Nucleotide polymorphism and phenotypic associations within and around the phytochrome B2 Locus in European aspen (Populus tremula, Salicaceae)

We investigated the utility of association mapping to dissect the genetic basis of naturally occurring variation in bud phenology in European aspen (Populus tremula). With this aim, we surveyed nucleotide polymorphism in 13 fragments spanning an 80-kb region surrounding the phytochrome B2 (phyB2) locus. Although polymorphism varies substantially across the phyB2 region, we detected no signs for deviations from neutral expectations. We also identified a total of 41 single nucleotide polymorphisms (SNPs) that were subsequently scored in a mapping population consisting of 120 trees. We identified two nonsynonymous SNPs in the phytochrome B2 gene that were independently associated with variation in the timing of bud set and that explained between 1.5 and 5% of the observed phenotypic variation in bud set. Earlier studies have shown that the frequencies of both these SNPs vary clinally with latitude. Linkage disequilibrium across the region was low, suggesting that the SNPs we identified are strong candidates for being causally linked to variation in bud set in our mapping populations. One of the SNPs (T608N) is located in the "hinge region," close to the chromophore binding site of the phyB2 protein. The other SNP (L1078P) is located in a region supposed to mediate downstream signaling from the phyB2 locus. The lack of population structure, combined with low levels of linkage disequilibrium, suggests that association mapping is a fruitful method for dissecting naturally occurring variation in Populus tremula.     

Multilocus patterns of nucleotide polymorphism and the demographic history of Populus tremula

I have studied nucleotide polymorphism and linkage disequilibrium using multilocus data from 77 fragments, with an average length of fragments of 550 bp, in the deciduous tree Populus tremula (Salicaceae). The frequency spectrum across loci showed a modest excess of mutations segregating at low frequency and a marked excess of high-frequency derived mutations at silent sites, relative to neutral expectations. These excesses were also seen at replacement sites, but were not so pronounced for high-frequency derived mutations. There was a marked excess of low-frequency mutations at replacement sites, likely indicating deleterious amino acid-changing mutations that segregate at low frequencies in P. tremula. I used approximate Bayesian computation (ABC) to evaluate a number of different demographic scenarios and to estimate parameters for the best-fitting model. The data were found to be consistent with a historical reduction in the effective population size of P. tremula through a bottleneck. The timing inferred for this bottleneck is largely consistent with geological data and with data from several other long-lived plant species. The results show that P. tremula harbors substantial levels of nucleotide polymorphism with the posterior mode of the scaled mutation rate, = 0.0177 across loci. The ABC analyses also provided an estimate of the scaled recombination rate that indicates that recombination rates in P. tremula are likely to be 2-10 times higher than the mutation rate. This study reinforces the notion that linkage disequilibrium is low and decays to negligible levels within a few hundred base pairs in P. tremula.     

An excess of nonsynonymous polymorphism and extensive haplotype structure at the PtABI1B locus in European aspen (Populus tremula): a case of balancing selection in an obligately outcrossing plant?

Here, we describe an unusually pronounced haplotype structure at the PtABI1B locus in the obligately outcrossing tree Populus tremula. Both nucleotide diversity and divergence at PtABI1B was low compared to other P. tremula genes suggesting that the gene is located in a region with a low mutation rate. Despite this, PtABI1B shows a very marked excess of nonsynonymous polymorphisms across the entire coding region and linkage disequilibrium (LD) extending across the entire PtABI1B region of approximately 2.6 kb. Such extensive LD is normally not seen in P. tremula. The extensive LD at PtABI1B is caused by the presence of two distinct haplotypes. The haplotype structure is not caused by a lack of recombination in the region, because evidence of recombination can be detected. In addition, several statistical tests strongly reject neutrality for the PtABI1B region, suggesting that the unusual haplotype structure could be actively maintained by balancing selection.     

Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa

Abstract Ecotilling was used as a simple nucleotide polymorphism (SNP) discovery tool to examine DNA variation in natural populations of the western black cottonwood, Populus trichocarpa, and was found to be more efficient than sequencing for large-scale studies of genetic variation in this tree. A publicly available, live reference collection of P. trichocarpa from the University of British Columbia Botanical Garden was used in this study to survey variation in nine different genes among individuals from 41 different populations. A large amount of genetic variation was detected, but the level of variation appears to be less than in the related species, Populus tremula, based on reported statistics for that tree. Genes examined varied considerably in their level of variation, from PoptrTB1 which had a single SNP, to PoptrLFY which had more than 23 in the 1000-bp region examined. Overall nucleotide diversity, measured as (Total), was relatively low at 0.00184. Linkage disequilibrium, on the other hand, was higher than reported for some woody plant species, with mean r2 equal to 0.34. This study reveals the potential of Ecotilling as a rapid genotype discovery method to explore and utilize the large pool of genetic variation in tree species.     

A set of novel DNA polymorphisms within candidate genes potentially involved in ecological divergence between Populus alba and P. tremula, two hybridizing European forest trees

We have identified 53 DNA (single nucleotide, microsatellite, and insertion-deletion) polymorphisms within 12 candidate genes potentially involved in ecological differences between Populus alba and P. tremula, two hybridizing European forest trees. The genes represent candidates for functional roles associated with abiotic or biotic stress response, cross-talk, phenology, and leaf development. Distributions within sequences, intraspecific levels of diversity, and genetic divergence (F(ST) ) between species are reported for each polymorphism, as are haplotype frequencies for each gene. The markers will be used for population genomic studies of the barrier to gene flow between these two ecologically divergent forest trees.     

Molecular population genetics of herbivore-induced protease inhibitor genes in European aspen (Populus tremula L., Salicaceae)

Plants defend themselves against the attack of natural enemies by using an array of both constitutively expressed and induced defenses. Long-lived woody perennials are overrepresented among plant species that show strong induced defense responses, whereas annual plants and crop species are underrepresented. However, most studies of plant defense genes have been performed on annual or short-lived perennial weeds or crop species. Here I use molecular population genetic methods to survey six wound-inducible protease inhibitors (PIs) in a long-lived woody, perennial plant species, the European aspen (Populus tremula), to evaluate the likelihood of either recurrent selective sweeps or balancing selection maintaining amino acid polymorphisms in these genes. The results show that none of the six PI genes have reduced diversities at synonymous sites, as would be expected in the presence of recurrent selective sweeps. However, several genes show some evidence of nonneutral evolution such as enhanced linkage disequilibrium and a large number of high-frequency-derived mutations. A group of at least four Kunitz trypsin inhibitor genes appear to have experienced elevated levels of nonsynonymous substitutions, indicating allelic turnover on an evolutionary timescale. One gene, TI1, has enhanced levels of intraspecific polymorphism at nonsynonymous sites and also has an unusual haplotype structure characterized by two divergent haplotypes occurring at roughly equal frequencies in the sample. One haplotype has very low levels of intraallelic nucleotide diversity, whereas the other haplotype has levels of diversity comparable to other genes in P. tremula. Patterns of sequence diversity at TI1 do not fit a simple model of either balancing selection or recurrent selective sweeps. This suggests that selection at TI1 is more complex, possibly involving allelic cycling.     

Molecular evolution of two linked genes, Est-6 and Sod, in Drosophila melanogaster.

We have obtained 15 sequences of Est-6 from a natural population of Drosophila melanogaster to test whether linkage disequilibrium exists between Est-6 and the closely linked Sod, and whether natural selection may be involved. An early experiment with allozymes had shown linkage disequilibrium between these two loci, while none was detected between other gene pairs. The Sod sequences for the same 15 haplotypes were obtained previously. The two genes exhibit similar levels of nucleotide polymorphism, but the patterns are different. In Est-6, there are nine amino acid replacement polymorphisms, one of which accounts for the S-F allozyme polymorphism. In Sod, there is only one replacement polymorphism, which corresponds to the S-F allozyme polymorphism. The transversion/transition ratio is more than five times larger in Sod than in Est-6. At the nucleotide level, the S and F alleles of Est-6 make up two allele families that are quite different from each other, while there is relatively little variation within each of them. There are also two families of alleles in Sod, one consisting of a subset of F alleles, and the other consisting of another subset of F alleles, designed F(A), plus all the S alleles. The Sod F(A) and S alleles are completely or nearly identical in nucleotide sequence, except for the replacement mutation that accounts for the allozyme difference. The two allele families have independent evolutionary histories in the two genes. There are traces of statistically significant linkage disequilibrium between the two genes that, we suggest, may have arisen as a consequence of selection favoring one particular sequence at each locus.     

Nucleotide variation in Quercus crispula Blume

Quercus is attractive for evolutionary studies, primarily for developing the concepts of the species, speciation and adaptation; however, remarkably little is known about levels of nucleotide polymorphism in the nuclear functional genes of this genus. This article provides the first characterization of levels of nucleotide polymorphism in 11 gene fragments in natural populations of a Quercus species, Quercus crispula Blume. Results show that the level of nucleotide variation in this oak is generally higher than that in conifers, as high as that in a European oak, but lower than that in an aspen. The level of population recombination is relatively high. Within-population inbreeding is negligible and between-population differentiation is modest. The decay of linkage disequilibrium is significantly faster in the species-wide samples and the three northernmost populations than in the other populations. Statistical tests support the hypothesis of a recent bottleneck for several populations in the southern part of Japan. The amounts and patterns of nucleotide variation, recombination and linkage disequilibrium, and genetic differentiation observed among populations of this species are contradictory to our expectations, given the recent colonization history of the northern Japan populations.     

中国山杨遗传多样性及遗传结构分析

利用6个单拷贝核基因标记,对中国山杨(Populus davidiana Dode)14个自然居群的遗传多样性和遗传分化水平进行了研究。结果表明,中国山杨表现出较高的遗传多样性水平,各居群间基因流(Nm)为0.66;居群内的变异占总变异的百分比(71.82%)大于居群间(28.18%)。Mantel test检验结果显示居群遗传距离和地理距离间没有相关性。失配分布检测显示中国山杨历史上曾经历过种群的扩张。复杂的种群历史动态、高度异交和较高的碱基突变速率是中国山杨遗传多样性水平较高的原因;而较强的花粉和种子扩散能力及中国山杨的连续分布可能是其居群间遗传分化较小的原因。     

Barrier to gene flow between two ecologically divergent Populus species, P. alba (white poplar) and P. tremula (European aspen): the role of ecology and life history in gene introgression

The renewed interest in the use of hybrid zones for studying speciation calls for the identification and study of hybrid zones across a wide range of organisms, especially in long-lived taxa for which it is often difficult to generate interpopulation variation through controlled crosses. Here, we report on the extent and direction of introgression between two members of the "model tree" genus Populus: Populus alba (white poplar) and Populus tremula (European aspen), across a large zone of sympatry located in the Danube valley. We genotyped 93 hybrid morphotypes and samples from four parental reference populations from within and outside the zone of sympatry for a genome-wide set of 20 nuclear microsatellites and eight plastid DNA restriction site polymorphisms. Our results indicate that introgression occurs preferentially from P. tremula to P. alba via P. tremula pollen. This unidirectional pattern is facilitated by high levels of pollen vs. seed dispersal in P. tremula (pollen/seed flow = 23.9) and by great ecological opportunity in the lowland floodplain forest in proximity to P. alba seed parents, which maintains gene flow in the direction of P. alba despite smaller effective population sizes (N(e)) in this species (P. alba N(e)c. 500-550; P. tremula N(e)c. 550-700). Our results indicate that hybrid zones will be valuable tools for studying the genetic architecture of the barrier to gene flow between these two ecologically divergent Populus species.     

单核苷酸多态性在林木中的研究进展

摘要: 单核苷酸多态性(Single nucleotide polymorphisms, SNPs)是许多生物体最丰富的遗传变异形式。林木是重要的植物类群和陆地植物生态系统的重要组成部分, SNP作为新的分子标记已应用于松、杨、黄杉、桉和云杉等属的多个树种的遗传育种学研究, 获得了包括核苷酸多样性、连锁不平衡及群体结构等相关的遗传信息, 这些研究主要建立在对候选基因序列进行测序分析的基础上。基于SNP的关联遗传学分析或连锁不平衡(Linkage disequilibrium, LD)作图, 已成为研究林木复杂数量性状的理想工具, 对桉树和火炬松的关联遗传学研究发现, 多个基因内的SNP位点与不同的木材性状相关联。利用SNP标记对林木遗传参数的估算从不同程度上揭示了林木群体进化规律及其生态学意义。SNP标记在林木中应用的不断深入, 必将极大地推动林木遗传育种学研究的发展。     

Linkage disequilibrium in the North American Holstein population

Linkage disequilibrium was estimated using 7119 single nucleotide polymorphism markers across the genome and 200 animals from the North American Holstein cattle population. The analysis of maternally inherited haplotypes revealed strong linkage disequilibrium ( r ²   >   0.8) in genomic regions of ∼50 kb or less. While linkage disequilibrium decays as a function of genomic distance, genomic regions within genes showed greater linkage disequilibrium and greater variation in linkage disequilibrium compared with intergenic regions. Identification of haplotype blocks could characterize the most common haplotypes. Although maximum haplotype block size was over 1 Mb, mean block size was 26–113 kb by various definitions, which was larger than that observed in humans (∼10 kb). Effective population size of the dairy cattle population was estimated from linkage disequilibrium between single nucleotide polymorphism marker pairs in various haplotype ranges. Rapid reduction of effective population size of dairy cattle was inferred from linkage disequilibrium in recent generations. This result implies a loss of genetic diversity because of the high rate of inbreeding and high selection intensity in dairy cattle. The pattern observed in this study indicated linkage disequilibrium in the current dairy cattle population could be exploited to refine mapping resolution. Changes in effective population size during past generations imply a necessity of plans to maintain polymorphism in the Holstein population.     

Molecular population genetics of the Arabidopsis CLAVATA2 region. The genomic scale of variation and selection in a selfing species

The Arabidopsis thaliana CLAVATA2 (CLV2) gene encodes a leucine-rich repeat protein that regulates the development of the shoot meristem. The levels and patterns of nucleotide variation were assessed for CLV2 and 10 flanking genes that together span a 40-kb region of chromosome I. A total of 296 out of 7959 sequenced nucleotide sites were polymorphic. The mean levels of sequence diversity of the contiguous genes in this region are approximately twofold higher than those of other typical Arabidopsis nuclear loci. There is, however, wide variation in the levels and patterns of sequence variation among the 11 linked genes in this region, and adjacent genes appear to be subject to contrasting evolutionary forces. CLV2 has the highest levels of nucleotide variation in this region, a significant excess of intermediate frequency polymorphisms, and significant levels of intragenic linkage disequilibrium. Most alleles at CLV2 are found in one of three haplotype groups of moderate (>15%) frequency. These features suggest that CLV2 may harbor a balanced polymorphism.     

Comparative Nucleotide Diversity Across North American and European Populus Species

Nucleotide polymorphisms in two North American balsam poplars (Populus trichocarpa Torr. & Gray and P. balsamifera L.; section Tacamahaca), and one Eurasian aspen (P. tremula L.; section Populus) were compared using nine loci involved in defense, stress response, photoperiodism, freezing tolerance, and housekeeping. Nucleotide diversity varied among species and was highest for P. tremula (θ(w) = 0.005, π(T) = 0.007) as compared to P. balsamifera (θ(w) = 0.004, π(T) = 0.005) or P. trichocarpa (θ(w) = 0.002, π(T) = 0.003). Across species, the defense and the stress response loci accounted for the majority of the observed level of nucleotide diversity. In general, the studied loci did not deviate from neutral expectation either at the individual locus (non-significant normalized Fay and Wu's H) or at the multi-locus level (non-significant HKA test). Using molecular clock analysis, section Tacamahaca probably shared a common ancestor with section Populus approximately 4.5 million year ago. Divergence between the two closely related balsam poplars was about 0.8 million years ago, a pattern consistent with an isolation-with-migration (IM) model. As expected, P. tremula showed a five-fold higher substitution rate (2 × 10(-8) substitution/site/year) compared to the North American species (0.4 × 10(-8) substitution/site/year), probably reflecting its complex demographic history. Linkage disequilibrium (LD) varied among species with a more rapid decay in the North American species (<400 bp) in comparison to P. tremula (?400 bp). The similarities in nucleotide diversity pattern and LD decay of the two balsam poplar species likely reflects the recent time of their divergence.     

Contrasting evolutionary histories of two introns of the duchenne muscular dystrophy gene, Dmd, in humans

The Duchenne muscular dystrophy (Dmd) locus lies in a region of the X chromosome that experiences a high rate of recombination and is thus expected to be relatively unaffected by the effects of selection on nearby genes. To provide a picture of nucleotide variability at a high-recombination locus in humans, we sequenced 5. 4 kb from two introns of Dmd in a worldwide sample of 41 alleles from Africa, Asia, Europe, and the Americas. These same regions were also sequenced in one common chimpanzee and one orangutan. Dramatically different patterns of genetic variation were observed at these two introns, which are separated by >500 kb of DNA. Nucleotide diversity at intron 44 pi = 0.141% was more than four times higher than nucleotide diversity at intron 7 pi = 0.034% despite similar levels of divergence for these two regions. Intron 7 exhibited significant linkage disequilibrium extending over 10 kb and also showed a significant excess of rare polymorphisms. In contrast, intron 44 exhibited little linkage disequilibrium and no skew in the frequency distribution of segregating sites. Intron 7 was much more variable in Africa than in other continents, while intron 44 displayed similar levels of variability in different geographic regions. Comparison of intraspecific polymorphism to interspecific divergence using the HKA test revealed a significant reduction in variability at intron 7 relative to intron 44, and this effect was most pronounced in the non-African samples. These results are best explained by positive directional selection acting at or near intron 7 and demonstrate that even genes in regions of high recombination may be influenced by selection at linked sites.     

Complex signatures of selection and gene conversion in the duplicated globin genes of house mice

Results of electrophoretic surveys have suggested that hemoglobin polymorphism may be maintained by balancing selection in natural populations of house mice, Mus musculus. Here we report a survey of nucleotide variation in the adult globin genes of house mice from South America. We surveyed nucleotide polymorphism in two closely linked alpha-globin paralogs and two closely linked beta-globin paralogs to test whether patterns of variation are consistent with a model of long-term balancing selection. Surprisingly high levels of nucleotide polymorphism at the two beta-globin paralogs were attributable to the segregation of two highly divergent haplotypes, Hbbs (which carries two identical beta-globin paralogs) and Hbbd (which carries two functionally divergent beta-globin paralogs). Interparalog gene conversion on the Hbbs haplotype has produced a highly unusual situation in which the two paralogs are more similar to one another than either one is to its allelic counterpart on the Hbbd haplotype. Levels of nucleotide polymorphism and linkage disequilibrium at the two beta-globin paralogs suggest a complex history of diversity-enhancing selection that may be responsible for long-term maintenance of alternative protein alleles. The alternative two-locus beta-globin haplotypes are associated with pronounced differences in intraerythrocyte glutathione and nitric oxide metabolism, suggesting a possible mechanism for selection on hemoglobin function.     

Genome-wide analysis in endangered populations: a case study in Barbaresca sheep

Analysis of genomic data is becoming increasingly common in the livestock industry and the findings have been an invaluable resource for effective management of breeding programs in small and endangered populations. In this paper, with the goal of highlighting the potential of genomic analysis for small and endangered populations, genome-wide levels of linkage disequilibrium, measured as the squared correlation coefficient of allele frequencies at a pair of loci, effective population size, runs of homozygosity (ROH) and genetic diversity parameters, were estimated in Barbaresca sheep using Illumina OvineSNP50K array data. Moreover, the breed’s genetic structure and its relationship with other breeds were investigated. Levels of pairwise linkage disequilibrium decreased with increasing distance between single nucleotide polymorphisms. An average correlation coefficient <0.25 was found for markers located up to 50 kb apart. Therefore, these results support the need to use denser single nucleotide polymorphism panels for high power association mapping and genomic selection efficiency in future breeding programs. The estimate of past effective population size ranged from 747 animals 250 generations ago to 28 animals five generations ago, whereas the contemporary effective population size was 25 animals. A total of 637 ROH were identified, most of which were short (67%) and ranged from 1 to 10 Mb. The genetic analyses revealed that the Barbaresca breed tended to display lower variability than other Sicilian breeds. Recent inbreeding was evident, according to the ROH analysis. All the investigated parameters showed a comparatively narrow genetic base and indicated an endangered status for Barbaresca. Multidimensional scaling, model-based clustering, measurement of population differentiation, neighbor networks and haplotype sharing distinguished Barbaresca from other breeds, showed a low level of admixture with the other breeds considered in this study, and indicated clear genetic differences compared with other breeds. Attention should be given to the conservation of Barbaresca due to its critical conservation status. In this context, genomic information may have a crucial role in management of small and endangered populations.     

Evolution at the tip and base of the X chromosome in an African population of Drosophila melanogaster

Hitchhiking effects of advantageous mutations have been invoked to explain reduced polymorphism in regions of low crossing-over in Drosophila. Besides reducing DNA heterozygosity, hitchhiking effects should produce strong linkage disequilibrium and a frequency spectrum skewed toward an excess of rare polymorphisms (compared to the neutral expectation). We measured DNA polymorphism in a Zimbabwe population of D. melanogaster at three loci, yellow, achaete, and suppressor of forked, located in regions of reduced crossing-over. Similar to previously published surveys of these genomic regions in other populations, we observed low levels of nucleotide variability. However, the frequency spectrum was compatible with a neutral model, and there was abundant evidence for recombination in the history of the yellow and ac genes. Thus, some aspects of the data cannot be accounted for by a simple hitchhiking model. An alternative hypothesis, background selection, might be compatible with the observed patterns of linkage disequilibrium and the frequency spectrum. However, this model cannot account for the observed reduction in nucleotide heterozygosity. Thus, there is currently no satisfactory theoretical model for the data from the tip and base of the X chromosome in D. melanogaster.      

Admixture in European Populus hybrid zones makes feasible the mapping of loci that contribute to reproductive isolation and trait differences

The use of admixed human populations to scan the genome for chromosomal segments affecting complex phenotypic traits has proved a powerful analytical tool. However, its potential in other organisms has not yet been evaluated. Here, we use DNA microsatellites to assess the feasibility of this approach in hybrid zones between two members of the 'model tree' genus Populus: Populus alba (white poplar) and Populus tremula (European aspen). We analyzed samples of both species and a Central European hybrid zone (N=544 chromosomes) for a genome-wide set of 19 polymorphic DNA microsatellites. Our results indicate that allele frequency differentials between the two species are substantial (mean delta=0.619+/-0.067). Background linkage disequilibrium (LD) in samples of the parental gene pools is moderate and should respond to sampling schemes that minimize drift and account for rare alleles. LD in hybrids decays with increasing number of backcross generations as expected from theory and approaches background levels of the parental gene pools in advanced generation backcrosses. Introgression from P. tremula into P. alba varies strongly across marker loci. For several markers, alleles from P. tremula are slightly over-represented relative to neutral expectations, whereas a single locus exhibits evidence of selection against P. tremula genotypes. We interpret our results in terms of the potential for admixture mapping in these two ecologically divergent Populus species, and we validate a modified approach of studying genotypic clines in 'mosaic' hybrid zones.     

Chromosomal inversion polymorphism leads to extensive genetic structure: a multilocus survey in Drosophila subobscura

The adaptive character of inversion polymorphism in Drosophila subobscura is well established. The O(ST) and O(3+4) chromosomal arrangements of this species differ by two overlapping inversions that arose independently on O(3) chromosomes. Nucleotide variation in eight gene regions distributed along inversion O(3) was analyzed in 14 O(ST) and 14 O(3+4) lines. Levels of variation within arrangements were quite similar along the inversion. In addition, we detected (i) extensive genetic differentiation between arrangements in all regions, regardless of their distance to the inversion breakpoints; (ii) strong association between nucleotide variants and chromosomal arrangements; and (iii) high levels of linkage disequilibrium in intralocus and also in interlocus comparisons, extending over distances as great as approximately 4 Mb. These results are not consistent with the higher genetic exchange between chromosomal arrangements expected in the central part of an inversion from double-crossover events. Hence, double crossovers were not produced or, alternatively, recombinant chromosomes were eliminated by natural selection to maintain coadapted gene complexes. If the strong genetic differentiation detected along O(3) extends to other inversions, nucleotide variation would be highly structured not only in D. subobscura, but also in the genome of other species with a rich chromosomal polymorphism.