Linkage disequilibrium (LD) mapping using natural populations results in higher resolution of markertrait associations compared to family-based quantitative trait locus (QTL) studies. Depending on the extent of LD, it is possible to identify alleles within candidate genes associated with a trait. Analysis of a natural mutant in Arabidopsis has shown that mutations in cinnamoyl CoA reductase (CCR), a key lignin gene, affect physical properties of the secondary cell wall such as stiffness and strength. Using this gene, we tested whether LD mapping could identify alleles associated with microfibril angle (MFA), a wood quality trait affecting stiffness and strength of wood. We identified 25 common single-nucleotide polymorphism (SNP) markers in the CCR gene in Eucalyptus nitens. Using single-marker and haplotype analyses in 290 trees from a E. nitens natural population, two haplotypes significantly associated with MFA were found. These results were confirmed in two full-sib families of E. nitens and Eucalyptus globulus. In an effort to understand the functional significance of the SNP markers, we sequenced the cDNA clones and identified an alternatively spliced variant from the significant haplotype region. This study demonstrates that LD mapping can be used to identify alleles associated with wood quality traits in natural populations of trees.

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Populations with low linkage disequilibrium (LD) offer unique opportunities to study functional variants influencing quantitative traits. We exploited the low LD in forest trees to identify functional polymorphisms in a Eucalyptus nitens COBRA-like gene (EniCOBL4A), whose Arabidopsis homolog has been implicated in cellulose deposition. Linkage analysis in a full-sib family revealed that EniCOBL4A is the most strongly associated marker in a quantitative trait locus (QTL) region for cellulose content. Analysis of LD by genotyping 11 common single-nucleotide polymorphisms (SNPs) and a simple sequence repeat (SSR) in an association population revealed that LD declines within the length of the gene. Using association studies we fine mapped the effect of the gene to SNP7, a synonymous SNP in exon 5, which occurs between two small haplotype blocks. We observed patterns of allelic expression imbalance (AEI) and differential binding of nuclear proteins to the SNP7 region that indicate that SNP7 is a cis-acting regulatory polymorphism affecting allelic expression. We also observed AEI in SNP7 heterozygotes in a full-sib family that is linked to heritable allele-specific methylation near SNP7. This study demonstrates the potential to reveal functional polymorphisms underlying quantitative traits in low LD populations. I N plants, gene function is typically investigated byreverse genetic approaches such as knockout mutants and overexpression transgenics. Functional analysis of subtle allele effects under native conditions is difficult. The large extent of linkage disequilibrium (LD) is one of the obstacles to fine mapping of functional variants in many plant species. In this context forest trees offer unique opportunities for fine mapping of candidate genes and functional analysis of gene variants, as LD and population structure are generally low (Brown et al. 2004;Thumma et al. 2005;Neale 2007). In inbreeding species LD typically extends over much larger distances compared to outcrossing species (Nordborg 2000). In Arabidopsis, average LD extends to $10 kb (Kim et al. 2007), while in the outcrossing maize and forest tree species, rapid decline in LD (within #1 kb) was observed in a number of candidate genes (Thornsberry et al. 2001;Savolainen and Pyhajarvi 2007). Some of the factors that are likely to have contributed to the low LD in forest trees include large effective population size, their outcrossing habit, and long history of recombination.The functional significance of nonsynonymous polymorphisms may be linked to protein phenotype while synonymous and noncoding single-nucleotide polymorphisms (SNPs) can affect phenotype by influencing alternative splicing, altering mRNA stability and/or allelic expression imbalance (AEI). Allelic expression imbalance or allele-specific expression analysis in heterozygous individuals is a powerful method for identifying cis-acting regulatory variants. Expression of a particular allele is controlled by both cis-acting factors such as DNA polymorphisms and methylation and trans...