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

Corresponding author: Wout Boerjan, Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium., woboe{at}gengenp.rug.ac.be (E-mail)

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Populus 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 maps were constructed for these three species by analyzing progeny of two controlled crosses sharing the same female parent, Populus deltoides cv. S9-2 x P. nigra cv. Ghoy and P. deltoides cv. S9-2 x P. trichocarpa cv. V24. The two-way pseudotestcross mapping strategy was used to construct the maps. Amplified fragment length 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 and to merge linkage groups within the individual maps. Linkage analysis and alignment of the homoeologous groups 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 over 28 groups for P. nigra covering 61%. Several tests for randomness showed that the AFLP markers were randomly distributed over the genome.

BECAUSE of its fast growth, ease for clonal propagation, and strong heterosis upon interspecific hybridization, Populus has become a tree of prime economic importance. Poplar wood has many end uses, including pulp and paper, timber, plywood, pallets, soft board, and hard board. There is also an increasing interest for cultivation of poplar as a biomass crop (PEARCE 1995 ). For the same reasons and because of its small genome size (550 Mb; 2C = 1.1 pg; 19 chromosomes) and its amenability for genetic transformation, poplar has become a model system for fundamental research on trees (STETTLER et al. 1996 ; KLOPFENSTEIN et al. 1997 ). Large expressed sequence tag (EST) databases have been constructed (STERKY et al. 1998 ; MELLEROWICZ et al. 2001 ) that will catalyze the development of efficient functional genomics research programs in trees.

Populus deltoides, P. nigra, and P. trichocarpa are the most important species for poplar breeding in Europe. Most of the commercial clones planted throughout Europe are derived from interspecific crosses between P. deltoides and P. trichocarpa and between P. deltoides and P. nigra and their backcrosses. Selection and breeding strategies have been oriented mainly toward resistance to leaf rust (caused by the fungus Melampsora larici-populina) and bacterial canker (caused by Xanthomonas populi), enhanced growth, rooting ability to improve clonal propagation, adaptation to latitude, and superior wood quality (CEULEMANS et al. 1987 ).

Tree breeding is a time-consuming process, mainly because of the long generation intervals and the fact that productivity and quality can best be evaluated at rotation age, which varies between 7 and 20 years. The development of polymerase chain reaction (PCR)-based molecular markers has facilitated the construction of genetic linkage maps to study the architecture of polygenic traits. Linkage maps also constitute the framework for the use of genetic markers in breeding programs via marker-assisted selection (MAZUR and TINGEY 1995 ) and facilitate map-based cloning (TANKSLEY et al. 1995 ). For many tree species, genome mapping projects have been initiated, which are mostly based on restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers (for references, see CERVERA et al. 1999 ). The first linkage groups identified in poplar were obtained with alloenzymes (MULLER-STARCK 1992 ; LIU and FURNIER 1993 ) and RFLPs (LIU and FURNIER 1993 ). A linkage map covering 50% of the genome was constructed based on RFLP, sequence-tagged site (STS), and RAPD markers (BRADSHAW et al. 1994 ). Molecular markers, associated with qualitative resistance to M. larici-populina and M. medusae, have been identified (CERVERA et al. 1996 ; NEWCOMBE et al. 1996 ; VILLAR et al. 1996 ; LEFEVRE et al. 1998 ; TABOR et al. 2000 ) and quantitative trait loci (QTL) for stem growth and shape, bud phenology, leaf variation, and resistance to M. medusae and Septoria populicola have been mapped (WU et al. 1992 , WU et al. 1997 ; WU and STETTLER 1994 , WU and STETTLER 1997 ; BRADSHAW and STETTLER 1995 ; NEWCOMBE and BRADSHAW 1996 ; NEWCOMBE et al. 1996 ; WU 1998 ; FREWEN et al. 2000 ).

Here we report on the construction of linkage maps for P. deltoides, P. nigra, and P. trichocarpa based on a two-way pseudotestcross strategy (RITTER et al. 1990 ; GRATTAPAGLIA and SEDEROFF 1994 ; HEMMAT et al. 1994 ) and amplified fragment length polymorphism (AFLP) markers (ZABEAU and VOS 1993 ; VOS et al. 1995 ). Microsatellites and STS markers were added to the set of AFLP markers to allow direct identification of homoeologous loci and to merge linkage groups of the individual maps. After the homoeologous loci were aligned, 19 linkage groups were found for P. deltoides and P. trichocarpa and 28 for P. nigra. The genetic maps covered 86, 73, and 61% of the total genome, respectively.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material and DNA extraction:
Two full-sib families were used to generate the genetic maps. One full-sib family (87001) consisted of 127 individuals and resulted from an interspecific cross between two elite trees, P. deltoides cv. S9-2 and P. nigra cv. Ghoy (CERVERA et al. 1996 ). The second full-sib family (87002) consisted of 105 individuals and was generated from an interspecific cross involving the same female P. deltoides cv. S9-2 and P. trichocarpa cv. V24. These crosses were designed initially to introduce the M. larici-populina resistance gene present in P. deltoides cv. S9-2 into the breeding program (CERVERA et al. 1996 ). Genomic DNA was extracted from frozen young leaves using the procedure described by DELLAPORTA et al. 1983 or the DNeasy plant miniprep kit (QIAGEN, Helden, Germany).

AFLP analysis and marker nomenclature:
AFLP analysis was performed according to VOS et al. 1995 with some modifications (CERVERA et al. 1996 ). Primer combinations (EcoRI + 3/MseI + 3) were selected on the basis of the total number of bands and the level of polymorphism observed when analyzing the three parents and eight progeny from each family (data not shown). The sequences of the primers used in the preamplifications and the selective amplifications as well as their codes are indicated in Table A, available on the world wide web (http://www.plantgenetics.rug.ac.be/~vesto). The AFLP marker name refers to the primers used: E followed by two numbers refers to the EcoRI primer and G or F followed by two numbers to the MseI primer (Fig 1 Fig 2 Fig 3). Polymorphic bands were numbered serially in ascendant order of molecular weight; thus the last two numbers of the AFLP marker code refer to the relative position of the polymorphic band on the gel. Linkage groups were constructed by using markers both in coupling and in repulsion phase. For this purpose, the matrix was duplicated and the duplicated part was subsequently inverted (absent/present and present/absent). An "r" indicates an inverted marker and represents a marker in repulsion. Uppercase letters were used to denote markers that had <10% missing data.

View larger version (211K): In this window In a new window Download PPT slide   Figure 1. Linkage map of P. deltoides. The linkage groups at the left of the linkage group number result from the cross 87001 and those at the right from the cross 87002. Framework markers are in boldface. Accessory markers that fit into single intervals are in roman type and the others are placed in their most likely position and are in italics. Uppercase letters were used to denote markers 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 homoeologous groups were found in either the map of P. nigra or of P. trichocarpa are denoted by a roman numeral.

View larger version (93K): In this window In a new window Download PPT slide   Figure 2. Linkage map of P. nigra. The symbols are the same as in Fig 1.

View larger version (89K): In this window In a new window Download PPT slide   Figure 3. Linkage map of P. trichocarpa. For symbols, see legend to Fig 1.

STS analysis and marker nomenclature:
STS marker analysis was performed according to BRADSHAW et al. 1994 . A selection of primers and their corresponding enzymes were tested: P164, P753, P754, P755, P757, P762, P763, P767, P770, P771, P781, P783, P832, P856, P869, P991, P993, P1010, P1013, P1018, P1027, P1046, P1054, P1079, and P1086. The STS markers are denoted by STS followed by the number given by BRADSHAW et al. 1994 . On the linkage maps (Fig 1 Fig 2 Fig 3), uppercase letters were used to denote markers with <10% missing data.

Microsatellite analysis and marker nomenclature:
Primer sequences for microsatellite analysis were obtained from the website of the Poplar Molecular Genetics Cooperative (PMGC; http://poplar2.cfr.washington.edu/PMGC/). All 153 microsatellites were analyzed. The names of the microsatellite markers were taken from the PMGC website with the prefix PMGC. A second set of microsatellites was provided by the Centre for Plant Breeding and Reproduction Research (Plant Research International, The Netherlands) and has the prefix wpms or pkhomta (VAN DER SCHOOT et al. 2000 ). On the linkage maps (Fig 1 Fig 2 Fig 3), uppercase letters were used to denote markers with <10% missing data. Most of the microsatellite markers were analyzed on half of the family only and were, therefore, not placed in the framework.

Initially, microsatellites were analyzed with [-³³P]ATP- labeled primers. In that case, the PCR reaction was performed in a total volume of 20 µl containing 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl₂; 200 µM each of dATP, dCTP, dGTP, and dTTP; 1 unit Taq Polymerase (Roche Diagnostics, Brussels, Belgium); 50 ng kinated forward primer; 50 ng reverse primer; and 30 ng DNA. The kinase reaction was performed in a total volume of 10 µl containing 10 mM Tris-acetate, pH 7.5; 10 mM Mg-acetate; 50 mM K-acetate; 500 ng forward primer; 1 µl [-³³P]ATP (3000 Ci/mmol); and 0.06 units T4 kinase (Amersham Pharmacia Biotech, Little Chalfont, UK). The mixture was incubated for 30 min at 37° followed by 10 min at 80° to inactivate the kinase. The primers were synthesized on a DNA/RNA synthesizer model 394 (Perkin-Elmer-Applied Biosystems, Foster City, CA), followed by purification on an oligonucleotide purification cartridge (Perkin-Elmer-Applied Biosystems). Alternatively, microsatellites were analyzed with fluorescent dye-labeled primers. For this method, each PCR reaction was performed in a total volume of 15 µl containing 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 2.5 mM MgCl₂; 250 µM each of dATP, dCTP, dGTP, and dTTP; 0.6 units Taq Polymerase (Amplitaq Gold; Perkin-Elmer, Norwalk, CT); 30 ng labeled forward primer; 30 ng reverse primer; and 30 ng DNA. The forward primers were labeled with fluorescent dyes [NED (Perkin-Elmer), HEX (Genset, Paris), and FAM (Genset)]. For both methods, the DNA was aliquoted separately into 0.2-ml tubes and a master mix of the other components was added, mixed, and centrifuged briefly. The reactions were transferred to a Gene AMP thermocycler 9600 (Perkin-Elmer). The amplification conditions were as described on the PMGC website or by VAN DER SCHOOT et al. 2000 . The ³³P-labeled amplification products were denatured and heated as described for AFLP analysis (CERVERA et al. 1996 ). The samples were loaded on 6% acrylamide/bisacrylamide 19:1, 7.5 M urea, and 1x Tris-borate (TBE; 0.1 M Tris-base, 0.1 M boric acid, 2 mM EDTA, pH 8.0) gels. For the dye-labeled amplification products, 2.4 µl loading buffer [60% formamide, 23% Perkin-Elmer loading dye containing 50 ng/µl blue dextran and 5 mM EDTA, and 17% internal standard Genescan 500 ROX (Perkin-Elmer)] was added to 0.2-ml tubes to which 0.2, 0.24, and 0.4 µl of FAM-, HEX-, and NED-labeled amplification products were added, respectively. The samples were heated for 7 min at 95°. One microliter of each sample was loaded on 4.25% acrylamide/bisacrylamide 19:1, 6 M urea, and 1x TBE gels. Gels were electrophoresed on an ABI 377 apparatus (Perkin-Elmer-Applied Biosystems) in 1x TBE buffer for 3 hr at 200 W. Gels were processed and scored using the Genescan and Genotyper software (Perkin-Elmer).

Scoring and sequencing of markers:
All markers were scored as dominant markers and scored visually independently by two persons to minimize scoring and interpretation errors.

Heterozygosity levels:
The average heterozygosity was estimated by analyzing the parents and 20 individuals of both families, using a subset of 10 AFLP primer combinations, and by analyzing all microsatellites. For the estimate based on AFLP, the average heterozygosity was defined as the ratio of bands segregating in the F₁ progeny compared to the total number of bands observed. For the estimate on microsatellites, the average heterozygosity was defined as the ratio of polymorphic microsatellites to all microsatellites (polymorphic and monomorphic).

Segregation analysis and map construction:
Maps were constructed according to the two-way pseudotestcross strategy (RITTER et al. 1990 ; GRATTAPAGLIA and SEDEROFF 1994 ; HEMMAT et al. 1994 ). AFLP, STS, and microsatellite markers segregating 1:1 in the offspring were used for the construction of genome maps of both parents of each progeny. For each marker, a ² test (d.f. = 1, P < 0.01, and P < 0.05) was used to identify deviations from Mendelian ratios. AFLP markers deviating at the 1% significance level were excluded for the linkage analysis. Four matrices were created, one for each parent of the two crosses. To detect linkages in repulsion phase, the data set was inverted and added to the original data. Linkage analysis was performed by MAPMAKER Unix version 3.0 (LANDER et al. 1987 ). The "triple error detection" and the "error detection" features were used to recognize the circumstance when an event was more probably the result of error than of recombination. These features avoid map expansion (LINCOLN and LANDER 1992 ). Initially, a logarithm of odds (LOD) of minimum 3.5 and a maximum recombination fraction of 0.30 (corresponding to a maximum Kosambi distance of 34.7 cM) were established as thresholds for grouping markers. One anchor marker was chosen from each linkage group for subsequent mapping using the genome features of MAPMAKER. A subset of informative markers defined as those with <5% missing data, separated at 5 cM from each other, were ordered at an initial LOD score of 3.0. Additional markers were subsequently added by lowering the LOD threshold to 2.0 ("order" and "ripple" commands) to obtain a framework map. Markers that could not be ordered with equal confidence were indicated as accessory markers linked to a specific marker on the map. Markers that showed a departure from the 1:1 ratio (0.01 < P < 0.05) were also incorporated as accessory markers on the map, except for 10 cases where this marker was unique in an area of >30 cM. Maps were constructed with the program DrawMap (version 1.1) developed by VAN OOIJEN 1994 .

Estimated and observed genome length:
The estimated genome length was determined from partial linkage data according to G_e = with a confidence interval of with N the number of framework markers and thus N(N - 1) the number of pairwise comparisons, X the maximum distance between two adjacent framework markers in centimorgans at a certain minimum LOD score, and K the number of marker pairs at the same minimum LOD score (HULBERT et al. 1988 ; CHAKRAVARTI et al. 1991 , method 3). A minimum LOD score of 3.5 was chosen to estimate the genome length. Only framework markers were used to avoid an overestimation of the genome size because of clustered markers.

To calculate the observed genome length, the total length of the framework map was calculated (G_of), as well as the total length when considering all markers (G_oa). In addition, the observed genome length was calculated by the formula of NELSON et al. 1994 that takes into account all markers, linked and unlinked: G_on = G_of + X(L - R), with X the observed maximum distance between two framework markers; L the total number of linkage groups, triplets, doublets, and unlinked markers; and R the haploid number of chromosomes.

Expected and observed map coverage:
The expected genome map coverage was calculated from the equation: C_el = 1 - e (LANGE and BOEHNKE 1982 ), adjusted for chromosomal ends and compared with the result from the following equation, which accounts for linear chromosomes (BISHOP et al. 1983 ):

In this equation, N is the number of framework markers; X, the maximum distance between two adjacent framework markers in centimorgans at a certain minimum LOD score (in this case 3.5); G_e, the estimated genome length; and R, the haploid number of chromosomes. Only framework markers were considered because these equations refer to randomly distributed markers.

The observed map coverage is defined as the ratio of the observed genome length G_of to the estimated genome length G_e. For the observed genome length, the observed framework map distance (G_of) was used also because for the expected map coverage only framework markers were taken into account.

Marker distribution:
To evaluate whether the AFLP markers were randomly distributed, all linkage groups were divided into 10-cM intervals. Intervals at the end of a linkage group were taken into account only when >7.5 cM. The number of intervals that contained no markers and one to nine markers were counted. The observed frequencies were compared to the expected binomial frequencies. Subsequently, a runs test was performed (SOKAL and ROHLF 1981 ). The relationship between the variance and the mean [the coefficient of dispersion (CD) = ] is a rapid method to verify whether the observed frequency distribution is distributed in Poisson fashion. A value >1 indicates that there are more markers than expected in a given interval of 10 cM (clustering); a value <1 means that there are less markers than expected in a given interval of 10 cM.

The AFLP marker distribution was also analyzed by calculating the Pearson correlation coefficient between the number of AFLP markers in the linkage groups and the size of the linkage groups (SOKAL and ROHLF 1981 ; SPSS version 7.5). The marker distribution was tested on both AFLP framework markers and all AFLP markers.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Analysis of AFLP markers:
The two-way pseudotestcross strategy was used to generate genetic maps for P. deltoides cv. S9-2, P. nigra cv. Ghoy, and P. trichocarpa cv. V24 based on the analysis of two full-sib families 87001 and 87002 (MATERIALS AND METHODS). The mapping program was initiated with AFLP markers. A total of 50 and 41 AFLP primer combinations were used to analyze the progeny 87001 and 87002, respectively (Table A, http://www.plantgenetics.rug.ac.be/~vesto). Illegitimate progeny trees were scored within 87001 and 87002 by identifying those individuals that did not show the monomorphic AFLP fragments present in the parental lines. Four (family 87001) and three (family 87002) individuals were detected and eliminated from linkage analysis. The total numbers of markers scored as heterozygous in one parent and absent in the other were 438 for P. deltoides (87001) and 321 for P. deltoides (87002; based on crosses 87001 and 87002, respectively), 383 for P. nigra, and 314 for P. trichocarpa (Table 1). The average number of scored markers per primer combination varied from 7.7 to 8.8 for the four maps (Table 1). The heterozygosity levels based on AFLP markers were 26% for P. deltoides (87001), which might explain the best map coverage (see below), and 21% for P. deltoides (87002). Heterozygosity levels for P. nigra and P. trichocarpa were both 20%.

Analysis of STS and microsatellite markers:
STS and microsatellite markers are very useful as genetic bridges for comparative mapping because they are locus specific and codominantly inherited. The STS markers published by BRADSHAW et al. 1994 were analyzed to align the three genetic maps. By using the PCR conditions described, only 1 STS primer combination (P1054) of the 25 tested resulted in a single band of the expected size. By increasing the annealing temperature to 60°, 3 more STS primer combinations yielded a single PCR product (P767, P856, and P1010), whereas a single PCR product was obtained at 65° for primer combination P991. The other primer combinations showed either no or more than one amplification product. After digestion, only P767 and P991 revealed a polymorphism. P767 segregated in the two families, whereas P991 only segregated in 87002. Microsatellite markers (153), obtained from PMGC and Plant Research International (MATERIALS AND METHODS), were tested on the three parents. The results of the analysis are presented in Table 2 and Table B, http://www.plantgenetics.rug.ac.be/~vesto. The heterozygosity levels based on microsatellites were 63, 58, and 75% for P. deltoides, P. nigra, and P. trichocarpa, respectively.

In the course of our mapping study with microsatellites, three aneuploids were observed in the progeny: 87001#25, 87001#136, and 87002#141. This aneuploidy was confirmed by flow cytometry (GALBRAITH et al. 1983 ). The two clones 87001#136 and 87002#141 had a 1.5-fold higher nuclear content than their diploid parents, whereas that of clone 87001#25 was only slightly higher. These individuals were discarded for the microsatellite and linkage analyses.

Segregation distortion and linkage analysis:
A ² test (d.f. = 1) was performed to test the null hypothesis of a 1:1 segregation of the AFLP markers. At the 1% significance level, 63 AFLP markers had aberrant segregation ratios in the cross 87001 (8%; 35 from P. deltoides and 28 from P. nigra) and 39 in 87002 (6%; 12 from P. deltoides and 27 from P. trichocarpa). This value is higher than the 1% expected to occur by chance alone. These markers were excluded from linkage analysis with the exception of 8 AFLP markers in P. deltoides (87002). Previously, 3 markers closely linked to the M. larici-populina resistance gene had been found in P. deltoides (87001; CERVERA et al. 1996 ), 2 of which segregated also in family 87002, but with a departure from the 1:1 segregation ratio. This distorted ratio of the resistant/susceptible progeny taken from cross 87002 was provoked by the death of rust-susceptible trees. When the significance level was lowered to 5%, 68 more markers showed a deviation in the cross 87001 (37 from P. deltoides and 31 from P. nigra) and 44 in the cross 87002 (13 from P. deltoides and 31 from P. trichocarpa). These markers were retained for linkage analysis. The AFLP markers showing a significant segregation distortion (0.01 < P < 0.05) in P. deltoides (87001) did not deviate in P. deltoides (87002) and vice versa. For the four maps, there was no preference in the direction of the deviation (i.e., more bands present than absent).

The STS markers were also tested against a 1:1 segregation, but none showed a segregation distortion (P < 0.05). Microsatellite markers polymorphic in both parents were tested against a 1:1:1:1 (d.f. = 3) segregation ratio. When polymorphic in only one parent, they were tested against a 1:1 segregation (Table B, http://www.plantgenetics.rug.ac.be/~vesto). All STS and microsatellite markers were retained for linkage analysis.

Linkage analysis in P. deltoides (87001) was based on 403 AFLP markers, 61 microsatellites, the resistance marker against M. larici-populina race E1, E2, and E3 (mer), and one STS marker (Fig 1; Table 1). Initially (MATERIALS AND METHODS), 20 major groups, one doublet, and 7 unlinked markers were obtained. The ordering of the markers of 1 group could not be determined accurately and was split into 2 groups, resulting in 21 linkage groups. This separation was also obtained at a minimum LOD score of 4.0 and a corresponding maximum recombination fraction of 0.288. During ordering, 9 other markers belonging to different groups could not be placed. Unlinked markers are either artifacts segregating in Mendelian ratios by chance or they represent regions with very few markers. The distorted AFLP markers (37, 0.01 < P < 0.05) were distributed over 12 linkage groups. Several clusters of distorted markers were found on groups I, IV, V, and VII (Fig 1). In the framework, 53% of the markers were retained and the map consisted of 238 markers distributed over 196 unique loci.

In P. nigra, linkage analysis was based on 355 AFLP markers and 49 microsatellites (Fig 2; Table 1). Initially, 32 major linkage groups, four triplets, two doublets, and 15 unlinked markers were obtained. Markers of 2 groups could not be ordered accurately and were split into 2 groups, resulting in 34 linkage groups. This separation into 2 groups was obtained at a minimum LOD score of 5.0 and a corresponding maximum recombination fraction of 0.254. The decision to split these groups was also based on the alignment with P. deltoides (see below). During ordering, 4 other markers belonging to 2 groups could not be placed. Of distorted markers (0.01 < P < 0.05), 26 were distributed over 11 groups and 5 belonged to triplets or were unlinked. Only two clusters were found (groups IV and B; Fig 2). In the framework, 60% of the markers were retained and the map consisted of 222 markers distributed over 190 unique loci.

Linkage analysis in P. deltoides (87002) was based on 309 AFLP markers, 63 microsatellites, the resistance marker mer, and 1 STS marker (Fig 1; Table 1). Initially, 21 major linkage groups, three doublets, and 11 unlinked markers were obtained. For the ordering, 2 groups were split in two and resulted in 23 major linkage groups. This result was confirmed at a minimum LOD score of 4.0 and a corresponding maximum recombination fraction of 0.288. All distorted markers (13, 0.01 < P < 0.05) were scattered over 4 groups. In group XV, 7 distorted markers grouped together (Fig 1). In the framework, 50% of the markers were retained and the map consisted of 179 markers distributed over 153 unique loci.

Linkage analysis in P. trichocarpa was based on 287 AFLP markers, 76 microsatellites, and 1 STS marker (Fig 3; Table 1). Initially, 20 major linkage groups, two doublets, three triplets, and 12 unlinked markers were obtained. For the ordering, 1 group was split into two and another group into three. This result is in agreement with the ordering at a minimum LOD score of 4.0 and a corresponding maximum recombination fraction of 0.288. Twenty-nine distorted markers (0.01 < P < 0.05) were scattered over 13 groups. Two clusters were found (groups I and V; Fig 3). Two distorted markers (0.01 < P < 0.05) were unlinked. In the framework, 57% of the markers were retained. The framework map consisted of 194 markers distributed over 168 unique loci.

For the four genetic maps, the average length of a group based on the framework markers, the smallest group, the largest group, the average distance between two framework markers, and the number of intervals >10, 20, and 30 cM is indicated in Table 1.

The PGRI software version 1.0 (LIU 1998 ), which is based on a bootstrap approach, was used to verify the correct locus ordering in P. deltoides, P. nigra, and P. trichocarpa and thus establish the degree of confidence on marker order in the framework map (>80%). Other data concerning the map constructions are summarized in Table 1.

Map comparisons:
The two maps of P. deltoides were compared with each other and with the maps of P. deltoides, P. nigra, P. trichocarpa, and the family 331 linkage map (BRADSHAW et al. 1994 ).

P. deltoides: Because two maps were constructed for the same parent on the basis of the analysis of two different progeny, the reliability of the map could be evaluated. Two hundred and thirty-seven markers (193 AFLP markers, 43 microsatellites, and the resistance marker) were found in common (Fig 1). The order of the markers was the same for 208 markers. For 20 markers, disorder occurred within an interval of <5 cM. Thus, in the two maps the order was the same for 96% of the framework markers. After the two P. deltoides maps had been aligned, 19 linkage groups were obtained, corresponding to the haploid number of chromosomes in poplar.

P. deltoides, P. nigra, P. trichocarpa, and family 331 linkage map (Bradshaw et al. 1994): To further reduce the number of linkage groups and to identify homoeologous linkage groups among the three maps, microsatellite and STS marker analyses were performed. These markers are ideal for aligning maps because they are locus specific and codominantly inherited. An overview of all the alignments is given in Table 3 (for more details, see Table C, http://www.plantgenetics.rug.ac.be/~vesto). After alignment, the number of linkage groups in P. nigra could be reduced by 6, finally resulting in 28 groups. The number of groups for P. trichocarpa was reduced to 19.

Some discrepancies were also found (Fig 1 Fig 2 Fig 3 and Table C, http://plantgenetics.rug.ac.be/~vesto). PMG-C14 and PMGC420 are on a single linkage group in P. nigra (group XIV), but on separate linkage groups in P. deltoides (XIII and XIV). A spurious linkage of marker PMGC14 in P. nigra is suggested here because, at a LOD score of 4.0, marker PMGC14 remained unlinked.

A second discrepancy occurred at the position of marker PMGC61. Markers PMGC61 and PMGC409 are located on the same linkage group in P. nigra (VIII) and P. trichocarpa (VIII) and also on the linkage map reported by BRADSHAW et al. 1994 (group C). On P. deltoides, they are located on two different linkage groups (VI and VIII). A very high number of double crossovers were observed at the position of marker PMGC61 in P. deltoides and at the position of marker PMGC409 in P. nigra, suggesting a possible gene conversion event (LIU 1998 ), or a specific chromosomal arrangement.

A third discrepancy was caused by the position of marker PMGC2020. In P. trichocarpa PMGC2020 is located on the same group as PMGC2881 and PMGC2826 (group IV), whereas this is not the case for P. deltoides and P. nigra, on which PMGC2020 is on group IX and PMGC2881 and PMGC2826 on group IV. However, PMGC2020 amplified two loci according to the PMGC website (indicated as PMGC2020 and PMGC2021). Only one locus was amplified, so there is no certainty that the locus amplified in P. trichocarpa is the same as in P. deltoides and P. nigra. Twelve groups of the linkage map of BRADSHAW et al. 1994 could be aligned to the maps described here.

Estimated and observed genome lengths:
The estimated and observed genome lengths for the four linkage maps are presented in Table 4. The values for the estimated genome length for P. deltoides and P. trichocarpa were in the same range but that for P. nigra was much higher. The number of marker pairs with a minimum LOD score of 3.5 was low compared to the total number of framework markers in P. nigra. A good estimate for P. nigra could not be obtained because of the high number of linkage groups. The observed map distance calculated according to the formula of NELSON et al. 1994 (G_on; MATERIALS AND METHODS) that takes into account all linked and unlinked markers gave a result that was very close to the estimated genome length.

Expected and observed map coverages:
The expected and observed map coverages for the four linkage maps are presented in Table 4. The estimate according to the equation of LANGE and BOEHNKE 1982 was very similar to that of BISHOP et al. 1983 . The difference between the expected and the observed map coverage gives an estimate of the marker distribution. In all cases, the observed map coverage was lower than the expected map coverage.

Marker distribution:
AFLP markers are expected to be randomly distributed (VOS et al. 1995 ) when polymorphisms are randomly distributed. This assumption was evaluated in three ways (see MATERIALS AND METHODS). The Pearson correlation coefficient between the number of markers in the linkage groups and the total size of the linkage groups indicated a significant correlation at the 1% significance level for all maps; thus, the markers are randomly distributed. The coefficient of dispersion of the markers was in all cases very close to one (Tables D and E, http://www.plantgenetics.rug.ac.be/~vesto), also pointing to a random distribution. The frequency table for the framework markers (Table D, http://www.plantgenetics.rug.ac.be/~vesto) showed that there were more intervals containing only one marker than expected. Regions with clustered markers were also observed. The numbers of intervals of 10 cM, containing at least four markers, that were detected on the framework maps were as follows: nine for P. deltoides (87001), four for P. deltoides (87002), two for P. nigra, and three for P. trichocarpa. The same regions of clusters were found back in both maps of P. deltoides. The frequency table for all markers (Table E, http://www.plantgenetics.rug.ac.be/~vesto) showed that many more clusters appeared than for framework markers only, except for P. nigra. The runs test, however, does not indicate significant deviations from randomness.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Heterozygosity levels:
The efficiency of constructing a genetic linkage map in outbred forest trees with the two-way pseudotestcross strategy depends on the level of genetic heterozygosity of the species and the marker system. Heterozygosity levels of P. trichocarpa and P. deltoides based on RFLP markers were 30 and 15% and, based on RAPD markers, 36 and 30%, respectively (BRADSHAW et al. 1994 ). The estimates in our study based on AFLP markers were 26% for P. deltoides (87001), 21% for P. deltoides (87002), and 20% for P. nigra and P. trichocarpa. Estimates based on dominant AFLP markers are minimum estimates because a band that is present in both parents and does not segregate in the F₁ progeny can still be heterozygous in one parent. In Eucalyptus, heterozygosity levels based on AFLP markers were 30.5% for Eucalyptus tereticornis and 22.4% for E. globulus (MARQUES et al. 1998 ). The estimates based on microsatellites are much higher, but then hypervariable noncoding regions are analyzed.

Segregation distortion:
Segregation distortion has often been observed in forest trees and fruit trees (NELSON et al. 1993 ; BRADSHAW and STETTLER 1994 ; CAI et al. 1994 ; GRATTAPAGLIA and SEDEROFF 1994 ; LANAUD et al. 1995 ; MUKAI et al. 1995 ; VIRUEL et al. 1995 ; BARRENECHE et al. 1998 ; KRUTOVSKII et al. 1998 ; MARQUES et al. 1998 ; PAGLIA et al. 1998 ). The results obtained here suggest a biological basis for the deviations. Markers with a deviation from the expected segregation ratio are generally believed to be linked to genes that are subject to direct selection. BRADSHAW and STETTLER 1994 found that a lethal allele in Populus sp. affecting embryo development was the cause of segregation distortion of markers linked to it. In the P. deltoides (87002) map, we observed that markers cosegregating with the Melampsora resistance gene also showed a significant deviation because of death of susceptible trees. If the reason for this distortion had not been known, the markers would have been rejected and part of the linkage group would be missing. Based on this principle, all markers should be used in the mapping process. However, including markers with segregation distortion increases the chance of type I errors (i.e., rejection of the null hypothesis) of false linkage. Moreover, map distances between markers with skewed segregation ratios may be inaccurate (GERBER and RODOLPHE 1994 ; CLOUTIER et al. 1997 ). Therefore, only markers that deviated at the 5% and not at the 1% level were included for linkage analysis. Distorted markers that appear in clusters suggest that these areas contain genes that affect viability (STRAUSS and CONKLE 1986 ; DURHAM et al. 1992 ; JARRELL et al. 1992 ; LANDRY et al. 1992 ; SALL and NILSSON 1994 ; FOOLAD et al. 1995 ; LANAUD et al. 1995 ; CHENG et al. 1996 ; VERHAEGEN and PLOMION 1996 ; BARRENECHE et al. 1998 ; KUANG et al. 1999 ). No difference was observed in the percentage of markers with segregation distortion between the female and the male parents.

The fact that markers with a deviation from the expected 1:1 ratio in the map of P. deltoides (87001) segregate 1:1 in the other map of P. deltoides (87002) may be explained by the presence of a lethal recessive allele in the heterozygous condition in this region in P. deltoides and P. nigra, but not in P. trichocarpa. The same reasoning applies for markers with a segregation distortion in P. deltoides (87002) and not in P. deltoides (87001).

Map construction:
Between the LOD score and the ² value for independence, a relation exists for a population of infinite size: ² = 2 LOD/log e (LANDER and BOTSTEIN 1989 ). A minimum LOD score of 3.0 was chosen for linkage analysis. The recombination fraction can be calculated from the equation = with N the number of progeny. The smallest population consisted of 101 progeny, resulting in a recombination fraction of 0.301 and a corresponding Kosambi map distance of 34.7 cM. From these equations, it is obvious that the recombination fraction increases with a decreasing LOD value. The recombination fraction also depends on the progeny size. For the same recombination fraction, the LOD increases with an increasing number of progeny. The maps were based on 121 progeny for P. deltoides (87001) and P. nigra and on 101 progeny for P. deltoides (87002) and P. trichocarpa. These population sizes are among the largest of all published maps on trees.

The 19 chromosomes of Populus sp. are represented in the genome maps of P. deltoides, P. nigra, and P. trichocarpa by 19, 34, and 23 linkage groups, respectively. The comparison of the order of markers and levels of recombination between the two maps of P. deltoides proves the robustness of the maps. Errors in locus ordering may invalidate further analysis, such as QTL analysis or map-based cloning, but the locus order is correct for 96% of the common framework markers when both maps of P. deltoides are compared. Similar results have been obtained by PLOMION et al. 1995 , who compared two genome maps of a unique Pinus pinaster genotype built on a common set of 263 RAPD markers. The linkage maps of two different progenies, consisting of 62 megagametophytes each, which had been obtained from a self cross and an open-pollinated cross, shared the order of 98% framework markers. Similar results were also obtained by SEWELL et al. 1999 , who made a consensus map for loblolly pine by integrating two individual maps from two outbred three-generation pedigrees.

The map of P. deltoides (87001) is the best-covered map. Although the map of P. nigra is based on 404 markers instead of 466 and 364 for P. deltoides (87002) and P. trichocarpa, respectively, linkage analysis resulted in 34 linkage groups. The estimated heterozygosity levels of P. nigra and P. trichocarpa were in the same range. A possible explanation is that the genome of P. nigra contains large regions of highly homologous sequences.

A direct approach to align the 19 chromosomes with 19 linkage groups would be to map telomere-proximal sequences at the distal ends of the chromosomes (BURR et al. 1992 ; GANAL et al. 1992 ). To date, telomere sequences are still unknown in poplar.

Map comparisons:
Comparative mapping is a useful technique for investigating chromosomal evolution and allows the import of genetic information (such as map positions of qualitative or quantitative traits) from one species to a related species. In a first attempt to align homoeologous groups, markers, which were either heterozygous in both parents (segregating 3:1) or heterozygous in P. nigra and P. trichocarpa, and null in P. deltoides (segregating 1:1 in both families), were looked for. The number of markers segregating 3:1 was 8 for both families 87001 and 87002. Twelve common markers were found between P. nigra and P. trichocarpa. For AFLP markers, however, sequence data are needed to prove that markers of the same size represent the same locus (QI and LINDHOUT 1997 ; ROUPPE VAN DER VOORT et al. 1997; WAUGH et al. 1997 ). Therefore, the selected AFLP markers were cloned from each parent and 5 clones were sequenced. Fourteen markers were analyzed in this way. However, in none of the cases was the sequence of all 10 clones the same due to the coamplification of underlying nonvisible bands. In six cases, one-half of the clones showed the same sequence. Very few AFLP markers (2%) segregated 3:1 compared to 1:1. This low frequency was also observed in a cross of Eucalyptus (GRATTAPAGLIA and SEDEROFF 1994 ) and almond (VIRUEL et al. 1995 ). Most of these markers remained unlinked because of the low information content between marker pairs segregating 1:1 and 3:1 (RITTER et al. 1990 ). Therefore, the efficiency and reliability of these markers to align the genetic maps are low.

Multiallelic codominant markers, such as STS markers and microsatellites, are the most efficient for map comparisons. In contrast to the microsatellite primers, however, most of the primers for detection of STS markers described by BRADSHAW et al. 1994 did not amplify the expected bands in the parents of the two families under study.

The alignments resulted finally in 19 groups for P. deltoides, 19 for P. trichocarpa, and 28 for P. nigra. Twelve groups of P. deltoides were aligned with 18 groups of P. nigra and 15 groups of P. deltoides with 19 groups of P. trichocarpa. The alignments are based on 27 microsatellites in common between P. deltoides and P. nigra, 34 between P. deltoides and P. trichocarpa, and 16 between P. nigra and P. trichocarpa; only 14 were found in common between P. deltoides, P. nigra, and P. trichocarpa. This corresponds with 17, 22, 11, and 9% of the total number (153) of available microsatellites. Therefore, a large number of microsatellites are necessary for a successful comparative analysis.

In general, the corresponding groups between P. deltoides, P. nigra, and P. trichocarpa are of comparable size, considering the differences in map coverage. Groups VIII of P. nigra and P. trichocarpa, however, are larger than group VIII of P. deltoides, supporting the idea of a rearrangement of the region around the microsatellite marker PMGC61. Indeed, the inconsistency for the marker PMGC61 may point to the presence of chromosomal rearrangements as proposed for apple (HEMMAT et al. 1994 ) and Prunus (FOOLAD et al. 1995 ). To prove this hypothesis, markers in the vicinity of this microsatellite should be identified and mapped both on P. deltoides and P. nigra.

Genome length estimates and map coverage:
BRADSHAW et al. 1994 estimated that the total genome map size for P. trichocarpa was 2400 cM at a minimum LOD score of 3.0. This size is very close to our estimates for P. deltoides and P. trichocarpa. The map of P. deltoides (87001) is well covered. The difference between the expected map coverage and the observed framework map coverage may be due to clusters or may simply result from the process of framework map construction (ECHT and NELSON 1997 ). The markers retained in the framework were 53 and 50% for the two maps of P. deltoides, 60% for the P. nigra map, and 57% for the P. trichocarpa map. These values are underestimated by 3% (for P. deltoides and P. nigra) to 7% (for P. trichocarpa) because most of the microsatellites were retained from the framework because they were analyzed on only half of the families. Taking this limitation into consideration, typical values were found (GRATTAPAGLIA and SEDEROFF 1994 ; ECHT and NELSON 1997 ; PAGLIA et al. 1998 ).

Marker distribution:
The presence of large gaps may be explained in two ways. First, there may be hot spots of recombination. Second, because only EcoRI and MseI were used as restriction enzymes for AFLP analysis, there is a chance that the AFLP markers are directed toward AT-rich regions, leaving a void of markers in GC-rich regions. Small clusters were also observed, indicating regions with suppressed recombination.

Future perspectives:
The construction of relatively dense framework maps, such as those presented here, will facilitate the dissection of complex inherited traits and enable us to study the genetic basis of QTL (DARVASI et al. 1993 ; FREWEN et al. 2000 ; WU et al. 2000 ). To locate the QTL more precisely, more markers could be searched for in the region of interest by using bulked segregant analysis (MICHELMORE et al. 1991 ). Accurate mapping of monogenic and multigenic traits may open possibilities to develop strategies for marker-assisted selection but is also the starting point for gene cloning. These AFLP framework maps constitute the skeleton on which codominant microsatellites and STS markers can be mapped progressively to construct a saturated "species consensus map," which will be a useful tool for evolution studies and breeding purposes (CERVERA et al. 1999 ).

Note:
Cuttings, DNA, and genotypic data of the two mapping populations are available to the scientific community.

	FOOTNOTES

¹ These authors contributed equally to this work.
² Present address: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología (CSIC), Campus de la Universidad Autónoma de Madrid, E-28049 Madrid, Spain.
³ Present address: Laboratório de Biodiversidade Molecular, Departamento de Genética, Instituto de Biologia, UFRJ, Bloco A, CCS, Ilha do Fundão, 21941-490, Rio de Janeiro, Brazil.

	ACKNOWLEDGMENTS

The authors thank Vic and Marijke Steenackers, An Vanden Broeck, and Boudewijn Michiels for a long-standing fruitful collaboration; Ron Sederoff, Christophe Plomion, and Carlos A. Malpica for their valuable comments during the project; Gerry Tuskan, Toby Bradshaw Jr., and Mitchell Sewell for their helpful information; Tom Gerats and Peter Breyne for critical reading of the manuscript; and Martine De Cock for help in preparing it. This work was supported by grants from the Flemish Government (BNO/BB/6/1994, 1995; IBW/3/1995–2000) and the Commission of the European communities AIR program (AIR1-CT92-0349). M.-T.C. is indebted to the European Union for an individual fellowship from the Human Capital Mobility program (41AS8694).

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

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