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  • Nucleotide diversity and linkage disequilibrium in balsam poplar (Populus balsamifera)

    Matthew S. Olson1, Amanda L. Robertson1, Naoki Takebayashi1, Salim Silim2, William R. Schroeder2 and

    Peter Tiffin3

    1Institute of Arctic Biology and Department of Biology and Wildlife, University of Alaska Fairbanks, AK 99775, USA; 2Agriculture and Agri-Food Canada,

    Agri-Environment Services Branch, Indian Head, SK, Canada; 3Department of Plant Biology, University of Minnesota, St Paul, MN, USA

    Author for correspondence: Matthew S. Olson

    Tel: +1 907 4742766


    Received: 14 October 2009 Accepted: 8 December 2009

    New Phytologist (2010) 186: 526–536 doi: 10.1111/j.1469-8137.2009.03174.x

    Key words: effective population size, genomic diversity, insertion deletion diversity, poplar, purifying selection, single nucleotide polymorphisms (SNPs).


    • Current perceptions that poplars have high levels of nucleotide variation, large effective population sizes, and rapid decay of linkage disequilibrium are based pri-

    marily on studies from one poplar species, Populus tremula.

    • We analysed 590 gene fragments (average length 565 bp) from each of 15 indi- viduals from different populations from throughout the range of Populus balsamif-


    • Nucleotide diversity (htotal = 0.0028, p = 0.0027) was low compared with other trees and model agricultural systems. Patterns of nucleotide diversity and site fre-

    quency spectra were consistent with purifying selection on replacement and intron

    sites. When averaged across all loci we found no evidence for decay of linkage dis-

    equilibrium across 750 bp, consistent with the low estimates of the scaled recombi-

    nation parameter, q = 0.0092. • Compared with P. tremula, a well studied congener with a similar distribution, P. balsamifera has low diversity and low effective recombination, both of which indi-

    cate a lower effective population size in P. balsamifera. Patterns of diversity and

    linkage indicate that there is considerable variation in population genomic patterns

    among poplar species and unlike P. tremula, association mapping techniques in

    balsam poplar should consider sampling single nucleotide polymorphisms (SNPs) at

    well-spaced intervals.


    Multilocus surveys of nucleotide diversity and linkage dis- equilibrium (LD) provide valuable insights into a species history and the genomic imprint of selection (Rosenberg et al., 2002; Wright & Andolfatto, 2008). To date, how- ever, genome-wide estimates of nucleotide diversity and linkage disequilibrium have allowed for detailed analysis of demographic history, selection and contemporary popula- tion structure in only a limited number of model plant spe- cies (Nordborg et al., 2005; Caicedo et al., 2007; Francois et al., 2008; Ingvarsson, 2008b). These, and other studies have leveraged information on the outliers of empirical distributions of diversity, frequency spectra, and LD among sites to identify likely targets of species-wide (Bustamante et al., 2005; Gojobori et al., 2007; Shapiro et al., 2007; Wright & Andolfatto, 2008; Cai et al., 2009) and local adaptation (Caicedo et al., 2004; Kane &

    Rieseberg, 2008; Moeller & Tiffin, 2008; McCracken et al., 2009). Multi-locus sampling also aids in estimation of the roles that selective constraint, background selection, and hitchhiking have played in shaping nucleotide diversity in regions of the genome with different func- tional characteristics (Clark et al., 2007; Andolfatto, 2008; Makinen et al., 2008; Wright & Andolfatto, 2008; McVicker et al., 2009; Sella et al., 2009). Finally, gen- ome-scale data are necessary for evaluating the potential power of marker-based approaches for identifying genes underlying phenotypic variation. Although association mapping techniques are not without their problems (McCarthy et al., 2008), association techniques remain attractive to forest tree breeders and researchers because they offer the potential to identify genomic regions (per- haps even individual nucleotides) underlying phenotypic variation without the time-intensive aspects associated with QTL mapping population development (Mackay, 2001;

    New PhytologistResearch

    526 New Phytologist (2010) 186: 526–536

    � The Authors (2010) Journal compilation � New Phytologist Trust (2010)

  • Brunner & Nilsson, 2004; Neale & Savolainen, 2004; Neale & Ingvarsson, 2008).

    Poplars (Populus spp.) have become a model system for studies of angiosperm tree physiology and genetics (Tuskan et al., 2006) offering a set of study organisms that span a wide range of habitats and community interactions (Stettler et al., 1996; Martin et al., 2004; LeRoy et al., 2006). Fur- ther development of population genetic resources for poplar species not only provides data from a long-lived perennial for comparison against existing short-lived population genetic models (e.g. Arabidopsis, Zea, Oryza), but also sets the stage for investigating the genetic basis of wood forma- tion, perenniality and dormancy (Brunner et al., 2004). Surveys of nucleotide diversity in Populus tremula are more extensive than those conducted for any other woody angio- sperm. An investigation of 77 gene fragments from the European portions of P. tremula’s range revealed a species with relatively high diversity (h = 0.018) and low LD (r2

    dropped to < 0.1 within 200 bp); both properties are con- sistent with a large effective population size (Ingvarsson, 2008b). Nucleotide diversity and LD in P. tremula suggest that association mapping approaches in this species may require dense sampling of single nucleotide polymorphisms (SNPs), but also may be effective at identifying very small genomic regions underlying phenotypic variation, perhaps even causative SNPs. Indeed, association mapping trials in P. tremula have identified candidate quantitative trait nucle- otides for influencing the timing of seasonal dormancy (Hall et al., 2007; Ingvarsson et al., 2008). Because few species of poplar are characterized for nucleotide diversity across many loci, it is unclear whether patterns of diversity and LD are common properties of the genus or historical contingencies have resulted in differences among species.

    Here, we present analyses of nucleotide polymorphism found in a survey of 590 genome regions sequenced from each of 15 balsam poplar (Populus balsamifera) trees sam- pled from different populations across the species’ range. This sampling scheme was designed to minimize effects of population structure in order to best discern species-wide patterns of diversity (Wakeley & Lessard, 2003; Staedler et al., 2009). Although the inclusion of two samples from each population (by sampling both chromosomes from each individual) may introduce minor effects of population structure, because population structure tends to be low in P. balsamifera (Keller et al., 2010) we expect that these effects are weak. We characterize species-wide genetic diversity, patterns of linkage disequilibrium decay and compare site frequency distributions among different functional catego- ries of nucleotide polymorphisms. Our data provide an in- trageneric comparison to patterns found in P. tremula; these two species have similar ecological roles in contemporary communities and may have been affected in similar ways by Quaternary climate fluctuations. Because balsam poplar is common throughout Canada and Alaska, it has garnered

    intense interest for silvicultural applications including car- bon sequestration, windrows, biofuels and wood products. For this reason, identification of functionally important genetic variants may not only provide insight into past adaptation into the species’ current niche, but also be valu- able in breeding programs.

    Materials and Methods

    Populus balsamifera L. is a dioecious (obligately outcrossing) species with a range from Newfoundland to Alaska at lati- tudes from 42�N to nearly 70�N. It is a key component of boreal forests, with natural populations in the Arctic (Bock- heim et al., 2003), further north than any other North American tree. Balsam poplar is the sister species to Populus trichocarpa (black cottonwood), with c. 25% shared ances- tral polymorphism (N. Levsen, pers. comm.). Although the USDA plants database ( classifies these taxa as subspecies because of morphological similarity, we refer to them as separate species to follow the precedent in recent scientific literature.

    For this study we sampled a single individual from each of 15 populations that spanned the geographical range and ecological environments in which P. balsamifera grows (Fig. 1). Most of these trees are part of the live AgCanBaP collection that is maintained at the Prairie Farm Rehabilita- tion Administration Shelterbelt Centre in Indian Head Canada. These trees were originally collected from the natu- ral populations as stem cuttings from trees separated from one another by > 200 m when possible, to minimize the possibility of sampling clonal stems. Stems were rooted and transplanted into common gardens in Indian Head Saskatchewan, Canada (latitude 50�N), Vancouver British Columbia Canada (49�N), and Fairbanks Alaska USA (65�N). Leaf or bud tissues for this study were collected from the Indian Head common garden and dried in silica gel before extraction in Qiagen DNeasy Plant Maxi kits using the manufacturer’s protocols.

    Primers for 881 gene fragments were designed using primer3 v1.0.0 (see the Supporting Information, Table S1; Rozen & Skaletsky, 2000). The target genes for these fragments were randomly s