BACKGROUND: QTL cloning for the discovery of genes underlying polygenic traits has historically been cumbersome in long-lived perennial plants like Populus. Linkage disequilibrium-based association mapping has been proposed as a cloning tool, and recent advances in high-throughput genotyping and whole-genome resequencing enable marker saturation to levels sufficient for association mapping with no a priori candidate gene selection. Here, multiyear and multienvironment evaluation of cell wall phenotypes was conducted in an interspecific P. trichocarpa x P. deltoides pseudo-backcross mapping pedigree and two partially overlapping populations of unrelated P. trichocarpa genotypes using pyrolysis molecular beam mass spectrometry, saccharification, and/ or traditional wet chemistry. QTL mapping was conducted using a high-density genetic map with 3,568 SNP markers. As a fine-mapping approach, chromosome-wide association mapping targeting a QTL hot-spot on linkage group XIV was performed in the two P. trichocarpa populations. Both populations were genotyped using the 34 K Populus Infinium SNP array and whole-genome resequencing of one of the populations facilitated marker-saturation of candidate intervals for gene identification. RESULTS: Five QTLs ranging in size from 0.6 to 1.8 Mb were mapped on linkage group XIV for lignin content, syringyl to guaiacyl (S/G) ratio, 5- and 6-carbon sugars using the mapping pedigree. Six candidate loci exhibiting significant associations with phenotypes were identified within QTL intervals. These associations were reproducible across multiple environments, two independent genotyping platforms, and different plant growth stages. cDNA sequencing for allelic variants of three of the six loci identified polymorphisms leading to variable length poly glutamine (PolyQ) stretch in a transcription factor annotated as an ANGUSTIFOLIA C-terminus Binding Protein (CtBP) and premature stop codons in a KANADI transcription factor as well as a protein kinase. Results from protoplast transient expression assays suggested that each of the polymorphisms conferred allelic differences in the activation of cellulose, hemicelluloses, and lignin pathway marker genes. CONCLUSION: This study illustrates the utility of complementary QTL and association mapping as tools for gene discovery with no a priori candidate gene selection. This proof of concept in a perennial organism opens up opportunities for discovery of novel genetic determinants of economically important but complex traits in plants.

BioEnergy Science Center Oak Ridge National Laboratory Oak Ridge TN 37831 USA

Bioscience Center National Renewable Energy Laboratory 15013 Denver West Parkway Golden CO 80401 USA

Current address Department of Plant Biology Carnegie Institute for Science Stanford CA 94305 USA

Department of Biology West Virginia University Morgantown WV 26506 USA

Department of Botany University of British Columbia Vancouver BC V6T 1Z4 Canada

Department of Forest and Conservation Sciences Faculty of Forestry University of British Columbia Forest Sciences Centre 2424 Main Mall Vancouver BC V6T 1Z4 Canada

Department of Genetics and Physiology of Forest Trees Faculty of Forestry and Wood Sciences Czech University of Life Sciences Prague Kamýcká 129 165 21 Praha 6 Czech Republic

Department of Wood Science Faculty of Forestry University of British Columbia Forest Sciences Centre 2424 Main Mall Vancouver BC V6T 1Z4 Canada

Institute of Biological Environmental and Rural Sciences Aberystwyth University Aberystwyth SY23 3EB UK

U S Department of Energy Joint Genome Institute Walnut Creek CA 94598 USA

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Dinus RJ, Payne P, Sewell MM, Chiang VL, Tuskan GA. Genetic modification of short rotation poplar wood: Properties for ethanol fuel and fiber production. Crit Rev Plant Sci. 2001;20:51–69. doi: 10.1016/S0735-2689(01)80012-5. DOI

Studer MH, DeMartini JD, Davis MF, Sykes RW, Davison B, Keller M, et al. Lignin content in natural Populus variants affects sugar release. Proc Natl Acad Sci U S A. 2011;108:6300–6305. doi: 10.1073/pnas.1009252108. PubMed DOI PMC

Lynd LR, Cushman JH, Nichols RJ, Wyman CE. Fuel ethanol from cellulosic biomass. Science. 1991;251:1318–1323. doi: 10.1126/science.251.4999.1318. PubMed DOI

Vermerris W, Saballos A, Ejeta G, Mosier NS, Ladisch MR. Molecular breeding to enhance ethanol production from corn and sorghum stover. Crop Sci. 2007;47:S142–S153. doi: 10.2135/cropsci2007.04.0013IPBS. DOI

Fu C, Mielenz J, Xiao X, Ge Y, Hamilton CY, Rodriguez M, Jr, et al. Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proc Natl Acad Sci U S A. 2011;108:3803–3808. doi: 10.1073/pnas.1100310108. PubMed DOI PMC

Novaes E, Osorio L, Drost DR, Miles BL, Boaventura-Novaes CRD, Benedict C, et al. Quantitative genetic analysis of biomass and wood chemistry of Populus under different nitrogen levels. New Phytol. 2009;182:878–890. doi: 10.1111/j.1469-8137.2009.02785.x. PubMed DOI

Yin T, Zhang X, Gunter L, Priya R, Sykes R, Davis M, et al. Differential detection of genetic loci underlying stem and root lignin content in Populus. PLoS ONE. 2010;5:e14021. doi: 10.1371/journal.pone.0014021. PubMed DOI PMC

Rafalski JA. Association genetics in crop improvement. Curr Opin Plant Biol. 2010;13:174–180. doi: 10.1016/j.pbi.2009.12.004. PubMed DOI

Wegrzyn JL, Eckert AJ, Choi M, Lee JM, Stanton BJ, Sykes R, et al. Association genetics of traits controlling lignin and cellulose biosynthesis in black cottonwood (Populus trichocarpa, Salicaceae) secondary xylem. New Phytol. 2010;188:515–532. doi: 10.1111/j.1469-8137.2010.03415.x. PubMed DOI

Guerra FP, Wegrzyn JL, Sykes R, Davis MF, Stanton BJ, Neale DB. Association genetics of chemical wood properties in black poplar (Populus nigra) New Phytol. 2013;197:162–176. doi: 10.1111/nph.12003. PubMed DOI

Du Q, Pan W, Tian J, Li B, Zhang D. The UDP-Glucuronate decarboxylase gene family in Populus: Structure, expression and association genetics. PLoS ONE. 2013;8:e60880. doi: 10.1371/journal.pone.0060880. PubMed DOI PMC

Porth I, Klápště J, Skyba O, Hannemann J, Mckown AD, Guy R, et al. Genome-wide association mapping for wood characteristics in Populus identifies an array of candidate single nucleotide polymorphisms. New Phytol. 2013;200:710–726. doi: 10.1111/nph.12422. PubMed DOI

Slavov GT, DiFazio SP, Martin J, Schackwitz W, Muchero W, et al. Genome resequencing reveals multiscale geographic structure and extensive linkage disequilibrium in the forest tree Populus trichocarpa. New Phytol. 2012;196:713–725. doi: 10.1111/j.1469-8137.2012.04258.x. PubMed DOI

Geraldes A, DiFazio S, Slavov GT, Priya R, Muchero W, et al. A 34 K SNP genotyping array for Populus trichocarpa: Design, application to the study of natural populations and transferability to other Populus species. Mol Ecol Resour. 2013;13:306–323. doi: 10.1111/1755-0998.12056. PubMed DOI

Porth I, Klápště J, Skyba O, Lai BSK, Geraldes A, Muchero W, et al. Populus trichocarpa cell wall chemistry and ultrastructure trait variation, genetic control and genetic correlations. New Phytol. 2013;197:777–790. doi: 10.1111/nph.12014. PubMed DOI

Faix O, Meier D, Fortmann Thermal degradation products of wood: Gas chromatographic separation and mass spectrometric characterization of monomeric lignin derived products. Holz als Rohund Werkstoff. 1990;48:281–285. doi: 10.1007/BF02639897. DOI

Sun Y-P, Nguyen KL, Wallis AFA. Ring-opened products from reaction of lignin model compounds with UV-assisted peroxide. Holzforschung. 1998;52:61–66. doi: 10.1515/hfsg.1998.52.1.61. DOI

Evans RJ, Milne TA. Molecular characterization of the pyrolysis of biomass. 1. Fundamentals. Energy Fuels. 1987;1:123–137. doi: 10.1021/ef00002a001. DOI

Tuskan G, West D, Bradshaw HD, Neale D, Sewell M, et al. Two high-throughput techniques for determining wood properties as part of a molecular genetics analysis of hybrid poplar and loblolly pine. Appl Biochem Biotechnol. 1999;77:55–65. doi: 10.1385/ABAB:77:1-3:55. DOI

Sykes R, Yung M, Novaes E, Kirst M, Peter G, Davis M. High-throughput screening of plant cell-wall composition using pyrolysis molecular beam mass spectroscopy. In: Mielenz JR, editor. Biofuels: Methods and Protocols. New York: Humana Press; 2009. pp. 169–183. PubMed

Ranjan P, Yin T, Zhang X, Kalluri UC, Yang X, Jawdy S, et al. Bioinformatics-based identification of candidate genes from QTLs associated with cell wall traits in Populus. Bioenergy Res. 2010;3:172–182. doi: 10.1007/s12155-009-9060-z. DOI

Plavcová L, Hacke UG, Almeida-Rodriguez AM, Li E, Douglas CJ. Gene expression patterns underlying changes in xylem structure and function in response to increased nitrogen availability in hybrid poplar. Plant Cell Environ. 2013;36:186–199. doi: 10.1111/j.1365-3040.2012.02566.x. PubMed DOI

Chen L, Ortiz-Lopez A, Jung A, Bush DR. ANT1, an aromatic and neutral amino acid transporter in Arabidopsis. Plant Physiol. 2001;125:1813–1820. doi: 10.1104/pp.125.4.1813. PubMed DOI PMC

Lu S, Li Q, Wei H, Chang M-J, Tunlaya-Anukit S, Kim H, et al: Ptr-miR397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa. Proc Natl Acad Sci USA 2013, doi:10.1073/pnas.1308936110. PubMed PMC

Díaz J, Bernal A, Pomar F, Merino F. Induction of shikimate dehydrogenase and peroxidase in pepper (Capsicum annuum L.) seedlings in response to copper stress and its relation to lignifications. Plant Sci. 2001;161:179–188. doi: 10.1016/S0168-9452(01)00410-1. DOI

Kováčik J, Klejdus B. Dynamics of phenolic acids and lignin accumulation in metal-treated Matricaria chamomilla roots. Plant Cell Rep. 2008;27:605–615. doi: 10.1007/s00299-007-0490-9. PubMed DOI

Downes GM, Ward JV, Turvey ND. Lignin distribution across tracheid cell walls of poorly lignified wood from deformed copper deficient Pinus radiata (D. Don) Wood Sci Technol. 1991;25:7–14. doi: 10.1007/BF00195552. DOI

Manley HA, Lennon VA. Endoplasmic reticulum membrane-sorting protein of lymphocytes (BAP31) is highly expressed in neurons and discrete endocrine cells. J Histochem Cytochem. 2001;49:1235–1243. doi: 10.1177/002215540104901005. PubMed DOI

Kalluri UC, Hurst GB, Lankford PK, Priya R, Pelletier DA. Shotgun proteome profile of Populus developing xylem. Proteomics. 2009;9:4871–4880. doi: 10.1002/pmic.200800854. PubMed DOI

Atanesyan L, Günther V, Dichtl B, Georgiev O, Schaffner W. Polyglutamine tracts as modulators of transcriptional activation from yeast to mammals. Biol Chem. 2012;393:63–70. doi: 10.1515/BC-2011-252. PubMed DOI

Chinnadurai G. CtBP family proteins: More than transcriptional corepressors. Bioessays. 2002;25:9–12. doi: 10.1002/bies.10212. PubMed DOI

Kim G-T, Shoda K, Tsuge T, Cho K-H, Uchimiya H, et al. The ANGUSTIFOLIA gene of Arabidopsis, a plant CtBP gene, regulates leaf-cell expansion, the arrangement of cortical microtubules in leaf cells and expression of a gene involved in cell-wall formation. EMBO J. 2002;21:1267–1279. doi: 10.1093/emboj/21.6.1267. PubMed DOI PMC

IIegems M, Douet V, Meylan-Bettex M, Uyttewaal M, Brand L, Bowman JL, et al. Interplay of auxin, KANADI and Class III HD-ZIP transcription factors in vascular tissue formation. Development. 2010;137:975–984. doi: 10.1242/dev.047662. PubMed DOI

Hepler PK. Calcium: A central regulator of plant growth and development. Plant Cell. 2005;17:2142–2155. doi: 10.1105/tpc.105.032508. PubMed DOI PMC

Lautner S, Ehlting B, Windeisen E, Rennenberg H, Matyssek R, et al. Calcium nutrition has a significant influence on wood formation in poplar. New Phytol. 2007;173:743–752. doi: 10.1111/j.1469-8137.2007.01972.x. PubMed DOI

Vanholme B, Cesarino I, Goeminne G, Kim H, Marroni F, Van Acker R, et al. Breeding with rare defective alleles (BRDA): a natural Populus nigra HCT mutant with modified lignin as a case study. New Phytol. 2013;198:765–776. doi: 10.1111/nph.12179. PubMed DOI

Libby WJ, Cockerham CC. Random non-contiguous plots in interlocking field layouts. Silvae Genet. 1980;29:183–190.

Van Ooijen JW. MapQTL®6, Software for the mapping of quantitative trait loci in experimental populations of diploid species Kyazma BV. Netherlands: Wageningen; 2009.

Churchill GA, Doerge RW. Empirical threshold values for quantitative trait mapping. Genetics. 1994;138:963–971. PubMed PMC

Yu J, Pressoir G, Briggs WH, Bi IV, Yamasaki M, et al. A unified mixed-model method for association mapping accounting for multiple levels of relatedness. Nat Genet. 2005;38:203–208. doi: 10.1038/ng1702. PubMed DOI

Bonferroni CE. II calcolo delle assicurazioni su gruppi di teste chapter “Studi in Onore del Professore Salvatore ortu Carboni” Rome 13–60. 1935.

Analytical software . Statistix 8 User’s manual: Analytical software Tallahassee, FL. 2003.

Sexual homomorphism in dioecious trees: extensive tests fail to detect sexual dimorphism in Populus †