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Keywords:

  • genetic mapping;
  • physical mapping;
  • poplar rust;
  • positional cloning;
  • qualitative resistance;
  • quantitative resistance;
  • R-gene cluster

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • RUS is a major dominant gene controlling quantitative resistance, inherited from Populus trichocarpa, whereas R1 is a gene governing qualitative resistance, inherited from P. deltoides.
  • Here, we report a reiterative process of concomitant fine-scale genetic and physical mapping guided by the P. trichocarpa genome sequence. The high-resolution linkage maps were developed using a P. deltoides × P. trichocarpa progeny of 1415 individuals. RUS and R1 were mapped in a peritelomeric region of chromosome 19. Markers closely linked to RUS were used to screen a bacterial artificial chromosome (BAC) library constructed from the P. trichocarpa parent, heterozygous at the locus RUS.
  • Two local physical maps were developed, one encompassing the RUS allele and the other spanning rUS. The alignment of the two haplophysical maps showed structural differences between haplotypes. The genetic and physical maps were anchored to the genome sequence, revealing genome sequence misassembly. Finally, the RUS locus was localized within a 0.8-cM interval, whereas R1 was localized upstream of RUS within a 1.1-cM interval.
  • The alignment of the genetic and physical maps with the local reorder of the chromosome 19 sequence indicated that RUS and R1 belonged to a genomic region rich in nucleotide-binding site leucine-rich repeat (NBS-LRR) and serine threonine kinase (STK) genes.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Leaf rust, caused by the fungus Melampsora larici-populina (Mlp), is the most devastating disease in European poplar plantations, credited with premature defoliation, reduced growth and 50% yield loss (Paillassa, 2006). Infections over successive years can result in the complete loss of economic value. Monoclonal plantations involving a limited number of cultivars have favored the emergence of new strains and virulences in Mlp populations and, so far, eight virulences have been defined (Pinon & Frey, 2005). Since 1984, no interspecific Populus deltoides × P. trichocarpa or P. deltoides ×P. nigra cultivars have remained free from rust damage for more than 5 yr after commercial deployment (Gumez et al., 2000).

The genetics of poplar resistance to Mlp have been studied in intra- and interspecific progenies involving P. deltoides, P. trichocarpa and P. nigra species, and both qualitative and quantitative resistances have been found. Qualitative resistances were inherited only from P. deltoides (Pichot & Teissier du Cros, 1993a) and segregations observed in P. deltoides × P. nigra and P. deltoides × P. trichocarpa progenies revealed relatively simple genetic determinism (Lefèvre et al., 1994, 1998; Dowkiw & Bastien, 2007). To date, Mer and R1, two loci controlling qualitative resistance to different Mlp strains, have both been mapped on the linkage group (LG) XIX of two different P. deltoides parental maps (Cervera et al., 1996, 2001; Jorge et al., 2005). Mer is overcome by the virulence factor 7 and R1 by the virulence factor 1 identified in different Mlp strains (Dowkiw & Bastien, 2004; Pinon & Frey, 2005).

Quantitative resistances, measured in field tests under natural infection and in leaf disk bioassay under artificial inoculation, have been identified in the three poplar species P. deltoides, P. trichocarpa and P. nigra, and their interspecific hybrids (Pichot & Teissier du Cros, 1993b; Lefèvre et al., 1998; Legionnet et al., 1999; Dowkiw & Bastien, 2004, 2007). In a reference P. deltoides × P. trichocarpa mapping progeny, quantitative resistance measured by sporulation intensity (uredinia size, US) appears to be mostly controlled by two genetic factors. The first, referred to as RUS, inherited from P. trichocarpa, was mapped on a small unassigned LG containing three markers and spanning 28.3 cM by Jorge et al. (2005). RUS has been shown to explain up to 80% of the variation for three components of quantitative resistance (latent period, size of uredinia and number of uredinia) after inoculation with seven Mlp strains. Recently, Dowkiw et al. (2010) identified several RUS-defeating strains which overcame the effect of RUS in different poplar genetic backgrounds. The other major locus is inherited from the P. deltoides parent and is associated either by pleiotropy or linkage with the qualitative resistance R1. Three other defeated qualitative resistances inherited from different P. deltoides parents could also be associated with quantitative resistance to Mlp (Dowkiw & Bastien, 2007). Villar et al. (1996) and Lefèvre et al. (1998) first suggested the existence of the key region involved in the control of Mlp rust resistance. Such genomic regions are frequently reported in plants and correspond to clusters of nucleotide-binding site leucine-rich repeat (NBS-LRR) genes (Meyers et al., 2003). In poplar, clusters of these genes have been found in the vicinity of the Mer locus (Zhang et al., 2001). Kohler et al. (2008) and Yang et al. (2008) counted c. 400 NBS-LRR genes in the genome v1.1 (Tuskan et al., 2006), some of them being arranged in super clusters.

To further elucidate the relationships between R1 and RUS loci, we used an iterative process of genetic and physical mapping with the help of the complete genome sequence of P. trichocarpa Nisqually-1. At the beginning of our work, we used the JAZZ assembly v1.1 (Tuskan et al., 2006) and later the ARACHNE assembly v2.0 (http://www.phytozome.org/). For physical mapping, we constructed a deep coverage (15-fold redundant) bacterial artificial chromosome (BAC) library for the individual P. trichocarpa 101-74 heterozygous at the RUS locus. The availability of BAC clones allowed the design of tightly closely linked markers through BAC end sequencing. An extension of the P. deltoides × P. trichocarpa segregating population up to 1415 individuals and its scoring for qualitative resistance and quantitative resistance traits allowed us to fine map the R1 and RUS resistance loci. Further characterization of the genomic regions surrounding R1 and RUS provided new insight into poplar genome organization. The optimal strategy for the identification of genes underlying R1 and RUS loci using a reference sequenced genome deprived of these alleles is discussed. Positional gene cloning is still a major challenge in heterozygous species with a duplicated genome and in complex regions such as clusters of R genes.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant material

The studied material comprised the following: P. deltoides 73028-62; P. trichocarpa 101-74; exhaustive or selected sets of genotypes from four P. deltoides × P. trichocarpa F1 families generated by controlled hybridization with this male genitor (Table 1); and the reference sequenced P. trichocarpa genotype ‘Nisqually-1’. The 73028-62 × 101-74 progeny comprises 1415 full-sib genotypes that were generated in two distinct years (1990 and 2003). The set of genotypes generated in 1990 crosses had already served as a mapping and quantitative trait locus detection pedigree in a previous study (Jorge et al., 2005). All genotypes were grown from cuttings under glasshouse rust-free conditions. Growth condition details can be found in Dowkiw et al. (2003). Populus trichocarpa 101-74 was later transferred to the dark to generate etiolated leaves for BAC library construction according to the protocol of Chalhoub et al. (2004). DNA extractions for genotyping were made from the youngest leaves, whereas rust resistance bioassays involved 3-cm-diameter excised leaf disks sampled from the fifth to the eighth fully expanded leaves below the apex. To ascertain the recombinant status of a few genotypes, both their phenotype and genotype were checked on leaves collected directly from original rootstocks in the nursery before rust epidemics.

Table 1.   List of Populus deltoides × P. trichocarpa progeny material used in this study
Female parentMale parentNumber of genotypes (selected phenotype)UseSource/reference for phenotypes
  1. BSA, bulk segregant analysis.

73028-62101-74336 (all)Local genetic map and high-resolution mappingJorge et al. (2005)
1079 (all)High-resolution mappingThis study
    12 (r1Rus)BSAJorge et al. (2005)
12 (r1rus)  
    12 (r1Rus)BSAJorge et al. (2005)
12 (r1rus)  
M170-3101-74    12 (Rus)BSADowkiw & Bastien (2007)
12 (rus)
L155-079101-74    11 (Rus)BSADowkiw & Bastien (2007)
11 (rus)
L150-089101-74     9 (Rus)BSAA. Dowkiw & C. Bastien (unpublished)
10 (rus)

Studied resistance segregating loci

The R1-mediated qualitative resistance to Mlp, inherited from P. deltoides 73028-62, has been shown to segregate 1 : 1 in the 73028-62 × 101-74 F1 progeny (Lefèvre et al., 1998; Dowkiw et al., 2003). The RUS-mediated quantitative resistance, inherited from P. trichocarpa 101-74, segregates independently from R1, also with a 1 : 1 ratio, and generates clear bimodal distributions of genotypic means for US in the considered progeny (Dowkiw & Bastien, 2004). The 1 : 1 segregation pattern of RUS has been found in all hybrid progenies derived from 101-74 until now (Dowkiw & Bastien, 2007). In this work, we followed the nomenclature proposed by Dowkiw et al. (2010): F1 individuals [RUS/rUS] and [rUS/rUS] are designated [RUS] and [rUS]. The same rule was applied for the R1 locus.

Complementary rust resistance assessments

Genotypes from the 73028-62 × 101-74 progeny that had not been characterized previously by Jorge et al. (2005) as R1 vs r1 and RUS vs rUS were phenotyped using Mlp strains 93ID6 (virulences 3-4) and 98AG69 (virulences 1-3-4-5-7), respectively.

The large number of genotypes studied involved spreading them into three sub-experiments for each strain. Each experimental design consisted of five randomized complete blocks, where each genotype was represented by one disk per block. Nisqually-1 was represented by three disks per block in one sub-experiment for each strain. Details of the inoculation procedure can be found in Dowkiw et al. (2003). The temperature was set at 17°C. The mean inoculum pressure ranged between 132 and 343 urediniospores per leaf disk. US was measured using the 1–5 fast scoring method given in Dowkiw et al. (2010).

The R1 phenotype was defined as the absence of any uredinia on any leaf disk after inoculation with strain 93ID6. Genotypes showing only one disk with uredinia during the first test had their phenotypes later validated in one to three complementary inoculation tests. The RUS and rUS phenotypes were defined graphically, by setting an upper (respectively lower) limit to the small (respectively large) mode of the bimodal distribution of clonal means observed for US in each experiment. Genotypes falling between these two limits were defined as ambiguous and tested again with strain 98AG69, together with some reference genotypes for RUS and rUS phenotypes.

Identification of markers linked to resistance locus through bulk segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) screening

A BSA-AFLP approach (Michelmore et al., 1991) was carried out to identify markers closely linked to RUS. In order to limit the number of false positives and to overcome some problems linked to the inheritance of dominant markers from the female parent, five independent pairs of RUS and rUS bulks were constituted (Table 1): The individuals from each bulk were also selected on their genotype at three RUS/rUS flanking markers (Jorge et al., 2005). Equal amounts of DNA from 10 genotypes were pooled in each bulk, and the AFLP procedure was applied as described in Jorge et al. (2005). The polymorphic bands were further analyzed at single genotype level. The confirmed bands were finally cloned and sequenced. Sequences were used for BLASTn alignment against the genome sequence of P. trichocarpa Nisqually-1 v1.1 (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html) to determine their physical location. Sequence characterized amplified region (SCAR) markers were developed from the AFLP sequences with the help of genome sequence v1.1.

Identification of PCR markers linked to resistance loci through genome sequence analysis

First, 13 simple sequence repeat (SSR) markers were retrieved from former studies (Tuskan et al., 2004; Yin et al., 2004). SSR motifs were also searched in the region of the genome sequence surrounding the cloned AFLP markers. Second, sequence-tagged site (STS) primers inside and outside the gene spaces were also generated. STS in NBS-LRR genes were generated not only from the candidate genomic region, but also from genes located on other scaffolds but showing sequence similarity. In some cases, in order to develop specific primer pairs in NBS-LRR genes, a consensus primer was designed in the Toll interleukin 1 receptor (TIR) or LRR domain, and the specific primer was designed in the 5′ or 3′ noncoding flanking region, respectively. Third, primers were also designed in three TIR-NBS-LRR genes located on a published P. deltoides BAC sequence near the Mer locus (Lescot et al., 2004).

Primer pairs were defined with Primer 3 software using default parameters, except for the primer length (22 nucleotides) and annealing temperature (59°C) (http://fokker.wi.mit.edu/primer3). The specificity of candidate primer pairs was checked against the genome sequence using both electronic polymerase chain reaction (ePCR) (Schuler, 1997) and BLASTn alignments. When necessary, cleaved amplified polymorphic sequences (CAPS), derived cleaved amplified polymorphic sequences (dCAPS) and Allele-specific polymerase chain reaction (AS-PCR) markers were also developed, as described by Konieczny & Ausubel (1993), Michaels & Amasino (1998) and Bundock et al. (2006), to recover polymorphism.

Primer pairs were tested for amplification and segregation on the two parents and six progenies of the mapping pedigree. The PCRs consisted of one denaturing step of 5 min at 94°C, followed by 35 cycles of 20 s at 94°C, 30 s at variable temperature according to the primer sequences, 40 s at 72°C and a final elongation step of 7 min at 72°C.

Construction of local genetic maps

Genetic markers were first employed to construct local genetic maps with 336 genotypes, 326 of which had already been used by Jorge et al. (2005). The mapping work was then extended to 1415 individuals with a subset of markers for high-resolution mapping. Markers with more than 40% of missing data and/or with more than 3.5% of genotyping errors were removed from the mapping analysis. The final order of the genetic markers was established manually starting with the physical order retrieved from the genome sequence v1.1 (http://genome.jgi-psf.org/poplar/). The raw data from recombinant individuals in the target region were then inspected and the order was corrected to minimize the number of recombination events. The map distances were calculated with MAPMAKER/EXP 3.0 using the Kosambi map function (Lander et al., 1987). LGs were drawn using MapChart v2.2 (Voorrips, 2002). The consensus genetic map of LG XIX was constructed using the map projection procedure implemented in Biomercator v2.1(Arcade et al., 2004) of the P. deltoides parental map on the P. trichocarpa parental map.

Construction of a P. trichocarpa (clone 101-74) BAC library, insert size, pools and BAC end sequences (BES)

A BAC library was constructed from the P. trichocarpa male parent (clone 101-74) according to the procedure developed by Chalhoub et al. (2004). Briefly, young etiolated leaves were used to isolate high-molecular-weight nuclear DNA, which was partially digested with HindIII and cloned into pIndigo BAC – vector (Epicentre Technologies, Madison, WI, USA). The library was arrayed in 384-well microtiter plates. The insert size of BAC clones was determined according to Chalhoub et al. (2004). Two types of BAC pool were prepared for PCR screening: plate pools and row pools. This genomic resource was completed by BAC end sequencing using the same procedure as in Tuskan et al. (2006) and Kelleher et al. (2007) of the clones from the first 20 plates.

Construction of physical maps

PCR screening of plate pools from the BAC library, representing 10X genome coverage, was performed and, if necessary, PCRs were also made with the remaining plate pools. Row pools corresponding to the positive plates were then screened and the 24 candidate clones of a positive row were individually tested for the identification of the positive BAC clones. The extremities of some positive BAC clones were sequenced and aligned onto the genome sequence by BLASTn. Con-currently, the BES of the first 20 plates were also aligned by BLASTn (e-value = 10−50) on the genome sequence. All the selected alignments of BES in the region of interest were manually inspected using MEGA4 software (Tamura et al., 2007). The construction of the BAC contigs and the orientation of BAC into the contigs were inferred from the genetic mapping of new markers designed in BES and/or from the alignment of BES onto the genome sequence.

This approach allowed the construction of two physical maps, one for each haplotype. The haplotype carrying the RUS allele was named haplotype hRUS and the haplotype carrying the rUS allele was named haplotype hrUS.

Domain identification of NBS-LRR genes in the target region

The NBS-LRR gene MEME motifs, established by Meyers et al. (2003) and Kohler et al. (2008), were used as tBLASTn queries to determine whether gene models encoded nuclear localization sequence (NLS), TIR, coiled coil (CC), BEAF and DREF DNA-binding finger (BED), NBS or LRR domains.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Complementary phenotyping for qualitative and quantitative resistances

The segregations of both R1 and RUS fitted 1 : 1 ratios in the complementary set of 1079 genotypes from the 73028-62 × 101-74 progeny studied here; 702 genotypes could be defined as R1 and 645 as r1 (P > X2, 1 : 1 = 0.120). Ambiguous genotypes for the presence of qualitative resistance were all declared r1 after several inoculations with strain 93ID6 at high inoculum pressures. Graphical analysis of the clonal mean distributions for US allowed us to declare 696 genotypes as RUS, and 656 as rUS (P > X2, 1 : 1 = 0.277), whereas 22 genotypes remained ambiguous for their RUS vs rUS status after multiple testing with strain 98AG69. The numbers of genotypes in the four categories R1RUS, R1rUS, r1RUS and r1rUS were 348, 337, 327 and 301, respectively, thus indicating independent segregation ratios for both resistance factors (P > X2, 1 : 1 : 1 : 1 = 0.297).

Nisqually-1 expressed high susceptibility with US performances close to those observed for rUS genotypes. Average US scores in square millimeters were 0.1768 ± 0.0002, 0.8169 ± 0.0008 and 0.806 ± 0.030 for 210 RUS genotypes, 235 rUS genotypes and Nisqually-1, respectively.

Identification of markers linked to resistance locus through BSA screening

One hundred and fourteen AFLP primer–enzyme combinations were tested to identify polymorphic bands between the four female parents and the male parent and between resistant and susceptible bulks derived from the four progenies. Twelve discriminating bands were found among parental clones and all the contrasting bulks. Seven polymorphic bands were confirmed by testing the segregation in 90 F1 individuals. These bands, together with two previously mapped AFLP markers (E4M2-7 and E5M5-7; Jorge et al., 2005) and the random amplification of polymorphic DNA (RAPD) marker M03/04_480 (Villar et al., 1996), were cloned and sequenced (Supporting Information Table S1). BLASTn alignments of the AFLP sequenced fragments onto the Populus genome sequence (v1.1) showed that six aligned with a cluster of NBS-LRR genes at the peritelomeric region of chromosome 19 (E5ME-7, E21M18, E23M2, E15M14_a, E25M14 and E6M5), whereas three aligned to scaffold 117 (E1M4, E25M14 and M03/04_480). Only three of these AFLP and RAPD markers could be converted into SCAR genetic markers; the others were monomorphic.

Genetic linkage maps of the RUS and R1 regions

For the construction of the P. trichocarpa genetic map surrounding the RUS region, three individuals showing a trisomic or triploid pattern were excluded. A total of 333 individuals was used to construct a local genetic map with 68 molecular markers expected on the LG XIX (5.4% of missing data). The map resulted in 27 distinct loci in an interval bounded by two SSR markers: I_524 and I_2879 (Fig. 1a). The total length of the interval studied was 23.6 cM. Average map distances between loci were 0.87 cM, ranging between 0.3 and 3.6 cM. Marker segregation type and phase were indicated in S2. RGAm4-1 designed from the P. deltoides BAC sequence near the Mer locus did not amplify in our P. deltoides parent, but was located 0.6 cM away from RUS on the P. trichocarpa map. Twenty-three robust markers were subsequently employed for fine mapping using 1078 additional progenies. The fine genetic map of the target region was constructed with 91% of data after the removal of 10 triploid or trisomic and two aneuploid individuals. The high-resolution map included 18 loci with RUS spanning 10 cM (Fig. 1a). New recombination events allowed the separation of previous co-segregating markers. However, among the 23 markers, four groups of markers still showed co-segregation. The RUS locus was mapped within a region of 0.5 cM flanked by the co-segregating markers RGAs135-1 and Is165-1 at 0.1 cM on one side and the marker 14N08-F at 0.4 cM on the other.

image

Figure 1. Local genetic maps of the target region and their alignment onto the scaffold 19 assembly v2.0. Local genetic maps of Populus trichocarpa derived from genotypic and phenotypic data for 333 and 1400 F1 individuals (a) and of P. deltoides derived from genotypic and phenotypic data for 335 and 1413 F1 individuals (c). Map distances in cM (Kosambi distances) are indicated on the left of the linkage groups (LGs). The P. trichocarpa and P. deltoides genetic maps were anchored onto the genome sequence v2.0 (b) through electronic polymerase chain reaction (ePCR) and BLASTn searches using sequences of primers as queries and the genome sequence as database. Sequence characterized amplified region (SCAR) markers originating from the bulk segregant analysis-amplified fragment length polymorphism (BSA-AFLP) approach are designated by the S_AFLP primer combination names (E, EcoRI, M, MseI) preceded by S_. Published simple sequence repeat (SSR) markers are named according to Yin et al. (2004); they begin with O_, G_ or P_. SSR markers developed in this study begin with I_ (for INRA). Markers developed from nucleotide-binding site leucine-rich repeat (NBS-LRR) or serine threonine kinase (STK) genes are designated RGA; the letter ‘m’ indicates that the marker was designed onto the BAC sequence located near the Mer locus (Lescot et al., 2004). Markers derived from gene models of the genome sequence carry the prefix PT. Markers derived from intergenic sequences begin with ‘nc’ for noncoding sequences. Markers derived from BES are named by plate&row letter&column number. The suffixes F and R designate left (Forward end) and right (Reverse end) ends of BAC clones, respectively. The letter ‘s’ in the marker names indicates that these markers were derived from scaffold sequence v1.1. AS, allele specific; dC, derived cleaved amplified polymorphic sequence (dCAPS).

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A local genetic map for R1 was constructed using a set of 335 individuals after removing one individual showing a trisomic/triploid pattern (17.4% of missing data). The local map covered 33.8 cM and consisted of 24 molecular markers distributed in 15 loci (Fig. 1c). Thirteen robust markers were subsequently employed for fine mapping using 1413 individuals (19.7% of missing data); two individuals showing an abnormal maternal meiosis pattern were removed. Finally, R1 was located in a 1.9-cM interval between two loci consisting, on one side, of the co-segregating markers I_1211 and G_79 (0.6 cM) and, on the other, marker I_920 (0.9 cM) (Fig. 1c).

Comparative mapping and anchoring onto the genome sequence

Thirteen markers (eight SSR, one resistance gene-analog (RGA), three STS, one SCAR) were shared between the P. trichocarpa and P. deltoides parental maps constructed using 333 and 335 F1 individuals, respectively. The marker order was perfectly conserved between the two maps (Figs 1, S1). Discrepancies were nevertheless observed among genetic distances between some marker pairs, recombination rates being, on average, 1.4 higher in P. deltoides than in P. trichocarpa in this genomic region (Fig. S1). Interestingly, the recombination rate between the markers flanking R1 (G_79 and I_920) was 2.3-fold greater in P. deltoides than in P. trichocarpa. Finally, the markers that were mapped in both P. trichocarpa and P. deltoides allowed us to determine that R1 and RUS were located at two different loci.

Sequences from all primers were subjected to BLASTn searches against the two versions of the poplar genomic sequence (v1.1 and v2.0; Fig. 1c and Table S2). Twelve genetic markers had no hits on genome sequence v1.1 and 17 had no hits on v2.0. For instance, the genetic markers 57L24-R and E6M5 had no homolog on v1.1, but were anchored on the superscaffold [i.e. chromosome] 19 of v2.0. The co-segregating markers E1M4/RGAs336 and PTs776-AS/PT1939-4/nc1432-1 had homologs on genome v1.1, but not on v2.0. Several major changes from the v1.1 to v2.0 versions of the genome sequence were confirmed by our genetic maps. In v2.0, the scaffold 117 bordered by the markers RGAs135-1 and 47N12-F was totally integrated into chromosome 19 (Figs S1, S2). This integration was confirmed by the alignment of our P. deltoides genetic map (Fig. 1). The genetic mapping of markers derived from the scaffolds 4600, 2139, 2701 and 283 of genome sequence v1.1 confirmed their integration into chromosome 19 in v2.0. The alignment of the P. trichocarpa genetic maps on v1.1 revealed an inversion of one co-linear block bordered by the markers 04J18-R-1 and RGA1159 (Fig. S2). This inversion was also found with the P. deltoides map, but was not found in v2.0 of the improved assembly (Fig. 1). Some discrepancies in marker order are, however, still observed between the v2.0 genome sequence and the genetic maps constructed here. Anchoring P. trichocarpa genetic markers on to v2.0 revealed a misassembly of five co-segregating markers (RGAs352–RGAs135-1). This misassembly was also retrieved after anchoring the P. deltoides map with the genome sequence. The closely linked markers RGAs2701-1, RGAs237-4 and RGAs237-1 appeared to be inverted at a long distance relative to their co-segregating markers. There were also three instances in which marker positions were inverted (PT2387, 47N12-F, 14F23-F). Finally, for several markers that were expected to be a multilocus, according to the results of BLASTn searches against the genome sequences v1.1 and v2.0, only one locus per marker could be unambiguously mapped. Nevertheless, in most cases, the order of the markers of P. trichocarpa and P. deltoides linkage maps was consistent with the order of the markers in the genome sequence.

Haplotype physical maps around RUS and R1 loci and anchoring onto the genome sequence

The 52 224 BAC clones of the BAC library were organized into 136 plate pools and 2176 row pools. The insert size of random sampling of 91 BAC clones allowed the estimation of an average insert size of 150 kb; 3% and 0.3% of the clones contained chloroplastic and mitochondrial DNA sequences, respectively. Based on an estimated genome size of 480 Mb, the BAC library was estimated to represent 15 Populus genome equivalents. End sequencing was performed on 7680 BAC clones from the first 20 plates. After removal of low-quality sequences, a set of 11 480 useful BES was available (GenBank: N280500-HN291979).

Ninety-five BAC clones were recovered by the PCR screening of our BAC library with 60 markers, and end sequenced. Concurrently, the 13 536 available BES were aligned onto the genome sequence, allowing the integration of 20 additional BAC clones in the physical mapping work. Finally, 133 BES coming from 71 BAC clones were used to anchor the physical map onto the genome sequence (Fig. 2; Tables S3, S4). Forty-five of these were used to derive new markers (Table S2). These markers were used to orientate individual clones and to confirm the entire subcontig grouping. Twenty-one were used as genetic markers to ensure that the walk had not jumped to other regions of the genome (Fig. 2; Table S2). The physical map of the hRUS haplotype consisted of 76 BAC clones arranged in two super-contigs (Figs 2, S3). The first, delimited by the markers RGA805 and 97G09-R-1, comprised 32 BAC and covered c. 850 kb. The second, delimited by the markers 4J18-1-R and 47N12-F, comprised 44 BAC and covered c. 900 kb. The physical map of the hrUS haplotype comprised 39 BAC clones also distributed in two contigs. The largest covered c. 1 Mb and consisted of 30 BAC clones; eight of these could not be orientated in the contig because of the alignment of BES to multiple locations on the genome sequence. The smallest comprised nine BAC clones and spanned c. 350 kb. The gap observed between the two physical maps was caused by the lack of BAC clones in our library.

image

Figure 2. Alignments between the physical and genetic maps and the chromosome 19 sequence v2.0. Local physical maps showing overlapping bacterial artificial chromosome (BAC) clones of the haplotype hRUS (blue) (b) and the haplotype hrUS (green) (d). BAC clones are represented as vertical bars; light blue and light green bars designate BAC clones for which the insert size is estimated; dark blue and dark green bars are used to show BAC clones for which the insert size is unknown. BAC end sequences (BES) represented by red rectangles show BLASTn alignment onto the chromosome 19 sequence, and orange rectangles indicate no BLASTn alignments on chromosome 19. BAC ends represented by black rectangles were found to be duplicated and were not assigned with high confidence. Local genetic maps of Populus trichocarpa derived from genotypic and phenotypic data for 335 F1 individuals (c). The chromosome 19 sequence v2.0 (a) was aligned with the physical map of the hRUS haplotype. Red blocks represent gaps in the genome assembly; black blocks show sequences of chromosome 19. Red and black connected lines designate alignments by BLASTn searches with BES or with marker primer sequences, respectively. Alignments of both BES and marker primer sequences are indicated by green connected lines. Markers in italic designate markers for which no additional BAC clones were found in our BAC library.

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Comparative analysis between the two haplophysical maps revealed a longer physical map of at least 100 kb for RUS than for rUS between the markers I_1211and 78I01-R-1. The 1.7-Mb hRUS contig spans a genetic distance of 9.5 cM. The overall ratio of physical to genetic distance was c. 180 kb cM−1. However, the ratio was not uniform across the region. It ranged from 650 kb cM−1 between the markers RGAs352 and PTs589 to 35 kb cM−1 between the markers RGAs682-dc and RGAs735-AS.

Anchoring the genetic and physical maps onto the genome sequence v2.0 revealed a global conservation of marker order and physical distances (Fig. 2). However, a discrepancy was confirmed for a 200-kb region with seven markers flanked by the markers RGAs135-1 and RGAs352, which was inverted and located 2 Mb further upstream than expected. In the second assembly, a 250-kb region, corresponding to the top of scaffold 117 from v1.1, appeared to be misassembled in v2.0 (Fig. S2).

Both physical and genome sequences showed complex regions of duplicated sequences between the markers S_E5M5 and INRA1211. The physical length of this region was estimated to be 300 kb, where all the markers were multilocus and BES had multiple alignments. The complexity of this genomic area is also visible on the alignment of the two physical maps (Fig. S3). Taking into account information from genetic and physical maps and alignment of the genome sequences v1.1 and v2.0, we showed that the large inversion initially identified on the genome assembly was corrected in v2.0 (Fig. S2).

Genes in the interval containing the RUS and R1 loci

The alignment of the consensus genetic map on the proposed local re-order allowed us to define homologous intervals for RUS and R1 on the genome sequence (Fig. 3). R1 was located between the two markers G_79 and I_920, and RUS was located between Is_165-1 and RGAs297 (Fig. 3). A tBLASTn alignment with MEME domains allowed us to precisely locate the candidate gene families in the two separate genomic regions. R1 was located in a region containing a cluster of complete or partial BED-NBS-LRR, TIR-NBS-LRR and serine threonine kinase (STK) genes, whereas the RUS region only contained genes from the TIR-NBS-LRR class.

image

Figure 3. Putative resistance genes in the interval of RUS and R1. The consensus genetic map (a) between the Populus trichocarpa map of 1400 F1 individuals and the P. deltoides map of 1413 F1 individuals was aligned onto the rearranged chromosome 19 v2.0 (b). Genetic distances in cM are noted on the left of the linkage groups. Markers in bold bordering RUS and R1 were mapped on the consensus map and aligned onto the chromosome 19 sequence. Underlined markers were found only on the P. trichocarpa map. Markers in italic were found only on the P. deltoides map. The gene models are designated by their number without the prefix POPTR_0019s. B, BEAF and DREF DNA-binding finger (BED) domain; L, leucine-rich repeat (LRR) domain; n, nuclear localization sequence (NLS) domain; N, nucleotide-binding site (NBS) domain; T, Toll interleukin 1 receptor (TIR) domain. STK, serine threonine kinase. Discrepancies in the alignment between the consensus genetic map and the rearranged local scaffold 19 sequence could be caused by length differences between individual genetic maps. For marker details, see Fig. 1 and Supporting Information Fig. S1.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

A previous mapping study placed a major Mlp quantitative resistance locus (QRL), named RUS, on an unassigned LG of a P. trichocarpa parental map and a qualitative resistance locus, R1, onto LG XIX of the P. deltoides parental map (Jorge et al., 2005). In the present study, we undertook comparative genetic mapping in order to determine the precise location and linkage relationships between the QRL identified in P. trichocarpa and the R1 gene localized on the LG XIX of P. deltoides. For this purpose, it was proposed to score the (phenotype) resistance conferred by the QRL as a Mendelian classification. Thus, we took advantage of the clear bimodal distribution of the average US after inoculation with mono-uredinial Mlp strains to categorize clones from the mapping progeny into two classes showing contrasting levels of quantitative resistance. This classification resulted in the accurate localization of RUS on one end of the LG XIX of the P. trichocarpa parent. Moreover, we concluded from the alignment on the reference P. trichocarpa sequence of genetic markers common to both parental maps that R1 and RUS were two distinct loci, R1 being located between RUS and the telomere.

In order to better determine the precise location of the two resistance loci, it was necessary to construct fine-scale, integrated, genetic and physical maps. For this purpose, a large segregating population of 1415 individuals and a deep genome coverage BAC library with an average insert size of 150 kb were created and used for an iterative process of physical mapping, fine-scale genetic mapping and alignment to the reference genome sequence of P. trichocarpa. Genetic linkage mapping requires polymorphic markers that segregate within the mapping population in order to estimate the recombination rate. Alternatively, physical mapping overcomes the need for the segregation of polymorphic loci and allows mapping in segments in which recombination is suppressed. In addition, the availability of BAC clones allowed the design of tightly linked genetic and physical markers derived from BES. The combination of these approaches allowed the resolution of both the heterozygous status of our model and the complex structure of the region of interest (Moroldo et al., 2008). An ordering based on only the genome sequence would have led to the establishment of erroneous BAC contigs as a result of misassembly caused by gene duplications, and to different genome organizations between P. trichocarpa cv Nisqually-1 and P. trichocarpa 101-74 at these regions. Our study showed that the development of a fine, dense genetic map was crucial for the ordering of the BAC clones. Moreover, as the poplar genome is heterozygous, genetic markers allowed the assignment of BAC clones to either hRUS or hrUS haplotypes. Nevertheless, the presence of highly redundant NBS-LRR encoding genes, both within a cluster and between different clusters, complicated the development of specific markers (Tang et al., 2010). Another constraint arose from the nonhomogeneous pattern of heterozygosity between the two haplotypes of P. trichocarpa 101-74. The variability in both the level of redundancy and the heterozygosity led to a succession of intervals, some having high redundancy with moderate levels of heterozygosity, others showing the opposite situation and, finally, some having both high redundancy and high heterozygosity. Specific strategies were used for each type of interval. When the heterozygosity was high, the design of markers in BES was generally efficient. When the redundancy was high, as in the large cluster of NBS-LRR encoding genes, intergenic areas were targeted for genetic marker development. On the whole, markers designed from BES were more successful than those derived from the genome sequence.

A comparison of the two haplotype physical maps revealed differences in their structural organization, with at least 100 kb indel. Structural variations have been reported previously in grape (Velasco et al., 2007) and rice (Zhou et al., 2007), and suspected in poplar by Yin et al. (2004) at the vicinity of the Mer locus.

The alignment of the 101-74 integrated genetic and physical map with the poplar genome sequence also suggested a genomic inversion between Nisqually-1 and 101-74 (Fig. 1). However, the marker order was co-linear between the P. deltoides and P. trichocarpa 101-74 genetic maps, reinforcing the hypothesis that the inversion was most probably the result of an error in the sequence assembly of Nisqually-1. Indeed, it has been shown that automatic assembly of BAC contigs based on BAC fingerprints (Moroldo et al., 2008; Scalabrin et al., 2010) and shotgun sequence assembly (Vinson et al., 2005) of highly heterozygous genomes can lead to errors in the final output, where the high heterozygosity rate observed in the corresponding genomic area could affect the assembly quality. The construction in this study of a detailed integrated genetic and physical map of the genome region encompassing the RUS and rUS alleles allowed us to determine a more precise organization of this complex genomic region.

The relationship between physical and genetic distances in Populus was estimated to be 200 kb cM−1 on average (Tuskan et al., 2006). Substantial variation in this estimated rate was observed in this study along the cluster of resistance genes, ranging from 28 to 600 kb cM−1. Moreover, some intervals showed a complete suppression of recombination, whereas others behaved like hot spots of recombination. A large variation in the genetic to physical distance ratio has also been observed in barley in the vicinity of Mla (Wei et al., 1999), in wheat around Lr10 (Stein et al., 2000) and in poplar, where a suppression of recombination occurred in the interval containing the Mxc3 resistance gene (Stirling et al., 2001). A high rate of polymorphism between allelic sequences could explain the suppression of recombination. Indeed, among the 13 genetic markers closely linked to RUS, all except one were dominant, suggesting an important sequence polymorphism between the two haplotypes.

The alignment of the P. trichocarpa local genetic map along the Nisqually-1 genomic sequence (v2.0 version) showed that the RUS locus co-localized in a peritelomeric region. This region comprises clusters of 83 potential NBS-LRR encoding genes, one of the largest gene families in the Populus genome (Kohler et al., 2008; Yang et al., 2008; Yin et al., 2008). A similar example of a large NBS-LRR gene cluster located near the telomere has been described in detail recently in common bean (Geffroy et al., 2009). The NBS-LRR gene cluster has been found associated with two knobs. David et al. (2009) suggested that location in a subtelomeric region, together with the presence of satellite DNA, could be favorable to NBS-LRR proliferation. No evidence of satellite DNA was found using the Tandem Repeat Finder Program and BLASTn against the genome sequence v2.0 in our dataset of BES (Benson, 1999). The annotation of BES included in the physical maps revealed the presence of transposable elements (Table S5). Retroelements were the most abundant (Tarailo-Graovac & Chen, 2009). Nevertheless, a comparison of the density of repeats between the random dataset of BES (GenBank HN280500HN291979) and BES within the physical maps showed no accumulation of repeat elements in the cluster (Table S6).

The closest flanking genetic markers of RUS define an interval of 116 kb that spans four NBS-LRR coding genes and two other genes (one potential NBS-LRR gene with no MEME motif and one gene with unknown function). Although the genotype containing RUS has not been sequenced to date, these findings strongly suggest that RUS could be a member of an allelic series of the NBS-LRR genes. QRLs co-localizing with mapped known R genes, with NBS-LRR-like genes, have been described in several plant species (Wu et al., 2004; Wisser et al., 2005; Ballini et al., 2008; Tan et al., 2008; Danan et al., 2009; Geffroy et al., 2009). We demonstrated that QRLs co-localizing with major R genes were also observed in poplar.

R1 and other R genes inherited from P. deltoides are frequently associated with improved levels of quantitative resistance against compatible (i.e. virulent) Mlp strains. However, co-localization can result either from pleiotropy (i.e. residual effect of defeated R genes themselves) or from linkage between defeated R genes and sensu stricto quantitative resistance factors (Dowkiw et al., 2003; Dowkiw & Bastien, 2007). As listed by Poland et al. (2009), several hypotheses on the molecular mechanisms underlying the RUS locus could be suggested. Consistent with the proposal of Jones & Dangl (2006), Zhang et al. (2010) demonstrated that effector-triggered immunity associated with qualitative resistance participates in the quantitative resistance of Arabidopsis to Pseudomonas syringae. Moreover, Rcg1, one of the rare map-based cloned QRLs, has been found to encode an NBS-LRR resistance gene (Broglie et al., 2007). However, we cannot completely exclude the possibility that RUS belongs to another class of defense genes. The QRL clone, pi21, is recessive and is a proline-rich gene of unknown function that lacks similarity to any known defense-related major gene (Fukuoka et al., 2009). Further characterization of RUS and QRL, and comparison of the corresponding sequences in different Populus species, will provide a better insight into the possible origins and evolution of disease resistance in this genus.

Less surprising is the fact that the alignment of the P. deltoides local genetic map along version v2.0 showed that the R1 locus falls into a cluster of NBS-LRR rich in BED-NBS-LRR encoding genes, where a marker closely linked to the Mer locus is also present (Zhang et al., 2001; Lescot et al., 2004; Yin et al., 2004; Germain & Seguin, 2011). In the same mapping pedigree, Lefèvre et al. (1998) suggested the presence of other R genes in the close vicinity of R1 in order to explain segregations for compatibility to 93CV1 Mlp strain. The identification of rare recombinants by Dowkiw et al. (2003) supported this hypothesis.

In order to identify which of these candidate genes encodes RUS, the full sequencing of one or two BAC clones that overlap the RUS locus will be initiated. It is expected that the number of genes present in the three haplotypes considered here (101-74 hRUS, 101-74 hrUS and Nisqually-1) may vary substantially, as is often observed for NBS-LRR gene clusters (Zhou et al., 2007). Moreover, the reference sequence from Nisqually-1 is a pseudomolecule that contains a chimeric mixture of maternal and paternal haplotypes. As such, the number of gene models proposed in the RUS region may not be complete or accurate. Finally, the observed co-linearity between P. deltoides and P. trichocarpa in the region should facilitate the future isolation of the R1 locus via comparative genome mapping.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors thank I. Le Clainche, F. Bitton, C. Guichard and C. Boussardon for technical assistance, J. Just and M. Villar for helpful discussions, P. Poursat and collaborators at the Experimental Unit of INRA Orléans for resistance assessments, and Anne Françoise Adam Blondon for the insightful comments on the manuscript. This work was supported by INRA (Program ECOGER), the French Ministry for Agriculture and Fisheries (Program 142, sous action 27) and the BRG (Bureau des Ressources Génétiques) AO 2007-2008.

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  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1 Local genetic maps anchored onto the genome sequence v1.1.

Fig. S2 Detailed alignment of the two versions of the Populus genome assembly (v1.1 and v2.0) of the region encompassing the super cluster of resistance genes.

Fig. S3 Alignment between the two haplotypical physical maps.

Table S1 Sequences of markers derived from the bulk segregant analysis (BSA) approach

Table S2 Primer pairs developed for genetic and physical marker development

Table S3 Bacterial artificial chromosome (BAC) insert sizes and location of BAC end sequences (BES) on the genome sequence v2.0

Table S4 Bacterial artificial chromosome (BAC) end sequences in the genomic target region of physical maps

Table S5 Bacterial artificial chromosome (BAC) end sequence analysis

Table S6 Repetitive and transposable element composition of bacterial artificial chromosome (BAC) end sequences (BES) (GenBank: HN280500–HN291979) and BES integrated to the physical maps

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