Dense Genetic Linkage Maps of Three Populus Species ( Populus … · and ‡Instituut voor Bosbouw en Wildbeheer, B-9500 Geraardsbergen, Belgium Manuscript received December 20, 1999

Copyright 2001 by the Genetics Society of America

Dense Genetic Linkage Maps of Three Populus Species (Populus deltoides,P. nigra and P. trichocarpa) Based on AFLP and Microsatellite Markers

Maria-Teresa Cervera,*,1,2 Veronique Storme,*,1 Bart Ivens,* Jaqueline Gusmao,*,3 Ben H. Liu,†

Vanessa Hostyn,* Jos Van Slycken,‡ Marc Van Montagu* and Wout Boerjan*

*Vakgroep Moleculaire Genetica en Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent,B-9000 Gent, Belgium, †Forest Biotechnology Group, Department of Forestry, North Carolina State University, Raleigh, North Carolina 27695

and ‡Instituut voor Bosbouw en Wildbeheer, B-9500 Geraardsbergen, Belgium

Manuscript received December 20, 1999Accepted for publication February 26, 2001

ABSTRACTPopulus deltoides, P. nigra, and P. trichocarpa are the most important species for poplar breeding programs

worldwide. In addition, Populus has become a model for fundamental research on trees. Linkage mapswere constructed for these three species by analyzing progeny of two controlled crosses sharing the samefemale parent, Populus deltoides cv. S9-2 3 P. nigra cv. Ghoy and P. deltoides cv. S9-2 3 P. trichocarpa cv.V24. The two-way pseudotestcross mapping strategy was used to construct the maps. Amplified fragmentlength polymorphism (AFLP) markers that segregated 1:1 were used to form the four parental maps.Microsatellites and sequence-tagged sites were used to align homoeologous groups between the maps andto merge linkage groups within the individual maps. Linkage analysis and alignment of the homoeologousgroups resulted in 566 markers distributed over 19 groups for P. deltoides covering 86% of the genome,339 markers distributed over 19 groups for P. trichocarpa covering 73%, and 369 markers distributed over28 groups for P. nigra covering 61%. Several tests for randomness showed that the AFLP markers wererandomly distributed over the genome.

BECAUSE of its fast growth, ease for clonal propaga- rope are derived from interspecific crosses between P.deltoides and P. trichocarpa and between P. deltoides andtion, and strong heterosis upon interspecific hy-P. nigra and their backcrosses. Selection and breedingbridization, Populus has become a tree of prime eco-strategies have been oriented mainly toward resistancenomic importance. Poplar wood has many end uses,to leaf rust (caused by the fungus Melampsora larici-popu-including pulp and paper, timber, plywood, pallets, softlina) and bacterial canker (caused by Xanthomonas pop-board, and hard board. There is also an increasing inter-uli), enhanced growth, rooting ability to improve clonalest for cultivation of poplar as a biomass crop (Pearcepropagation, adaptation to latitude, and superior wood1995). For the same reasons and because of its smallquality (Ceulemans et al. 1987).genome size (550 Mb; 2C 5 1.1 pg; 19 chromosomes)

Tree breeding is a time-consuming process, mainlyand its amenability for genetic transformation, poplarbecause of the long generation intervals and the facthas become a model system for fundamental researchthat productivity and quality can best be evaluated aton trees (Stettler et al. 1996; Klopfenstein et al. 1997).rotation age, which varies between 7 and 20 years. TheLarge expressed sequence tag (EST) databases havedevelopment of polymerase chain reaction (PCR)-basedbeen constructed (Sterky et al. 1998; Mellerowicz etmolecular markers has facilitated the construction ofal. 2001) that will catalyze the development of efficientgenetic linkage maps to study the architecture of poly-functional genomics research programs in trees.genic traits. Linkage maps also constitute the frameworkPopulus deltoides, P. nigra, and P. trichocarpa are thefor the use of genetic markers in breeding programsmost important species for poplar breeding in Europe.via marker-assisted selection (Mazur and Tingey 1995)Most of the commercial clones planted throughout Eu-and facilitate map-based cloning (Tanksley et al. 1995).For many tree species, genome mapping projects havebeen initiated, which are mostly based on restriction

Corresponding author: Wout Boerjan, Vlaams Interuniversitair Insti- fragment length polymorphism (RFLP) and randomtuut voor Biotechnologie, Universiteit Gent, K.L. Ledeganckstraat 35,

amplified polymorphic DNA (RAPD) markers (for ref-B-9000 Gent, Belgium. E-mail: [email protected], see Cervera et al. 1999). The first linkage1 These authors contributed equally to this work.groups identified in poplar were obtained with alloen-2 Present address: Departamento de Genetica Molecular de Plantas,

Centro Nacional de Biotecnologıa (CSIC), Campus de la Universidad zymes (Muller-Starck 1992; Liu and Furnier 1993)Autonoma de Madrid, E-28049 Madrid, Spain. and RFLPs (Liu and Furnier 1993). A linkage map

3 Present address: Laboratorio de Biodiversidade Molecular, Departa-covering 50% of the genome was constructed based onmento de Genetica, Instituto de Biologia, UFRJ, Bloco A, CCS, Ilha

do Fundao, 21941-490, Rio de Janeiro, Brazil. RFLP, sequence-tagged site (STS), and RAPD markers

Genetics 158: 787–809 ( June 2001)

788 M.-T. Cervera et al.

repulsion. Uppercase letters were used to denote markers that(Bradshaw et al. 1994). Molecular markers, associatedhad ,10% missing data.with qualitative resistance to M. larici-populina and M.

STS analysis and marker nomenclature: STS marker analysismedusae, have been identified (Cervera et al. 1996; was performed according to Bradshaw et al. (1994). A selec-Newcombe et al. 1996; Villar et al. 1996; Lefevre et tion of primers and their corresponding enzymes were tested:

P164, P753, P754, P755, P757, P762, P763, P767, P770, P771,al. 1998; Tabor et al. 2000) and quantitative trait lociP781, P783, P832, P856, P869, P991, P993, P1010, P1013,(QTL) for stem growth and shape, bud phenology, leafP1018, P1027, P1046, P1054, P1079, and P1086. The STS mark-variation, and resistance to M. medusae and Septoria popu-ers are denoted by STS followed by the number given by

licola have been mapped (Wu et al. 1992, 1997; Wu and Bradshaw et al. (1994). On the linkage maps (Figures 1–3),Stettler 1994, 1997; Bradshaw and Stettler 1995; uppercase letters were used to denote markers with ,10%

missing data.Newcombe and Bradshaw 1996; Newcombe et al. 1996;Microsatellite analysis and marker nomenclature: PrimerWu 1998; Frewen et al. 2000).

sequences for microsatellite analysis were obtained from theHere we report on the construction of linkage mapswebsite of the Poplar Molecular Genetics Cooperative (PMGC;

for P. deltoides, P. nigra, and P. trichocarpa based on a http://poplar2.cfr.washington.edu/PMGC/). All 153 micro-two-way pseudotestcross strategy (Ritter et al. 1990; satellites were analyzed. The names of the microsatellite mark-

ers were taken from the PMGC website with the prefix PMGC.Grattapaglia and Sederoff 1994; Hemmat et al.A second set of microsatellites was provided by the Centre for1994) and amplified fragment length polymorphismPlant Breeding and Reproduction Research (Plant Research(AFLP) markers (Zabeau and Vos 1993; Vos et al. 1995).International, The Netherlands) and has the prefix wpms or

Microsatellites and STS markers were added to the set pkhomta (van der Schoot et al. 2000). On the linkage mapsof AFLP markers to allow direct identification of homo- (Figures 1–3), uppercase letters were used to denote markers

with ,10% missing data. Most of the microsatellite markerseologous loci and to merge linkage groups of the indi-were analyzed on half of the family only and were, therefore,vidual maps. After the homoeologous loci were aligned,not placed in the framework.19 linkage groups were found for P. deltoides and P.

Initially, microsatellites were analyzed with [g-33P]ATP-trichocarpa and 28 for P. nigra. The genetic maps covered labeled primers. In that case, the PCR reaction was performed86, 73, and 61% of the total genome, respectively. in a total volume of 20 ml containing 10 mm Tris-HCl, pH 8.3;

50 mm KCl; 2.5 mm MgCl2; 200 mm each of dATP, dCTP,dGTP, and dTTP; 1 unit Taq Polymerase (Roche Diagnostics,Brussels, Belgium); 50 ng kinated forward primer; 50 ng re-MATERIALS AND METHODSverse primer; and 30 ng DNA. The kinase reaction was per-formed in a total volume of 10 ml containing 10 mm Tris-Plant material and DNA extraction: Two full-sib familiesacetate, pH 7.5; 10 mm Mg-acetate; 50 mm K-acetate; 500 ngwere used to generate the genetic maps. One full-sib familyforward primer; 1 ml [g-33P]ATP (3000 Ci/mmol); and 0.06(87001) consisted of 127 individuals and resulted from anunits T4 kinase (Amersham Pharmacia Biotech, Little Chal-interspecific cross between two elite trees, P. deltoides cv. S9-2

and P. nigra cv. Ghoy (Cervera et al. 1996). The second font, UK). The mixture was incubated for 30 min at 378 fol-lowed by 10 min at 808 to inactivate the kinase. The primersfull-sib family (87002) consisted of 105 individuals and was

generated from an interspecific cross involving the same fe- were synthesized on a DNA/RNA synthesizer model 394 (Per-kin-Elmer-Applied Biosystems, Foster City, CA), followed bymale P. deltoides cv. S9-2 and P. trichocarpa cv. V24. These crosses

were designed initially to introduce the M. larici-populina resis- purification on an oligonucleotide purification cartridge (Per-kin-Elmer-Applied Biosystems). Alternatively, microsatellitestance gene present in P. deltoides cv. S9-2 into the breeding

program (Cervera et al. 1996). Genomic DNA was extracted were analyzed with fluorescent dye-labeled primers. For thismethod, each PCR reaction was performed in a total volumefrom frozen young leaves using the procedure described by

Dellaporta et al. (1983) or the DNeasy plant miniprep kit of 15 ml containing 10 mm Tris-HCl, pH 8.3; 50 mm KCl; 2.5mm MgCl2; 250 mm each of dATP, dCTP, dGTP, and dTTP;(QIAGEN, Helden, Germany).

AFLP analysis and marker nomenclature: AFLP analysis was 0.6 units Taq Polymerase (Amplitaq Gold; Perkin-Elmer, Nor-walk, CT); 30 ng labeled forward primer; 30 ng reverse primer;performed according to Vos et al. (1995) with some modifica-

tions (Cervera et al. 1996). Primer combinations (EcoRI 1 and 30 ng DNA. The forward primers were labeled with fluo-rescent dyes [NED (Perkin-Elmer), HEX (Genset, Paris), and3/MseI 1 3) were selected on the basis of the total number

of bands and the level of polymorphism observed when analyz- FAM (Genset)]. For both methods, the DNA was aliquotedseparately into 0.2-ml tubes and a master mix of the othering the three parents and eight progeny from each family

(data not shown). The sequences of the primers used in the components was added, mixed, and centrifuged briefly. Thereactions were transferred to a Gene AMP thermocycler 9600preamplifications and the selective amplifications as well as

their codes are indicated in Table A, available on the world (Perkin-Elmer). The amplification conditions were as de-scribed on the PMGC website or by van der Schoot et al.wide web (http://www.plantgenetics.rug.ac.be/zvesto). The

AFLP marker name refers to the primers used: E followed by (2000). The 33P-labeled amplification products were dena-tured and heated as described for AFLP analysis (Cerveratwo numbers refers to the EcoRI primer and G or F followed

by two numbers to the MseI primer (Figures 1–3). Polymorphic et al. 1996). The samples were loaded on 6% acrylamide/bisacrylamide 19:1, 7.5 m urea, and 13 Tris-borate (TBE; 0.1 mbands were numbered serially in ascendant order of molecular

weight; thus the last two numbers of the AFLP marker code Tris-base, 0.1 m boric acid, 2 mm EDTA, pH 8.0) gels. For thedye-labeled amplification products, 2.4 ml loading buffer [60%refer to the relative position of the polymorphic band on the

gel. Linkage groups were constructed by using markers both formamide, 23% Perkin-Elmer loading dye containing 50 ng/ml blue dextran and 5 mm EDTA, and 17% internal standardin coupling and in repulsion phase. For this purpose, the

matrix was duplicated and the duplicated part was subse- Genescan 500 ROX (Perkin-Elmer)] was added to 0.2-ml tubesto which 0.2, 0.24, and 0.4 ml of FAM-, HEX-, and NED-labeledquently inverted (absent/present and present/absent). An

“r” indicates an inverted marker and represents a marker in amplification products were added, respectively. The samples

789Linkage Maps of Poplar

were heated for 7 min at 958. One microliter of each sample To calculate the observed genome length, the total lengthof the framework map was calculated (Gof), as well as the totalwas loaded on 4.25% acrylamide/bisacrylamide 19:1, 6 m urea,

and 13 TBE gels. Gels were electrophoresed on an ABI 377 length when considering all markers (Goa). In addition, theobserved genome length was calculated by the formula ofapparatus (Perkin-Elmer-Applied Biosystems) in 13 TBE

buffer for 3 hr at 200 W. Gels were processed and scored Nelson et al. (1994) that takes into account all markers, linkedand unlinked: Gon 5 Gof 1 X(L 2 R), with X the observedusing the Genescan and Genotyper software (Perkin-Elmer).

Scoring and sequencing of markers: All markers were scored maximum distance between two framework markers; L thetotal number of linkage groups, triplets, doublets, and un-as dominant markers and scored visually independently by

two persons to minimize scoring and interpretation errors. linked markers; and R the haploid number of chromosomes.Expected and observed map coverage: The expected ge-Heterozygosity levels: The average heterozygosity was esti-

mated by analyzing the parents and 20 individuals of both nome map coverage was calculated from the equation:Cel 5 1 2 e2XN/1.25Ge (Lange and Boehnke 1982), adjusted forfamilies, using a subset of 10 AFLP primer combinations, and

by analyzing all microsatellites. For the estimate based on chromosomal ends and compared with the result from thefollowing equation, which accounts for linear chromosomesAFLP, the average heterozygosity was defined as the ratio of

bands segregating in the F1 progeny compared to the total (Bishop et al. 1983):number of bands observed. For the estimate on microsatellites,the average heterozygosity was defined as the ratio of polymor- Ceb 5 1 2 3 2R

N 1 1 111 2X

2Ge2N11

2 11 2XGe

2N11

2 1 11 2RXGe

211 2XGe

2N

4.phic microsatellites to all microsatellites (polymorphic andmonomorphic). In this equation, N is the number of framework markers;

Segregation analysis and map construction: Maps were con- X, the maximum distance between two adjacent frameworkstructed according to the two-way pseudotestcross strategy markers in centimorgans at a certain minimum LOD score(Ritter et al. 1990; Grattapaglia and Sederoff 1994; Hem- (in this case 3.5); Ge, the estimated genome length; and R,mat et al. 1994). AFLP, STS, and microsatellite markers segre- the haploid number of chromosomes. Only framework mark-gating 1:1 in the offspring were used for the construction of ers were considered because these equations refer to randomlygenome maps of both parents of each progeny. For each distributed markers.marker, a x2 test (d.f. 5 1, P , 0.01, and P , 0.05) was used The observed map coverage is defined as the ratio of theto identify deviations from Mendelian ratios. AFLP markers observed genome length Gof to the estimated genome lengthdeviating at the 1% significance level were excluded for the Ge. For the observed genome length, the observed frameworklinkage analysis. Four matrices were created, one for each map distance (Gof) was used also because for the expected mapparent of the two crosses. To detect linkages in repulsion coverage only framework markers were taken into account.phase, the data set was inverted and added to the original Marker distribution: To evaluate whether the AFLP markersdata. Linkage analysis was performed by MAPMAKER Unix were randomly distributed, all linkage groups were dividedversion 3.0 (Lander et al. 1987). The “triple error detection” into 10-cM intervals. Intervals at the end of a linkage groupand the “error detection” features were used to recognize the were taken into account only when .7.5 cM. The number ofcircumstance when an event was more probably the result intervals that contained no markers and one to nine markersof error than of recombination. These features avoid map were counted. The observed frequencies were compared toexpansion (Lincoln and Lander 1992). Initially, a logarithm the expected binomial frequencies. Subsequently, a runs testof odds (LOD) of minimum 3.5 and a maximum recombina- was performed (Sokal and Rohlf 1981). The relationshiption fraction u of 0.30 (corresponding to a maximum Kosambi between the variance and the mean [the coefficient of disper-distance of 34.7 cM) were established as thresholds for group- sion (CD) 5 s2/Y] is a rapid method to verify whether theing markers. One anchor marker was chosen from each link- observed frequency distribution is distributed in Poisson fash-age group for subsequent mapping using the genome features ion. A value .1 indicates that there are more markers thanof MAPMAKER. A subset of informative markers defined as expected in a given interval of 10 cM (clustering); a value ,1those with ,5% missing data, separated at 5 cM from each means that there are less markers than expected in a givenother, were ordered at an initial LOD score of 3.0. Additional interval of 10 cM.markers were subsequently added by lowering the LOD thresh- The AFLP marker distribution was also analyzed by calculat-old to 2.0 (“order” and “ripple” commands) to obtain a frame- ing the Pearson correlation coefficient between the numberwork map. Markers that could not be ordered with equal of AFLP markers in the linkage groups and the size of theconfidence were indicated as accessory markers linked to a linkage groups (Sokal and Rohlf 1981; SPSS version 7.5).specific marker on the map. Markers that showed a departure The marker distribution was tested on both AFLP frameworkfrom the 1:1 ratio (0.01 , P , 0.05) were also incorporated markers and all AFLP markers.as accessory markers on the map, except for 10 cases wherethis marker was unique in an area of .30 cM. Maps wereconstructed with the program DrawMap (version 1.1) devel- RESULTSoped by van Ooijen (1994).

Estimated and observed genome length: The estimated ge- Analysis of AFLP markers: The two-way pseudotest-nome length was determined from partial linkage data ac- cross strategy was used to generate genetic maps for P.cording to Ge 5 N(N 2 1)X/K with a confidence interval of

deltoides cv. S9-2, P. nigra cv. Ghoy, and P. trichocarpa cv.Ge/(1 6 1.96/√K) with N the number of framework markersV24 based on the analysis of two full-sib families 87001and thus N(N 2 1) the number of pairwise comparisons, Xand 87002 (materials and methods). The mappingthe maximum distance between two adjacent framework mark-

ers in centimorgans at a certain minimum LOD score, and K program was initiated with AFLP markers. A total of 50the number of marker pairs at the same minimum LOD score and 41 AFLP primer combinations were used to analyze(Hulbert et al. 1988; Chakravarti et al. 1991, method 3). the progeny 87001 and 87002, respectively (Table A,A minimum LOD score of 3.5 was chosen to estimate the

http://www.plantgenetics.rug.ac.be/zvesto). Illegiti-genome length. Only framework markers were used to avoidmate progeny trees were scored within 87001 and 87002an overestimation of the genome size because of clustered

markers. by identifying those individuals that did not show the


TABLE 1

Data on the genome maps

87001 87002

P. deltoides P. nigra P. deltoides P. trichocarpaFeatures cv. S9-2 cv. Ghoy cv. S9-2 cv. V24

Average AFLP markers per primer combinationbefore and after x2 analysis (P , 0.01) 8.8/8.1 7.7/7.1 7.8/7.5 7.7/7.0

Total AFLP markers scored before x2 analysis 438 383 321 314AFLP markers used for linkage analysis 403 355 309 287Microsatellite markers used for linkage analysis 61 49 63 76Other markers used for linkage analysis

(STS and/or resistance markers) 2 0 2 1Total markers used for linkage analysis 466 404 374 364Markers assigned to groups .3 markers 448 369 355 339Markers in the framework map 238 222 179 194Accessory markers 210 147 176 145Groups .3 markers, before making alignments 21 34 23 23Groups .3 markers, after making alignments 19 28 19 19Triplets 0 4 0 3Doublets 1 2 3 2Unlinked markers 16 19 11 12Smallest group (cM) of the framework 7.0 10.5 8.9 9.0Largest group (cM) of the framework 183.8 252.1 169.3 178.3Average length of a group (cM) based on the

framework map 6 SD 103.7 6 47.1 69.3 6 57.8 70.7 6 46.5 83.5 6 46.6Average distance between 2 framework markers

(cM) 6 SD 10.0 6 8.8 12.5 6 9.5 10.4 6 8.5 11.2 6 8.3Total framework intervals 217 188 156 171Framework marker intervals .10 cM 89 104 65 88Framework marker intervals .20 cM 26 43 18 26Framework marker intervals .30 cM 8 11 5 6Percentage missing data (AFLP markers only) 3.5 3.0 1.7 1.4Percentage distorted AFLP markers

(x2, d.f. 5 1, P 5 0.01) 7.8 7.3 3.7 8.6Percentage distorted AFLP markers

(x2, d.f. 5 1, P 5 0.05) 16.2 15.4 7.8 18.8

SD, standard deviation.

monomorphic AFLP fragments present in the parental the three genetic maps. By using the PCR conditionsdescribed, only 1 STS primer combination (P1054) oflines. Four (family 87001) and three (family 87002)

individuals were detected and eliminated from linkage the 25 tested resulted in a single band of the expectedsize. By increasing the annealing temperature to 608, 3analysis. The total numbers of markers scored as hetero-

zygous in one parent and absent in the other were 438 more STS primer combinations yielded a single PCRproduct (P767, P856, and P1010), whereas a single PCRfor P. deltoides (87001) and 321 for P. deltoides (87002;

based on crosses 87001 and 87002, respectively), 383 product was obtained at 658 for primer combinationP991. The other primer combinations showed eitherfor P. nigra, and 314 for P. trichocarpa (Table 1). The

average number of scored markers per primer combina- no or more than one amplification product. After diges-tion, only P767 and P991 revealed a polymorphism.tion varied from 7.7 to 8.8 for the four maps (Table 1).

The heterozygosity levels based on AFLP markers were P767 segregated in the two families, whereas P991 onlysegregated in 87002. Microsatellite markers (153), ob-26% for P. deltoides (87001), which might explain the

best map coverage (see below), and 21% for P. deltoides tained from PMGC and Plant Research International(materials and methods), were tested on the three(87002). Heterozygosity levels for P. nigra and P. tricho-

carpa were both 20%. parents. The results of the analysis are presented inTable 2 and Table B, http://www.plantgenetics.rug.Analysis of STS and microsatellite markers: STS and

microsatellite markers are very useful as genetic bridges ac.be/zvesto. The heterozygosity levels based on micro-satellites were 63, 58, and 75% for P. deltoides, P. nigra,for comparative mapping because they are locus specific

and codominantly inherited. The STS markers pub- and P. trichocarpa, respectively.In the course of our mapping study with microsatel-lished by Bradshaw et al. (1994) were analyzed to align


TABLE 2

Number of polymorphic and monomorphic microsatellites

P. deltoidesMarkers (87001 and 87002) P. nigra P. trichocarpa

Microsatellites analyzed 153 153 153Polymorphic 64 49 76Monomorphic 37 36 25Linked polymorphic 59 40 60

lites, three aneuploids were observed in the progeny: marker against M. larici-populina race E1, E2, and E3(mer), and one STS marker (Figure 1; Table 1). Initially87001#25, 87001#136, and 87002#141. This aneuploidy

was confirmed by flow cytometry (Galbraith et al. (materials and methods), 20 major groups, one dou-blet, and 7 unlinked markers were obtained. The order-1983). The two clones 87001#136 and 87002#141 had

a 1.5-fold higher nuclear content than their diploid ing of the markers of 1 group could not be determinedaccurately and was split into 2 groups, resulting in 21parents, whereas that of clone 87001#25 was only slightly

higher. These individuals were discarded for the micro- linkage groups. This separation was also obtained at aminimum LOD score of 4.0 and a corresponding maxi-satellite and linkage analyses.

Segregation distortion and linkage analysis: A x2 test mum recombination fraction of 0.288. During ordering,9 other markers belonging to different groups could(d.f. 5 1) was performed to test the null hypothesis

of a 1:1 segregation of the AFLP markers. At the 1% not be placed. Unlinked markers are either artifactssegregating in Mendelian ratios by chance or they repre-significance level, 63 AFLP markers had aberrant segre-

gation ratios in the cross 87001 (8%; 35 from P. deltoides sent regions with very few markers. The distorted AFLPmarkers (37, 0.01 , P , 0.05) were distributed over 12and 28 from P. nigra) and 39 in 87002 (6%; 12 from P.

deltoides and 27 from P. trichocarpa). This value is higher linkage groups. Several clusters of distorted markerswere found on groups I, IV, V, and VII (Figure 1). Inthan the 1% expected to occur by chance alone. These

markers were excluded from linkage analysis with the the framework, 53% of the markers were retained andthe map consisted of 238 markers distributed over 196exception of 8 AFLP markers in P. deltoides (87002).

Previously, 3 markers closely linked to the M. larici- unique loci.In P. nigra, linkage analysis was based on 355 AFLPpopulina resistance gene had been found in P. deltoides

(87001; Cervera et al. 1996), 2 of which segregated markers and 49 microsatellites (Figure 2; Table 1). Ini-tially, 32 major linkage groups, four triplets, two dou-also in family 87002, but with a departure from the 1:1

segregation ratio. This distorted ratio of the resistant/ blets, and 15 unlinked markers were obtained. Markersof 2 groups could not be ordered accurately and weresusceptible progeny taken from cross 87002 was pro-

voked by the death of rust-susceptible trees. When the split into 2 groups, resulting in 34 linkage groups. Thisseparation into 2 groups was obtained at a minimumsignificance level was lowered to 5%, 68 more markers

showed a deviation in the cross 87001 (37 from P. del- LOD score of 5.0 and a corresponding maximum recom-bination fraction of 0.254. The decision to split thesetoides and 31 from P. nigra) and 44 in the cross 87002

(13 from P. deltoides and 31 from P. trichocarpa). These groups was also based on the alignment with P. deltoides(see below). During ordering, 4 other markers belong-markers were retained for linkage analysis. The AFLP

markers showing a significant segregation distortion ing to 2 groups could not be placed. Of distorted mark-ers (0.01 , P , 0.05), 26 were distributed over 11 groups(0.01 , P , 0.05) in P. deltoides (87001) did not deviate

in P. deltoides (87002) and vice versa. For the four maps, and 5 belonged to triplets or were unlinked. Only twoclusters were found (groups IV and B; Figure 2). In thethere was no preference in the direction of the deviation

(i.e., more bands present than absent). framework, 60% of the markers were retained and themap consisted of 222 markers distributed over 190The STS markers were also tested against a 1:1 segre-

gation, but none showed a segregation distortion (P , unique loci.Linkage analysis in P. deltoides (87002) was based on0.05). Microsatellite markers polymorphic in both par-

ents were tested against a 1:1:1:1 (d.f. 5 3) segregation 309 AFLP markers, 63 microsatellites, the resistancemarker mer, and 1 STS marker (Figure 1; Table 1).ratio. When polymorphic in only one parent, they were

tested against a 1:1 segregation (Table B, http://www. Initially, 21 major linkage groups, three doublets, and11 unlinked markers were obtained. For the ordering,plantgenetics.rug.ac.be/zvesto). All STS and microsa-

tellite markers were retained for linkage analysis. 2 groups were split in two and resulted in 23 majorlinkage groups. This result was confirmed at a minimumLinkage analysis in P. deltoides (87001) was based on

403 AFLP markers, 61 microsatellites, the resistance LOD score of 4.0 and a corresponding maximum recom-


bination fraction of 0.288. All distorted markers (13, had been aligned, 19 linkage groups were obtained,corresponding to the haploid number of chromosomes0.01 , P , 0.05) were scattered over 4 groups. In group

XV, 7 distorted markers grouped together (Figure 1). in poplar.P. deltoides, P. nigra, P. trichocarpa, and family 331 linkageIn the framework, 50% of the markers were retained

and the map consisted of 179 markers distributed over map (Bradshaw et al. 1994): To further reduce thenumber of linkage groups and to identify homoeolo-153 unique loci.

Linkage analysis in P. trichocarpa was based on 287 gous linkage groups among the three maps, microsatel-lite and STS marker analyses were performed. TheseAFLP markers, 76 microsatellites, and 1 STS marker

(Figure 3; Table 1). Initially, 20 major linkage groups, markers are ideal for aligning maps because they arelocus specific and codominantly inherited. An overviewtwo doublets, three triplets, and 12 unlinked markers

were obtained. For the ordering, 1 group was split into of all the alignments is given in Table 3 (for more details,see Table C, http://www.plantgenetics.rug.ac.be/zvesto).two and another group into three. This result is in agree-

ment with the ordering at a minimum LOD score of After alignment, the number of linkage groups in P.nigra could be reduced by 6, finally resulting in 284.0 and a corresponding maximum recombination frac-

tion of 0.288. Twenty-nine distorted markers (0.01 , groups. The number of groups for P. trichocarpa wasreduced to 19.P , 0.05) were scattered over 13 groups. Two clusters

were found (groups I and V; Figure 3). Two distorted Some discrepancies were also found (Figures 1–3 andTable C, http://plantgenetics.rug.ac.be/zvesto). PMG-markers (0.01 , P , 0.05) were unlinked. In the frame-

work, 57% of the markers were retained. The framework C14 and PMGC420 are on a single linkage group in P.nigra (group XIV), but on separate linkage groups inmap consisted of 194 markers distributed over 168

unique loci. P. deltoides (XIII and XIV). A spurious linkage of markerPMGC14 in P. nigra is suggested here because, at a LODFor the four genetic maps, the average length of a

group based on the framework markers, the smallest score of 4.0, marker PMGC14 remained unlinked.A second discrepancy occurred at the position ofgroup, the largest group, the average distance between

two framework markers, and the number of intervals marker PMGC61. Markers PMGC61 and PMGC409 arelocated on the same linkage group in P. nigra (VIII).10, 20, and 30 cM is indicated in Table 1.

The PGRI software version 1.0 (Liu 1998), which is and P. trichocarpa (VIII) and also on the linkage mapreported by Bradshaw et al. (1994; group C). On P.based on a bootstrap approach, was used to verify the

correct locus ordering in P. deltoides, P. nigra, and P. deltoides, they are located on two different linkage groups(VI and VIII). A very high number of double crossoverstrichocarpa and thus establish the degree of confidence

on marker order in the framework map (.80%). Other were observed at the position of marker PMGC61 in P.deltoides and at the position of marker PMGC409 in P.data concerning the map constructions are summarized

in Table 1. nigra, suggesting a possible gene conversion event (Liu1998), or a specific chromosomal arrangement.Map comparisons: The two maps of P. deltoides were

compared with each other and with the maps of P. A third discrepancy was caused by the position ofmarker PMGC2020. In P. trichocarpa PMGC2020 is lo-deltoides, P. nigra, P. trichocarpa, and the family 331 link-

age map (Bradshaw et al. 1994). cated on the same group as PMGC2881 and PMGC2826(group IV), whereas this is not the case for P. deltoidesP. deltoides: Because two maps were constructed for

the same parent on the basis of the analysis of two and P. nigra, on which PMGC2020 is on group IX andPMGC2881 and PMGC2826 on group IV. However,different progeny, the reliability of the map could be

evaluated. Two hundred and thirty-seven markers (193 PMGC2020 amplified two loci according to the PMGCwebsite (indicated as PMGC2020 and PMGC2021). OnlyAFLP markers, 43 microsatellites, and the resistance

marker) were found in common (Figure 1). The order one locus was amplified, so there is no certainty thatthe locus amplified in P. trichocarpa is the same as in P.of the markers was the same for 208 markers. For 20

markers, disorder occurred within an interval of ,5 cM. deltoides and P. nigra. Twelve groups of the linkage mapof Bradshaw et al. (1994) could be aligned to the mapsThus, in the two maps the order was the same for 96%

of the framework markers. After the two P. deltoides maps described here.

c

Figure 1.—Linkage map of P. deltoides. The linkage groups at the left of the linkage group number result from the cross 87001and those at the right from the cross 87002. Framework markers are in boldface. Accessory markers that fit into single intervalsare in roman type and the others are placed in their most likely position and are in italics. Uppercase letters were used to denotemarkers with ,10% missing data. Markers in common between the two maps of P. deltoides are indicated with allelic bridges.Microsatellite markers are in lightface type. Markers between brackets are markers cosegregating with the Melampsora larici-populina resistance locus, but deviating from the 1:1 segregation ratio. Loci with a distorted segregation ratio are marked by one(0.01 , P , 0.05) or two (P . 0.01) asterisks. Recoded markers are marked by “r.” Linkage groups for which the homoeologousgroups were found in either the map of P. nigra or of P. trichocarpa are denoted by a roman numeral.



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TABLE 3

Alignment of the homoeologous groups

P. deltoides P. nigra P. trichocarpaFamily 331c

Groupa Sizeb Group Size Group Size Group

I 233.7, 238.5 I 179.0 I 192.0 AII 191.2, 131.5 II 220.4 II 128.1 MIII 170.0, 93.1 III 220.5 III 100.9IV 154.5, 140.0 IV 146.4 IV 165.6 IV 134.9, 150.4 V 132.3 V 167.5 BVI 154.3, 161.0 VI 122.8 VI 150.6VII 146.3, 70.3 VII 107.5 VII 76.4 HVIII 137.9, 136.2 VIII 269.2 VIII 183.1 L 1 CIX 137.0, 30.8 IX 39.1 IX 145.3 DX 93.5, 117.1 X 220.8 JXI 104.3, 83.1 XI 76.5 XI 68.2 EXII 102.9, 49.7 XII 54.7 XII 91.8XIII 99.5, 72.8 XIII or XIVd 63.1 XIII 99.8XIV 98.3, 79.6 XIII or XIVd XIV 119.4 XXV 72.3, 57.6 XV 69.2XVI 62.5, 31.6 XVI 135.5

B O

a Only those linkage groups for which an alignment could be made with a linkage group of either of thetwo other maps are presented.

b Two sizes are given for the linkage groups in P. deltoides : the first is for the linkage group based on family87001, and the second is for family 87002. When two groups should link into one linkage group, the size ofboth groups was summed.

c Bradshaw et al. (1994).d The microsatellite markers PMGC14 and PMGC420 belong to one group in P. nigra, but belong to group

XIII and XIV in P. deltoides, respectively.

Estimated and observed genome lengths: The esti- phisms are randomly distributed. This assumption wasevaluated in three ways (see materials and methods).mated and observed genome lengths for the four link-

age maps are presented in Table 4. The values for the The Pearson correlation coefficient between the num-ber of markers in the linkage groups and the total sizeestimated genome length for P. deltoides and P. tricho-

carpa were in the same range but that for P. nigra was of the linkage groups indicated a significant correlationat the 1% significance level for all maps; thus, the mark-much higher. The number of marker pairs with a mini-

mum LOD score of 3.5 was low compared to the total ers are randomly distributed. The coefficient of disper-sion of the markers was in all cases very close to onenumber of framework markers in P. nigra. A good esti-

mate for P. nigra could not be obtained because of (Tables D and E, http://www.plantgenetics.rug.ac.be/zvesto), also pointing to a random distribution. Thethe high number of linkage groups. The observed map

distance calculated according to the formula of Nelson frequency table for the framework markers (Table D,http://www.plantgenetics.rug.ac.be/zvesto) showed thatet al. (1994) (Gon; materials and methods) that takes

into account all linked and unlinked markers gave a there were more intervals containing only one markerthan expected. Regions with clustered markers wereresult that was very close to the estimated genome length.

Expected and observed map coverages: The expected also observed. The numbers of intervals of 10 cM, con-taining at least four markers, that were detected on theand observed map coverages for the four linkage maps

are presented in Table 4. The estimate according to the framework maps were as follows: nine for P. deltoides(87001), four for P. deltoides (87002), two for P. nigra,equation of Lange and Boehnke (1982) was very similar

to that of Bishop et al. (1983). The difference between and three for P. trichocarpa. The same regions of clusterswere found back in both maps of P. deltoides. The fre-the expected and the observed map coverage gives an

estimate of the marker distribution. In all cases, the quency table for all markers (Table E, http://www.plantgenetics.rug.ac.be/zvesto) showed that many moreobserved map coverage was lower than the expected

map coverage. clusters appeared than for framework markers only, ex-cept for P. nigra. The runs test, however, does not indi-Marker distribution: AFLP markers are expected to

be randomly distributed (Vos et al. 1995) when polymor- cate significant deviations from randomness.


TABLE 4

Genome length and map coverage

P. deltoides P. deltoidesGenome length (87001) (87002) P. nigra P. trichocarpa

Observeda

Gof (cM) 2178 1626 2356 1920Goa (cM) 2304 1838 2791 2326X (cM) 39.7 35.4 35.1 33.5L 38 47 59 39R 19 19 19 19Gon (cM) 2932 2618 3760 2590

Estimatedb

N 238 179 222 194X (cM) 34.8 34.7 34.7 34.7K 779 465 440 496Ge (cM) 2520 2375 3869 2616Lower bound 2354 2177 3539 2405Higher bound 2710 2612 4268 2869

Observed and estimated genome coveragec

Cof (%) 86.4 68.5 60.9 73.4Cel (%) 92.8 87.6 79.7 87.2Ceb (%) 94.8 90.6 84.8 90.5

a X, observed maximum distance between two framework markers; L, total number of linkage groups, pairs,and unlinked markers; R, haploid number of chromosomes in poplar; Gof, total length of the map based onall framework markers; Goa, total length based on all markers; Gon, takes into account the maximum distanceX of detectable linkages to unlinked loci and the ends of linkage groups, as well as the reduction in coverage,0.5x, expected at the ends of chromosomes.

b N, number of framework markers; K, observed number of locus pairs with minimum LOD scores of 3.5;X, corresponding maximum distance between the locus pairs; Ge, estimated genome length.

c Cof, observed map coverage based on Gof; Cel and Ceb, theoretical map coverages according to Lange andBoehnke (1982) and Bishop et al. (1983), respectively. For all the equations, see materials and methods.

DISCUSSION et al. 1998; Krutovskii et al. 1998; Marques et al. 1998;Paglia et al. 1998). The results obtained here suggestHeterozygosity levels: The efficiency of constructinga biological basis for the deviations. Markers with a devi-a genetic linkage map in outbred forest trees with theation from the expected segregation ratio are generallytwo-way pseudotestcross strategy depends on the levelbelieved to be linked to genes that are subject to directof genetic heterozygosity of the species and the markerselection. Bradshaw and Stettler (1994) found thatsystem. Heterozygosity levels of P. trichocarpa and P. del-a lethal allele in Populus sp. affecting embryo develop-toides based on RFLP markers were 30 and 15% and,ment was the cause of segregation distortion of markersbased on RAPD markers, 36 and 30%, respectivelylinked to it. In the P. deltoides (87002) map, we observed(Bradshaw et al. 1994). The estimates in our studythat markers cosegregating with the Melampsora resis-based on AFLP markers were 26% for P. deltoidestance gene also showed a significant deviation because(87001), 21% for P. deltoides (87002), and 20% for P.of death of susceptible trees. If the reason for this distor-nigra and P. trichocarpa. Estimates based on dominanttion had not been known, the markers would have beenAFLP markers are minimum estimates because a bandrejected and part of the linkage group would be missing.that is present in both parents and does not segregateBased on this principle, all markers should be used inin the F1 progeny can still be heterozygous in one parent.the mapping process. However, including markers withIn Eucalyptus, heterozygosity levels based on AFLPsegregation distortion increases the chance of type Imarkers were 30.5% for Eucalyptus tereticornis and 22.4%errors (i.e., rejection of the null hypothesis) of falsefor E. globulus (Marques et al. 1998). The estimateslinkage. Moreover, map distances between markers withbased on microsatellites are much higher, but then hy-skewed segregation ratios may be inaccurate (Gerberpervariable noncoding regions are analyzed.and Rodolphe 1994; Cloutier et al. 1997). Therefore,Segregation distortion: Segregation distortion has of-only markers that deviated at the 5% and not at theten been observed in forest trees and fruit trees (Nelson1% level were included for linkage analysis. Distortedet al. 1993; Bradshaw and Stettler 1994; Cai et al.markers that appear in clusters suggest that these areas1994; Grattapaglia and Sederoff 1994; Lanaud et al.

1995; Mukai et al. 1995; Viruel et al. 1995; Barreneche contain genes that affect viability (Strauss and Conkle


1986; Durham et al. 1992; Jarrell et al. 1992; Landry map. Although the map of P. nigra is based on 404markers instead of 466 and 364 for P. deltoides (87002)et al. 1992; Sall and Nilsson 1994; Foolad et al. 1995;and P. trichocarpa, respectively, linkage analysis resultedLanaud et al. 1995; Cheng et al. 1996; Verhaegen andin 34 linkage groups. The estimated heterozygosity lev-Plomion 1996; Barreneche et al. 1998; Kuang et al.els of P. nigra and P. trichocarpa were in the same range.1999). No difference was observed in the percentage ofA possible explanation is that the genome of P. nigramarkers with segregation distortion between the femalecontains large regions of highly homologous sequences.and the male parents.

A direct approach to align the 19 chromosomes withThe fact that markers with a deviation from the ex-19 linkage groups would be to map telomere-proximalpected 1:1 ratio in the map of P. deltoides (87001) segre-sequences at the distal ends of the chromosomes (Burrgate 1:1 in the other map of P. deltoides (87002) may beet al. 1992; Ganal et al. 1992). To date, telomere se-explained by the presence of a lethal recessive allele inquences are still unknown in poplar.the heterozygous condition in this region in P. deltoides

Map comparisons: Comparative mapping is a usefuland P. nigra, but not in P. trichocarpa. The same reason-technique for investigating chromosomal evolution anding applies for markers with a segregation distortion inallows the import of genetic information (such as mapP. deltoides (87002) and not in P. deltoides (87001).positions of qualitative or quantitative traits) from oneMap construction: Between the LOD score and thespecies to a related species. In a first attempt to alignx2 value for independence, a relation exists for a popula-homoeologous groups, markers, which were either het-tion of infinite size: x2 5 2 LOD/log e (Lander anderozygous in both parents (segregating 3:1) or heterozy-Botstein 1989). A minimum LOD score of 3.0 was chosengous in P. nigra and P. trichocarpa, and null in P. deltoidesfor linkage analysis. The recombination fraction can be(segregating 1:1 in both families), were looked for. Thecalculated from the equation u 5 (N 2 √x2N)/2N withnumber of markers segregating 3:1 was 8 for both fami-N the number of progeny. The smallest population con-lies 87001 and 87002. Twelve common markers weresisted of 101 progeny, resulting in a recombination frac-found between P. nigra and P. trichocarpa. For AFLPtion of 0.301 and a corresponding Kosambi map dis-markers, however, sequence data are needed to provetance of 34.7 cM. From these equations, it is obvious thatthat markers of the same size represent the same locus

the recombination fraction increases with a decreasing(Qi and Lindhout 1997; Rouppe van der voort et al.

LOD value. The recombination fraction also depends 1997; Waugh et al. 1997). Therefore, the selected AFLPon the progeny size. For the same recombination frac- markers were cloned from each parent and 5 clonestion, the LOD increases with an increasing number of were sequenced. Fourteen markers were analyzed inprogeny. The maps were based on 121 progeny for P. this way. However, in none of the cases was the sequencedeltoides (87001) and P. nigra and on 101 progeny for of all 10 clones the same due to the coamplification ofP. deltoides (87002) and P. trichocarpa. These population underlying nonvisible bands. In six cases, one-half ofsizes are among the largest of all published maps on the clones showed the same sequence. Very few AFLPtrees. markers (2%) segregated 3:1 compared to 1:1. This low

The 19 chromosomes of Populus sp. are represented frequency was also observed in a cross of Eucalyptusin the genome maps of P. deltoides, P. nigra, and P. (Grattapaglia and Sederoff 1994) and almond (Vir-trichocarpa by 19, 34, and 23 linkage groups, respectively. uel et al. 1995). Most of these markers remained un-The comparison of the order of markers and levels of linked because of the low information content betweenrecombination between the two maps of P. deltoides marker pairs segregating 1:1 and 3:1 (Ritter et al.proves the robustness of the maps. Errors in locus order- 1990). Therefore, the efficiency and reliability of theseing may invalidate further analysis, such as QTL analysis markers to align the genetic maps are low.or map-based cloning, but the locus order is correct for Multiallelic codominant markers, such as STS mark-96% of the common framework markers when both ers and microsatellites, are the most efficient for mapmaps of P. deltoides are compared. Similar results have comparisons. In contrast to the microsatellite primers,been obtained by Plomion et al. (1995), who compared however, most of the primers for detection of STS mark-two genome maps of a unique Pinus pinaster genotype ers described by Bradshaw et al. (1994) did not amplifybuilt on a common set of 263 RAPD markers. The link- the expected bands in the parents of the two familiesage maps of two different progenies, consisting of 62 under study.megagametophytes each, which had been obtained The alignments resulted finally in 19 groups for P.from a self cross and an open-pollinated cross, shared deltoides, 19 for P. trichocarpa, and 28 for P. nigra. Twelvethe order of 98% framework markers. Similar results groups of P. deltoides were aligned with 18 groups of P.were also obtained by Sewell et al. (1999), who made nigra and 15 groups of P. deltoides with 19 groups of P.a consensus map for loblolly pine by integrating two trichocarpa. The alignments are based on 27 microsatel-individual maps from two outbred three-generation lites in common between P. deltoides and P. nigra, 34pedigrees. between P. deltoides and P. trichocarpa, and 16 between P.

nigra and P. trichocarpa; only 14 were found in commonThe map of P. deltoides (87001) is the best-covered


between P. deltoides, P. nigra, and P. trichocarpa. This on which codominant microsatellites and STS markerscan be mapped progressively to construct a saturatedcorresponds with 17, 22, 11, and 9% of the total number

(153) of available microsatellites. Therefore, a large “species consensus map,” which will be a useful tool forevolution studies and breeding purposes (Cervera etnumber of microsatellites are necessary for a successful

comparative analysis. al. 1999).Note: Cuttings, DNA, and genotypic data of the twoIn general, the corresponding groups between P. del-

toides, P. nigra, and P. trichocarpa are of comparable size, mapping populations are available to the scientific com-munity.considering the differences in map coverage. Groups

VIII of P. nigra and P. trichocarpa, however, are larger The authors thank Vic and Marijke Steenackers, An Vanden Broeck,than group VIII of P. deltoides, supporting the idea of a and Boudewijn Michiels for a long-standing fruitful collaboration;

Ron Sederoff, Christophe Plomion, and Carlos A. Malpica for theirrearrangement of the region around the microsatellitevaluable comments during the project; Gerry Tuskan, Toby Bradshawmarker PMGC61. Indeed, the inconsistency for theJr., and Mitchell Sewell for their helpful information; Tom Geratsmarker PMGC61 may point to the presence of chromo-and Peter Breyne for critical reading of the manuscript; and Martine

somal rearrangements as proposed for apple (Hemmat De Cock for help in preparing it. This work was supported by grantset al. 1994) and Prunus (Foolad et al. 1995). To prove from the Flemish Government (BNO/BB/6/1994, 1995; IBW/3/

1995–2000) and the Commission of the European communities AIRthis hypothesis, markers in the vicinity of this microsatel-program (AIR1-CT92-0349). M.-T.C. is indebted to the Europeanlite should be identified and mapped both on P. deltoidesUnion for an individual fellowship from the Human Capital Mobilityand P. nigra.program (41AS8694).

Genome length estimates and map coverage: Brad-shaw et al. (1994) estimated that the total genome mapsize for P. trichocarpa was 2400 cM at a minimum LOD

LITERATURE CITEDscore of 3.0. This size is very close to our estimates forBarreneche, T., C. Bodenes, C. Lexer, J.-F. Trontin, S. Fluch etP. deltoides and P. trichocarpa. The map of P. deltoides

al., 1998 A genetic linkage map of Quercus robur L. (pedunculate(87001) is well covered. The difference between theoak) based on RAPD, SCAR, microsatellite, minisatellite, isozyme

expected map coverage and the observed framework and 5S rDNA markers. Theor. Appl. Genet. 97: 1090–1103.Bishop, D. T., C. Cannings, M. Skolnick and J. A. Williamson,map coverage may be due to clusters or may simply

1983 The number of polymorphic DNA clones required to mapresult from the process of framework map constructionthe human genome, pp. 181–200 in Statistical Analysis of DNA

(Echt and Nelson 1997). The markers retained in the Sequence Data, edited by B. S. Weir. Marcel Dekker, New York.Bradshaw, H. D. Jr., and R. F. Stettler, 1994 Molecular geneticsframework were 53 and 50% for the two maps of P.

of growth and development in Populus. II. Segregation distortiondeltoides, 60% for the P. nigra map, and 57% for the P.due to genetic load. Theor. Appl. Genet. 89: 551–558.

trichocarpa map. These values are underestimated by 3% Bradshaw, H. D. Jr., and R. F. Stettler, 1995 Molecular geneticsof growth and development in Populus. IV. Mapping QTLs with(for P. deltoides and P. nigra) to 7% (for P. trichocarpa)large effects on growth, form, and phenology traits in a forestbecause most of the microsatellites were retained fromtree. Genetics 139: 963–973.

the framework because they were analyzed on only half Bradshaw, H. D. Jr., M. Villar, B. D. Watson, K. G. Otto, S.of the families. Taking this limitation into consideration, Stewart et al., 1994 Molecular genetics of growth and develop-

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Dense Genetic Linkage Maps of Three Populus Species ( Populus … · and ‡Instituut voor Bosbouw en Wildbeheer, B-9500 Geraardsbergen, Belgium Manuscript received December 20, 1999