past two decades the fields of molecular ecology and population genetics have been dominated by the use of putatively neutral DNA markers, primarily to resolve spatio-temporal patterns of genetic variation to inform our understanding of population structure, gene flow and pedigree. Recent emphasis in comparative functional genomics, however, has fuelled a resurgence of interest in functionally important genetic variation that underpins phenotypic traits of adaptive or ecological significance. It may prove a major challenge to transfer genomics information from classical model species to examine functional diversity in non-model species in natural populations, but already multiple gene-targeted candidate loci with major effect on phenotype and fitness have been identified. Here we briefly describe some of the research strategies used for isolating and characterising functional genetic diversity at candidate gene-targeted loci, and illustrate the efficacy of some of these approaches using our own studies on red grouse (Lagopus lagopus scoticus). We then review how candidate gene markers have been used to: (1) quantify genetic diversity among populations to identify those depauperate in genetic diversity and requiring specific management action; (2) identify the strength and mode of selection operating on individuals within natural populations; and (3) understand direct mechanistic links between allelic variation at single genes and variance in individual fitness.     

Interpretation of variation across marker loci as evidence of selection

Population structure and history have similar effects on the genetic diversity at all neutral loci. However, some marker loci may also have been strongly influenced by natural selection. Selection shapes genetic diversity in a locus-specific manner. If we could identify those loci that have responded to selection during the divergence of populations, then we may obtain better estimates of the parameters of population history by excluding these loci. Previous attempts were made to identify outlier loci from the distribution of sample statistics under neutral models of population structure and history. Unfortunately these methods depend on assumptions about population structure and history that usually cannot be verified. In this article, we define new population-specific parameters of population divergence and construct sample statistics that are estimators of these parameters. We then use the joint distribution of these estimators to identify outlier loci that may be subject to selection. We found that outlier loci are easier to recognize when this joint distribution is conditioned on the total number of allelic states represented in the pooled sample at each locus. This is so because the conditional distribution is less sensitive to the values of nuisance parameters.     

Estimation of mating system parameters in an evolving gynodioecous population of cultivated sunflower (Helianthus annuus L.)

Cultivated plants have been molded by human-induced selection, including manipulations of the mating system in the twentieth century. How these manipulations have affected realized parameters of the mating system in freely evolving cultivated populations is of interest for optimizing the management of breeding populations, predicting the fate of escaped populations and providing material for experimental evolution studies. To produce modern varieties of sunflower (Helianthus annuus L.), self-incompatibility has been broken, recurrent generations of selfing have been performed and male sterility has been introduced. Populations deriving from hybrid-F1 varieties are gynodioecious because of the segregation of a nuclear restorer of male fertility. Using both phenotypic and genotypic data at 11 microsatellite loci, we analyzed the consanguinity status of plants of the first three generations of such a population and estimated parameters related to the mating system. We showed that the resource reallocation to seed in male-sterile individuals was not significant, that inbreeding depression on seed production averaged 15-20% and that cultivated sunflower had acquired a mixed-mating system, with ～50% of selfing among the hermaphrodites. According to theoretical models, the female advantage and the inbreeding depression at the seed production stage were too low to allow the persistence of male sterility. We discuss our methods of parameter estimation and the potential of such study system in evolutionary biology.     

Genome scan to detect genetic structure and adaptive genes of natural populations of Cryptomeria japonica

We investigated 29 natural populations of Cryptomeria japonica using 148 cleaved amplified polymorphic sequence markers to elucidate their genetic structure and identify candidate adaptive genes of this species. In accordance with the inferred evolutionary history of the species during and after the last glacial episode, the genetic diversity was higher in western populations than in northern populations. The results of phylogenetic and genetic structure analyses suggest that populations of the two main varieties of the species have clearly diverged from each other and that two of the examined loci are strongly associated with the differentiation between the two varieties. Using a coalescent simulation based on F(ST) and H(e) values, we detected five genes that had higher, and two that had lower, values than the respective 99% confidence intervals (C.I.s) that are theoretically expected intervals under a neutral infinite-island model. We also detected 13 outlier loci using a coalescent simulation based on the assumption that the 2 varieties originated from the splitting of an ancestral population. Four of these loci were detected by both methods, two of which were detected in a genetic structure analysis as loci associated with differentiation between the two varieties of the species, and are strong candidates for genes that have been subject to selection.     

The genomic basis of adaptation to high‐altitude habitats in the eastern honey bee (Apis cerana)

The eastern honey bee (Apis cerana) is of central importance for agriculture in Asia. It has adapted to a wide variety of environmental conditions across its native range in southern and eastern Asia, which includes high‐altitude regions. eastern honey bees inhabiting mountains differ morphologically from neighbouring lowland populations and may also exhibit differences in physiology and behaviour. We compared the genomes of 60 eastern honey bees collected from high and low altitudes in Yunnan and Gansu provinces, China, to infer their evolutionary history and to identify candidate genes that may underlie adaptation to high altitude. Using a combination of F_ST‐based statistics, long‐range haplotype tests and population branch statistics, we identified several regions of the genome that appear to have been under positive selection. These candidate regions were strongly enriched for coding sequences and had high haplotype homozygosity and increased divergence specifically in highland bee populations, suggesting they have been subjected to recent selection in high‐altitude habitats. Candidate loci in these genomic regions included genes related to reproduction and feeding behaviour in honey bees. Functional investigation of these candidate loci is necessary to fully understand the mechanisms of adaptation to high‐altitude habitats in the eastern honey bee.     

High-resolution mapping of quantitative trait loci affecting increased life span in Drosophila melanogaster

Limited life span and senescence are near-universal characteristics of eukaryotic organisms, controlled by many interacting quantitative trait loci (QTL) with individually small effects, whose expression is sensitive to the environment. Analyses of mutations in model organisms have shown that genes affecting stress resistance and metabolism affect life span across diverse taxa. However, there is considerable segregating variation for life span in nature, and relatively little is known about the genetic basis of this variation. Replicated lines of Drosophila that have evolved increased longevity as a correlated response to selection for postponed senescence are valuable resources for identifying QTL affecting naturally occurring variation in life span. Here, we used deficiency complementation mapping to identify at least 11 QTL on chromosome 3 that affect variation in life span between five old (O) lines selected for postponed senescence and their five base (B) population control lines. Most QTL were sex specific, and all but one affected multiple O lines. The latter observation is consistent with alleles at intermediate frequency in the base population contributing to the response to selection for postponed senescence. The QTL were mapped with high resolution and contained from 12 to 170 positional candidate genes.     

A scan for signatures of positive selection in candidate loci for skin pigmentation in humans

Although the combination of pale skin and intense sun exposure results in an important health risk for the individual, it is less clear if at the population level this risk has possessed an evolutionary meaning. In this sense, a number of adaptive hypotheses have been put forward to explain the evolution of human skin pigmentation, such as photoprotection against sun-induced cancer, sexual selection, vitamin D synthesis or photoprotection of photolabile compounds, among others. It is expected that if skin pigmentation is adaptive, we might be able to see the signature of positive selection on some of the genes involved. In order to detect this signature, we analyze a battery of 81 candidate loci by means of phylogenetic and population genetic tests. Our results indicate that both light and dark skin may possess adaptive value. Of the main loci presenting this signature, TP53BP1 shows clear evidence of adaptive selection in Africans, whereas TYRP1 and SLC24A5 show evidence of adaptive selection in Caucasians. Although we cannot offer a mechanism that based on these genes explains the advantage of light skin, if TP53BP1, and perhaps RAD50, have truly conferred an adaptive value to the African population analyzed, photoprotection against sun-induced skin damage/cancer might be proposed as a mechanism that has driven the evolution of human skin pigmentation.     

Parallel evolution of gene classes,but not genes: Evidence from Hawai'ian honeycreeper populations exposed to avian malaria

Adaptation in nature is ubiquitous, yet characterizing its genomic basis is difficult because population demographics cause correlations with nonadaptive loci. Introduction events provide opportunities to observe adaptation over known spatial and temporal scales, facilitating the identification of genes involved in adaptation. The pathogen causing avian malaria, Plasmodium relictum, was introduced to Hawai'i in the 1930s and elicited extinctions and precipitous population declines in native honeycreepers. After a sharp initial population decline, the Hawai'i ‘amakihi (Chlorodrepanis virens) has evolved tolerance to the parasite at low elevations where P. relictum exists, and can sustain infection without major fitness consequences. High‐elevation, unexposed populations of ‘amakihi display little to no tolerance. To explore the genomic basis of adaptation to P. relictum in low‐elevation ‘amakihi, we genotyped 125 ‘amakihi from the island of Hawai'i via hybridization capture to 40,000 oligonucleotide baits containing SNPs and used the reference ‘amakihi genome to identify genes potentially under selection from malaria. We tested for outlier loci between low‐ and high‐elevation population pairs and identified loci with signatures of selection within low‐elevation populations. In some cases, genes commonly involved in the immune response (e.g., major histocompatibility complex) were associated with malaria presence in the population. We also detected several novel candidate loci that may be implicated in surviving malaria infection (e.g., beta‐defensin, glycoproteins and interleukin‐related genes). Our results suggest that rapid adaptation to pathogens may occur through changes in different immune genes, but in the same classes of genes, across populations.     

Genome-wide SNP discovery and genetic linkage map construction in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) using a genotyping by sequencing (GBS) approach

Recently developed plant genomics approaches (LD mapping and genome-wide selection) require many molecular markers distributed throughout the plant genome. As a result, the availability of an increasing number of markers is essential for maintaining highly efficient and accurate plant breeding programs. In this study, we identified SNP loci in sunflower using a genotyping by sequencing (GBS) approach in an intraspecific F₂ mapping population. A total of 271,445,770 reads were generated by the Genome Analyzer II next-generation sequencing platform and 29.2 % of the reads were aligned to unique locations in the genome. A total of 46,278 SNP loci were identified and 7646 SNP loci were validated in an F₂ population. In addition, a SNP-based linkage map was constructed. This is the first report of SNP discovery in sunflower by GBS. The SNP markers and SNP-based linkage map will be valuable molecular genetics tools for sunflower breeding.