A physical map of the highly heterozygous Populus genome: integration with the genome sequence and...

A physical map of the highly heterozygous Populus genome:integration with the genome sequence and genetic map andanalysis of haplotype variation

Colin T. Kelleher1, Readman Chiu2, Heesun Shin2, Ian E. Bosdet2, Martin I. Krzywinski2, Chris D. Fjell2, Jennifer Wilkin1,

TongMing Yin3, Stephen P. DiFazio3,†, Johar Ali2, Jennifer K. Asano2, Susanna Chan2, Alison Cloutier2, Noreen Girn2, Stephen

Leach2, Darlene Lee2, Carrie A. Mathewson2, Teika Olson2, Katie O’Connor2, Anna-Liisa Prabhu2, Duane E. Smailus2, Jeffery M.

Stott2, Miranda Tsai2, Natasja H. Wye2, George S. Yang2, Jun Zhuang1, Robert A. Holt2, Nicholas H. Putnam4, Julia Vrebalov5,

James J. Giovannoni5, Jane Grimwood6, Jeremy Schmutz6, Daniel Rokhsar4, Steven J.M. Jones2, Marco A. Marra2, Gerald A.

Tuskan3, Jorg Bohlmann1,7,8, Brian E. Ellis1, Kermit Ritland7, Carl J. Douglas8,* and Jacqueline E. Schein2

1Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z3, Canada,2Genome Sciences Centre, 100-570 West 7th Avenue, Vancouver, BC V5Z 4S6, Canada,3Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6422, USA,4US Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598, USA,5Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853, USA,6Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5329, USA,7Department of Forest Sciences, University of British Columbia, Vancouver, BC, V6T 1Z4, Canada, and8Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada

Received 1 October 2006; revised 9 February 2007; accepted 23 February 2007.*For correspondence (fax +1 604 822 6089; e-mail [email protected]).†Present address: Department of Biology, West Virginia University, Morgantown, WV 26506, USA.

Summary

As part of a larger project to sequence the Populus genome and generate genomic resources for this emerging

model tree, we constructed a physical map of the Populus genome, representing one of the few such maps of

an undomesticated, highly heterozygous plant species. The physical map, consisting of 2802 contigs, was

constructed from fingerprinted bacterial artificial chromosome (BAC) clones. The map represents approxi-

mately 9.4-fold coverage of the Populus genome, which has been estimated from the genome sequence

assembly to be 485 � 10 Mb in size. BAC ends were sequenced to assist long-range assembly of whole-

genome shotgun sequence scaffolds and to anchor the physical map to the genome sequence. Simple

sequence repeat-based markers were derived from the end sequences and used to initiate integration of the

BAC and genetic maps. A total of 2411 physical map contigs, representing 97% of all clones assigned to contigs,

were aligned to the sequence assembly (JGI Populus trichocarpa, version 1.0). These alignments represent a

total coverage of 384 Mb (79%) of the entire poplar sequence assembly and 295 Mb (96%) of linkage group

sequence assemblies. A striking result of the physical map contig alignments to the sequence assembly was

the co-localization of multiple contigs across numerous regions of the 19 linkage groups. Targeted sequencing

of BAC clones and genetic analysis in a small number of representative regions showed that these co-aligning

contigs represent distinct haplotypes in the heterozygous individual sequenced, and revealed the nature of

these haplotype sequence differences.

Keywords: Populus trichocarpa, physical map, genome integration, BAC end sequences, poplar genomics,

haplotype diversity.

Introduction

Black cottonwood (Populus trichocarpa Torr. & Gray) is a

genetically highly variable outbreeding tree species that

primarily inhabits floodplains and river margins. It is wind-

pollinated and propagates sexually through minute wind-

borne seeds, often dispersed long distances along river

corridors (Braatne et al., 1996; Farmer, 1996). The species

ª 2007 The Authors 1063Journal compilation ª 2007 Blackwell Publishing Ltd

The Plant Journal (2007) 50, 1063–1078 doi: 10.1111/j.1365-313X.2007.03112.x

extends through approximately 30 degrees of latitude along

western North America, and ranges inland to the Rocky

Mountains (Burns and Honkala, 1990). Populus (poplar)

species are economically important crops in temperate cli-

mates throughout the world for a variety of purposes,

including wood pulp, paper and biomass, and for use in

phyto-remediation and waste water treatment.

Because of its relatively small genome size (485 Mb), the

availability of genetic and genomic resources, and ease of

propagation and genetic manipulation, Populus provides a

useful model system to study a number of biological

processes of importance to woody perennial plants, such

as dormancy, secondary xylem (wood) development, meta-

bolism and responses to environmental stress (Strauss and

Martin, 2004; Taylor, 2002). As commercial species, poplars

are of most interest with respect to wood production, and

Populus has thus been the focus of numerous studies

examining the molecular biology of wood and secondary

wall formation (Plomion et al., 2001; Schrader et al., 2004;

Sterky et al., 1998, 2004). Poplar hybrids, such as P. tricho-

carpa · P. deltoides L., grow much faster than either paren-

tal species alone, and are widely used in plantations as a

fast-growing source of wood and fiber. In terms of its

ecological adaptation, poplar is also a valuable study

subject. For example, due to its size and longevity, poplar

provides an ideal system to study spatial and temporal

patterns of local and systemic defenses against herbivores

(Arimura et al., 2004). A large number of genetic adaptations

are likely to explain its ecological success over broad

geographical and climatic ranges (Cronk, 2005). It forms

hybrid zones with multiple sympatric species from the

Tacamahaca and Aigeiros sections of the genus (Eckenwal-

der, 1996), making it useful for studying factors involved in

species distinction and the biological species concept

(Rieseberg et al., 1999).

To further expand the use of Populus as a model woody

perennial species, the development of genomic tools and

resources is essential. Primary among those recently made

available is the full Populus genome sequence, derived from

a wild P. trichocarpa individual (named Nisqually-1), and the

accompanying genome annotation (Tuskan et al., 2006). A

multitude of additional resources, including controlled cross

populations, cross-species molecular markers, EST collec-

tions and full-length cDNAs, have been developed and

employed to further poplar genomics (Ralph et al., 2006;

Strauss and Martin, 2004; Tuskan et al., 2006). Populus has

been used extensively in experimental and population

biology studies, and dense genetic maps are available for

a number of species within the genus (Cervera et al., 2001;

Yin et al., 2004). With the growth in available resources,

Populus is becoming increasingly attractive as a model

organism for tree biology (Tuskan et al., 2004a).

Clone-based physical maps have been shown to be useful

in providing a framework to aid in the generation and

validation of genome sequence assemblies and as a valu-

able resource for map-based cloning (Chen et al., 2002;

Gregory et al., 2002; Krzywinski et al., 2004; McPherson

et al., 2001; Mozo et al., 1999; Nelson et al., 2005; Wallis

et al., 2004). To enhance the resources available for poplar

genomics and to assist assembly of the poplar whole-

genome shotgun sequence and its integration with the

genetic map, we undertook the generation of a poplar

physical map by large-scale fingerprinting of a bacterial

artificial chromosome (BAC) library. The library was con-

structed using DNA from the P. trichocarpa Nisqually-1

individual and so has exact correspondence with the DNA

used for the genome sequence assembly. In physical map

construction, similarities in large insert clone fingerprint

patterns are used to identify clones derived from overlap-

ping regions of the genome, and this information is used to

create a series of ordered, overlapping clones representing

contiguous genomic regions (contigs). End sequence reads

from physical map clones [e.g. BAC end sequence (BES)

reads] can be used to align physical map contigs to

sequence assemblies, thereby integrating physical maps to

the genome sequence. The integration of genetic and

physical maps has also been shown to be a useful genomic

resource for map-based cloning (Chen et al., 2002).

Poplar presents a particular challenge for physical map-

ping and genome sequencing efforts due to its high level of

heterozygosity and its gene and genome duplications

(Sterck et al., 2005; Tuskan et al., 2006). These two phenom-

ena (heterozygosity and duplication) could confound both

sequence and fingerprint contig assembly. A high level of

heterozygosity could lead to independent assembly of

haplotypes in hyper-variable genomic regions. Likewise,

duplicated regions could lead to mis-assembly due to the

presence of genomic regions of high sequence similarity at

multiple locations within the genome. Other plant species

subject to physical mapping efforts to date include Arabid-

opsis (Mozo et al., 1999), maize (Fang et al., 2003; Nelson

et al., 2005), rice (Chen et al., 2002) and soybean (Wu et al.,

2004). Maize, rice and soybean have undergone consider-

able domestication and inbreeding, which has led to a more

homogenized genetic complement (Buckler et al., 2001;

Wang et al., 1999), and Arabidopsis is an inbreeding species

with consequent low heterozygosity (Abbott and Gomes,

1989; Bustamante et al., 2002). In contrast, Populus is an

obligate dioecious outcrosser, with high levels of gene flow

due its wind-pollinated habit. Analysis of 4.2 million

phred 20 shotgun sequence end reads used for the P. tricho-

carpa Nisqually-1 genome assembly supports the high level

of heterozygosity and haplotype diversity in the Nisqually-1

individual, with an overall rate of approximately 2.6 poly-

morphisms (SNPs and insertion/deletions) per kb (Tuskan

et al., 2006).

In this paper, we report generation of the poplar BAC

fingerprint physical map, tanchoring of the physical map to

1064 Colin T. Kelleher et al.

ª 2007 The AuthorsJournal compilation ª 2007 Blackwell Publishing Ltd, The Plant Journal, (2007), 50, 1063–1078

the sequence assembly through BES alignments, and

integration of the genetic and physical maps using markers

common to both. Unexpectedly, alignment of physical map

contigs onto the poplar genome sequence assembly

revealed a consistent pattern of co-aligning BAC contigs.

These were presumed to represent haplotypes, a finding

confirmed by genetic analysis of representative examples.

Targeted sequencing of representative BAC clones uncov-

ered the details of extensive indel and SNP haplotype

polymorphisms within this P. trichocarpa individual, but

the haplotypes sequences were otherwise co-linear, and we

found no evidence of haplotype-specific gene complements,

as has been found in maize. The physical map and its

integration with the poplar genome and genetic map are

core resources important in establishing poplar as a model

system for tree biology.

Results

BAC library and fingerprint map assembly

A BAC library of 48 384 clones created from the Nisqually-1

individual was used to construct the physical map. The BACs

were fingerprinted with HindIII, and successful fingerprints

were obtained for 46 025 (95%) of the clones. On average,

fingerprints contained 31 restriction fragments within the

range of 600–30 000 bp. The average insert size of the

clones, based on the fingerprint data, was 100 kb, thus the

fingerprinted clones represented approximately 9.4-fold

coverage of the 485 � 10 Mb Populus genome, as estimated

from the genome sequence (Tuskan et al., 2006).

An initial automated assembly of the clone fingerprints

was performed using FPC (fingerprinted contigs) software

(Soderlund et al., 1997, 2000). This assembly was performed

at relatively high stringency to avoid binning together clones

from unrelated regions of the genome. All contigs contain-

ing >10 clones (representing approximately 40% of all clones

in contigs) were manually edited to refine the clone order

derived by FPC, using clone and contig editing tools

available within the FPC software. During this process, a

total of 515 clones were identified as having fingerprints

resulting from either cross-well contamination or partial

enzyme digestion, and these were removed from the

available clone set. Contig merges were performed manu-

ally where supported by the fingerprint data. Contigs with

£10 clones had their clone order refined using our automa-

ted contig ordering application, CORAL (Flibotte et al., 2004),

which became available after manual editing had been

partially completed. During the manual review phase, we

identified small subsets of overlapping clones internal to

some contigs that, while otherwise highly similar in their

restriction fragment patterns to their closest neighbors in the

contig, contained restriction fragment pattern irregularities

in the form of missing and extra fragments. These irregular

fragments were commonly shared within the small subset of

clones but not with the larger group of clones in the contig,

suggesting underlying, biologically relevant DNA differ-

ences. These clones potentially represented restriction frag-

ment differences resulting from sequence polymorphisms

between the two parental haplotypes (i.e. the same genomic

region), or clones containing duplicated or repetitive ge-

nomic sequences (i.e. similar sequence but from different

regions of the genome). In the absence of any orthogonal

evidence suggesting the underlying nature of the observed

differences, and wishing to avoid potentially collapsing

independent regions of the genome into the same map

contig, we removed these clone subsets from the larger

contigs and placed them into independent contigs.

Following completion of manual editing of the largest

contigs and automated clone ordering for the remaining

contigs, automated scripts were employed to compare

clones at contig ends to identify additional contig merges.

This was performed at a reduced stringency from that of the

initial fingerprint assembly. Seven rounds of automated

merging were performed with varying parameters for

required fingerprint similarity at the merge point (see

Experimental procedures). This included two rounds in

which singleton clones (those that did not assemble into

contigs with the parameters used for the initial automated

assembly) were assessed for their potential to bridge contigs

that otherwise would have insufficient similarity between

their end clones to permit a merge, and one round in which

contigs with £3 clones were inserted internally into larger

contigs where supported by fingerprint similarity. Following

each round of merging, a subset of the merges was

manually reviewed to ensure the parameters were suffi-

ciently stringent to prevent incorrect merges being per-

formed. At the end of this process, the map consisted of 3471

contigs. On average, contigs contained 11 clones, ranging

from 2 to 128 clones per contig (excluding a single excep-

tional contig containing >1000 clones, described below).

When the sequence assembly became available (September

2004, JGI Populus trichocarpa, version 1.0), additional con-

tig merges were performed based on supporting evidence

from the BES alignments to the sequence scaffolds (see

below and Experimental procedures).

BAC end sequencing

To facilitate integration of the BAC map with both the Pop-

ulus sequence assembly and genetic maps, end reads were

obtained from the clones in the BAC library. A total of 81 904

BES reads passed quality filters (see Experimental proce-

dures), with an average phred 20 (Ewing and Green, 1998;

Ewing et al., 1998) read length of 504 bp, corresponding to

more than 41 Mb of poplar genomic DNA sequence, nearly

10% of the total genome size. These reads represented

44 422 BAC clones, 37 482 (84%) of which had reads from

Physical mapping of the Populus genome 1065


both ends. The BESs provided a clone-linked sequence re-

source for use in aligning BACs to the genomic sequence

and for identification of simple sequence repeats (SSRs) to

be used in genetic mapping experiments. In addition, the

BESs were employed during assembly of the Populus gen-

ome sequence, providing a powerful aid to long-range

contiguity of the assembled scaffolds (Tuskan et al., 2006).

The availability of the BESs also allowed us to decipher

the nature of one unusual contig in the physical map. This

contig contains 1271 clones, approximately 10 times more

than the 128 clones in the next-most populated contig, and

well above the map average of 11 clones per contig. The vast

majority of these clones are entirely redundant, suggesting

that they potentially represent a duplicated sequence within

the genome or cloning bias resulting in the over-represen-

tation of a small genomic region. Analysis of BESs derived

from clones in this contig showed that they had high

similarity to Arabidopsis and poplar chloroplast DNA, sug-

gesting that the genomic DNA used in the construction of

the BAC library was contaminated with chloroplast DNA,

and that BAC clones derived from the chloroplast DNA had

assembled into a deeply redundant contig in the map. This

phenomenon was also encountered during genome sequen-

cing efforts, where it was revealed that, in some of the DNA

libraries used, the poplar total genomic DNA was contam-

inated with chloroplast genome DNA, leading to separate

assembly of the poplar chloroplast genome at a very high

level of sequence depth (Tuskan et al., 2006).

Integration of genetic and physical maps

To anchor the physical map to poplar genetic maps, we used

a P. trichocarpa Nisqually-1 genetic map based on simple

sequence repeat (SSR) and AFLP (amplified fragment length

polymorphisms) markers in a pedigree (family 545) in which

Nisqually-1 was the female parent. The SSR markers were

primarily developed from the BES reads. The remaining ten

SSRs have been used in other poplar genetic map studies

(Cervera et al., 2001; van der Schoot et al., 2000; Tuskan

et al., 2004b; Yin et al., 2004) or were designed from

assembled shotgun sequence. In total, 122 BES-derived

SSRs were used for construction of the Nisqually-1 genetic

map. In addition, 123 dominant AFLP markers (Vos et al.,

1995) were added to the genetic mapping analysis. This

map, and a consensus Populus map derived from merges

with maps derived from other Populus pedigrees, including

P. trichocarpa family 13 (Yin et al., 2004) will be described in

detail elsewhere (T. Yin et al., unpublished).

We evaluated use of the in silico identified SSRs derived

from the BESs as genetic markers for the purpose of

integrating the physical map contigs onto the family 545

genetic map. This evaluation was based solely on markers

from the Nisqually-1 pedigree. SSRs were selected based on

contig size to map large physical map contigs to the genetic

map. From the 122 BES-derived SSRs on the genetic map,

119 of the corresponding contigs were mapped to the

Nisqually-1 pedigree (family 545). An example of this

physical–genetic map integration is shown in Figure 1 for

LG X. Table 1 summarizes the total number of contigs

mapped onto family 545 using BES-derived SSRs and the

percentage of each LG covered by the contigs. The average

contig coverage of the LGs using these SSRs alone was 22%,

a significant percentage given the relatively small number of

SSRs tested. Given the success of this approach, we next

evaluated a much larger pool of 3506 potential SSR markers,

derived from in silico analysis of BESs. Of these, 1769 passed

the BLAST criteria, based on primer sequence alignment to

the LG sequence assembly, and 352 were mapped in family

13 while 392 failed mapping. These data will be presented in

detail elsewhere (T. Yin et al., unpublished). Given the

success of mapping physical map contigs to LGs using the

BES-derived SSRs (Figure 1 and Table 1), and the 50%

success rate of mapping additional BES-derived SSRs, it

would be possible to anchor a substantial fraction of

physical map contigs to the genetic map using the larger

set of BES-derived SSR markers. However, due to parallel

work on aligning the physical map and the genome

sequence assembly, we decided to concentrate on a more

Figure 1. The position of physical map contigs on the LG X genetic map.

Individual contigs are represented as different colored sections along the

linkage group. Contigs were mapped to the LGs using BES-derived SSRs

(markers beginning with G). Approximately 29% of the LG is covered by the

contigs.



high-throughput and automated approach to linking the

physical map and the genome assembly – namely, align-

ment through BESs.

Alignment of the BAC map to the genome sequence

assembly

Alignment of the BESs to version 1.0 of the JGI Populus

trichocarpa assembly (http://genome.jgi-psf.org/Poptr1/

Poptr1.home.html) enabled large-scale integration of the

physical map with the genome sequence. A total of 73 374

end sequence reads derived from 42 809 unique clones

passed the alignment filters (see Experimental procedures).

A total of 34 770 clones (76% of all map clones) had

informative BES alignments, of which 22 526 (65% of clones

with informative reads) had paired-end alignments. Using

these clone alignments, we positioned the contigs on the

genome sequence assembly. Examination of the order and

orientation of the map contigs with respect to the sequence

assembly identified adjacent map contigs with closely jux-

taposed assembly coordinates, indicating candidate contig

merges that had not met the stringency requirements for

automated merges based on fingerprint similarities alone

(probably due to insufficient overlap between the contig

ends). With the supporting sequence coordinate evidence,

and where substantiated by the fingerprint data, these

merges were performed, producing a final map with 2802

contigs and 5746 singletons. The average contig size is

466 kb, with some contigs larger than 1 Mb. The distribution

of fingerprint contig sizes is shown in Figure 2.

A total of 2226 of the 2802 contigs aligned to unique

regions in the genome assembly. An additional 185 contigs

mapped to multiple sequence regions, with the majority of

these (87%) mapping to two regions. The remaining 391

contigs could not be positioned on the sequence assembly

using our criteria. Thus, 86% of the physical map contigs

were aligned to the genome sequence assembly, and these

contigs contained 97% of all clones assigned to map contigs.

The scaffolds in the version 1.0 Populus genome assem-

bly (Tuskan et al., 2006; Yin et al., unpublished) contain in

total 485 Mb of genomic DNA, of which 308 Mb are

anchored to the 19 Populus LG assemblies. BAC contigs

aligned to the genome sequence represent 384 Mb (79%) of

the entire assembled poplar sequence and 295 Mb (96%) of

the LG assemblies. As shown in Table 2, all LG assemblies

with the exception of LG XIX have >90% coverage in the BAC

map, based on the contig alignments. A graphical display of

the contig alignment results for all 19 LGs is provided in

Figure S1. An example of this display, showing LG X, is

provided in Figure 3. In a small number of cases, contigs

were mapped to two genomic regions within a linkage group

(represented by internal arcs linking contigs in Figure 3 and

Figure S1). These could potentially indicate regions of

repetitive DNA or genome duplication. They may also arise

from fingerprints derived from mixed DNAs, resulting from

cross-well contamination in the library plates, which may

cause a mis-assembly in the fingerprint map (contigs joined

in error).

Of obvious note in Figure 3 are the complex contig

alignment patterns in many regions, with multiple contigs

aligning to the same sequence region. These alignment

patterns are also observed in the other LG alignments

Table 1 Summary of the integration of the genetic and physicalmaps through BAC end sequence derived SSR markers

Linkagegroup

No. contigs mappedto the Nisqually-1 pedigree

Estimated %coverage of LG1

LG_I 11 16LG_II 9 23LG_III 10 26LG_IV 4 12LG_V 8 29LG_VI 7 18LG_VII 4 14LG_VIII 7 36LG_IX 5 25LG_X 10 28LG_XI 6 28LG_XII 4 13LG_XIII 7 29LG_XIV 8 19LG_XV 5 26LG_XVI 5 13LG_XVII 3 30LG_XVIII 5 19LG_XIX 1 6Totals 119 Average 22 %

The data presented is for those markers mapped in the Nisqually-1pedigree and used to evaluate the integration of the BAC physicalmap and the genetic map.1The % LG cover values given are only for those physical map contigsmapped in Nisqually-1. The percentage coverage of the LG by contigswas calculated a posteriori of the poplar sequence assembly and isbased on the contig size estimates.

Figure 2. A summary of contig size distribution within the physical map.

The number of clones per contig varied from 2 to 189, with an average of 14.



(Figure S1). These patterns suggest two theoretical possi-

bilities. First, the genome sequence may be mis-assembled

in these locations, for example by artifactual collapse of

duplicated but physically distinct regions during the gen-

ome assembly process. However, the large-scale co-linearity

of the genetic map and sequence assembly (Tuskan et al.,

Table 2 Summary of integration of thesequence assembly linkage groups (LGs)with the physical map

Linkagegroup

No. alignedmap contigs

No. alignedBACs in contigs

No. alignedsingletons

Estimated %coverage of LG1

LG_I 219 2443 344 95.2LG_II 170 1840 266 97.0LG_III 103 1290 145 92.8LG_IV 106 1125 170 95.5LG_V 104 1291 182 97.0LG_VI 121 1425 186 96.3LG_VII 84 950 107 97.0LG_VIII 84 1388 161 99.0LG_IX 66 1010 128 99.9LG_X 98 1708 175 96.0LG_XI 93 996 112 95.6LG_XII 81 953 152 96.8LG_XIII 70 904 106 92.3LG_XIV 86 1211 115 97.8LG_XV 64 830 92 96.7LG_XVI 81 918 140 97.7LG_XVII 35 390 42 94.4LG_XVIII 83 901 135 95.7LG_XIX 76 615 105 87.1Totals 1831 22188 2863 95.8

1The % coverage was calculated using the distances between aligned clones, obtained from thesequence assembly.

LGX

Figure 3. Fingerprinted BAC clone and contig

layout on the LG X sequence assembly.

Clone placement is based on BES alignments to

the genome sequence. The ideogram of LG X is

composed circularly (outermost ring), with 1 Mb

spans colored in alternating black and white

strips. The innermost histogram track (black)

illustrates the depth of aligned BAC clone cover-

age, with each concentric circle representing a

fivefold clone depth. The next outer histogram

track (red) shows the coverage provided by

aligned BAC map clones not assigned to contigs

(singletons). The next track shows the extent of

anchored contigs, coded with an alternating

color scheme. The final track inside the ideogram

circle shows the sequence alignment position of

individual aligned clones in each contig, colored

by map contig assignment. Fingerprint contigs

aligning to two different regions of the sequence

assembly are linked by arcs. Genetic markers

(SSRs) derived from BESs are indicated by

triangles on the sequence track. Green triangles

indicate SSRs mapped in the Nisqually-1 pedi-

gree, blue triangles indicate those mapped in

another P. trichocarpa pedigree (family 13), pink

triangles indicate those for which mapping

failed, and gray triangles indicate SSRs that have

not yet been tested, illustrating that a large

number of well-spaced SSRs remain for use in

future use in integration of the genetic and

physical maps. The outer track of triangles shows

those SSRs used for preliminary mapping of

contigs to LGs. The diagram was prepared using

Circos (http://mkweb.bcgsc.ca/circos/).



2006) suggests that mis-assembly at such a large scale is

unlikely. A second possibility is that the clones in overlap-

ping BAC contigs represent individual Nisqually-1 haplo-

types that were assembled independently into distinct

haplotype-specific contigs in the physical map due to

haplotype-specific sequence diversity. This hypothesis was

investigated further, as described below.

Haplotype sequence diversity

To assess the possibility that distinct physical map contigs

aligning to the same locations in the sequence assembly

represented the two Nisqually-1 haplotypes, we selected

and sequenced eight BAC clones representing four exam-

ples of putative haplotype differences, based on fingerprint

pattern discrepancies and presence in contigs co-aligning to

the genome. The BAC clones represent genomic regions on

LGs I and XIV. We compared the sequences of the BACs in

detail to ascertain the nature of the differences and to iden-

tify nucleotide variations that could be genetically mapped

to test for allelism. We describe below representative results

for two of the four examples. Table S1 contains a summary

of the polymorphisms found in all four pairs of sequenced

BAC clones.

On LG I, contig 846, a small contig consisting of seven

clones, has sequence assembly coordinates spanning

105 kb of this LG (http://genome.jgi-psf.org/Poptr1/Poptr1.-

home.html). These coordinates are contained entirely within

the LG I alignment coordinates of contig 8, a larger contig

containing 127 clones and spanning 1263 kb (Figure 4a). The

contig 8 clones that lie within the region of co-alignment

share consistent HindIII fingerprint patterns, suggesting that

they represent a single haplotype. The corresponding BAC

clones in contig 846 have HindIII restriction patterns highly

similar to, but distinct from, those in contig 8, suggesting

that the clones in contig 846 potentially represent the

alternative haplotype for this region. The LG I sequence

assembly alignment coordinates for clone T0048O04 from

contig 846 overlap by 89 kb with the LG I alignment coordi-

nates for clone T0068B19 from contig 8 (Figure 4a), which is

98% of the alignment length for T0068B19. However, the two

clones differ in >20 restriction fingerprint fragments (Fig-

ure 4b). Comparison of the clone sequences revealed they

were substantially co-linear, sharing a high degree of

identity (84%) in the overlap region, consisting of segments

of complete identity interspersed with localized differences

in the form of indels and nucleotide substitutions, some of

which affect HindIII recognition sites. Figure 5 illustrates the

differences between the sequences relative to the resulting

restriction fingerprint patterns of the two BAC clones. One

large (11 kb) and two small (1244 bp and 189 bp) indels were

identified, in addition to five nucleotide differences, that

impact HindIII recognition sequences such that these recog-

nition sites are present in one clone but absent in the other.

As shown in Figure 5, these five SNPs, together with the

three indels noted above, account for the anomalous HindIII

fingerprint patterns that resulted in these BAC clones

assembling into distinct contigs. Additional analysis of the

sequences revealed a further 888 single nucleotide differ-

ences between the clones, and 217 smaller indels, ranging

Figure 4. Illustration of two of the four putative

haplotype-specific contigs analyzed.

(a) Schematic representation of the relationship

between contigs 8 and 846 on LG I and contigs

160 and 162 on LGX IV. Sequence assembly

coordinates are from version 1.0 of the P. tricho-

carpa genome assembly (Tuskan et al., 2006).

The approximate locations of BAC clones

T0068B19 (contig 8), T0048O04 (contig 846),

T0021 J18 (contig 160) and T0033M07 (contig

162) are shown.

(b) HindIII fingerprint images of the BAC clone

pairs aligning to LGs I and XIV. Thin lines drawn

through the DNA bands indicate restriction frag-

ments identified by BandLeader software; red

lines indicate fragments that are unique to each

potential haplotype.



from 1 to 275 bp. In total, 14.8% of the clone overlap region

contains alignment gaps. Sequence analysis of two addi-

tional BAC clone pairs identified within contig 8, and

displaying restriction fragment differences also suggestive

of haplotype differences, revealed a similar pattern of large

regions of sequence identity interrupted by several indels

ranging in size from 10–1000 bp and SNPs at HindIII

recognition sites.

A similar analysis was carried out on representative BAC

clones T0021J18 and T0033M07 from contigs 160 and 162,

respectively, two small contigs that have overlapping align-

ments on LG XIV (Figure 4a). Sequence comparison of these

two BAC clones revealed that they were co-linear, but

differed by 440 single nucleotide changes and 83 indels,

ranging in size from 1 to 129 bp. However, only 0.5% of the

clone overlap region contains alignment gaps. Two of the

SNPs and one 20 bp indel affect HindIII recognition sites.

The observed HindIII fingerprint pattern differences in the

clones correlated with the fragment sizes predicted by the

BAC sequences (data not shown).

The results of these BAC sequence comparisons were

compatible with haplotypic variation as the cause of the

observed restriction fingerprint pattern variation. To test the

hypothesis that LG I clones T0068B19 and T0048O04 repre-

sent two Nisqually-1 haplotypes, and thus that the contigs

into which they were placed are haplotype-specific, one SNP

site in a HindIII recognition sequence in T0068B19 was

mapped in parallel with the corresponding alternative SNP

from T0048O04 in family 545. As shown in Figure 6, the

alternative SNPs mapped to the same location on LG I, but in

reverse phase. This illustrates that they are alleles of the

same locus, consistent with the interpretation that BAC

clones T0068B19 and T0048O04 are derived from distinct

Nisqually-1 haplotypes. Similarly, genetic mapping in family

545 of putative alternative alleles at a HindIII SNP in clones

T0021J18 and T0033M07 showed that they map to the same

location on LG XIV. These results indicate that considerable

haplotype sequence variation exists in the Nisqually-1

genome, including numerous small to large indels and

SNPs, and that in regions where the variation sufficiently

perturbs the fingerprint patterns, this resulted in the creation

of haplotype-specific contigs during the map assembly

process, as indicated in Figure 3 and Figure S1.

To examine the manner in which these haplotype-specific

sequences were assembled into the genome sequence,

which is derived from reads generated from both haplo-

types, we compared the sequences of the BAC clones to

those of the corresponding region from the version 1.1

poplar genome assembly (http://genome.jgi-psf.org/

Poptr1_1/Poptr1_1.home.html). An example of this analysis

is shown in Table 3, which illustrates the sequence align-

ment of BAC T0068B19 (contig 8) to the corresponding

region of the assembly. The genome assembly in this region

is a mosaic of the sequences of T0068B19 and the corres-

ponding alternative haplotype BAC T0048O04, as recognized

by diagnostic indels specific to each clone sequence.

Notably, the 11 kb region deleted in T0048O04 relative to

T0068B19 is also absent in the genome assembly. Analysis

of this haplotype-specific 11 kb sequence revealed numer-

ous short open reading frames (ORFs), and two large ORFS

of 229 and 154 amino acids. However, no matches of the

ORFs to poplar ESTs were found, and none had significant

Figure 5. Depiction of the sequence differences between clone T0068B19 (contig 8) and clone T0048O04 (contig 846) affecting the HindIII fragment patterns.

Individual restriction fragments for each clone are represented by colored boxes, and numbered underneath from left to right using the sequence of clone T0068B19

as the reference. Matching fragments are represented in light green, and are assigned a consensus fragment number in black text. Unmatched fragments in

T0048O04 are assigned alphabetical identifiers and colored differentially based on the nature of the sequence difference. The various types of sequence differences

are classified in the key on the lower left. The region from fragments 10 to 15 of T0068B19 is expanded in the center for a more detailed depiction of the alignment

result.



similarity to predicted proteins in the non-redundant

sequence database.

Sequence analysis of all four pairs of haplotype-specific

BAC clones showed that, in each case, the haplotype-specific

sequences in regions of clone overlap were co-linear, but

contained extensive indel polymorphisms (Figures 4 and 5,

Table 3 and Table S1). This led to alignment gaps affecting

between 0.5 and 14.8% of the overlapping sequences

(Table S1). Despite large-scale haplotype sequence co-lin-

earity, these results raised the possibility that, in addition to

ORFs in the large 11 kb haplotype-specific sequence des-

cribed above, gene content and/or order could be distinct in

the two haplotypes, as has been described for several

regions of the maize genome (Brunner et al., 2005; Song and

Messing, 2003; Wang and Dooner, 2006).

To determine whether indel polymorphisms could affect

gene content and/or order in a haplotype-specific manner,

we examined in detail the locations and potential effects of

31 haplotype-specific indels ranging in size from 19 to

1244 bp, across a total of >320 kb of sequence from each

haplotype, as summarized in Table 4. We first examined the

genome assembly within the regions represented by the

sequenced BAC clones (http://genome.jgi-psf.org/Poptr1_1/

Poptr1_1.home.html), and found a total of 25 annotated

genes (Table 4). When the locations of the selected indels

were mapped relative to the genome assembly and anno-

tated genes, we found that in 30 of 31 cases, the indel fell

either within an intergenic region lacking an ORF (29 cases)

or within an intron (two cases). In one case, a 24 bp

haplotype-specific indel (absent in the genome assembly)

resulted in a predicted haplotype-specific eight amino acid

insertion in a predicted gene product (eugene3.00141429).

However, as this short gene appears to encode only a

fragment of an ammonium transporter protein, and lacks

EST expression support (http://genome.jgi-psf.org/Poptr1_1/

Poptr1_1.home.html), it is questionable whether it is func-

tional. In summary, our data show that extensive haplotype-

specific polymorphism exists in poplar, ranging from SNPs

to indels of variable size, up to >10 kb. However, haplotype-

specific sequences were largely co-linear, and we found no

evidence for differences in gene content or order between

the two haplotypes in the regions we examined.

Discussion

The Populus trichocarpa genome represents that of an un-

domesticated and highly heterozygous plant species. We

Figure 6. Genetic maps of LG I, showing mapping of contig 8/846 SNPs.

Positions of LG I genetic markers are shown on the right, with distances in

centimorgans (cM) on the left. The mapped locations of two putative

alternative SNP alleles (SNP2_1 and SNP2_2) polymorphic between clones

T0068B19 and T0048O04 are shown.

Table 3 Comparison of haplotype content in the poplar genomeassembly with respect to the haplotype-specific restriction frag-ments represented in BACs T0068B19 and T0048O04

HindIII restriction fragment size (bp)

T0048O04 T0068B19 P. trichocarpa assembly90 90 90

211 211 2112878 2878 28781374 1374 13744369 2674 2674152 152 152

1384 803 1403– 985 –

105 105 10562 251 251

1897 826 8262327 3701 3701

Fragments shared in common between the two haplotypes areindicated in green; fragments specific to the haplotype representedby clone T0048O04 are indicated in blue; fragments specific to thehaplotype represented by clone T0068B19 are in red. The assemblycoordinates were determined using BES alignment coordinates forclone T0068B19. The analysis indicates that the assembled sequencein this region is a mosaic of the 2 haplotypes embodied in T0048O04and T0068B19. Note that the 1403 bp fragment in the assembly,labeled in grey, is a combination of the two haplotypes. It lacks theHindIII restriction site present in T0068B19 (thus reflecting thehaplotype represented in T0048O04) but contains a 24 bp insertionmissing in T0048O04 (thus reflecting the haplotype represented inT0068B19).



have aligned the physical map contigs to the sequence

assembly, and integrated the physical and genetic maps

through SSR markers. This work illustrates the power of an

integrated approach to assembling a physical map that is

anchored to both a whole-genome assembly and a genetic

map. These represent complementary resources, each hav-

ing the ability to inform the others, and their integration

provides added utility to the research community. For

example, the BESs provided an essential link between the

physical map and the genome sequence. Through these, the

physical map aided long-range contiguity of the sequence

assembly and resolution of repetitive regions (Tuskan et al.,

2006). In turn, alignment of the physical map contigs to the

linkage groups and sequence scaffolds provided informa-

tion useful for improvement of the physical map assembly.

Physical map coverage and genome representation

The sum of the estimated sizes of the BAC contigs is

approximately 577 Mb. This is approximately 20% larger

than the current estimated genome size of 485 Mb, derived

from the sequence assembly. The difference in the map-

based estimate is probably due at least in part to unrecog-

nized overlap between the ends of map contigs, as has been

reported in the soybean physical map study (Wu et al.,

2004), which would result to some extent in an inflated size

estimate. However, a large contributing factor to the differ-

ence in genome size estimates is the existence of haplotype-

specific contigs in the BAC map. If we consider only a single

haplotype contig for each of the co-aligning contigs an-

chored to the genome sequence, then the overall genome

size estimate represented by the map is reduced to 478 Mb,

which is in very good agreement with the 485 Mb estimated

derived from the sequence assembly. However, it is also

possible, given the duplicated nature of the genome (Sterck

et al., 2005; Tuskan et al., 2006), that in some cases dis-

persed, duplicated sequences have been collapsed within

the sequence assembly. At present, 187 BAC contigs map to

multiple regions of the genome based on the BES align-

ments. The discrepancies between the physical map and the

sequence assembly will require further investigation to

elucidate the underlying reasons for the assembly differ-

ences, providing an excellent opportunity to understand the

structure of sequence duplications and the nature of haplo-

typic differences in this species.

The physical map provides good coverage of the

sequence assembly. Approximately 384 Mb (79%) of the

entire poplar sequence assembly and 295 Mb (96%) of

linkage group assemblies were anchored to the physical

map (Table 2). This is probably an underestimate as it is

based only on contiguous regions of aligned BESs. It does

not take into account the presence of any BAC clones lacking

sequence alignments that flank the aligned regions within

contigs, or map contigs that could not be anchored to the

sequence assembly. The number of anchoring clones per

aligned contig averaged 10 and ranged from 2 to 138. These

contigs varied in size, with the majority being above 200 kb

and some extending to over 1 Mb (Figure 2). These larger

contigs are extremely useful for long-range sequence integ-

ration, providing a framework on which to orientate

sequence scaffolds. However, it is important to note that

the physical map was derived from a single BAC library

constructed by HindIII partial digestion, and thus would not

contain regions of the genome where HindIII sites are

separated by distances larger than can be typically cloned

into a BAC vector. Based on size analysis of P. trichocarpa

genomic DNA digested to completion with HindIII (data not

shown), we estimate that as much as 10% of the genome

could be missing from the BAC library, and thus the physical

map would lack coverage for these regions of the genome

assembly.

In comparison to other plant physical maps, the number

of contigs in the poplar map is still large and requires further

resolution. The physical map of rice contains 458 contigs

Table 4 Summary of haplotype-specific indels examined for effects on predicted gene content

BAC pair1 FPC2Overlap(bp)3

No. predictedgenes4

No. indelsexamined

Indel sizerange5

Indel locationIndel effecton genes6Coding Intron Inter-gentic

T0068B19 T0048O04 Ctg 8 Ctg846 88,954 3 8 40–244 0 1 7 noneT0021J18 T0033M07 Ctg 160 Ctg 162 94,123 7 5 19–29 1 1 3 17

T0053A03 T0011N15 Ctg 836 101,155 13 11 24–32 0 0 11 noneT0017N13 T0065A01 Ctg 1158 38,020 2 7 40–81 0 0 7 none

1Sequenced haplotype-specific BAC clones, as described in text and Table S1.2BAC fingerprint contig number or numbers.3Overlap between the pair of haplotype-specific BAC clones.4Genes predicted in the corresponding region of the Populus genome assembly (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html).5Size range of the indels investigated, in bp.6Number of genes with changes in structure or location that could be affected by an indel.7Eight amino acid insertion into predicted gene eugene3.00141429, annotated as an incomplete coding sequence and without EST expressionsupport (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html).



representing approximately 90% of the 430 Mb genome at

an estimated 20-fold coverage (Chen et al., 2002). For

Arabidopsis, a physical map consisting of 27 contigs

covered the majority of the 125 Mb genome (Mozo et al.,

1999). Rice and Arabidopsis have more homogenized

genomes due to domestication in the case of rice (Buckler

et al., 2001) and inbreeding in the case of Arabidopsis

(Bustamante et al., 2002). In contrast to this, the physical

mapping efforts in soybean are more comparable with those

in poplar, in terms of gross genome size and chromosome

number. The soybean genome is more complex than that of

rice or Arabidopsis, having a larger chromosome number

(soybean n = 20, poplar n = 19, rice n = 12, Arabidopsis

n = 5) and larger genome size (approximately 1115 Mb), and

being tetraploid (Wu et al., 2004). However, it is autogamous

and has been domesticated for approximately 3000 years.

These factors have combined to result in reduced haplotypic

diversity (Zhu et al., 2003). The soybean BAC- and BIBAC

(binary BAC)-based physical map consisted of 2905 contigs,

representing a 9.6-fold redundancy (Wu et al., 2004). How-

ever, this physical map did not include a sequence compar-

ison, which was possible with the poplar genome and which

facilitated an approximate 12% decrease in the contig

number. Considering the reduced diversity within soybean,

the poplar map compares well due to the larger haplotypic

diversity inherent in poplar as an obligate outbreeding

species.

Options for improving the P. trichocarpa physical map

include creation of additional BAC libraries using different

enzymes, as it has been shown that two-enzyme methods

outperform single-enzyme methods in simulations (Xu

et al., 2004), and optimization of library construction proto-

cols to obtain BACs with larger inserts.

Haplotype sequence diversity and effect on gene content

An unanticipated outcome of the alignment of the physical

map with the poplar genome sequence was the detection of

haplotype-specific map contigs, which resulted from high

levels of haplotype variation in some regions of the genome.

Analysis of the sequence differences between haplotype-

specific BAC clones suggests that haplotypes are charac-

terized by numerous small to large indel polymorphisms, in

addition to SNPs, raising the possibility of differences in the

repertoire of genes between haplotypes. Such differences

would not be apparent from the genome assembly because,

as illustrated in Table 3, the genome assembly represents a

mosaic of the two haplotypes.

In maize, detailed analysis of haplotype-specific DNA

sequences has revealed striking examples of non-co-linear-

ity in DNA sequence between haplotypes and, in some

cases, haplotype-specific gene complements. For example,

near the bz locus, Wang and Dooner (2006) observed

extensive DNA sequence non-co-linearity in eight maize

haplotypes as the result of massive differences in the

insertion sites and numbers of mobile DNA elements

surrounding and within eight genes in the region examined.

In another example, at a locus containing multiple z1C

genes, haplotype-specific differences in z1C gene number

and order were found in two haplotypes (Song and Messing,

2003). Finally, Brunner et al. (2005) observed extensive

breakdown of sequence co-linearity between two maize

haplotypes at four loci on different maize chromosomes.

This lack of co-linearity is largely caused by differential

insertion of long-terminal repeat retrotransposons in a

haplotype-specific manner, but, surprisingly, there were

also a number of haplotype-specific genes at three of the

four loci (i.e. genes that are present in one haplotype but

absent in the other).

Our data from the sequences of four pairs of BAC clones

representing over 320 kb of haplotype-specific DNA se-

quence in poplar allowed us to compare the extent and

consequences of haplotype-specific DNA polymorphisms in

this species. In contrast to maize, the pairs of poplar

haplotype sequences were largely co-linear, punctuated by

an assortment of small to large indels, indicating a lack of

large-scale sequence rearrangement in the haplotypes rel-

ative to each other. Our analysis of an 11 kb region specific

to one haplotype on LG I, but absent in the genome

assembly, failed to find support for expressed genes in this

region. Moreover, almost all haplotype-specific indels were

in intergenic regions where they have no impact on gene

content or order (Table 4). While small indels in coding

regions such as observed in one gene in haplotype-specific

contigs 160 and 162 (Table 4) may be relatively common in

the poplar genome (Tuskan et al., 2006), many of the larger

indels appeared in gene-poor regions (data not shown).

These data do not exclude the possibility of haplotype-

specific differences in gene content in poplar, but the fact

that no such differences were found in a total of 320 kb of

haplotype-specific sequence containing 25 annotated genes

at four loci suggests such differences may be relatively rare

or confined to certain regions of the poplar genome. Indeed,

extensive haplotype diversity, including haplotype-specific

differences in gene content appear to be present on LGXIX

(T.-M. Yin, G.A. Tuskan and S.P. DiFazio, unpublished data),

an interesting LG with relatively poor genome sequence

assembly (Tuskan et al., 2006) and physical map coverage

(Figure S1). However, based on our sampling of the poplar

genome, the extensive and widespread haplotype-specific

genome organization and gene content found in maize do

not appear to be general phenomena in angiosperms, and

may be related to extremely active families of mobile

elements in that species. Further analysis of haplotype

variation in poplar and other plants will provide more

definitive data on whether the variation we observed in

poplar is more typical of angiosperms. The large numbers of

apparently haplotype-specific BAC contigs revealed by the



physical map present an opportunity for more detailed

analysis of the nature and functional consequences of

haplotype sequence diversity in Populus.

Integration of genetic and physical maps

A total of 122 SSRs developed from the BESs were mapped

onto the poplar genome assembly using the Nisqually-1

pedigree (Tuskan et al., 2006; T. Yin et al., unpublished). The

markers are spread across the linkage groups, and those

mapped initially were chosen to span each linkage group

relatively evenly (approximately 20–30 cM spacing) and also

to represent large physical map contigs. These SSRs were

used to map contigs onto genetic positions (Figure 1). Using

122 BES-derived SSR markers, a total of 22% of the LG

assemblies were covered by 119 contigs mapped to the

genetic map (Table 1). These markers enable a direct

association of genetic loci with physical map BAC clones.

They also provide a resource for comparative genomics in

the genus Populus and related species. Additional micro-

satellites and SNP markers are being designed based on

observed polymorphisms in the genome sequence, and

these will be used to complete the map-based genome

assembly (T. Yin et al., unpublished). Integration of the

physical and genetic maps, by mapping BES-derived SSRs

and genome assembly, also provides a genome-wide data

set of comparative genetic and physical distances across the

19 linkage groups, from which potential differences in

recombination rates can be inferred. These data will be

presented in detail elsewhere (T. Yin et al., unpublished).

Map utility

The framework of the physical map, the sequence assembly

and the genetic markers provide a considerable collection of

resources for poplar research. The current map already

provides a considerable resource in terms of genomic

interrogation. The combined integration of physical map,

genetic map and genome sequence will be of use in detailed

studies on QTLs for traits of interest in tree biology, such as

wood quality, biomass production, responses to environ-

mental cues, and responses to biotic and abiotic stresses

(Frewen et al., 2000; Tagu et al., 2005). Once QTLs of interest

have been mapped to intervals, use of the BES-derived

markers will allow identification of specific BACs in these

intervals, providing a source of cloned Populus genomic

DNA of known sequence location for functional studies on

selected candidate genes. As an example, we have used a

QTL map of poplar wood quality traits to identify markers in

regions of interest, and subsequently used the physical map

BAC clones to target these regions for further characteriza-

tion (C.T. Kelleher et al., unpublished data).

The Nisqually-1 physical map BAC clones also provide a

reference point for Populus genome organization. The

genus Populus contains 29 species, distributed among six

sections (Eckenwalder, 1996), and many of these species

have unique ecological adaptations (Cronk, 2005). While all

species contain 19 chromosomes, the extent to which small-

scale genome rearrangements or insertions/deletions occur

between species, perhaps contributing to changes in gene

complement and adaptation, is unknown. The Nisqually-1

physical map, combined with the genome sequence, will

serve as a reference for comparative studies on gene

synteny and genome structure within the genus, using

BAC libraries prepared from other Populus or related

species, by comparative BAC mapping and hybridization

strategies to the Nisqually-1 BAC contigs.

In summary, the physical map and other resources

available for poplar genomics should significantly aid the

advance of research into the biology of woody perennials,

and help establish this poplar as a model system for tree

biology. The complete fingerprint map is available for

download in FPC format from the Genome Sciences Centre

website (http://www.bcgsc.bc.ca/lab/mapping/data). The

map may also be viewed using Internet Contig Explorer

(iCE) (Fjell et al., 2003), a Java-based application that allows

viewing of FPC-based maps (http://www.bcgsc.ca/ice/), and

copies of the BAC library containing all clones may be

obtained by contacting the corresponding author (CJD).

Experimental procedures

BAC clone fingerprinting and map construction

The BAC library was constructed from Nisqually-1 genomic DNApartially digested with HindIII, and consisted of 48 384 BAC clones.The procedure for BAC library construction has been describedelsewhere (Stirling et al., 2001). BAC clones were fingerprinted byHindIII digestion and fragment separation on agarose gels (Marraet al., 1997; McPherson et al., 2001; Schein et al., 2004). Restrictionfragment identification, fragment mobility and size determinationwere performed automatically using BandLeader software (Fuhr-mann et al., 2003). Automated fingerprint map assembly was per-formed using FPC version 5.0.1 (Soderlund et al., 1997, 2000), withan initial assembly performed using default parameters and aSulston cut-off score of 1e-15 (Sulston et al., 1988).

Additional processing of the map contigs was achieved by acombination of manual review using tools within FPC and externalautomated tools. CORAL (Flibotte et al., 2004) is an automatedapplication for improving clone order within FPC-assembled con-tigs, and was applied to contigs containing £10 clones. The majorityof contig merging was achieved by the use of automated scripts.Multiple rounds of analysis were performed, with varying param-eters used to identify valid merges. Clone fingerprint comparisonswere performed only between clones at the ends of contigs, orbetween singleton clones (clones that did not assemble intocontigs) and clones at the ends of contigs. Fingerprint similaritieswere first calculated using the Sulston score (Sulston et al., 1988),and those falling below the set cut-off score for the round wereidentified as candidate merges. The candidate merges were furtherinterrogated for the number of unconfirmed fragments across themerge point, where an unconfirmed fragment is one that is present



in a clone at a contig end, but is not present elsewhere in that contigend nor in the clones at the end of the candidate merging contig.Those candidate merges not exceeding the allowed number ofunconfirmed fragments for the round were considered valid mergesand the contigs were joined. The Sulston score cut-off and thepermitted number of unmatched fragments were varied for eachround, with the parameters balanced in order to avoid promiscuousmerges. Sulston score cut-offs varied from 9e-10 to 9e-6, andpermitted unconfirmed fragments varied from 0 to 3, with the latterparameter allowing for minor errors in BandLeader fragmentidentification. A single exceptional round of merging was per-formed in which very small contigs were merged internally to largercontigs only if all clones in the smaller contig matched a group ofneighboring clones in a larger contig, with a maximum Sulstonscore cut-off of 9.99e-10 and a maximum of two unconfirmedfragments. A copy of the BAC library containing all clones in thephysical map may be obtained by contacting the correspondingauthor (CJD).

Contig size estimation

To estimate contig sizes based on fingerprint data, an automatedalgorithm was used to compare the restriction fragments of over-lapping clone pairs in the canonical clone set for each contig.Canonical clones are the set of non-redundant overlapping clonesspanning a contig that each represent a unique complement ofrestriction fragments in their fingerprint, such that the remainingnon-canonical clones within the contig are subsumed by thecanonical clones (i.e. all the restriction fragments in the fingerprintof a non-canonical clone are completely represented in one of thecanonical clones). The unique fragments for each canonical clonewere identified, and their sizes were summed to estimate the overallsize of the contigs. Specifically, the algorithm performed the fol-lowing procedure for each contig: (i) sum the sizes of all the frag-ments in the left-most canonical clone in the contig to create acumulative size estimate; (ii) identify the next canonical cloneimmediately to the right and identify its unique fragments (anyfragments not shared with the two previous canonical clones to theleft or the next two canonical clones to the right), then add the sizesof these unique fragments to the cumulative size estimate; (iii) re-peat step 2 until all unique fragments in the canonical clones havebeen identified and summed to give a total size estimate for thecontig. Two fragments were considered the same if their calculatedstandard mobilities were within 10 mobility units of each other.

Genetic and physical map integration

A genetic map of the Nisqually-1 pedigree was constructed throughthe collaborative effort of Oak Ridge National Laboratory, Tennes-see, USA, and the Treenomix group at the University of BritishColumbia, Canada (T. Yin et al., unpublished). The pedigree wasfamily 545, an inter-specific F1 population obtained from a crossbetween P. trichocarpa (Nisqually-1 as mother) and P. deltoides L.(as father) (Stirling et al., 2001). The data obtained from the geneticmap were merged with another P. trichocarpa pedigree map, basedon family 13 (Yin et al., 2004). Young buds were collected and theDNA was isolated using Qiagen DNeasy plant mini kits (http://www.qiagen.com/). A total of 94 individuals were used for the AFLPanalysis and 87 for the SSR analysis. The markers from the geneticmap were used to integrate the physical map with the genetic map.SSR markers primarily developed from the BESs (http://www.ornl.gov/sci/ipgc/ssr_resource.htm) and those used in otherPopulus mapping studies (Cervera et al., 2001; van der Schoot et al.,

2000; Yin et al., 2004) were used by both laboratories. At theUniversity of British Columbia, additional dominant AFLP markers(Vos et al., 1995) were analyzed for the genetic mapping, and bothSSRs and AFLPs were visualized by addition of an M13 sequence onthe forward primer and subsequent addition of M13 IRD-labeledprimer (for details see Oetting et al., 1995).

For PCR amplification of SSR loci, 20 ng of total genomic DNAwas added to a 10 ll reaction volume of 1· Roche PCR buffer,250 lM dNTPs, 0.2 lM forward and reverse primers, 0.05 lM M13IRD-labeled primer and 1 U of AmpliTaq DNA polymerase (Roche;http://www.rochecanada.com). Reactions were carried out on anMJ Research PT-100 thermal cycler (http://www.bio-rad.com) withthe following program: 95�C for 4 min, followed by 30 cycles of 95�Cfor 1 min, 60�C for 30 sec and 72�C for 1 min, then a final extensionfor 4 min at 72�C. AFLP reactions involved a restriction digestion, apre-selective amplification and a final selective amplification step(Vos et al., 1995). A 30 ll restriction–ligation reaction was incubatedat 37�C for 4 h. The reaction contained 250 ng total genomic DNA ina reaction mix of buffer (giving final concentrations of 10 mM TrisHCL, 10 mM MgAc, 50 mM KAC and 5 mM DTT), 12 U EcoRI or PstIand 8 U Tru9I, 2.5 pmol EcoRI/PstI adaptor, 25 pmol Tru9I adaptor,0.15 mM ATP and 0.25 U of T4 DNA ligase (Invitrogen; http://www.invitrogen.com/). The restriction–ligation reactions were dilu-ted 1:10, and 5 ll of this reaction was used in a pre-selective PCRamplification together with 1· Roche PCR buffer, 200 lM dNTPs,0.15 lM EcoRI/PstI/Tru9I pre-selective primers and 1 U of AmpliTaqDNA polymerase (Roche). PCR amplification was performed in anMJ Research PT-100 thermal cycler with the following cycles: 94�Cfor 1 min, followed by 30 cycles of 94�C for 30 sec, 65�C for 30 sec,72�C for 1 min, and a final extension for 5 min at 72�C. Pre-selectivePCR product was diluted 1:40, and 5 ll were used in a 20 ll reactioncomprising 1· Roche PCR buffer, 400 lM dNTPs, 0.3 lM EcoRI/PstI/Tru9I selective primers and 1 U AmpliTaq DNA polymerase (Roche).Selective amplification reactions were the same as for the pre-selective amplification, except that M13 IRD-labeled primer wasadded to a concentration of 0.05 lM and a drop-down annealingtemperature was used, starting at 65�C and decreasing by 0.7�C foreach cycle until a final set of 22 cycles at an annealing temperatureof 56�C.

SSRs and AFLPs were analyzed on LI-COR 4300 DNA analyzers(http://www.licor.com) with 6% polyacrylamide gels, and on anABI3730 capillary sequencer (Applied Biosystems; http://www.appliedbiosystems.com). Images were processed using SagaMX

AFLP�, SagaGT (LI-COR Biosciences; http://www.licor.com) andanalyzed using JoinMap (Van Ooijen and Voorrips, 2001) andMAPMAKER to determine linkage groups (Lander et al., 1987).Details on the genetic mapping analysis will be presented else-where (T. Yin et al., unpublished). MapChart 2.1 was used to drawlinkage group diagrams (Voorrips, 2002).

A number of BES-derived SSRs (122) were used to positionphysical map contigs on the genetic map. In addition, the remainingSSRs were shown to be useful in integrating other LG in silico ontothe physical map by using BLAST hits from SSR primer sequencesagainst the BESs. The BLAST results were screened for e valuesbelow 1.0, and size ranges between 50 and 300 bp. Those thatpassed these criteria were used to illustrate the integration of thephysical and genetic maps.

BAC end sequencing and alignment of map contigs to

sequence assembly

The BAC DNA isolated for fingerprinting was also used to generateend sequence data for the clones. The protocol for BAC end



sequencing reactions was provided by S. Zhao of the Institute forGenomic Research (Rockville, MD, USA). The primers used were)21 M13 forward (TGTAAAACGACGGCCAGT) and M13 reverse(CAGGAAACAGCTATGAC).

The data were collected on ABI Prism 3700 DNA analyzersequencing instruments. The trace data were processed by theprogram phred (Ewing and Green, 1998; Ewing et al., 1998), usingdefault parameters, and the sequence trimmed for quality and vectorsequences. Reads that contained <15 bp of sequence followingprocessing were removed from the data set. Average read lengthswere calculated from the quality length reported by phred for eachread. The BES traces are available from the trace archives at theNational Center for Biotechnology Information (NCBI) (Ti numbers1439871865–1439912628, 1439111083–1439151202, or query with‘species_code = ‘POPULUS TRICHOCARPA’’’).

Comparisons of BESs to the whole-genome shotgun assemblyscaffolds (JGI Populus trichocarpa genome assembly, version 1.0)were performed using BLAST (Altschul et al., 1990). Those align-ments satisfying the criteria of either (a) >99% identity ande-value < 10)50, or (b) >95% identity for >95% of the read lengthwith an alignment length >50 bp, were used to anchor fingerprintmap contigs to the sequence assembly. Where alignments for bothend sequences of a clone were available, the paired reads wererequired to have alignments with opposite orientation. Groups oftwo or more clones with overlapping end sequence alignmentswere used to map the contigs to the sequence. In cases wherecontigs mapped to multiple sequence regions, these were filtered asfollows. The region with the most clones aligning to it was accepted.The region with the next largest number of alignments was requiredto have at least three aligned clones, the next four, and so on. In thisway, some small contig loci that passed the initial two-cloneminimum were rejected because the presence of larger loci resultedin an increased minimum clone cut-off.

BAC insert sequencing, assembly and analysis

BAC clones T0021J18 and T0033M07 were sequenced using a ran-dom in vitro transposon insertion approach. BAC DNA was pre-pared as per the fingerprinting protocol described by Schein et al.(2004), and transposon libraries were generated using the TemplateGeneration System� I Kit (Finnzymes; http://www.finnzymes.fr),following the manufacturer’s recommended protocol for BACclones, and the Kan(R) Entranceposon (Finnzymes). BACs withinserted transposons were cultured and DNA-purified in a 96-wellformat (Schein et al., 2004). Sequencing reactions were assembledin 384-well clear optical reaction plates (Applied Biosystems; http://www.appliedbiosystems.com/) using a Biomek FX workstation(Beckman-Coulter; http://www.beckmancoulter.com) (Yang et al.,2005). To each 8 ll reaction (total volume), the following were ad-ded: 5 ll of purified BAC DNA, 0.7 ll of sequencing primer (5 pmol/ll, Invitrogen), 0.3 ll of Ultrapure water (Gibco; http://www.invitrogen.com) and 2 ll of BigDye v.3.1 ready reaction mix(Applied Biosystems). Sequence reads were performed on trans-posed BAC clones using primers SeqA2 (5¢-GAATTCTCTAGA-TGATCAGCGGC-3¢) and SeqB2 (5¢-CGAACTTTATTCGGTCGAA-AAGG-3¢). Cycling was performed on PTC-225 thermal cyclers (MJResearch) with parameters of 95�C for 2 sec, followed by 85 cyclesof 96�C for 30 sec, 56.6�C for 5 sec using SeqA2 primer or 56.0�C for5 sec using SeqB2 primer and 60�C for 3 min, followed by incuba-tion at 4�C. Reaction products were precipitated using 2 ll of125 mM EDTA (pH 8) and 18 ll of 95% ethanol per well, followed bycentrifugation at 2750 g for 30 min at 4�C. The supernatant wasdecanted by inverting the plate and firmly shaking liquid from thewells. Plates were left to air-dry for 15 min. Samples in each well

were then resuspended in 10 ll of Ultrapure water and analyzedusing a 3730XL DNA analyzer (Applied Biosystems).

Transposon-directed sequenced reads were base-called usingphred (Ewing and Green, 1998; Ewing et al., 1998). The base-calledreads were imported and checked for contamination againstEscherichia coli, vector and transposon sequences. BAC vectorand inserted Mu transposon sequences were removed, and theremaining sequences were assembled together using PHRAP(http://www.phrap.org/). After initial assemblies, CONSED (Gordonet al., 1998) was used to view the data to check for any possibleerrors and make appropriate corrections/edits. CONSED navigationtools were utilized to check for low-quality (phred quality below 30)consensus sequences and high-quality discrepancies (mismatchesof phred base quality of 20 and above between/among individualreads) in the assembled reads. All the repeat regions were manuallyassembled using single base pair mismatches and read pairsinformation. Mononucleotide and dinucleotide runs were sortedby making 4–5 bp overlapping joins between the read pairs. Forruns with not enough read pairs information to tilepath, the contigswere joined together by making minimum appropriate joins. All thefinished assemblies were re-examined if any misplaced high-qualityread pairs were found and fixed accordingly. Final confirmation ofthe finished assemblies was made by comparing their in silicoHindIII restriction enzyme digests to the respective experimentalrestriction enzyme digests, and any deviation was manually exam-ined and corrected

To correlate the sequence differences with the resulting finger-print differences, each pair of BAC sequences was first in silico-digested using the HindIII restriction enzyme motif, and theresulting fragments mapped to corresponding fragments in theirexperimental fingerprints, using a size tolerance of 10 bp. The twoclone sequences were then aligned against each other and againstthe corresponding region of the genome assembly (based on BESalignments) using Dotter software (Sonnhammer and Durbin,1995). The sources of variation in DNA sequences were identifiedand checked for existence in the corresponding genome assemblyregion. Potential effects of haplotype-specific indel polymorphismson gene order and content were investigated by comparing indelsand surrounding sequences to the poplar genome assembly andannotation (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html).

Acknowledgements

This project was supported by Genome Canada, Genome BritishColumbia and the Province of British Columbia (Treenomix project)with funds to C.J.D., B.E.E., J.B. and K.R., and by a Natural Sciencesand Engineering Research Council of Canada (NSERC) DiscoveryGrant to C.J.D. We thank Scott Paper Inc. for maintenance of thefamily 545 material used for the Nisqually-1 genetic map. Fundingfor the Oak Ridge National Laboratory portion of this research wasprovided by the US Department of Energy, Office of Science,Biological and Environmental Research Carbon SequestrationProgram, the Basic Energy Sciences Program, and National ScienceFoundation grant 0421743 to G.A.T. Oak Ridge National Laboratoryis managed by UT-Battelle, for the US Department of Energy undercontract number DE-AC05-00OR22725. M.A.M., S.J.M.J. and R.A.H.are scholars of the Michael Smith Foundation for Health Research;J.B. is a Steacie Fellow of the Natural Sciences and EngineeringResearch Council of Canada.

Supplementary Material

The following supplementary material is available for this articleonline:



Figure S1. Fingerprinted BAC clone and contig layout on thesequence assemblies of each of 19 Populus trichocarpa linkagegroups (LG) based on BES alignments to the genome sequence(http://mkweb.bcgsc.ca/poplar/supplementary/060515).Table S1 Summary of haplotype-specific DNA polymorphismsbased on BAC sequence comparisonsThis material is available as part of the online article from http://www.blackwell-synergy.com

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