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Genetic Analysis of Early Adventitious Root Development in a Pseudo-Backcross Population of Populus.

Permanent Link: http://ufdc.ufl.edu/UFE0042613/00001

Material Information

Title: Genetic Analysis of Early Adventitious Root Development in a Pseudo-Backcross Population of Populus.
Physical Description: 1 online resource (78 p.)
Language: english
Creator: Silva, Cynthia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: adventitious, cuttings, deltoides, expressed, genes, genetic, genetics, genome, greenwood, microarray, poplar, poplars, populus, pseudobackcross, qtl, quantitative, root, rooting, trichocarpa
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Forest Resources and Conservation thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The ability of stem cuttings to form adventitious roots (AR) is critical for propagation of trees species that are cultivated for bioenergy, pulp and paper, and timber production. However, genes and molecular mechanisms that control AR formation are largely unknown. To identify quantitative trait loci (QTLs) that regulate AR development in poplar ? a tree model species ? we measured rooting response in a pseudo-backcross population derived from a cross between a hybrid female Populus trichocarpa times Populus deltoides, and an unrelated male parent P. deltoides. Apical cuttings were collected from 234 individuals and grown in a hydroponic system for 18 days. The number of ARs was recorded daily. After 18 days, roots were harvested and scanned to measure length, volume, and surface area of total, primary (first order) roots, and root branches, as well as dry biomass and average diameter. QTLs for number of roots and root architectural traits were identified using composite interval mapping performed on the progeny's mother map. Significant QTLs for most of these traits mapped consistently in two regions of linkage groups II and XIV. Next, each genotype was categorized depending on the allele carried in each of the two main QTL regions identified previously. Genotypes carrying P. trichocarpa alleles in both QTL regions generally had higher number of roots than genotypes carrying the P. deltoides alleles. Three genotypes were selected within each QTL category for gene expression analysis using whole-transcriptome microarrays, at multiple time points. This study enabled the detection of significant differences in transcript abundance across time points and between extreme genotypes, and identification of candidate genes that control number of ARs. Of particular importance among these was the gene superroot2 (SUR2), which was more highly expressed in poor rooters. Mutants for SUR2 have been reported to cause overproduction of auxin and higher number of adventitious roots in Arabidopsis. This study also allowed insights into the genes expressed during different phases of AR formation in Populus.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Cynthia Silva.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Kirst, Matias.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042613:00001

Permanent Link: http://ufdc.ufl.edu/UFE0042613/00001

Material Information

Title: Genetic Analysis of Early Adventitious Root Development in a Pseudo-Backcross Population of Populus.
Physical Description: 1 online resource (78 p.)
Language: english
Creator: Silva, Cynthia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: adventitious, cuttings, deltoides, expressed, genes, genetic, genetics, genome, greenwood, microarray, poplar, poplars, populus, pseudobackcross, qtl, quantitative, root, rooting, trichocarpa
Forest Resources and Conservation -- Dissertations, Academic -- UF
Genre: Forest Resources and Conservation thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The ability of stem cuttings to form adventitious roots (AR) is critical for propagation of trees species that are cultivated for bioenergy, pulp and paper, and timber production. However, genes and molecular mechanisms that control AR formation are largely unknown. To identify quantitative trait loci (QTLs) that regulate AR development in poplar ? a tree model species ? we measured rooting response in a pseudo-backcross population derived from a cross between a hybrid female Populus trichocarpa times Populus deltoides, and an unrelated male parent P. deltoides. Apical cuttings were collected from 234 individuals and grown in a hydroponic system for 18 days. The number of ARs was recorded daily. After 18 days, roots were harvested and scanned to measure length, volume, and surface area of total, primary (first order) roots, and root branches, as well as dry biomass and average diameter. QTLs for number of roots and root architectural traits were identified using composite interval mapping performed on the progeny's mother map. Significant QTLs for most of these traits mapped consistently in two regions of linkage groups II and XIV. Next, each genotype was categorized depending on the allele carried in each of the two main QTL regions identified previously. Genotypes carrying P. trichocarpa alleles in both QTL regions generally had higher number of roots than genotypes carrying the P. deltoides alleles. Three genotypes were selected within each QTL category for gene expression analysis using whole-transcriptome microarrays, at multiple time points. This study enabled the detection of significant differences in transcript abundance across time points and between extreme genotypes, and identification of candidate genes that control number of ARs. Of particular importance among these was the gene superroot2 (SUR2), which was more highly expressed in poor rooters. Mutants for SUR2 have been reported to cause overproduction of auxin and higher number of adventitious roots in Arabidopsis. This study also allowed insights into the genes expressed during different phases of AR formation in Populus.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Cynthia Silva.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Kirst, Matias.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0042613:00001


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GENETIC ANAL YSIS OF EARLY ADVENT ITIOUS ROOT DEVELOPMENT IN A PSEUDO-BACKCROSS POPULATION OF Populus By CYNTHIA M. SILVA A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORID A IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010 1

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2010 Cy nthia M. Silva 2

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To my Mom, Dad, and sisters 3

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ACK NOWLEDGMENTS I would like to thank to my advisor, Dr. Matias Kirst, for a ccepting me in his research group, for his guidance and patience. I also want to thank my committee members, Dr. John Davis and Dr. Timothy Martin, for their c onstructive suggestions and guidance during my graduate studies and resear ch work. I am grateful to Dr. Luis Osorio and Dr. Dudley Huber for all the help provided in the statistical analysis of my data. Special thanks to Juan Acosta, for hel ping me with the analysis and interpretation of the microarray data, and Carolina Novaes, with the RNA extraction and labeling. This project could have never been c oncluded without the generous help of several colleagues to whom I will be eternally grateful: Chris Dervinis, Kathy Smith, Evandro and Carolina Novaes, Derek Drost, Ryan Brown, Tania Q uesada, Catherine Benedict, Leandro Neves, Barbar a Kahn, Juan Acosta, Cintia Ribeiro, and Luis Osorio. Special thanks to Chris Dervinis, for assi sting me during almost all steps of my experiment, and to Tania Quesada and Ryan Brown for helping me collect cuttings under the rain, a day that I will never for get! Thanks to Carolina Novaes, Fatima Barreto, Juliana Teixeira, and Cintia Ribeiro fo r the emotional support during the course of my graduate program. Many thanks to Juliana Teixeira, fo r the uncountable times she spent teaching, encouraging, and guiding me not only during my undergrad, but mostly after that, during my Master 's program. Thanks to my fo rmer advisors Dr. Luis Camargo and Dr. Elizabeth Ann Veasey, for believing and motivating me. I am also grateful to my best friend, Ma rio Blanco, for being such a good listener, even though I am a bad speaker. Most specially, thanks to my fam ily that made all this possible. 4

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TABL E OF CONTENTS page ACKNOWLEDG MENTS..................................................................................................4 LIST OF TABLES............................................................................................................7 LIST OF FI GURES..........................................................................................................8 ABSTRACT .....................................................................................................................9 CHAPTER 1 LITERATURE REVIEW..........................................................................................11 Introducti on.............................................................................................................11 Interspecific Hybrids in Populus ..............................................................................14 Quantitative Genetics of Growth and De velopment................................................15 P. trichocarpa P. deltoides Hybrid s......................................................................16 Adventitious R oot Forma tion...................................................................................18 Factors Regulat ing ARF...................................................................................19 Genes Related to ARF.....................................................................................22 Genetic Control of Gene Expre ssion ......................................................................24 2 GENETIC ANALYSIS OF EARLY ADVE NTITIOUS ROOT DEVELOPMENT IN A PSEUDO-BACKCROSS POPULA TION OF PO PULUS......................................28 Introducti on.............................................................................................................28 Material and Methods.............................................................................................30 Plant Mate rials..................................................................................................30 Phenotypic Meas urement s...............................................................................30 Statistical Analys is............................................................................................31 QTL Anal ysis....................................................................................................32 Selection of Genotypes wit h Alternative Alleles fo r Genomic Regions that Control Adventitious Root Fo rmation............................................................32 Tissue samp ling.........................................................................................33 RNA extraction, cDNA syn thesis and l abeling ...........................................33 Microarray experimental design and data analysis ....................................34 Annotatio n..................................................................................................35 Result s....................................................................................................................35 Analysis of Par ental Genot ypes.......................................................................35 Phenotypic Va riation........................................................................................35 Genetic Control of Earl y Root-Relat ed Trai ts...................................................36 Phenotypic Corre lations...................................................................................36 QTL Analysis of Nu mber of Root ......................................................................36 QTL Analysis of Root Architectura l Trai ts.........................................................37 QTL Analysis of Root Biom ass.........................................................................37 5

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Genotypes with Alternativ e Rooting A lleles ...................................................... 37 Transcriptome Analysis of Advent itious Root De velopment.............................38 Gene expression analysis of time effect....................................................38 Gene expression analysis of genotype effect.............................................39 Gene expression comparison between extreme genotypes within time point ........................................................................................................40 Cluster ana lysis..........................................................................................41 Genetical genomic analysis of QTLs for r oot num ber................................43 Discussio n..............................................................................................................44 QTL Identific ation.............................................................................................45 Adventitious Root ing Anal ysis..........................................................................45 Successive Phases in Advent itious Root Formation........................................46 Conclusi ons............................................................................................................48 LIST OF RE FERENCES...............................................................................................68 BIOGRAPHICAL SKETCH ............................................................................................78 6

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LIST OF TABLES Table page 1-1 Broad-sense heritabilities of adventitious root-related traits of different types of stem cuttings of Populus species under different conditions of growth and type of mate rial...................................................................................................27 2-1 Clonal repeatability estimates for all adventitious root-related phenotypes measur ed........................................................................................................... 64 2-2 Pair-wise estimates of phenotypic correlations between all trai ts.......................65 2-3 Phenotypic variance explained by each QT L interval identified for number of root traits and the respective linkage group (LG), flanking markers location, LOD peak and origin of positive a llele................................................................66 2-4 Phenotypic variance explained by eac h QTL interval detected for root architecture traits and root biomass with the respective linkage group (LG), flanking marker location, LOD score and origin of posit ive alle le........................67 7

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LIST OF FIGURES Figure page 2-1 Subset of the microarray experimental design showing only two time points.....49 2-2 LSM of number of adventitious root s developed on the female hybrid parent Populus trichocarpa P. deltoides 52-225 ( TD ), and the unrelated male parent P. deltoides D124 ( D ) maintained in hydroponic solution for 25 days....50 2-3 Distribution of Least Square Mean (LSM) estimates for number of root traits of 236 individuals of family 52-124 and their parents P. deltoides' (D) and P. trichocarpa P.deltoides) P.deltoides (TD).....................................................51 2-4 Cumulative percentage of genotypes rooted during the experiment in each replicatio n...........................................................................................................53 2-5 Distribution of Least Square Mean (LSM) values for root architectural and biomass traits of 225 individuals of family 52-124 and their parents P. deltoides' (D) and ( P. trichocarpa P.deltoides ) P.deltoides (TD), measured after 18 days in hydroponic so lution..................................................54 2-6 Localization of 11 quantit ative trait loci (QTLs) detected on the mother map for number of roots.............................................................................................56 2-7 Localization of 15 quantit ative trait loci (QTLs) detected on the mother map for root architectural trai ts and root bi omass......................................................57 2-8 Least square means (LSM) of number of adventitious roots developed on selected genotypes for each day meas ured.......................................................58 2-9 Least Square Means (LSM) of number of adventitious roots developed on extreme genotypes selected fo r expression a nalysis. ........................................59 2-10 Number of genes differ entially expressed when contrasting consecutive time points. .................................................................................................................60 2-11 Total number of genes differentially expressed between genotypes in the Pt QTL and Pd QTL categories within each time point.........................................61 2-13 Average of absolute val ues of signal differences between genotypes in the Pt QTL and Pd QTL categories across each time point for the only cluster with a linear increase in the difference in transcript abundance between the two categorie s...........................................................................................................63 8

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Abstract of Thesis Pres ented to the Graduate School of the University of Fl orida in Partial Fulf illment of the Requirements for t he Degree of Master of Science GENETIC ANALYSIS OF EARLY ADVENT ITIOUS ROOT DEVELOPMENT IN A PSEUDO-BACKCROSS POPULATION OF Populus By Cynthia M. Silva December 2010 Chair: Matias Kirst Major: Forest Resources and Conservation The ability of stem cuttings to form adventitious roots (AR) is critical for propagation of trees species that are cultivated for bioenergy, pulp and paper, and timber production. However, genes and mo lecular mechanisms that control AR formation are largely unknown. To identify quantit ative trait loci (QTL s) that regulate AR development in poplar a tree model spec ies we measured rooting response in a pseudo-backcross population de rived from a cross between a hybrid female Populus trichocarpa Populus deltoides and an unrelated male parent P. deltoides Apical cuttings were collected from 234 individual s and grown in a hydr oponic system for 18 days. The number of ARs was recorded daily. After 18 days, roots were harvested and scanned to measure length, volume, and surfac e area of total, pr imary (first order) roots, and root branches, as well as dr y biomass and average diameter. QTLs for number of roots and root architectural traits were identified using composite interval mapping performed on the progeny's mother m ap. Significant QTLs for most of these traits mapped consistently in two r egions of linkage groups II and XIV. Next, each genotype was categorized depending on the allele carried in each of the two main QTL regions identified previously. Genotypes carrying P. trichocarpa 9

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10 alleles in both QTL regions generally had higher number of roots than genotypes carrying the P. deltoides alleles. Three genotypes were selected within each QTL category for gene expression analysis using whole-transcriptome microarrays, at multiple time points. This study enabled the detection of signifi cant differences in transcript abundance across time points and between extreme genot ypes, and identification of candidate genes that control number of ARs. Of parti cular importance among these was the gene superroot2 (SUR2), which was more highly expr essed in poor rooters. Mutants for SUR2 have been reported to cause overpr oduction of auxin and higher number of adventitious roots in Arabidopsis. This study also allowed insights into the genes expressed during different phas es of AR formation in Populus

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CHA PTER 1 LITERATURE REVIEW Introduction Species of the genus Populus comprise hardwood trees that belong to the Salicaceae family and include poplars, cottonwoods and aspens (Eckenwalder, 1996). Generally referred to as poplars (Heilman, 1999), the genus Populus consist of 29 species grouped into six sections: Abaso, Turanga, Leucoides, Aigeiros, Tacamahaca and Populus (Eckenwalder, 1996). Although there is no doubt about the existence of the genus Populus, the definition of species within the genus is still not well established, as well as their classification into each section. Problems in defining species and delineating sections are due to the great morphological diversity and extensive interspecific hybridization (Eckenwalder, 1996, Zsuffa, 1975). Despite these difficulties, poplar species have been traditionally placed into sections according to morphological and reproductive characteristics, as well as by their hybridization potential (Zsuffa, 1975). Species within a section can freely interc ross, but crosses of species of different sections are more difficult to occur. One of the remarkable characteristics of Populus species is their adaptability to a variety of ecological habitats. Poplars are widely distributed in the Northern Hemisphere, from far north to the tr opics (Dickmann, 2001, Dickmann and Kuzovkina, 2008). They are found naturally in riparian site s, but also in well drained uplands, with few species doing well in both types of si tes (Farmer Jr, 1996). Except for section Turanga all sections have at least one species native to North America (Eckenwalder, 1996). The Tacamahaca section contains nine species, three of them native to North America ( P. balsamifera, P. trichocarpa, and P. angustifolia ), while the Aigeiros section 11

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consists of three species, of which two occur naturally in North America ( P. deltoides and P. fre montii ) (Dickmann, 2001). In 2007, indigenous Populus species occupied more than 17 million hectares in the United States, and nearly 70 million hectares in the world (FAO, 2008). The United States is the third country with the greatest reported area of indigenous poplars, after Canada ( approximately 28 million hectares) and the Russian federation, with around 21 million hect ares (Ball et al., 2005). Most of the world's explored poplars grow in natural forests and are used for wood production (Heilman, 1999). Significant annual remova ls are reported in the Russian Federation and Canada, which remove around 100 and 16 m illion cubic meters, respectively (Ball et al., 2005). All species in the genus Populus except for P. lasiocarpa Oliv., are dioecious, thus obligate outcrossers (Eckenwalder 1996) The vast natural geographic range of poplars can be explained by their pollination and seed dispersal mechanisms: Populus species are wind-pollinated, producing very small and light seeds (300 to 16000 seeds per gram) that contain many cottony hairlike structures that are easily dispersed (Dickmann and Kuzovkina, 2008). These structur es allow them to be carried for more than ten kilometers by the wind, or even mo re distant on the surface of moving water (Dickmann and Kuzovkina, 2008). Poplars colonizing ability is also facilitat ed by their remarkable growth rates, which have attracted attention to the commercial use of these species. In forest plantations, rotations of 6-7 years are used for harvest ing pulp, and 10 years or longer for solid wood products (Heilman, 1999). In addition, poplars are easy to propagate vegetatively, a critical characteristic for deployment of superior genotypes in forestry plantations. 12

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Commercially, the wood of poplars has num erous uses: paper, veneer, plywood, and engineered wood products lik e oriented strand board (Heilm an, 1999). The country with the largest area of poplar plant ations is Chin a (4.9 million hectares, representing more than 70 % of the global popl ar plantation area), wher e poplars are used for wood production and environmental pur poses (Ball et al., 2005). In addition to their economic relevanc e, poplars play an im portant role in environmental restoration. Mo st poplar species inhabit ri parian sites (Eckenwalder, 1996), where they provide shelter to wildlif e and protect the margins of the rivers against soil erosion (Braatne et al., 1996) and runoff of agricultural chemicals (Ball et al., 2005). Moreover, poplars are used in refo restation of degraded areas due to their fast growth rates and short lifespan (Braatne et al., 1996). In several countries, including the United States, poplars have been used for phytoremediation purposes: decreasing pollutant concentrations in contaminated soils (Ball et al., 2005). Poplars are also used in carbon sequestration strategi es due to their rapid growth. More recently, poplars have become importa nt in scientific research as model organisms for tree biology studies. Populus trichocarpa Torr. &Gray was the first woody perennial plant to have its genome sequenced (Tuskan et al., 2006), and only the second dicotyledon, after Arabidopsis thaliana The Populus genome size is estimated to be approximately 485 MB, with 45,555 putative genes (Tuskan et al., 2006). Another important characte ristic of poplars is thei r relatedness with the model plant Arabidopsis (Bradshaw et al., 2000). Divergence of Arabidopsis and Populus is recent (100 to 120 million years ago [Mya], Tuskan et al., 2006) when compared to the divergence of angiosperm and gymnosperms (~ 300 Mya, Bowe et al 2000). The 13

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availability of both genome seque nces facilitat es comparative and evolutionary genomic studies, as well as the application of highthroughput genomic technology (Yang et al., 2009). Developmental processes such as wood formation, seasonality of growth and adaptability of perennial pl ants cannot be studied in Arabidopsis but are well suited for analysis in Populus (Bradshaw et al., 2000). A rema rkable similarity between Arabidopsis and poplar is described in the study of Bohlenius et al. (Bohlenius et al., 2006), where it was shown that th e CONSTANS/FLOWERING LOCUS T ( CO/FT ) regulatory module, which cont rols flowering time in response to the day length in Arabidopsis also controls flowering time in Populus However, this module also induces growth cessation and regulates bud set in the late season in Populus, a process absent in Arabidopsis In addition, as it has been pointed out by Wullschleger et al., ecological questions can also be answered using Populus as a model, because of the wide distribution of this genus and its adaptability a variety of sites (Wullschleger et al., 2002). Interspecific Hybrids in Populus Interspecific hybridization among poplars oc curs in nature between species of the same section and, eventually, between some species of different sections, as long as their range overlaps. The only exception occurs with species of the section Populus which are reproductively isolated from spec ies of other sections (Eckenwalder, 1996). For example, intersectional hybrids between species in the Aigeiros and Tacamahaca sections occur readily in nature (Dickm ann and Kuzovkina, 2008). Most hybrids are fertile because all species of this genus are diploid, with the same number of chromosome (2n=38) (Rae et al., 2007). Therefore, interspecific hybridization has been 14

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widely used to combine desirable traits of different species in selected genotypes (Stettler et al., 1996). Also, F1 hybrids have shown grea ter performance than the parents, a phenomenon known as heterosis or hybrid vigor (Heilman and Stettler 1985; Stettler et al 1988). In addition, in terspecific hybridizatio n has an important func tion in the stability of phenotypic variance of the hybrids in a vari ety of ecological habitats since hybrids combine traits of trees from different habitats (US EPA 1999). There are eight native species of Populus in North America, where several regions of natural hybridization occur (Eckenwalder 1996). These zones are usually relatively narrow (10-15 km), containing F 1 individuals and subsequent interspecific generations, such as F 2 and backcrosses (US EPA, 1999). The most common interspecific hybrids originate from crosses between members of the Aigeiros and Tacamahaca sections of Populus Selective crosses between Asian and North American species are known as Asian-american hybrids, bet ween Asian and European species as Eurasian hybrids', between European and North America specie s as Euroamerican', and between North America species as Intra-american' hybrids. The Euramerican hybrid between P. deltoides P. nigra is the most extensively used cross in forest plantations of poplars in North America. Hybrids between P. trichocarpa P. deltoides have also become important, mostly in commercial plantations of northwestern No rth America (Dickmann 2008). Quantitative Genetics of Growth and Development Assuming genetic variability among parental s pecies, interspecific hybrid crosses can provide insight into the genetic basis of species differences (Stettler et al., 1996) based on the analysis of trait segregation in F 2 progeny and backcrosses. These 15

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multigener ation pedigrees can be used in genet ic mapping experiments, to identify genes that regulate phenotypes (R ae et al., 2007). However, most traits of interest in forest trees, like growth and root ability, do not follo w simple Mendelian monogenic inheritance, being controlled by several genes with high influence of the environment. These traits are often referr ed to as quantitative traits, due to continuous distribution of values measured in segregating populations. T hus, the loci associated to these traits are known as quantitative trai t loci (QTL) (McClean, 1998). QTL analysis is used to study the genet ic basis of quantitative phenotypic variation, determining the position of a lo cus causing variation in the genome, and estimating the effect of the alleles and mode of action (Mackay et al., 2009). The goal of QTL mapping is to identify markers in close genetic distance to the causal loci (Mackay, 2001) that, consequently segr egate together due to lower pr obability of recombination. Therefore, this probab ility tends to increase with the physical distance (Mackay et al., 2009). Several traits have been evaluated through QTL analysis in Populus hybrids, including important adaptive traits that enhanc e survival in forest trees, like bud set and bud flush (Frewen et al., 2000), commercially im portant traits, like stem growth and form (Bradshaw and Stettler, 1995), and wood composit ion and biomass traits (Novaes et al., 2009). P. trichocarpa P. deltoides Hybrids Hybrids between P. trichocarpa and P. deltoides are commonly used in commercial plantations because of their s uperior growth perfo rmance (Heilman and Stettler, 1985; Stettler et al., 1988) The commonly called black cottonwood P. trichocarpa is the largest hardwood tree found in western North America and is the 16

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largest of the American poplars (DeBell, 1990). P. deltoides or eastern cottonwood, is one of the fastest-growing commercial species in North America and one of the largest eastern hardwoods, found mostly in the entire eastern region to the arid west of the United States (US EPA, 1999). Under favorable conditions, growth of these hybrids is superior to any other temperate tree species (Eckenwalder 2001). The superior properties of these hybrids relativ e to t he pure species may be due to their accelerated radial growth (Heilman and Stettler, 1985) Among the intersectional crosses between Tacahamaca and Aigeiros, the cross between P. trichocarpa and P. deltoides subsp. deltoides (southern cottonwood) is the most vigorous (Eckenwalder, 2001). The success of Populus hybrids is not only due to their remarkable growth rate and good performance, but also to the ease of their vegetative propagation which facilitates breeding and operatio nal deployment of superior individuals (White et al., 2007). Vegetative propagation enabl es the replication of s uperior genotypes, therefore capturing the additive and non-additive vari ance that contributes to the higher phenotypic value (Bradshaw 2000). Populus individuals have been vegetatively propagated as hardwood cuttings for centuries (Ritchie, 1994). The existence of root primordia in the inner bark allows the rapi d root development. However, only species and hybrids from sections Aigeiros and Tacahamaca have root primordia. Species form other sections do not typically develop adv entitious roots and r oot poorly (Dickmann 2008). All species of the section Tacamahaca are easily propagated by hardwood cuttings. Most species of the section Aigeiros also root well, wit h the exception of P. deltoides which is not easily propagated by hardw ood cuttings (Rae et al., 2007). This limitation is significantly improved in hybrids of P. deltoides and P. trichocarpa 17

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Adventitious Root Formation Adventitious roots are form ed in parts of the plant ot her than the embryonic root produced during embryogenes is (Barlow, 1986). Adventitious roots can develop from a variety of tissues. North American aspens ( Populus tremuloides and P. gradidentata ), for example, regenerate largely by vegetative suckering from the residual root systems (Pregitzer and Friend, 1996). Commercial popl ar plantations are based mostly on the propagation through adventiti ous rooting of hardwood cuttings (Ritchie, 1994). Adventitious root formation (ARF) is an organized developmental process that is generally divided into phases according to physiological, histological and biochemical observations (Davies and Hartmann, 1988). Generally, ARF is divided into three phases: the dedifferentiation phase, before any histological event; the induction, where cells start to divide to form an internal r oot meristem; and elongat ion or differentiation phase, where the root-primordia grows and em erges from the stem (De Klerk et al., 1999). Lateral and adventitious r oot formation follow a si milar organogenesis process, except that the later roots need to acquire competence for cell proliferation in most of the species. This involves the dedifferentiation of cells that were committed to a different developmental process (Srivastava, 2002) But both processes share the second induction phase', and third elongation phas e' (Ozawa et al., 1998). Although the identification of each phase may not be strai ghtforward in all species, studies have shown that each phase has its own r equirements (De Klerk et al., 1999). Some species have pre-defined sites for t he formation of root primordia (Lovell and White, 1986). Usually, adventitious root primordia arise close to the phloem and cambium, at the ray cells, or in bud or leaf gaps. They might also arise in the pericycle, 18

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which is the tissue located between endoder mis and phloem in roots ( Lovell and White, 1986). ARF may have important function in suppor ting and anchoring plants, as can be observed in vine plants and fig trees wit h the banyan habit (Barlow, 1986). ARF contributes to water-use effi ciency and extraction of nutrients from the soil. In these cases, adventitious roots are formed with no severance. However, in the case of stem cuttings, there is an added dimension of wound response (Srivastava, 2002). The breakage of the connection with the root sys tem in a cutting exposes stem tissue, interrupts transport of substanc es to roots and from them, and activate a series of repair responses and systemic signaling cycles (Lovell and White, 1986). The timing of each phase of adventitious root formation varies among species and depends on external stimuli. Ahkami et al (Ahkami et al., 2009) studied anatomical changes during ARF in Petunia hybrid and found that the root initiation phase may occur in the first 72 hours after severance of the cutting. At 72 hours after excision, signaling for meristematic cells marks the tr ansition from initiation phase to induction phase. It was also observed that the first root meristem was visible after 96 hours, but the root primordium was only completely fo rmed after six days of excision. Roots were formed after eight days. This timing is very similar to that described in previous studies of De Klerk and colleagues (De Klerk et al., 1999, De Klerk, 2002), which identified three phases of adventitious root formation in apple microcuttings. Factors Regulating ARF Many environmental and endogenous fact ors regulate rooting. Critical endogenous factors in adventitio us root formation are phytohormones. They can act directly on cell division and growth, or indire ctly, interacting with other molecules or 19

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phytohormones (Correa and Fett-Neto, 2004). Au xin is the principal phytohormone that initiates rooting (Srivastava, 2002). A majo r advance in vegetative propagation was the discovery of the effect of aux in on the production of adventit ious roots from cuttings in the 1930s (Thimann and Went, 1934). Although au xins are required for rooting during the first and second phases of the adventitio us rooting process (the dedifferentiation and induction stages), they inhibit the proc ess during the elongation phase (De Klerk, 2002). Indole-3-acetic acid (IAA) is t he main endogenous auxin. However, Indole-3butyric acid (IBA), another endogenous auxin, is more stable than IAA (Epstein and Lavee, 1984). Therefore, I BA is most commonly used for rooting in commercial operations (De Klerk et al., 1999). There is significant evidence that ethylene interacts with auxin to control adventitious rooting in stems or stem cuttings (Srivastav a, 2002). Ethylene is a small, readily diffusible hormone that has an important role integrating developmental events with external stimuli (Klee, 2004). It influenc es developmental processes such as seed germination, fruit ripening, abscission, and senesc ence (Abeles et al., 1992). It is also an important stress hormone. Adverse biotic or abiotic stimuli usually lead to ethylene synthesis. Ethylene slows down plant growth unt il the stress is remov ed. At the level of gene expression, ethylene induces transcrip tion of many genes in response to a multitude of environmental and developmenta l stimuli (Klee, 2004). It seems that ethylene has a promotive effect only in the presence of an auxin source. Some studies suggest that auxin promotes dedifferentiation through stimulation of ethylene synthesis (Sun and Bassuk, 1993) Although ethylene is promotive during the dedifferentiation phase I, it is inhibitory during the inducti on phase (De Klerk, 2002). Application of 20

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inhibitors of ethylene biosynthesis or et hylene action also reduces the number of adventitious root formed. Another phytohormone class that plays a role in ARF are the cytokinins. The effect of cytokinins on rooting, however, varies with concentration as well as duration of treatment. Higher concentrations and longer tr eatments are inhibitory, whereas lower concentrations and shorter treatment times may have an enhancement effect (Srivastava, 2002). It is al so well known that exogenous as well as endogenous balances between auxins and cytokinins can favor a developmental pattern or orient an organogenic program (Gas par et al., 2003). Ramirez-Carvaj al et al. (Ramirez-Carvajal et al., 2009) have shown the effect of cytok inin on ARF through the alteration of the type-B response regulator PtRR13 in Populus plants. Their results suggest that cytokinin acts through PtRR13 to repress ad ventitious root development in intact Populus plants. Although cytokines have a negative effect on ARF, being inhibitory during the induction phase, they are required at low levels to promote dedifferentiation. Jasmonic acid (JA) and methyl ester methyl jasmonate (MeJA), usually referred to as jasmonates, are also plant growth regul ators whose role on adventitious root has been reported recently (Fattorini et al., 2009). It has been shown that MeJA, combined with root-inductive hormones, enhances adventit ious rooting in tobacco. Jasmonates are important in plant defense against mec hanical wounding (Srivastava, 2002). As a wounding response in vegetat ive propagation through stem cu ttings, the JA and MeJA have been suggested to affect the dedifferentia tion phase, responsible to make cells prompt to respond to auxin (De Klerk, 2002). 21

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Genes Related to ARF Few genes have been described to be specifically involved in ARF. Konishi and Sugiyama (Konishi and Sugiyama, 2006) have analyzed the phenotype of the ROOT PRIMORDIUM DEFE CTIVE 1 gene ( rpd1 ) in Arabidopsis thaliana mutants. Seeds of the Landsberg erecta strain of Arabidopsis were mutagenized by treatment with ethyl methanesulfonate (EMS). The mutation in the rpd1 gene prevents the de velopment of root primordia in adventitious root formation in hypocotyl segments. It was observed that rpd1 mutants form the initial r oot primordia, but development is retarded beyond twoto four-cell-layer stages. Sorin and colleagues (Sorin et al., 2005) used two classes of Arabidopsis mutants to study adventitious rooting, superroot (SUR1 and SUR2) and argonaute1 (AGO1) mutants described previously (Boerjan et al., 1995, Delarue et al., 1998, Bohmert et al., 1998). SUR1 and SUR2 are auxin overproducer s that develop excess adventitious and lateral roots. Both genes were isolated from an EMS-mutagenized Arabidopsis thaliana seed stock, described before by Boerj an and colleagues (Boerjan et al., 1992). Therefore, superrroot genes are probably negative regula tors of adventitious root formation. On the other hand, AGO1 mutant s are defective in adventitious root formation. Using an allelic series of AGO alleles and AGO SUR double mutants, alteration of auxin homeostasis and a hypers ensitivity to light were observed. Their results show mRNA accumulation of the Auxin Response Factor17 (ARF17) in hypocotyls of AGO1. This suggest that ARF 17 might be the princi pal negative regulator of adventitious root formati on, by regulating genes involv ed in auxin and light signaling during adventitious root develop ment. In a follow-up study, Guti errez et al. (Gutierrez et al., 2009) showed that Auxin Response Factor6 and 8 are positive regulators of 22

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adventitious root formation. These thr ee genes are regulated by light and act i n a complex regulatory network. With the aim to identify gene markers regul ating ARF in Arabidopsis, Sorin et al. (Sorin et al., 2006) used proteomic analysis of Arabidopsis mutants defective in ARF to identified 11 proteins that might infl uence endogenous auxin content, number of adventitious root primordi a developed, and/or num ber of adventitious roots formed. Analyzing molecular and biochem ical processes in ARF of Petunia hybrida Ahkami et al. (Ahkami et al., 2009) showed RNA accumula tion of cyclin B1 gene after 48 hours of excision that is not expressed in roots sugges ting that it can serve as a marker for root initiation phase of ARF. The gene LRP1 ( lateral root primordium-1 ) has been shown to be specifically expressed in lateral and advent itious root primordia of Arabidopsis thaliana (Smith and Fedoroff, 1995). Also, gene HRGPnt3 ( hydroxyproline-rich glyprotein ) is expressed in adventitious and lateral roots of tobacco (Vera et al., 1994). It is expressed after the first cell division of lateral root primordium formation. Adventitious rooting ability of woody perennial species has been suggested to be under strong genetic control (Borralho and Wilson, 1994, Ronnberg-Wastljung et al., 2005). However, large variation in broad-sense heritability of adventitious root-related traits has been reported in poplar (Table 11). In general, ARF is under moderate to strong genetic control in poplar. Variation can be due to high diversity among different species of poplar and because of genotype environment interactions. Table 1-1 suggests that the presence of a P. trichocarpa background is associated with lower 23

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heritability of adventitious root-rel ated traits in Populus but no conclusions can be made since growth conditions and type of cuttings used differ significantly among studies. Genetic Control of Gene Expression Functional genomics tools like DNA microarra ys are useful for studies of global gene expression. Through microarray technology, the expression levels of essentially all genes can be quantified simultaneously based on hy bridization of labeled transcripts to glass slides (Yang et al., 2009). The basic pr inciple of DNA microarrays is to bind an unknown sample to an ordered array of known DNA molecules fixed in a precise location of a two-dimensional surface (Ger shon, 2002). There are two main microarray platforms commonly used: cDNA microarrays in which amplified cDNA clones are printed directly onto slides. Al ternatively, in oligonucleot ide microarrays, shorter probe sequences developed based on gene sequence information is synthesized in situ or printed on a microarray (Murphy, 2002). DNA microarrays have broad application. Mi croarrays enable the i dentification of genes expressed in a certain time and under specific treatment. Furthermore, microarrays can be used to assess differentially expressed genes of a given tissue under different conditions (Wang et al., 2007b), as well as changes in gene expression at different stages of a devel opmental process (Hertzberg et al., 2001, Brinker et al., 2004). Applications of microarrays also in clude genotyping individuals for genetic differences, such as single-nucleotide polymor phisms (SNPs) associated with a specific phenotype (Cutler et al., 2001). Microarrays are also being used to discovery information about a gene's function through co mparison of patterns of gene expression among known genes (Alberts et al., 2002). 24

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The completion of the genome sequence of Populus has allowed the development of whole-genome oligonucleot ide microarrays for these species, which was first designed by Oak Ridge National Laboratory in collaboration with NimbleGen (Madison, Wi, USA) (Yang et al., 2009). Quesada and colleagues (Quesada et al., 2008) have used this array to compare transcr ibed genes in vegetative organs of P. trichocarpa genotype Nisqually-1 to A. thaliana orthologs. The greatest va riety of expressed genes occurred in woody stem, where was also observed the highest proportion of unknown transcripts. Populus whole-genome oligonucleotide mi croarrays were also used by Ramirez-Carvajal and colleagues (Ramirez -Carvajal et al., 2008) to investigate expression pattern of the cytokinin response regulator gene family in P. trichocarpa genotype Nisqually-1, and Bocock et al. (B ocock et al., 2008), to identify genes encoding invertase, an enzyme important in carbon utilization because it catalyzes the hydrolysis of sucrose in to glucose and fructose. Studies have shown that variation in tr anscript levels is genetically controlled and heritable. Because gene expression can assu me continuous values, it can be studied as a quantitative trait. As the poplar genome sequence and DNA microarrays are available, these two techniques c an now be combined with the QTL mapping information in an approach known as Genetic al Genomics (Jansen and Nap, 2001). The principle of this strategy is based on the genet ic variation between related individuals in a segregating population. Therefore, the expression profil e of all individuals for each gene is a phenotypic trait, hence quantitative trai t that is combined with a genetic map, enabling the assessment of t he genetic map positions of gene expression QTLs, known as eQTLs (Kirst and Yu, 2007). If the eQTL co-loc alizes with the physical position of 25

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26 genes that affect their own expression, they are called cis -acting eQTL. Otherwise, this gene expression QTL is trans-regulated, in other words, regulated by factors that are not the genes themselves (Jansen and Nap, 2001).

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Table 1-1. Broad-sens e heritabilities of adventitious root-related traits of different types of stem cuttings of Populus species under different conditions of growth and type of material. Growth conditions Type of cutting Number of adventitious roots Root dry weight Total length of primary roots Reference Population studied Sharkey clay and 1:1:2 sand:peat:loam substrates Apica l Hardwood 0.44-0.56 0.36-0.58 0.33-0.52 Wilcox and Farmer (1968) P. deltoides Sand Dormant hardwood 0.85-0.91 Ying and Bagley (1977) P. deltoides F 2 of BC (Family 331) in vitro Greenwood stems discs 0.32 0.29 Han et al (1994) Sand Hardwood 0.15-0.18 0.20-0.33 0.23-0.28 Riemenschneider and Bauer (1997) P. trichocarpa Field in three geographic locations Dormant hardwood 0.09-0.11 Zalesny Jr. et al (2005) BC*, D*, DM*, DN* and NM* P. deltoides P. canadensis syn euramericana Hydroponic Greenwood apical 0.80-0.85 0.58-0.71 Zhang et al (2009) pBC (Family 52-124) Fafard 4MIX soil Greenwood apical 0.28-0.33 Novaes et al (2009) *BC: Backcross ( P. trichocarpa P. deltoides ) P. deltoides ; D: P. deltoides ; DM: P. deltoides P. maximowiczii ; DN: P. deltoides P. nigra ; NM: P. nigra P. maximowiczii ; pBC: pseudo-backcross ( P. trichocarpa P. deltoides ) unrelated P. deltoides. 27

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CHA PTER 2 GENETIC ANALYSIS OF EARLY ADVENT ITIOUS ROOT DEVELOPMENT IN A PSEUDO-BACKCROSS POPULA TION OF POPULUS Introduction Adventitious roots are pos t-embryonic roots formed on plant shoots. Although adventitious and lateral roots have common dev elopmental properties adventitious root formation involves additional dedifferentiati on of already committed cells (Srivastava, 2002). Generally, the formation of adventitious roots occurs in three phases that may partially overlap. The first phase is of dedifferentiation, when cells are prepared to respond to rhizogenic stimuli from auxin, and further are going to originate root primordia' (De Klerk et al., 1999). In woody pl ants, the onset of adventitious roots may occur in several tissues. Although adventitious roots form more fr equently from young secondary phloem cells, they also originate fr om vascular rays, cambium or pith cells (Davies and Hartmann, 1988). The second stage in the formation of adventitious roots is the induction', when cells activated' in the first phase become committed to form root primordia, which elongates in the third phase (De Klerk et al., 1999). Adventitious root formation from cu ttings is used extensively for propagating superior genotypes vegetatively. Clonal propagation through rooted cuttings is used particularly in Eucalyptus and in species of the Salicac eae family, which includes poplars ( Populus ) and willows ( Salix ). The ability of poplars to readily form adventitious roots from hardwood cuttings is one of the reasons for thei r wide use in commercial plantations (Zsuffa et al., 1996). In 1994, mo re than 50 percent of all poplar plantings worldwide were represented by fewe r than 10 clones (Ritchie, 1994). Considerable variation in adventitious root producti on is found between species, cultivars, different ages and organs (Lovell and White, 1986). There are very few tree 28

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taxa that are able to produce adv entitious roots promptly on hardwood cuttings, which are segments of dormant stem w ood. Species and hybrids in the Aigeiros and Tacamahaca sections of the genus Populus are among them. But even in these sections, there is considerable variation in the degree and vigor of adventitious rooting (Dickmann and Hendrick, 1994). In poplar, most of the adventitio us roots originate from preformed root primordia in the periderm of the stem (Dickmann and Hendrick, 1994). Even though rooting ability is a complex trait with high phenotyp ic plasticity, it has been shown to be under strong genetic control in species of Eucalyptus (Borralho and Wilson, 1994) Salix (Ronnberg-Wastljung et al., 2005) and Populus (Wilcox and Farmer, 1968, Zhang et al., 2009, Rae et al., 2007). Poplar is a suitable model for studying the genetic control of root development because there is extensive variation for the trait and well-established genetic and genomic resources (Bradshaw et al., 2000, Taylor, 2002, Wullschleger et al., 2002, Brunner et al., 2004). Availabl e genomic resources include the genome sequence of the P. trichocarpa genotype Nisqually-1 (Tuskan et al. 2006) and whole-transcriptome microarrays (Jansson and Douglas, 2007), enabling the integration of genomic information from quantitative trait loci (Q TL) analysis and gene expression data (Jansen and Nap, 2001). QTL mapping allows the identi fication of genomic regions that regulate trait variation and, in combination with gene expression data, can be used to identify genes that underlie quantit ative trait variation (Schadt et al., 2003a). This integrative strategy, also called genetical genomics, was initially proposed by Jansen and Nap (Jansen and Nap, 2001) and is being extensively utilized in medical research (De Haan 29

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et al., 2003, Schadt et al., 2003b). In forest species, only a fe w studies utilized genetical genomics in Eucalypt us (Kirst et al., 2004) and in Populus (Street et al., 2006). The objective of this study was to di ssect the quantitative genetic control of adventitious root formation in Populus and the transcriptional program that differentiates individuals that carry alternative alleles for root development in the main genetic loci that control the trait. This analysis allowed the identification of two QTL involved in the regulation of root formation, and several c andidate genes co-located in these QTL intervals A transcriptome analysis identifi ed genes that are differentially regulated between genotypes with distinct rooting ability. Fi nally, the combination of QTL and transcriptome information was used to define genes possibly involved in initial adventitious root development in the hybrid population. Material and Methods Plant Materials The family (52-124) used in this study is a pseudo-backcross between the hybrid female parent 52-225 [ Populus trichocarpa (clone 93-968) P. deltoides (clone ILL101)] and the unrelated male parent D124 ( Populus deltoides ), established by the Natural Resources Research Institute of t he University of Minnesota. The parent D124 is from northern Minnesota. The P. trichocarpa parent of the hybrid came from western Washington, whereas the P. deltoides parent material originated in Illinois. Phenotypic Measurements Twelve centimeter long apical cuttings were collected from 234 individuals of family 52-124, as well as the parental genot ypes. Cuttings were placed in 23! 16.2 6 inch-containers, with up to 59 cuttings, which were maintained in hydroponic culture (H 2 O buffered at pH 5.7, with 0.5 g L -1 of MES) for the durati on of the experiment. The 30

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experimental design was an incomplete bl oc k design with four blocks and three replications, for a total of 708 cuttings. Root emergence was recorded daily at the same time (10 am), until the 18 th day of culture. After 18 days, roots from each cutting were excised and scanned using a Canon LiDE 600F scanner (in grayscale at 600 dpi). Images were analyzed with WinRHIZO TM (version 2007d, Regents Instruments Inc., Quebec, Canada) to measure root architectura l traits: length, volume, and surface area of total, primary (first order roots), and root branches (sec ond and third order roots), as well as average diameter. After being sc anned, the roots were dried in a 65 o C drying room. Dried biomass was weighed in a high-p recision scale after samples were placed for 72 hours at room temperatur e and humidity in the laborato ry. Table 2-1 describes all traits measured, as well as maximum and minimum phenotypic values, compared to parental values. Statistical Analysis Covariance parameters were estimated fo r all traits using PROC MIXED (SAS Institute Inc. 9.2¨ 2004, Cary, NC, USA), considering all vari ables random in the following model: where is the phenotypic value of the i th clone in the j th block within the k th replication, is the overall mean, is the random effe ct of the clone; is the random effect of replication, is the random effect of incomple te block (within replication) and is the residual error. 31

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Clonal repeatability was calculated usi ng the covariance parameter estimates in the followin g formula: where and are the variance components corresponding to clone and residual effect across the three replications, respectively. A log transformation was applied to all trai ts, except for number of roots. Leastsquare means used in the QTL analysis were ca lculated by including clone as a fixed effect in the model, using PROC MIXED. QTL Analysis QTLs for root-related traits were identified based on a linkage map previously described (Novaes et al., 2009). The linkage m ap consists of 181 markers distributed homogenously in the hybrid female parent, with an average density of one marker every 16 cM. QTLs were identified using composit e interval mapping (Zeng, 1993) in Windows QTL Cartographer v.2.5 (Wang et al., 2007a) using standard model 6 with walk speed of 2 cM. A genome-wide significance level of P<0.05 was established based on 1000 permutations (Churchill and Doerge, 1994). Selection of Genotypes with Alternative Alleles for Genom ic Regions that Control Adventitious Root Formation QTL for the trait number of roots were consistently mapped on LG II and XIV (see Results). We classified each ge notype depending on the allele ( P. trichocarpa or P. deltoides ) that was observed in both QTL regions Four categories were defined: (1) genotypes carrying P. deltoides or (2) P. trichocarpa alleles at both QTL, and (3) genotypes carrying P. trichocarpa alleles at the QTL in LGII and P. deltoides alleles in 32

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QTL on LG XIV, and (4) vice-versa. Genoty pes with recombination between markers flanking in each of the two QTL w ere not grouped into any of the categories. As expected, genotypes carrying P. trichocarpa alleles in both QTL regions generally had more roots than genotypes carrying the P. deltoides alleles in those intervals. These are referred hereafters as the Pt QTL and Pd QTL categories, respectively. Within each one of these two categories, we identified three genotypes with extreme phenotypes for number of roots. For these six genotypes we collected 12 centimeter-long cuttings and established them in the same hydroponic conditions used previously in the QTL detection experiment. The number of new roots formed in these genotypes was recorded daily for 12 days, and samples were collected for transcriptome analysis. Tissue sampling To measure gene expression during adventit ious root formation in the three selected genotypes from each QTL category ( Pt QTL and Pd QTL), a section of 1 centimeter, measured fr om the base of each cutting, was collected at 0, 1, 2, 4 and 8 days after placing them in the hydroponic solu tion. Samples were flash-frozen in liquid nitrogen for posterior RNA extrac tion. Four biological replic ates were collected from each genotype, at each time point. In addition, five biological replicates of each genotype were maintained in hydroponic growth conditions until day 12 to verify that the root development was consistent with the phenotype observed in the QTL detection experiment. RNA extraction, cDNA synthesis and labeling Total RNA was extracted (Chang et al., 1993) from the bottom 1 cm stem section collected from each sample. The sample included xylem, phloem and bark. RNA was purified using RNeasy Mini Kit columns (Qiagen), and DNase treated with RNase-Free 33

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DNase set (Qiagen). RNA qualit y was eval uated in 1% w/v agarose gels. RNA was amplified and cRNA synthesiz ed and labeled using Two Dyes Agilent Low Input Quick Amp Labeling Kit. The microarray platform us ed consisted of single 60-mer probes designed for each of 43,803 annotated gene models from the sequenced genome of P. trichocarpa These probes were previously select ed for being adequate for analysis of gene expression in this mapping population (Drost et al., 2009) Microarray experimental design and data analysis A total of 60 microarrays were used in the transcriptome analysis. Gene expression of each of si x genotypes was analyzed in five time points (0, 1, 2, 4 and 8 days), with four biological r eplicates per genotype and time point, following a "bird cage" design (Figure 2-1). Median values of si gnal intensities were quantile normalized (Bolstad et al., 2003) and log 2 transformed. Normalized signals were analyzed in SAS 9.2 (SAS Institute In c. 9.2¨ 2004, Cary, NC, USA) using a mixed-model ANOVA with genotype and genotype time interactions as fixed effects, and microarray as random effect. Differences in expression between the group of genotypes from the Pt QTL and Pd QTL categories were estimated at each time point, and the significance was determined based on a false discovery rate (F DR) of 5% (Storey and Tibshirani, 2003). Genes showing a similar pattern of expressi on differences between genotypes from the Pt QTL and Pd QTL categories, at all time points, were clustered using a Modulated Modularity Clustering graph-based technique using Sp earman correlation (Stone and Ayroles, 2009). 34

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Annotation Populus gene model transcript sequences were annotated by searching for sequence s imilarities using BLASTx against Populus (JGI v.1.1) and The Arabidopsis Information Resource (TAIR v8.0) gene models. Results Analysis of Parental Genotypes Adventitious roots developed almost simultaneously in both parental genotypes. However, the number of roots devel oped was significantly different ( P <0.01) until the 17 th day in hydroponic culture, with the female parent ( Populus trichocarpa P. deltoides hybrid) developing more roots than the pure P. deltoides male parent (Figure 2-2). After the 17 th day, the number of roots developed was no longer significantly different between the two parents. Theref ore, both parents appear to have a similar capacity to form roots, but development of advent itious roots appears to be delayed in P. deltoides relative to the hybrid. Phenotypic Variation Three classes of traits were measur ed in the progeny of Family 52-124: (1) number of roots, measured from day 9 to day 18, (2) root architecture and (3) total root biomass, measured in day 18 (Table 2-1). Number of roots. A Poisson distribution was obs erved for the trait number of roots, which displayed transgressive segregat ion in the hybrid pop ulation (Figure 2-3). The hybrid parent had higher number of roots in all days m easured, when compared to the pure P. deltoides parent. Considerable variation in the day of root emergence was also observed in the progeny. After nine da ys in the hydroponic so lution half of the genotypes had produced roots (Figure 2-4). 35

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Root architectural traits and biomass. After 18 days in hydroponic solution, roots were harvested to measur e architectural traits, includi ng total length, surface area, total volume of first order roots and root branches, and av erage diameter. Root architectural traits and total dry-weight al so showed transgressiv e segregation in the progeny (Figure 2-5). The hybrid P. trichocarpa P. deltoides parent presented higher values for most of the traits, with the ex ception of VOL, DIAM PRISA, PRIV and DRYWT (Figure 2-5). Genetic Control of Early Root-Related Traits Clonal repeatability ( H ) was estimated for all traits and was generally low, ranging from 0.115 for L1 to 0.342 for day16 (Table 2-1). The trait number of roots showed higher H ranging from 0.274 for day12, to 0.342 for day16, than root architectural traits Phenotypic Correlations Phenotypic correlations were calculated betw een all pairs of traits (Table 2-2). In general, root architectural traits and number of roots were highly co rrelated, except for DIAM, which had lower correlations (ranging fr om 0.17 to 0.52) rela tive to the root architectural traits. Also, phenotypic co rrelations among number of roots and root architectural traits were moderate to high, ranging from 0.35 to 0.68 (excluding DIAM). Phenotypic correlations were positive for all traits. QTL Analysis of Number of Root QTL analysis was performed for number of roots, measured after 9 days in hydroponic culture. Eleven QTLs for number of roots were identified on the mother map, using the 95 th percentile of 1000 permutations as a threshold ( P <0.05) (Table 2-3, Figure 2-6). An average of 1.83 QTLs was identified, each day that the number of roots was measured. QTLs for number of roots mapped consistently in LG II and LG XIV. The 36

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effects of the QTLs mapped in these two lin kage groups tended to decrease over time (Table 2-3). Each QTL explained between 6. 66 to 10.66% of the phenotypic variation, depending on the trait (Table 2-3). The total variance explained for each trait averaged 9%. QTL Anal ysis of Root Architectural Traits QTLs were detected for eight of the ten root architectural traits measured. In total, 15 significant QTLs were mapped, seven on LG XII and six on LG XIV (Table 2-4, Figure 2-7). The phenotypic variation expl ained by each QTL ranged from 5.8 to 10.87%, similar to the range explained by QTL for the number of r oot traits. All QTL alleles positively affecting the traits were derived from P. deltoides, grand-parent of the pedigree. QTL Analysis of Root Biomass A QTL for root dry weight was detected on LG XVII, the only trait to have a QTL detected in this interval (Table 2-4, Figure 2-7) In total, 7% of the variance in this trait was explained by this QTL. Alleles positively affecting this trait come from P. trichocarpa Genotypes with Alternative Rooting Alleles Genotypes carrying alternative alleles at both QTLs on LG II and XIV were selected for gene expression analysis (Figure 2-8). Genotypes in the Pt QTL category (UF352, UF498 and UF926) develope d higher number of roots ( P <0.1) than those in the Pd QTL category (UF717, UF209 and UF912). The rooting properties of these genotypes were confirmed in a separate exper iment using the same hydroponic growth conditions as those in the QTL det ection experiment (Figure 2-9). 37

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Transcriptome Anal ysis of Ad ventitious Root Development The transcriptome response of cuttings in hydroponic solution was analyzed to identify genes differentially expressed in di fferent experimental conditions. Based on the analysis of variance, genes differentiall y regulated across time points, between genotypes in the Pt QTL and Pd QTL categories across the entire experiment and in individual time points were identified. Gene expression analysis of time effect A total of 26,121 putative genes were i dentified as significantly differently expressed (FDR<0.001) between at least two time point s in the experiment. To identify the time point at which the most signifi cant changes in gene expression occurred, a contrast of the expression bet ween consecutive time points (i.e. time points 0 and 1, 1 and 2, 2 and 4, 4 and 8, and 8 and 0) was perfo rmed. Most differences in transcript levels occurred during the fi rst 24 hours of the experiment (i.e. time points 0 and 1, Figure 2-10). On the other hand, only ten genes were differentially regulated between 4 and 8 days in hydroponic culture. This suggest s that these two time points are within the same rooting phase. Among genes previously described to be in volved in root development in model plants, the gene euge ne3.153750001 (putative homolog of superroot2 ) was detected as being significantly more highly expressed in time point 0 compared to time point 1. A mutant of SUR2 has been described as c ausing auxin overproduction and abnormally high number of adventitious roots (Delarue et al., 1998). The expression of SUR2 also increases as a response to wounding (Barlier et al., 2000). Within the same contrast, but more highly expressed in time point 1 instead, is the gene CPC902 (Condensin complex components subunit C, eugene3.0005131 6 ) which is a homolog of the 38

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Arabidopsis gene SMC1 ( structu ral mainten ance of chromosomes 1). SMC1 encodes for one of the proteins of t he cohesion complex family (S chubert et al., 2009), necessary for correct chromosome segregation during nuclear divisions, possibly indicating the initiation of cell divisions necessary for root meristem organization. The contrast of time points 1 and 2 showed higher expressi on of gene grail3.00490111 01, homolog of Arabidopsis gene TOR1 ( tortifolia 1) in time point 1. TOR1 encodes a protein associated with microtubules, which regulates the directi on of organ growth (Furutani et al., 2000). In time point 2, the gene estExt_Genewise1_ v1.C_440031, a putativ e homolog of the Arabidopsis gene TPL ( topless ), was highly expressed. TPL is involved in transcriptional repression of root-promoting genes during the transition stage of embryogenesis (Osmont and Hardtke, 2008), Among genes more highly expressed in time point 4, fgenesh4_pm.C_LG_VIII000556, homolog of Arabidopsis gene PIN3, was detected. PIN3 is an auxin efflux regulator involved in root development and elongation (Blilou et al., 2005). Gene expression analysis of genotype effect A total of 1929 genes were identified as differentially expressed between genotypes in the Pt QTL and Pd QTL categories (FDR<0.05), in at least one time point (Figure 2-11a). Only 45 of these genes were differentially expressed across all time points. These included the genes estExt_fgenesh4_pg. C_LG_II0581 and gw1.XIV.1942.1, homologs of the Arabidosis genes RUB1 ( related to ubiquitin 1, AT1G31340) and OMT1 ( caffeic acid O-methyltransferase 1 ), respectively, which were expressed primarily in the Pt QTL category. RUB1 was found to be involved in response to auxin stimulus and regulation of ethyl ene production (Bostick et al., 2004). Gene OMT1 is involved in lignin biosynt hesis (Goujon et al., 2003). In the Pd QTL category, 39

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genes estExt_fgenesh4_pm.C_L G_IV0339 1 (putative homol og of the gene COL2) and gw1.XV.655.1 (putative DEAD-box protein) were more highly expressed, respectively. The gene COL2 is related to flowering time in Arabidopsis but known to control growth cessation in Populus (Bohlenius et al., 2006). Gene expression comparison between ex treme genotypes within time point In order to identify genes r egulating rooting ability, we analyzed genes differentially regulated between genotypes in the Pt QTL and Pd QTL categories at each time point. An interesting gene found to be signific antly more highly expressed in the Pd QTL category in time point 0 was gene gw1.XI.1499.1, homolog of STM Arabidopsis gene ( shoot meristemless ). Yanai et al (Yanai et al., 2005) de monstrated that the activation of STM proteins increase cytokinin levels dr amatically. Low levels of cytokinins are necessary to promote dedifferentiation of cell s in the first phase of adventitious root formation. Also in time point 0, but more highly expressed in the genotypes of the Pt QTL category, is gene eugene 3.00190357, a homolog of Arabidospsis gene APY2 ( apyrase 2 ). APY2 has been reported to be expressed in fast growing tissues, including meristematic region of root tips (Wu et al., 2007). In time point 1, the gene estExt_fgenesh4_pg.C_LG _IV1532, which in Arabidopsis encodes a basic chitinase (CHIB) was identified. CHIB is involved in the signaling pathway mediated by ethylene and jasmonic acid during pathogen response (Zhou et al., 2005). We found the gene grail3.1392000301, homolog of Arabidopsis gene SUR1 ( superroot1 ) to be expressed only in time point 2. This gene is hi ghly expressed in the genotypes of the Pt QTL category in our experiment. SUR1 was first isolated by Boerjan and collegues (Boerjan et al., 1995), and is also an auxin overproducer that causes development of an excess of adventitious roots. Also in time point 2, but more highly expressed in the genotypes of 40

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the Pt QTL category, is gene euge ne3.00012559, homolog of Arabidospsis gene AIR9 (auxin-induced in root cultur es 9). AIR9 doesn't have homology to any known proteins and was first isolated from cDNA library of au xin-treated root culture. In time point 4, gene gw1. VIII.2487.1, P5CS1 ( delta1-pyrroline-5-carboxylate synthase 1 ), involved in root elongation response (Szekely et al., 2008) is expressed in poor rooters. In time point 8, the gene fgenesh4_pg. C_LG_II000237, SUR2 ( superroot2 ) is expressed in the Pd QTL category. Several auxin-responsive genes were detec ted as differentially regulated in this study. Most of them were differentia lly expressed between genotypes in the Pt QTL and Pd QTL categories. Among genes involved in auxin signaling, we detected gene estExt_fgenesh4_pm.C_LG_VIII 0154, homolog of PGP1 Arabidopsis gene, differentially expressed in genotypes of the Pd QTL category in time point 1. Also gene grail3.0003074001, a homolog of Arabidopsis gene AUX1, expressed in the Pd QTL category in time point 8, estExt_fgenesh4_pg.C _LG_II0422, TSA1 in poor rooters in times 2 and 8. Cluster analysis A total of 1929 genes were differentia lly regulated between genotypes in the Pt QTL and Pd QTL categories at any time point. For these genes, the difference in transcript abundance between the set of genoty pes in each of the two QTL categories were estimated. The estimates were used to cluster genes with common transcriptional response differences between genotypes in the Pt QTL and Pd QTL categories, across all time point. Sixty clusters were identified, varyi ng from 2 to 148 transcripts in size, with eight unclustered genes (Figure 2-12). Further analysis showed one cluster with a noticeable pattern of expression, with a linear increase in the difference in transcript 41

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abundance between genotypes in the Pt QTL and Pd QTL categories across each time point (Figure 2-13). Of the 91 genes in this cluster, 36 showed an increase in express ion over time in genotypes of the Pd QTL category. A set of 55 genes showed the opposite pattern, with hi gher expression in the Pt QTL category. For example, the gene estExt_fgenesh4_pg.C _1630059, homolog of Arabidopsis gene ARF4 ( auxin response factor 4) was significantly more highly expressed in genotypes in the Pd QTL category. ARF4 is a member of the ARF gene family of transcription factors that mediates auxin responses. In Arabidopsis, ARF4 seems to have the same function of ARF3 in specifying abaxial cell identity (Pekker et al., 2005). In the group of genes more highly expressed among genotypes in the Pd QTL category, the gene fgenesh4_pg.C_LG_III001778, homolog of SGS3 Arabidopsis gene ( suppressor of gene silencing 3), was observed. A previous study (Peragine et al 2004) hypothesized that SGS3 promotes ma intenance of the juvenile phase by repressing the expression of adult-promoting genes. In the same study, the accumulation of ARF4 transcripts was detected in shoot apices of SGS3 mutants, i ndicating a possible function of this gene in ARF4 regulation. Among genotypes in the Pt QTL category, a couple of genes related to cell wall, cellulose biosynthesis and ligni n were identified as more hi ghly expressed. For instance, gene grail3.0594000101, CESA3 encodes cellulose synthase isomer (Burn et al., 2002). Also, gene eugene3.00110748, TBL38 (t richome birefringence-like), which encodes a member of the TBL gene fam ily containing a plant-specif ic DUF231 domain of unknown function. Two other genes of the same family have been shown to be involved in the synthesis and deposition of sec ondary wall cellulose (Bischoff et al., 2010). Also more 42

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highly expressed in genotypes of the Pt QTL category, COB-like (g w1.155.39.1) is a key regulator of the orientation of cell expansion in roots (Schindelman et al., 2001). The gene fgenesh4_pg.C_scaffold_246000012, homolog of Arabidopsis gene RHD3 (root hair defective 3), encodes a putative GTP-bi nding protein that might be involv ed in cell wall biosynthesis and actin or ganization (Wang et al., 1997). Genetical genomic analysis of QTLs for root number A set of 81 genes differentially ex pressed between genotypes in the Pd QTL and Pt QTL categories co-localize with the QTL interv al for number of root s in LG II and XIV. Of particular interest are two genes (estExt_fgenesh4_pg.C_LG_II0422 and fgenesh4_pg.C_LG_II000237) that are part of the tryptophan biosynthesis pathway. Tryptophan is a precursor of auxin, a phyt ohormone well known to affect production of adventitious rooting in cuttings (De Kler k et al., 1995). fgenesh4_ pg.C_LG_II000237 is the homolog of Arabidopsis gene SUR2 ( superroot2 ), also found to be significantly expressed in the contrast of time points 0 and 1, as previously described. The gene estExt_fgenesh4_pg.C _LG_II0422 is the homolog of TSA1 ( tryptophan synthase alpha chain ), which catalyzes the conversion of i ndole-3-glycerolphosphate to indole, the penultimate reaction in the biosynthesis of tryptophan. Several transcription factors located in t he QTL intervals were also identified as being differentially regul ated. Genes eugene3.00020393 and fgenesh4_pg.C_LG_III000900, are highly expressed in genotypes of the Pd QTL and Pt QTL categories, respectively. T he gene eugene3.00020393 is a homolog of Arabidopsis gene MYB4R1, a putative MYB protein containing four R1R2-like repeats, which is unusual for MYB proteins and whos e biological function has not been explored to date (Stracke et al., 2001). F genesh4_pg.C_LG_III000900 is a homolog of 43

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Arabidopsis gene WRKY21, whic h encodes WRKY DNAbinding protein 21. It is significantly expressed in time point 2. T he detailed function of WRKY proteins is still largely unknown, but several WRKY proteins have been associated to the response to pathogen infection and other stresses (Eulgem et al., 2000). Both genes are located in the QTL interval in Linkage Group II. Also with higher expressi on in genotypes of the Pd QTL category, but in time points 4 and 8, gene estExt_Genewise1_ v1.C_LG_II1725, homolog of Arabidopsis gene GASA1, is involved in response to gibberellin s stimulus, brassinosteroid, abscisic acid stimulus and unidimensional cell growth (B ouquin et al., 2001). Expressed in genotypes of the Pt QTL category in time poi nts 2 and 4, a homolog of Arabidopsis gene EOL1, estExt_Genewise1_v1.C_LG_II 2213, encodes a paralog of ETO1, which is a negative regulator of ACS5, a key enzyme in ethylene biosynthesis pathway (Christians et al., 2009). Discussion This study investigated the genetic contro l of adventitious root formation and early root-related traits of a ps eudo-backcross population (family 52124) originated from a cross between the hybrid Populus trichocarpa P. deltoides and an unrelated P. deltoides parent. Some previous quantitativ e genetics studies have shown that adventitious root formation is under strong genetic control in Populus species (Ying and Bagley, 1977, Zhang et al., 2009, Wilcox an d Farmer, 1968). However, we found weak to moderate genetic effect in rooting ( H 2 = 0.12 0.34), in line with a previous study in the same family ( H 2 = 0.28 0.33) (Novaes et al., 2009), and a few studies in other Populus species (Zalesny et al., 2005, Riemen schneider and Bauer, 1997). Differences 44

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might be attributed to the use of different growth conditions, the type of cuttings that were used or differences in the genetic control of the trait among species of Populus QTL Identification QTLs were discovered for a broad range of early root-rel ated traits. Few QTL mapping studies have been carried out to ident ify genes related to adventitious root formation, and most of them were carried out in crop species such as rice (Zheng et al., 2003), maize (Mano et al., 2005) and common bean (Ochoa et al., 2006). Only two QTL studies on adventitious rooting of Populus had been previously reported (Han et al., 1994, Zhang et al., 2009). Han et al. (1994) studied the quantitativ e genetic aspect of in vitro adventitious root formation and shoot regeneration in F 1 hybrids of P. trichocarpa and P. deltoides, and segregating populations (F 2 and BC 1 ). That study mapped two QTLs involved in control of organogenesis in vitro. However, no QTL was detected for root number or length. Zhang et al. (2009) used functional mapping to detect QTLs for number of roots and maximum root length measur ed at five time points. The population used was an interspecific hybrids between P. deltoides and P. euramericana from which cuttings were also grown in hydroponi cs. Three QTLs affecting maximum root length and total root number were detected, but no common QTL affected both traits. This result might be due to the fewer number of individuals used (93 genotypes, relative to 234 genotypes used in our experiment) a nd the mapping approach used. In that previous study, the proportion of the phenotypic variance explained by QTLs detected for root number and root length ranged from 0. 01 to 0.20 and 0.01 to 0.14, respectively. Adventitious Rooting Analysis Adventitious root development on t he bottom of apical cuttings has been suggested to be due to higher concentrations of auxin translocate d to that region, 45

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probably in response to wounding (Zalesny Jr et al., 2003). The work of Zalesny Jr. and Wiese (Zalesny Jr and Wiese, 2006) has shown that time of the year and ancestry, and their interaction, are highly significant factor s in determining the num ber of root and root dry weight of Populus hardwood cuttings. Also, it has been observ ed that superior rooting of apical cuttings occurs for species from the section Tacamahaca compared to those from Aigeros On the other hand, species from section Aigeiros such as P. deltoides and P. nigra rooted better when using middle and basal cuttings. Zalesny Jr. et al (Zalesny Jr et al., 2003) have also shown that stem position on the stool plant accounted for approximately 6% of the variation in rooting of different species of poplar, including P. deltoides Previous studies in adventitious rooting ability, that included several species of Populus (Zalesny et al., 2005), conclude d that dormant hardwood cuttings of P. deltoides don't root as well as other Populus species, possibly because fewer number of preformed root primor dia are present. Our study suggests that a slower development of roots in P. deltoides may be at the source of this di fference. Depending on the growth condition, this effect might be critical in t he survival of cuttings. Studies linking number of roots and existence of preformed root prim ordia are still lacking. Successive Phases in Adventitious Root Formation The phases of adventitious root formati on possibly follow similar pattern in Populus as in apple microcuttings, where the proc ess has been studies in much detail. Roots were observed in the present study after six days in culture. In apple microcuttings, roots are generally observed afte r five days (De Klerk et al., 1999). Wu (Wu, 2004) observed root outgrowth from the stem after six days of culture, in the hybrid Populus tremula P. alba Therefore, these woody species appear to follow 46

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approximately the same timing of adventitious root form ation. The transcriptome information also indic ates that in the fi rst 24 hours, described previously as the dedifferentiation phase (De Klerk et al., 1999), the largest number of genes is differentially regulated (Figure 2-10). This might be due to several internal changes such as hormonal changes and gene regulation. Also probably most of the wound response takes place during this phase. Cells st art to become less specialized and dedifferentiate to a meristematic stat e that is capable of cell division. Among the most interesting genes observed within each putative adventitious root formation phase, we found gene CP C902 (eugene3.00051316 ) involved in chromosome segregation during nuclear divi sions in time point 1. Probably due to meristematic root organization during the induction phase. Following induction phase, intense rhizogenesis activity in stem root primordia causes the mobilization of lipids reserves probably for energy source (Ciam porova, 1983). Interestingly, we found a number of lipid-related genes differentia lly expressed between genotypes in the Pt QTL and Pd QTL categories (fgenesh4_pg.C _LG_VII001302, eugene3.00640195, gw1.VII.1867.1, eugene3.01850030, fgenesh4_pg.C_LG_X000784, estExt_fgenesh4_pg.C_LG _XVIII0095, estExt_Genewise1_v1.C_LG_X3279, gw1.III.811.1, gw1.147.138.1, eugene3.00640241, fgenesh4_pg.C_scaffold_7085000001). Also genes P5CS1 and PIN3 (differentially expressed in time point 4), and gene LAC15, (d ifferentially expressed in time point 8), are known to influence root elongation, wh ich is in agreement with the respective rooting phase. Significantly expressed in genotypes of the PtQTL category during time 47

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point 8, gene gw1.15 5.39.1, homolog of Arabidopsis COB-like gene, is a key regulator of the orientation of ce ll expansion in roots. The most promising gene, howev er is the gene fgenesh4_pg.C_LG_II000237 superroot2 (SUR2). EMS Arabidopsis mutants of SUR2 result in overproduction of auxin hormone and abundant adventitious root devel opment. Therefore, this gene is a negative regulator of adventiti ous root. SUR2 is significa ntly overexpressed in poor rooters when compared to good r ooters in our experiment suggesting that this gene is also controlling adventitious root formation in Populus. This gene might act by negatively affect auxin biosynthesis, auxin conj ugate hydrolysis, sensitivity to auxins, or by increasing cytokinin levels. Cloning of gene SUR2 in Populus might be a valuable tool to study how this gene alters number of adventitious root in this species. Conclusions QTL mapping and microarray data was used to dissect adventitious root formation in poplar stem cuttings. Parental genotypes presented differential rooting response to early number of adventitious roots. T he pseudo-backcross progeny showed segregation for all phenotypic traits measured. This allo wed the detection of major QTLs related to early number of adventitious roots. Ev aluation of genotypes showing extreme phenotypes through whole-transcriptome analys is made possible the identification of candidate genes of particular importance to rooting that co-locates with major QTLs interval detected, as well as insights into root development processes in Populus species. 48

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Figure 2-1. Subset of the mi croarray experimental design s howing only two time points. Each arrow is a microarray chip. Arro ws indicate dye orientations: The sample at the tail of the arrow is l abeled with red (Cy5) dye, and sample at the head of the arrow is labeled with gr een (Cy3) dye. Dark gray color circles represent genotypes in Pt QTL category, while li ght gray represents genotypes in Pd QTL category. Comparisons were made for all consecutive time points as indicated in the figure. 49

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50 Figure 2-2. LSM of number of adventitious roots developed on the female hybrid parent Populus trichocarpa P. deltoides 52-225 ( TD ), and the unrelated male parent P. deltoides D124 ( D ) maintained in hydroponic solution for 25 days. Error bars show standard error. Parent s means are significantly diferent (p<0.05) from day 7 to day 19.

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Figure 2-3. Distribution of Least Square Mean (L SM) estimates for numbe r of root traits of 236 individuals of family 52-124 and their parents P. deltoides' (D) and P. trichocarpa P.deltoides) P.deltoides (TD). 51

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Figure 2-3. Continued. 52

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Figure 2-4. Cumulative perc entage of genotypes rooted duri ng the experiment in each replication. Each color bar represents a replication. 53

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Figure 2-5. Distribution of Least Square Mean (LSM) values for root architectural and biomass traits of 225 individuals of family 52-124 and their parents P. deltoides' (D) and ( P. trichocarpa P.deltoides ) P.deltoides (TD), measured after 18 days in hydroponic solution. 54

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55 Figure 2-5. Continued.

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Figure 2-6. Localization of 11 quantitative trait loci (QTLs) detected on the mother map for number of roots. QTLs were identified as in Table 2-3. 56

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57 Figure 2-7. Localization of 15 quant itative trait loci (QTLs) detec ted on the mother map for root architectural traits and root biomass. QTLs were identified as in Table 2-4.

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Figure 2-8. Least square means (LSM) of num ber of adventitious roots developed on selected genotypes for each day m easured. Genotypes UF352, UF498 and UF926 carry P. trichocarpa allele, whereas genotypes UF717, UF912 and UF209 carry P. deltoides allele. Number of roots is significantly different between extreme genotypes for all days. 58

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Figure 2-9. Least Square Means (LSM) of nu mber of adventitious roots developed on extreme genotypes selected for expre ssion analysis. Genotypes UF352, UF498 and UF926 carry P. trichocarpa allele, whereas genotypes UF717, UF912 and UF209 carry P. deltoides allele. 59

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Figure 2-10. Number of genes differentially expressed when contrasting consecutive time points. 60

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Figure 2-11. Total number of genes differentially expressed between genotypes in the Pt QTL and Pd QTL categories within each time point. 61

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Figure 2-12. Correlation matrix of the si gnal difference between genotypes in the Pt QTL and Pd QTL categories estimated for 1929 genes identified as differentially expressed (FDR<0.05) between the two ca tegories in at least one time point. 62

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63 Figure 2-13. Average of absolute values of si gnal differences between genotypes in the Pt QTL and Pd QTL categories across each time point for the only cluster with a linear increase in the difference in transcript abundance between the two categories.

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Table 2-1. Clonal repeatability estimates for a ll adventitious root-rel ated phenotypes measured. Phenotypic values Mean SD Trait Acronym P. trichocarpa P. deltoides P. deltoides Progeny Variation Clonal repeatability H 2 SD Root architectural traits Total root length (cm) L 56.5 86.4 123.1 94 0.153 0.051 Total root surface area (cm 2 ) SA 10.0 23.4 25.1 19 0.169 0.052 Total root volume (cm 3 ) VOL 0.14 0.51 0.42 0.3 0.185 0.053 Average diamet er (mm) DIAM 0.56 0.93 0. 70 0.2 0.236 0.055 Length of root branches (cm) L1 25.6 28.5 55.7 49 0.115 0.049 Surface area of root branches (cm 2 ) SA1 2.90 2.40 4.33 3.8 0.117 0.049 Volume of root branches (cm 3 ) V1 0.28 0.20 0.034 0.03 0.120 0.049 Total length of pr imary roots (cm) PRIL 30.8 57. 6 67.1 50 0.193 0.053 Surface area of primary roots (cm 2 ) PRISA 6.90 20.1 19.4 15 0.187 0.053 Volume of primary roots (cm 3 ) PRIV 0.19 0.61 0.49 0.42 0.177 0.053 Number of root traits Count of root s on day 9 day9 3.79 0.46 1. 76 1.97 0.294 0.052 Count of root s on day 10 day10 4.64 1.08 2. 36 2.35 0.297 0.052 Count of root s on day 11 day11 5.21 1.25 3. 05 2.72 0.281 0.052 Count of root s on day 12 day12 5.79 1.58 3. 69 3.09 0.274 0.052 Count of root s on day 13 day13 6.50 1.92 4. 24 3.35 0.289 0.053 Count of root s on day 14 day14 6.86 2.92 4. 62 3.55 0.304 0.054 Count of root s on day 15 day15 7.21 3.58 5. 05 3.66 0.311 0.054 Count of root s on day 16 day16 7.59 4.16 0. 14 0.47 0.342 0.056 Count of root s on day 17 day17 7.36 4.58 5. 71 3.88 0.336 0.056 Count of root s on day 18 day18 7.36 4.92 5. 92 3.95 0.341 0.056 Root biomass (mg) DRYWT 4.73 27. 5 19.3 19.7 0.144 0.051 64

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65 Table 2-2. Pair-wise estimates of phenot ypic correlations between all traits. Trait L SA DIAM VOL L1 SA1 V1 PRIL PRISA PRIV DRYWT day9 day10 day11 day12 day13 day14 day15 day16 day17 day18 L X 0.98 0.34 0.92 0.95 0.94 0.92 0.95 0.94 0.92 0.91 0.46 0.49 0.52 0.55 0.55 0.58 0.58 0.58 0.57 0.56 SA X 0.44 0.98 0.88 0.87 0.85 0.98 0.99 0.98 0.96 0.45 0.47 0.51 0.53 0.53 0.57 0.57 0.58 0.57 0.57 DIAM X 0.51 0.21 0.19 0.17 0.44 0.49 0.51 0.48 0.17 0.15 0.12 0.13 0.12 0.17 0.17 0.18 0.18 0.18 VOL X 0.78 0.77 0.76 0.97 0.99 0.99 0.96 0.42 0.45 0.47 0.49 0.50 0.54 0.55 0.56 0.55 0.55 L1 X 0.99 0.97 0.80 0.80 0.80 0.81 0.38 0.38 0.41 0.43 0.42 0.44 0.43 0.42 0.42 0.40 SA1 X 0.99 0.79 0.79 0.79 0.80 0.37 0.37 0.40 0.42 0.41 0.42 0.42 0.41 0.40 0.39 V1 X 0.77 0.77 0.77 0.78 0.38 0.37 0.40 0.41 0.41 0.42 0.41 0.41 0.40 0.39 PRIL X 0.99 0.96 0.91 0.50 0.54 0.58 0.62 0.63 0.66 0.67 0.68 0.67 0.67 PRISA X 0.99 0.95 0.45 0.48 0.52 0.54 0.55 0.59 0.59 0.60 0.60 0.59 PRIV X 0.97 0.39 0.42 0.44 0.46 0.47 0.50 0.51 0.52 0.52 0.51 DRYWT X 0.35 0.37 0.40 0.41 0.41 0.45 0.45 0.46 0.45 0.44 day9 X 0.92 0.87 0.80 0.76 0.75 0.72 0.70 0.69 0.67 day10 X 0.93 0.88 0.84 0.82 0.79 0.77 0.75 0.73 day11 X 0.96 0.93 0.91 0.88 0.85 0.83 0.81 day12 X 0.98 0.96 0.93 0.90 0.88 0.86 day13 X 0.98 0.96 0.93 0.92 0.89 day14 X 0.99 0.96 0.95 0.93 day15 X 0.99 0.97 0.96 day16 X 0.99 0.98 day17 X 0.99 day18 X

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Table 2-3. Phenotypic variance explained by eac h QTL interval identified for number of root traits and the respective linkage group (LG), flanking markers location, LOD peak and origin of positive allele. Flanking Markers Trait acronym LG Marker 1 Marker 2 LOD peak Origin of positive allele Phenotypic variance explained (%) QTL 1 day9 II G734 rG876 5.34 P. deltoides 10.12 2 day9 IV O349 G961 4.22 P. trichocarpa 7.94 3 day11 II S96 rG876 5.48 P. deltoides 10.34 4 day14 II S96 rG876 5.60 P. deltoides 10.66 5 day14 XIV rO386a G674 4.99 P. deltoides 9.67 6 day15 II S96 rG876 5.00 P. deltoides 9.33 7 day15 XIV rO386a G674 5.03 P. deltoides 8.93 8 day16 II S96 O461 3.72 P. deltoides 7.20 9 day16 XIV rO386a P2515 3.87 P. deltoides 7.86 10 day17 II S96 rG876 4.75 P. deltoides 9.19 11 day17 XIV rO386a P2515 3.63 P. deltoides 6.66 66

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67 Table 2-4. Phenotypic variance explained by each QTL interval detected for root architecture traits and root biomass with the respective linkage group (LG), flanking marker location, LOD score and origin of positive allele. Flanking markers QTL Trait acronym LG Marker 1 Marker 2 LOD peak Origin of positive allele Phenotypic variance explained (%) 1 L1 XII G2643 G2673 3.80 P. deltoides 7.64 2 L XII G2643 G2673 3.86 P. deltoides 7.18 3 L XIV rO386a P2515 4.30 P. deltoides 10.0 4 PRIL II S96 O461 3.16 P. deltoides 5.8 5 PRIL XII G2643 G674 4.45 P. deltoides 8.12 6 PRIL XIV rO386a G674 5.12 P. deltoides 10.87 7 PRISA XII G2643 G2673 4.56 P. deltoides 8.54 8 PRISA XIV rO386a G674 4.84 P. deltoides 10.84 9 PRIV XII G2643 G2673 3.17 P. deltoides 6.12 10 PRIV XIV rO386a P2515 3.18 P. deltoides 6.76 11 DRYWT XVII rG880 P648 3.08 P. trichocarpa 7.04 12 SA XII G2643 G2673 3.95 P. deltoides 7.40 13 SA XIV rO386a G674 4.64 P. deltoides 10.03 14 VOL XII G2643 G2673 3.61 P. deltoides 6.92 15 VOL XIV rO386a P2515 3.43 P. deltoides 7.22

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LIST OF REFERENCES Ahkami AH, Lischew ski S, Haensch KT, Po rfirova S, Hofmann J, Rolletschek H, Melzer M, Franken P, Hause B, Druege U, Hajirezaei MR. 2009 Molecular physiology of adventitious r oot formation in Petunia hybrida cuttings: involvement of wound response and primary metabolism. New Phytologist, 181 : 613-625. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P. 2002 Molecular biology of the cell, New York, Garland Science. Ball J, Carle J, Del Lungo A. 2005 Contributions of poplars and willows to sustainable forestry and rural development. Unasylva 221, 56 : 3-9. Barlier I, Kowalczyk M, Marchant A, Lj ung K, Bhalerao R, Bennett M, Sandberg G, Bellini C. 2000 The SUR2 gene of Arabidopsis thaliana encodes the cytochrome P450 CYP83B1, a modul ator of auxin homeostasis. Proc Natl Acad Sci U S A, 97 : 14819-24. Barlow PW. 1986. Adventitious roots of whole pl ants: their forms, functions, and evolution. In: Jackson MB ed. New root formation in plants and cuttings. Boston, MA, M. Nijhoff, Distributors for the U.S. and Canada, Kluwer Academic Publishers. Bischoff V, Nita S, Neumetzler L, Schinde lasch D, Urbain A, Eshed R, Persson S, Delmer D, Scheible WR. 2010 TRICHOME BIREFRINGENCE and Its Homolog AT5G01360 Encode Plant-Spec ific DUF231 Proteins Required for Cellulose Biosynthesis in Arabidopsis. Plant Physiology, 153 : 590-602. Blilou I, Xu J, Wildwater M, Willemsen V, Paponov I, Friml J, Heidstra R, Aida M, Palme K, Scheres B. 2005 The PIN auxin efflux faci litator network controls growth and patterning in Arabidopsis roots. Nature, 433 : 39-44. Bocock PN, Morse AM, Dervinis C, Davis JM. 2008 Evolution and diversity of invertase genes in Populus trichocarpa. Planta, 227 : 565-576. Boerjan W, Cervera MT, Delarue M, Beeckman T, Dewitte W, Bellini C, Caboche M, Van Onckelen H, Van Montagu M, Inze D. 1995 Superroot, a recessive mutation in Arabidopsis, confers auxin overproduction. Plant Cell, 7 : 1405-19. Boerjan W, den Boer B, van Montagu M. 1992 Molecular genetic approaches to plant development. Int J Dev Biol, 36 : 59-66. Bohlenius H, Huang T, Charbonnel-Camp aa L, Brunner AM, Jansson S, Strauss SH, Nilsson O. 2006 CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science, 312 : 1040-1043. 68

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Bohmert K, Camus I, Bellini C, Bouchez D, Caboche M, Benning C. 1998 AGO1 defines a novel locus of Arabidop sis controlling leaf development. EMBO J, 17 : 170-80. Bolstad BM, Irizarry RA, Astrand M, Speed TP. 2003 A comparison of normalization methods for high density oligonucleoti de array data based on variance and bias. Bioinformatics, 19 : 185-193. Borralho NMG, Wilson PJ. 1994 Inheritance of Initial Surviv al and Rooting Ability in Eucalyptus-Globulus Labill Stem Cuttings. Silvae Genetica, 43 : 238-242. Bostick M, Lochhead SR, Honda A, Palmer S, Callis J. 2004 Related to ubiquitin 1 and 2 are redundant and essential and r egulate vegetative growth, auxin signaling, and ethylene production in Arabidopsis. Plant Cell, 16 : 2418-2432. Bouquin T, Meier C, Foster R, Nielsen ME, Mundy J. 2001 Control of specific gene expression by gibberellin and brassinosteroid. Plant Physiology, 127 : 450-458. Braatne JH, Rood SB, Heilman PE. 1996. Life history, ecology, and conservation of riparian cottowoods. In: Stettler RF, Bradshaw HD, Heilman PE, Hinckley TM eds. Biology of Populus and its implications for management and conservation. Ottawa, Canada, NRC Research Press. Bradshaw HD, Ceulemans R, Davis J, Stettler R. 2000 Emerging model systems in plant biology: Poplar (Populu s) as a model forest tree. J. Plant Growth. Regul., 19 : 306-313. Bradshaw HD, Jr., Stettler RF. 1995 Molecular genetics of growth and development in populus. IV. Mapping QTLs with large effects on growth, form, and phenology traits in a forest tree. Genetics, 139 : 963-73. Brinker M, van Zyl L, Liu W, Craig D, Sederoff RR, Clapham DH, von Arnold S. 2004 Microarray analyses of gene expressi on during adventitious root development in Pinus contorta. Plant Physiol, 135 : 1526-39. Brunner AM, Busov VB, Strauss SH. 2004 Poplar genome sequence: functional genomics in an ecologically dominant plant species. Trends Plant Sci., 9 : 49-56. Burn JE, Hocart CH, Birch RJ, Cork AC, Williamson RE. 2002 Functional analys is of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis. Plant Physiology, 129 : 797-807. Chang S, Puryear J, Cairney J. 1993 A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol. Rep., 11 : 117-121. Christians MJ, Gingerich DJ, Hansen M, Binder BM, Kieber JJ, Vierstra RD. 2009 The BTB ubiquitin ligases ETO1, EOL1 and EOL2 act collectively to regulate 69

PAGE 70

ethylene biosynthesis in Arabidopsis by controlling type-2 ACC synthase levels Plant Journal, 57 : 332-345. Churchill GA, Doerge RW. 1994 Empirical Threshold Values for Quantitative Trait Mapping. Genetics, 138 : 963-971. Ciamporova M. 1983 An Ultrastructural-Study of Re serve Lipid Mobilization in Stem Root Primordia and Poplar. New Phytologist, 95 : 19-27. Correa LD, Fett-Neto AG. 2004 Effects of temperatur e on adventitious root development in microcuttings of Euca lyptus saligna Smith and Eucalyptus globulus Labill. Journal of Thermal Biology, 29 : 315-324. Cutler DJ, Zwick ME, Carrasquillo MM, Yohn CT, Tobin KP, Kashuk C, Mathews DJ, Shah NA, Eichler EE, Warrington JA, Chakravarti A. 2001 Highthroughput variation detection and genotyping using microarrays. Genome Research, 11 : 1913-1925. Davies FT, Hartmann HT. 1988 The physiological basis of adventitious root formation. Acta Horticulturae, 227 : 133-120. De Haan G, Weersing E, Dontje B, Sutton S, Vellenga E, Manly KF, Williams RW, Cooke M, Bystrykh LV. 2003 Identification of transcr iptional networks affecting hematopoietic stem cell functi on using "genetical genomics". Blood, 102 : 343A343A. De Klerk GJ. 2002 Rooting of microcuttings: Theory and practice. In Vitro Cellular & Developmental Biology-Plant, 38 : 415-422. De Klerk GJ, Keppel M, Terbrugge J, Meekes H. 1995 Timing of the Phases in Adventitious Root-Formati on in Apple Microcuttings. Journal of Experimental Botany, 46 : 965-972. De Klerk GJ, Van der Krieken W, De Jong JC. 1999 Review The formation of adventitious roots: New concepts, new possibilities. In Vitro Cellular & Developmental Biology-Plant, 35 : 189-199. DeBell DS. 1990. Populus trichocarpa Torr. & Gray: Black Cottowood. In: Burns RM, Honkala BH eds. Silvics of North Am erica: Hardwoods. Washington, D.C., USDA Agriculture Handbook 654. Delarue M, Prinsen E, Onckelen HV, Caboche M, Bellini C. 1998 Sur2 mutations of Arabidopsis thaliana define a new locu s involved in the control of auxin homeostasis. Plant J, 14 : 603-11. Dickmann DI. 2001. An overview of the genus Popul us. In: Dickmann DI, Isebrands JG, Eckenwalder JE, Richardson J eds. Poplar Culture in North America. Ottawa, Canada, NRC Research Press, Nati onal Research Council of Canada. 70

PAGE 71

Dickmann DI, Hendrick RL. 1994. Modeling adventitious root system development in trees: clonal poplars. In: Davis TD, Haissig BE eds. Biology of Adventitious Root For mation. New York, Plenum Press. Dickmann DI, Kuzovkina J. 2008 Poplars and willows of t he world, with the emphasis on silviculturally important species. Poplars and willows of the world. Rome, Italy, FAO: Food and Agriculture Or ganization of the United Nations. Drost DR, Novaes E, Boaventura-Novaes C, Be nedict CI, Brown RS, Yin T, Tuskan GA, Kirst M. 2009 A microarray-based genot yping and genetic mapping approach for highly heterozygous outcrossi ng species enables localization of a large fraction of the unassemble d Populus trichocarpa genome sequence. Plant J, 58 : 1054-67. Eckenwalder JE. 1996. Systematics and evolution of Populus. In: Stettler RF, Bradshaw HD, Heilman PE, Hinckley TM eds. Biology of Populus and its implications for management and conservation. Ottawa, Canada, NRC Research Press. Eulgem T, Rushton PJ, Robatzek S, Somssich IE. 2000 The WRKY superfamily of plant transcription factors. TRENDS in Plant Science, 5 : 199-206. FAO. 2008 Activities related to poplar and willow cultivation and utilization, 2004 through 2007. Poplars, Willows, and People's Wellbeing: 23rd Session of the International Poplar Commission. Rome, Food and Agriculture Organization (FAO), Forest Resources Division. Farmer Jr RE. 1996. Genecology of Populus In: Stettler RF, Bradshaw HD, Heilman PE, Hinckley TM eds. Biology of Populus and its implications for management and conservation. Ottawa, Canada, NRC Research Press. Fattorini L, Falasca G, Kevers C, Rocca LM, Zadra C, Altamura MM. 2009 Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers. Planta, 231 : 155-168. Frewen BE, Chen THH, Howe GT, Davis J, Rohde A, Boerjan W, Bradshaw HD. 2000 Quantitative trait loci and candidate gene mapping of bud set and bud flush in Populus. Genetics, 154 : 837-845. Furutani I, Watanabe Y, Prieto R, Masuka wa M, Suzuki K, Naoi K, Thitamadee S, Shikanai T, Hashimoto T. 2000 The SPIRAL genes are r equired for directional control of cell elongation in Aarabidops is thaliana. Development, 127 : 4443-53. Gaspar T, Kevers C, Faivre-Rampant O, Crevecoeur M, Penel C, Greppin H, Dommes J. 2003 Changing concepts in plant hormone action. In Vitro Cellular & Developmental Biology-Plant, 39 : 85-106. 71

PAGE 72

Gershon D. 2002 Microarray technology: An array of opportunities. Nature, 416 : 885891. Goujon T, Sibout R, Pollet B, Maba B, Nussaume L, Bechtold N, Lu FC, Ralph J, Mila I, Barriere Y, Lapierre C, Jouanin L. 2003 A new Arabidops is thaliana mutant deficient in the expression of O-methyltransferase impacts lignins and sinapoyl esters. Plant Molecular Biology, 51 : 973-989. Gutierrez L, Bussell JD, P acurar DI, Schwambach J, Pacurar M, Bellini C. 2009 Phenotypic plasticity of adventitious r ooting in Arabidopsis is controlled by complex regulation of AUXIN RESPONSE FACTOR transcripts and microRNA abundance. Plant Cell, 21 : 3119-32. Han K, Bradshaw HD, Jr., Gordon MP. 1994 Adventitious root and shoot regeneration in vitro is under major gene control in an F2 family of hybrid poplar (Populus trichocarpa P. deltoides). Forest Genetics, 1 : 139-146. Heilman PE. 1999 Planted forests: poplars. New Forests, 17 : 89-93. Hertzberg M, Aspeborg H, Schrader J, A ndersson A, Erlandsson R, Blomqvist K, Bhalerao R, Uhlen M, Teeri TT, Lundeberg J, Sundberg B, Nilsson P, Sandberg G. 2001 A transcriptional roadmap to wood formation. Proceedings of the National Academy of Sciences of the United Stat es of America, 98 : 1473214737. Jansen RC, Nap JP. 2001 Genetical genomics: the added value from segregation. Trends Genet., 17 : 388-391. Jansson S, Douglas CJ. 2007 Populus: A model system for plant biology. Annual Review of Plant Biology, 58 : 435-458. Kirst M, Myburg AA, De Leon JP G, Kirst ME, Scott J, Sederoff R. 2004 Coordinated genetic regulation of growth and lignin revealed by quantitative trait locus analysis of cDNA microarray data in an interspecific backcross of Eucalyptus. Plant Physiol., 135 : 2368-2378. Kirst M Yu Q. 2007. Genetical genomics: successes and prospects in plants. In: Varshney RK, Tuberosa R eds. Genomics-assisted crop improvement SringerVerlag. Klee HJ. 2004 Ethylene signal transduction. Moving beyond Arabidopsis. Plant Physiology, 135 : 660-667. Konishi M, Sugiyama M. 2006 A novel plant-specific family gene, ROOT PRIMORDIUM DEFECTIVE 1, is requir ed for the maintenance of active cell proliferation. Plant Physiol, 140 : 591-602. 72

PAGE 73

Lovell PH, White J. 1986. Anatomical changes during advent itious root formation. In: Jackson MB ed. New root form ation in plants and cuttings. Boston, MA, M. Nijhoff, Distributors for t he U.S. and Canada, Kluwer Academic Publishers. Mackay TF, Stone EA, Ayroles JF. 2009 The genetics of quantitative traits: challenges and prospects. Nat Rev Genet, 10 : 565-77. Mackay TFC. 2001 The genetic architecture of quantitative traits. Annual Review of Genetics, 35 : 303-339. Mano Y, Muraki M, Fujimori M, Takamizo T, Kindiger B. 2005 Identification of QTL controlling adventitious root formation during flooding conditions in teosinte (Zea mays ssp huehuetenangensis) seedlings. Euphytica, 142 : 33-42. Murphy D. 2002 Gene expression studies using micr oarrays: Principles, problems, and prospects. Advances in Physiology Education, 26 : 256-270. Novaes E, Osorio L, Drost DR, Miles BL, Boaventura-Novaes CRD, Benedict C, Dervinis C, Yu Q, Sykes R, Davis M, Martin TA, Peter GF, Kirst M. 2009 Quantitative genetic analysis of biomass and wood chemistry of Populus under different nitrogen levels. New Phytologist, 182 : 878-890. Ochoa IE, Blair MW, Lynch JP. 2006 QTL analysis of adventiti ous root formation in common bean under contrasting phosphorus availability. Crop Science, 46 : 1609-1621. Osmont KS, Hardtke CS. 2008 The topless plant developm ental phenotype explained! Genome Biology, 9 : -. Ozawa S, Yasutani I, Fukuda H, Komamine A, Sugiyama M. 1998 Organogenic responses in tissue culture of srd mutants of Arabidopsis thaliana. Development, 125 : 135-42. Pekker I, Alvarez JP, Eshed Y. 2005 Auxin response factors mediate Arabidopsis organ asymmetry via modulation of KANADI activity. Plant Cell, 17 : 2899-910. Pregitzer KS, Friend AL. 1996. The structure and function of Populus root systems. In: Stettler RF, Bradshaw HD, Heilman PE, Hinc kley TM eds. Biology of Populus and its implications for management and conservation. Ottawa, Canada, NRC Research Press. Quesada T, Li Z, Dervinis C, Li Y, Bo cock PN, Tuskan GA, Casella G, Davis JM, Kirst M. 2008 Comparative analysis of t he transcriptomes of Populus trichocarpa and Arabidopsis thaliana s uggests extensive evolution of gene expression regulation in angiosperms. New Phytol, 180 : 408-20. Rae AM, Tricker PJ, Bunn SM, Taylor G. 2007 Adaptation of tree growth to elevated CO2: quantitative trait loci for biomass in Populus. New Phytol, 175 : 59-69. 73

PAGE 74

Ramirez-Carvajal GA Morse AM, Davis JM. 2008 Transcript profiles of the cytokinin response regulator gene family in P opulus imply diverse roles in plant development. New Phytologist, 177 : 77-89. Ramirez-Carvajal GA, Morse AM, Dervinis C, Davis JM. 2009 The cytokinin type-B response regulator PtRR13 is a negativ e regulator of adv entitious root development in Populus. Plant Physiol, 150 : 759-71. Riemenschneider DE, Bauer EO. 1997. Quantitative genetic an alysis of adventitious root forming ability in Populus trichocarpa (Torr et. Gray). In: Altman A, Waisel Y eds. Biology of root forma tion and development. New York, Plenum Press. Ritchie GA. 1994. Commercial application of adventitious rooting to forestry. In: Davis TD, Haissig BE eds. Biology of Adventitious Root Formation. New York, Plenum Press. Ronnberg-Wastljung AC, Glynn C, Weih M. 2005 QTL analyses of drought tolerance and growth for a Salix dasyclados x Salix viminalis hybrid in contrasting water regimes. Theoretical and Ap plied Genetics, 110 : 537-549. Schadt EE, Monks SA, Drake TA, Lusis AJ, Che N, Colinayo V, Ruff TG, Milligan SB, Lamb JR, Cavet G, Linsley PS, Mao M, Stoughton RB, Friend SH. 2003a Genetics of gene expression survey ed in maize, mouse and man. Nature, 422 : 297-302. Schadt EE, Monks SA, Friend SH. 2003b A new paradigm for drug discovery: integrating clinical, genet ic, genomic and molecular phenotype data to identify drug targets. Biochemical Society Transactions, 31 : 437-443. Schindelman G, Morikami A, Jung J, Baskin TI, Carpita NC, Derbyshire P, McCann MC, Benfey PN. 2001 COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary fo r oriented cell expansion in Arabidopsis. Genes & Development, 15 : 1115-1127. Schubert V, Weissleder A, Ali H, Fuchs J, Lermonto va I, Meister A, Schubert I. 2009 Cohesin gene defects may impair sist er chromatid alignment and genome stability in Arabido psis thaliana. Chromosoma, 118 : 591-605. Smith DL, Fedoroff NV. 1995 LRP1, a gene expressed in lateral and adventitious root primordia of arabidopsis. Plant Cell, 7 : 735-45. Sorin C, Bussell JD, Camus I, Ljung K, Kowalczyk M, Geiss G, McKhann H, Garcion C, Vaucheret H, Sandberg G, Bellini C. 2005 Auxin and light control of adventitious rooting in Ar abidopsis require ARGONAUTE1. Plant Cell, 17 : 1343-59. Sorin C, Negroni L, Balliau T, Corti H, Jacquemot MP Davanture M, Sandberg G, Zivy M, Bellini C. 2006 Proteomic analysis of differ ent mutant genotypes of 74

PAGE 75

Arabidopsis led to the ident ification of 11 proteins co rrelating with adventitious root development. Plant Physiol, 140 : 349-64. Srivastava LM. 2002 Plant growth and development : hormones and environment, Amsterdam ; Boston, Academic Press. Stettler RF, Zsuffa L, Wu R. 1996. The role of hybridization in the genetic manipulation of Populus In: Stettler RF, Bradshaw HD, Heilman PE, Hinckley TM eds. Biology of Populus and its implications for management and conservation. Ottawa, Canada, NRC Research Press. Stone EA, Ayroles JF. 2009 Modulated modularity clustering as an exploratory tool for functional genomic inference. PLoS Genet, 5 : e1000479. Storey JD, Tibshirani R. 2003 Statistical significance for genomewide studies. Proc. Natl. Acad. Sci. USA, 100 : 9440-9445. Stracke R, Werber M, Weisshaar B. 2001 The R2R3-MYB gene family in Arabidopsis thaliana. Current Opinion in Plant Biology, 4 : 447-456. Street NR, Skogstrom O, Sjodin A, Tucker J, Rodriguez-Acosta M, Nilsson P, Jansson S, Taylor G. 2006 The genetics and genom ics of the drought response in Populus. Plant J., 48 : 321-341. Sun WQ, Bassuk NL. 1993 Auxin-Induced Ethylene Synthesis during Rooting and Inhibition of Budbreak of Royalty Rose Cuttings. Journal of the American Society for Horticultural Science, 118 : 638-643. Szekely G, Abraham E, Cselo A, Rigo G, Zsigmond L, Csiszar J, Ayaydin F, Strizhov N, Jasik J, Schmelzer E, Koncz C, Szabados L. 2008 Duplicated P5CS genes of Arabidopsis play dist inct roles in stress regulation and developmental control of proline biosynthesis. Plant Journal, 53 : 11-28. Taylor G. 2002 Populus: Arabidopsis for fore stry. Do we need a model tree? Ann. Bot., 90 : 681-689. Thimann KV, Went FW. 1934 On the chemical nature of the rootforming hormone. Proceedings of the Koni nklijke Ak ademie Van We tenschappen Te Amsterdam, 37 : 456-459. Tuskan GADiFazio SJansson SBohlmann JGrigoriev IHellsten UPutnam NRalph SRombauts SSalamov ASchein JSter ck LAerts ABhalerao RRBhalerao RPBlaudez DBoerjan WBrun ABrunner ABusov VCampbell MCarlson JChalot MChapman JChen GLCoope r DCoutinho PMCouturier JCovert SCronk QCunningham RDavis JDegroev e SDejardin ADepamphilis CDetter JDirks BDubchak IDuplessis SEhlti ng JEllis BGendler KGoodstein DGribskov MGrimwood JGroover AGunter LHamberger BHeinze BHelariutta YHenrissat BHolligan DHolt RHuang WIslam-Faridi NJones 75

PAGE 76

SJones-Rhoades MJorgensen RJoshi CKangasjar vi JKarlsson JKelleher CKirkpatrick RKirst MKohler AKalluri ULarimer FLeebens-Mack JLeple JCLocascio PLou YLucas SMartin FMontanini BNapoli CNelson DRNelson CNieminen KNilsson OPereda VPeter GPhilippe RPilate GPoliakov ARazumovskaya JRichardson PRinaldi CRitland KRouze PRyaboy DSchmutz JSchrader JSegerman BShi n HSiddiqui ASterky FTerry ATsai CJUberbacher EUnneberg PVahala JWall KWessler SYang GYin TDouglas CMarra MSandberg Gde Peer YV, Rokhsar D. 2006 The genome of black cottonwood, Populus trichoc arpa (Torr. & Gray). Science, 313 : 1596-1604. Vera P, Lamb C, Doerner PW. 1994 Cell-cycle regulation of hydroxyproline-rich glycoprotein HRGPnt3 gene expression during the init iation of lateral root meristems. Plant Journal, 6 : 717-727. Wang HY, Lockwood SK, Hoeltzel MF, Schiefelbein JW. 1997 The ROOT HAIR DEFECTIVE3 gene encodes an evolutiona rily conserved protein with GTPbinding motifs and is required for r egulated cell enlargement in Arabidopsis. Genes & Development, 11 : 799-811. Wang SC, Basten CJ, Zeng ZB. 2007a Windows QTL Cartograph er 2.5. Raleigh, NC., Department of Statistics, No rth Carolina State University. Wang Y, Yang T, Liu G, Zhao C, Wang P, Chen X. 2007b Differently expressed genes in tobacco leaves under osmotic stress. Acta Agronomica Sinica, 33 : 914920. Wilcox JR, Farmer RE. 1968 Heritability and C Effects in Early Root Growth of Eastern Cottonwood Cuttings. Heredity, 23 : 239-&. Wu J, Steinebrunner I, Sun Y, Butterfield T, Torres J, Ar nold D, Gonzalez A, Jacob F, Reichler S, Roux SJ. 2007 Apyrases (nucleoside triphosphatediphosphohydrolases) play a key role in growth control in arabidopsis. Plant Physiology, 144 : 961-975. Wu Q. 2004 Isolation of genes associated with adventitious root development in Populus, Master of Science, North Caro lina State University, Raleigh. Wullschleger SD, Jansson S, Taylor G. 2002 Genomics and forest biology: Populus emerges as the perennial favorite. Plant Cell, 14 : 2651-2655. Yanai O, Shani E, Dolezal K, Tarkowski P, Sablowski R, Sandberg G, Samach A, Ori N. 2005 Arabidopsis KNOXI proteins acti vate cytokinin biosynt hesis. Curr Biol, 15 : 1566-71. Yang XH, Kalluri UC, DiFazio SP, Wullsch leger SD, Tschaplinski TJ, Cheng ZM, Tuskan GA. 2009 Poplar Genomics: St ate of the Science. Critical Reviews in Plant Sciences, 28 : 285-308. 76

PAGE 77

77 Ying CC, Bagley WT. 1977 Variation in Rooting Capability of Populus deltoides Silvae Genetica, 26 : 204-207. Zalesny Jr RS, Hall RB, Bauer EO, Riemenschneider DE. 2003 Shoot position affects root initiation and growth of dormant unrooted cuttings of Populus Silvae Genetica, 52 : 273-279. Zalesny Jr RS, Wiese AH. 2006 Date of shoot collection, genotype, and original shoot position affect early rooting of do rmant hardwood cuttings of Populus. Silvae Genetica, 55 : 169-182. Zalesny RS, Riemenschneider DE, Hall RB. 2005 Early rooting of dormant hardwood cuttings of Populus: analysis of quantitative genetics and genotype x environment interactions. Canadian Journal of Fo rest Research-Revue Canadienne De Recherche Forestiere, 35 : 918-929. Zeng ZB. 1993 Theoretical basis for separation of multiple linked gene effects in mapping quantitative trait loci. Proc. Natl. Acad. Sci. USA, 90 : 10972-10976. Zhang B, Tong CF, Yin TM, Zhang XY, Zhuge QQ, Huang MR, Wang MX, Wu RL. 2009 Detection of quantitative trait loci influencing growth trajectories of adventitious roots in Populus using functional mapping. Tree Genetics & Genomes, 5 : 539-552. Zheng BS, Yang L, Zhang WP, Mao CZ, Wu YR, Yi KK, Liu FY, Wu P. 2003 Mapping QTLs and candidate genes for rice root traits under different watersupply conditions and comparativ e analysis across three populations. Theoretical and Applied Genetics, 107 : 1505-1515. Zhou C, Zhang L, Duan J, Miki B, Wu K. 2005 HISTONE DEACETYLASE19 is involved in jasmonic acid and ethyl ene signaling of pathogen response in Arabidopsis. Plant Cell, 17 : 1196-204. Zsuffa L. 1975 A summary review of interspecific breeding in the genus Populus L. Proceedings of the fourt eenth meeting of the Canadian Tree Improvement Association: part 2. Symposium on interspecific and interprovenanc e hybridization in forest trees. Ottawa, ON, Canadian Forestry Service. Zsuffa L, Giordano E, Pryor LD, Stettler RF. 1996. Trends in poplar culture: some global and regional perspecti ves. In: Stettler RF, Bradshaw HD, Heilman PE, Hinckley TM eds. Biology of Populus and its impl ications for management and conservation. Ottawa, Canada, NRC Research Press.

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BIOGRAPHICAL SKETCH Cynthia M. Silva was born in Santo AndrŽ (S‹o Paulo state, Braz il) in 1985. At the age of 17, she entered into Univ ersity of S‹o Paulo in Pi racica ba (ESALQ-USP, Brazil) to pursue her Bachelor of Science in Ag ronomic Engineering. As an undergrad, she joined the Ph.D. student Marines Karasawa, s upervised by Professor Dr. Elizabeth Ann Veasey, to study the genetic di versity of the rice species Oryza glumaepatula using microsatellites markers. When she conclude d her project in Dr. Veasey's lab, she started a new project with t he Ph.D. student Juliana Teixei ra on the development of molecular markers for disease resistance in Eucalyptus under the supervision of Dr. Luis Camargo. During her last year as an undergrad, Dr. Camargo in troduced her to Dr. Matias Kirst, who accepted her as an intern in his Forest Genomics lab at the University of Florida. During her three-month internship in the U.S., she first learned about the tree genus Populus and was introduced to the world of QTL mapping. She came back to Brazil to obtain her Bachelor of Science in January of 2008. She returned to the United States in August of 2008, to pursue her Master of Science degree in the School of Forest Resources and Conservation at the Univ ersity of Florida, as a member of Dr. Kirst's Forest Genomics research group. 78