Comparative Genome Mapping Among Populus Adenopoda, P

Genes Genet. Syst. (2011) 86, p. 257–268 Comparative genome mapping among Populus adenopoda, P. alba, P. deltoides, P. euramericana and P. trichocarpa

Yuanxiu Wang1,2*†, Bo Zhang1†, Xiaoyan Sun1, Biyue Tan1, Li-an Xu1*, Minren Huang1 and Mingxiu Wang1 1Key Laboratory of Forest Genetics & Biotechnology (Nanjing Forestry University), Ministry of Education 210037, China 2College of Life Science, the Key Laboratory of Molecular Evolution and Biodiversity and the Key Laboratory of Biotic Environment and Ecological Safety in Anhui Province, Anhui Normal University, Wuhu 241000, China

(Received 21 May 2011, accepted 10 November 2011)

Among the genus Populus, the sections Populus (white poplar), Aigeiros Duby (black poplar) and Tacamahaca Spach contain many tree species of economical and ecological important properties. Two parental maps for the inter-specific hybrid population of Populus adenopoda × P. alba (two species of Populus section) were constructed based on SSR and SRAP markers by means of a two-way pseudo- test cross mapping strategy. The same set of SSR markers developed from the P. trichocarpa (belonging to Tacamahaca section) genome which were used to construct the maps of P. deltoides and P. euramericana (two species of Aigeiros section) was chosen to analyze the genotype of the experimental population of P. adenopoda × P. alba. Using the mapped SSR markers as allelic bridges, the alignment of the white and black poplar maps to each other and to the P. trichocarpa physical map was conducted. The alignment showed high degree of marker synteny and colinearity and the closer relationship between Aigeiros and Tacamahaca sections than that of Populus and Tacamahaca. Moreover, there was evidence for the chromosomal duplication and inter-chromosomal reorganization involving some poplar linkage groups, suggesting a complicated course of fission or fusion in one of the lineages. A poplar consensus map based on the comparisons could be constructed will be useful in practical applications including marker assisted selection.

Key words: comparative map, Populus adenopoda, P. alba

genetic analyses of the family Salicaceae using nuclear INTRODUCTION rDNA, molecular markers and chloroplast DNA suggest The species of the genus Populus L. (Salicaceae) are that Populus is the oldest monophyletic group, and close widely distributed in the northern hemisphere from sub- relationship and introgression exist between Aigeiros and tropical to boreal forests (Hamzeh and Dayanandan, Tacamahaca (Azuma et al., 2000; Cervera et al., 2005; 2004). According to a recent classification (Eckenwalder, Hamzeh and Dayanandan, 2004; Hamzeh et al., 2006; 1996), the genus Populus is classified into 29 species in Leskinen and Alstrome-Rapaport, 1999; Rahman and six sections, and most of the economically and ecologically Rajora, 2002). important species are in the sections Populus syn. Leuce During the last decades, Populus has been established Duby (white poplar), Aigeiros Duby (black poplar) and as a favorable model system for trees and woody peren- Tacamahaca Spach (Rahman and Rajora, 2002). Phylo- nial plants due to its rapid growth, ease of vegetative and seed propagation, small genome (~480 Mbp), availability Edited by Yoshihiko Tsumura of efficient transformation systems, and conservation of * Corresponding author. E-mail: [email protected] chromosome number across the genus (n = 19) (Bradshaw E-mail: [email protected] et al., 2000; Taylor, 2002; Tuskan et al., 2004; Jansson † These authors contributed equally to this work Note: Supplementary materials in this article are at http:// and Douglas, 2007). These wonderful biological charac- www.jstage.jst.go.jp/browse/ggs ters make Populus ideal for experimental researches. 258 Y. WANG et al.

Many mapping populations have been produced for Woolbright et al., 2008; Pakull et al., 2009; Zhang et al., numerous geographically and ecologically distinct species 2009; Berlin et al., 2010; Paolucci et al., 2010; Liu et al., from diverse sections within the genus. These experi- 2011), and allow an alignment of these maps to each other mental populations provide an unique opportunity for and to the P. trichocarpa genomic sequence and the con- comparative mapping in the model system—an opportunity sensus map, providing much information for comparative that has been further enhanced by the whole genome genomic studies of different species in the genus. The sequence of a female P. trichocarpa tree released (http:// dominant markers (for example amplified fragment genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html and http:// length polymorphism (AFLP) and sequence related ampli- www.phytozome.net/poplar ) (Tuskan et al., 2006). Up to fied polymorphism (SRAP) (Vos et al., 1995; Li and now, 16 mapping works have been published in Populus, Quiros, 2001) are unvalued for comparative mapping but leading to the construction of 30 maps relating to 10 pop- can enrich the maps. Populus trees are allogamous spe- lar species in Populus, Aigeiros and Tacamahaca sections cies, with very long generation time. Natural poplar (Cervera et al., 2004; Gaudet et al., 2008; Woolbright et populations present a high level of heterozygosity and the al., 2008; Zhang et al., 2009; Pakull et al., 2009; Paolucci possibility to obtain many full-sib progenies where mark- et al., 2010). Several comparative maps with Populus ers can segregate efficiently. Consequently, classical consensus map (based on a P. trichocarpa and P. deltoids strategies used in grass genetic mapping are not feasible background) (Yin et al., 2004, 2008) or the physical map for them. The pseudo-testcross strategy described by of P. trichocarpa showed high degree of synteny and pro- Grattapaglia and Sederoff (1994) is usually used in the vided a direct link to P. trichocarpa genome sequence mapping of forest trees. In this research the genetic (Woolbright et al., 2008; Zhang et al., 2009; Pakull et al., maps of the white poplar were constructed using the same 2009; Paolucci et al., 2010). Most of these comparative mapping strategy and software as two kinds of black pop- maps are constructed for species of Populus or Aigeiros lar maps reported by Zhang et al. (2009) in order to com- section and Tacamahaca section, few comparative map- pare between them (here P. deltoides and P. euramericana ping was actualized based on these three sections alto- are regarded as deputies of the species in the section gether. Aigeiros Duby, because the maps of P. deltoides and P. P. adenopoda Maxim. (quaking aspen, a mountain euramericana were constructed based on the common set poplar species native to China) and P. alba L. (white of SSR markers in our laboratory). Therefore, the objec- poplar) belong to the Populus section, and are two kinds tives of the present study were: (1) to provide genetic link- of trees of high ecological and economic values in China. age maps for P. adenopoda and P. alba based on a P. adenopoda is distributed in the middle and low combination of SSR and SRAP markers data by the two- Yangtse valley up to elevations of 1000 m and a pioneer way pseudo-testcross strategy and Mapmarker3.0 species of ruin ecosystems, growing fast in warm and (Grattapaglia and Sederoff, 1994; Lander, 1987) (2) to moist environmental conditions but poorly rooting. align our white poplar maps and the black poplar maps Contrarily P. alba is widely distributed across the to the physical map of P. trichocarpa using BLAST and Eurasian continent and can well adapt to dry and infer- the sequence of SSR primers in order to evaluate the tile soil. The two species are much different on their degree of macro-synteny and macro-colinearity in Populus, phenotype and present high genetic diversity detected Aigeiros and Tacamahaca sections genomes. Genomic using RAPD markers (Yin et al., 2001). In 2006, we comparisons of three sections poplars will allow us to detect obtained more than 1000 F1 hybrids of the two species the nuance of genome structure in the genus Populus. through inter-specific crossing and seeds culture in green house in order to construct linkage maps, and meanwhile METHODS we expected to find some individuals combining merits of the two species in F1 population to use for environmental The white poplar maps The two parental species protection and industrial production. For these two used in this study were P. adenopoda Maxim. (Chinese kinds of poplar, Yin et al. (2001) once published prelimi- quaking poplar) and P. alba L. (white poplar). The nary RAPD-based linkage maps through the double female P. adenopoda parent selected for hybridization pseudo-test-cross strategy. However, the subsequent was a single tree planted at the Arboretum of Nanjing research came to an end due to the gradual reduction in Forestry University (118°46’E, 32°03’N). However the the size of the mapping population. male P. alba parent was from a natural region across a Poplar microsatellite (simple sequence repeat, SSR) plain forestry center of Manas County (86°13’E, 44°05’N), markers, abundantly available on the website (http:// Xinjiang, China. In spring 2006, 1100 resulting seed- www.ornl.gov/sci/ipgc/ssr_resources.htm) have been used lings were obtained by embryo culture (culture medium: for many genetic maps in both Populus and the related MS0, 75% alcohol 30 s + 1% HgCl 3.5 min sterilizing after genus Salix (Hanley et al., 2002; Cervera et al., 2004; Yin 1–2 h washing by water ), and subsequently were planted et al., 2004; Hanley et al., 2006; Gaudet et al., 2008; in nursery garden at Nanjing Forestry University. One Comparative genome mapping among Populus 259 hundred and eighty-nine of them were randomly selected Poplars in Italy and introduced to China in 1972. for map construction. Total DNA was extracted from fro- Approximately 2000 seedlings were generated by inter- zen young leaves for the 189 progeny and the two paren- specific cross between these two clones, from which 450 tal clones. In this study 1186 pairs of SSRs and 163 individuals were randomly selected to establish a field primer pairs of SRAPs were selected for marker trial. A total of 93 genotypes randomly selected from the analysis. For each marker, a χ2 test (P < 0.01 and P < 450 seedlings were used for map construction (Zhang et 0.05) was performed to identify alleles of each parent that al., 2009). deviated from Mendelian segregation ratios. Distorted As the white poplar maps, the pseudo-test cross strat- markers were not excluded from linkage analysis and noted egy and MapMaker 3.0 were used to construct two black with the suffix “d” and “dd” which deviating at 0.01 < P ≤ poplar maps based on AFLP, ISSR, RAPD, SSR and SNP 0.05 and P ≤ 0.01 respectively. Estimated and observed markers. A total of 329 markers (including 146 SSRs, genome length and map coverage were calculated accord- 131 AFLPs, 47 RAPDs, 3 ISSRs, and 2 SNPs) construct a ing to the previously reported method (Hulbert et al., map for the maternal P. deltoids “I-69” genome. This 1988; Grattapaglia and Sederoff, 1994). All details were map (denoted as D) is 2293 cM long and contains 22 link- described in Wang et al. (2010). age groups. The map for the paternal P. euramericana “I-45” genome was constructed with 300 markers (includ- Map construction The two-way pseudo-test-cross map- ing 150 SSRs, 108 AFLPs, 39 RAPDs, 2 ISSRs, and ping strategy (Grattapaglia and Sederoff, 1994) was 1SNP). This map (denoted as E) has a total length of applied with MapMaker software version 3.0 (Lander, 2345.7 cM, composed of 36 linkage groups (Zhang et al., 1987) generating two maps, one for each parent. 2009). Therefore, two data matrices were created with SSR and SRAP markers segregating 1:1 in the progeny. To detect Alignment of maps Comparisons among the maps of linkages in repulsion phase, the data set was inverted the three sections of the genus Populus were conducted and added to the original data. The inverted markers with based on the SSR markers. The availability of the are indicated by an “r” and represent markers in P. trichocarpa genome sequence allows us to align our repulsion. To determine the correct genetic order, the white and black poplar maps (Zhang et al., 2009) to the “triple error detection” and the “error detection” features physical map of P. trichocarpa. SSR markers mapped on were used to recognize the cases when an event was the maps of white and black poplar were searched in the resulted by an error rather than a recombination avoiding P. trichocarpa genome database (http://genome.jgi-psf. substantial map expansion and interference (Lincoln and orgPoptr1_1/Poptr1_1.home.html and http://www. phyto- Lander, 1992). Initially, markers were grouped by two- zome.net/poplar) using BLAST and the sequence of the point analysis using a default LOD of 3.0 and a maximal SSR primers. When the two primers are exactly located recombination. The most likely order of markers within on a scaffold (mainly refer to scaffold_1 – scaffold_19 in a linkage group was determined by multi-point analysis the P. trichocarpa genome database) and separated by as follows. For linkage groups with more than five about 100 to 500 bases, the sequence between the 2 prim- markers, the “three point” command was used to pre-com- ers are considered as a SSR marker. The P. trichocarpa pute the likelihood of all three-point crosses of each physical map was designed with the MapChart 2.1 group. Then, the “order” command was used to select a (Voorrips, 2002). To simplify the representation, only subset of markers ordered at a minimum LOD of 3.0. homologous markers are indicated in the P. trichocarpa Additional markers were added by the “try” command physical map. The start base of each SSR was taken as with a log-likelihood threshold of 2.0. The order of the reference. If one of two primers is found to align a marker subset was controlled with the “ripple” command. homology on the physical groups and the marker was New markers were added only if the new order obtained mapped on the corresponding group of the white and was confirmed with this command. For the linkage black maps, it was considered as a homologous marker groups with less than five markers, the “compare” com- and located it on P. trichocarpa physical map. The num- mand was used. The marker order of these groups was ber of bases per cM was estimated by the ratio physical equally supported by a log-likelihood of 2.0. Linkage length/genetic length as described by Gerber and Rodolphe maps were generated with the “map” command using the (1994). Kosambi mapping function. Maps were drawn with the program MapChart 2.1 (Voorrips, 2002). RESULTS The black poplar maps The female P. deltoides, I-69, The white poplar linkage maps construction The from a natural population in Illinois, and P. euramericana, SSR and SRAP markers tested in this study were well I-45, a natural hybrid between P. deltoides and P. nigra, defined in the reference (Wang et al., 2010). The pseudo- were selected in the 1950s at the Research Institute of test cross strategy was used to construct two parent-spe- 260 Y. WANG et al. cific maps with the segregating markers. A total of 140 orthologous markers of 206 common markers found in markers (including 116 SSRs and 24 SRAPs) constructed poplar genome database using BLAST. In the AD map, a map for the maternal P. adenopoda genome. This map 96 orthologous markers aligned 31 linkage groups with 15 (denoted as AD) spanned 2168.3 cM and contained 34 groups (IX, XII, XIV and XVIII excluded) of poplar phys- linkage groups (8 triplets and 11 doublets included), with ical map, and in the AL map, 111 orthologous markers an average length of 63.8 cM, ranging from 2.1 to 211.4 aligned 36 linkage groups with 19 groups. It occurred cM. The average distance between adjacent markers that several sub-groups were aligned to the same homol- was 20.5 cM, ranging from 2.1 to 36.2 cM. The map for ogous group of the physical map. The colinearity of the paternal P. alba genome was constructed with 175 SSRs between the white poplar maps and the physical markers (including 144 SSRs and 31 SRAPs). This map map was judged to be high since 91.6% and 90.0% of syn- (denoted as AL) had a total length of 2749.2 cM composed tenic markers respectively in the AD and AL maps con- of 38 linkage groups (15 triplets and 6 doublets included), served in the same order (Supplementary Fig. S1). with an average length of 72.3 cM ranging from 4.2 to The alignment of the white poplar maps to poplar phys- 330.8 cM. The average distance between adjacent mark- ical map revealed high marker synteny, however some ers was 20.2 cM, ranging from 4.2 to 36.6 cM (Supplemen- interesting characteristics were observed for several tary Fig. S1). homologous groups. SSR P_2818 located on ADII and The total genome length of P. adenopoda map was esti- ALII, and W_21 located on ALII and ADV were found on mated at 2443.2 cM (95% CI = 2154.6~2821.2 cM, p < the homologous group LGV of P. trichocarpa. SSR 0.05) , and the estimate of P. alba map was 2719.5 cM O_402 on group ADV was located on the homologous (95% CI = 2436.2~3077.4 cM, p < 0.05). Using the func- group LGXIV. On the first linkage groups of white pop- tion given by Lange and Boehnke (1992), the expected lar, two markers (G_2955 and O_30) existed on LGIII and genome coverage was 93.49% for P. adenopoda map and one marker(G_437) on LGXVII, SSR P_2861 of ALIV 94.85% for P. alba map. occurred on LGIX, and G_901 of ALXIII occurred on A total of 14 and 8 distorted markers were respectively LGXI. The primers of O_241 amplified successfully and mapped on the maternal and paternal maps. However, produced two markers, one was linked to homologous these markers were not distributed randomly across the group (ADI) and another was linked to ALIII which was whole genome. In P. adenopoda map, 4 distorted mark- the homologous group of LGIII. The same things hap- ers were clustered on linkage group ADI, and 3 on linkage pened on the primers of O_114 and P_2020, for O_114, groups ADVI and ADXVII. aIn P. alb map, there were one was linked to ALXVI and another to ALVI, and for 3 distorted marker clustered on linkage group ALXIX P_2020, one was linked to ADIV and ALIV and another (Supplementary Fig. S1). to ALIX. The marker G_4063 on different groups of the The female and male maps presented 39 allelic bridges. white poplar maps was mapped on the group LGXIX of Nine linkage groups, with more than two common SSR the physical map. The group ALXIX was regarded as loci, showed full colinearity. The discrepancies was the homologous group of LGXIX in poplar physical map found for SSR W_21, which mapped on linkage groups but four markers on this group were found on other dif- ADII and on ALV, and G_4063, which mapped on linkage ferent groups, G_801 on LGXVIII, G_4067 on LGX, groups ADXIII and ALXIX (Supplementary Fig. S1). G_1781 on LGV and G_3376 on LGI (Supplementary Fig. S1). Map comparison A physical map of SSR positions was constructed to facilitate the assignment of linkage groups Alignment of the black poplar maps to P. and the comparison of the white and the black poplar trichocarpa physical map P. deltoides (D) and P. maps with the P. trichocarpa genome. Three hundred euramericana (E) maps were aligned with the physical fifty-eight SSRs were positioned on the genomic sequence map of P. trichocarpa also based on the anchor SSR mark- resulting from a BLAST search of the primer sequence ers (detail in Supplementary Fig. S2). The result of our against the P. trichocarpa genome database (http://genome. study was different from that of Zhang et al. (2009), and jgi-psf.org/Poptr1_1/Poptr1_1.home.html and http://www. we obtained more orthologous markers between the two phytozome.net/poplar ). sections poplars. In our research all linkage groups of the D map were aligned with 19 homologous groups of the Alignment of the white poplar maps to P. poplar physical map based on 142 orthologous SSR trichocarpa physical map P. adenopoda (AD) and P. markers. In the E map, also 139 orthologous markers alba (AL) maps were aligned to P. trichocarpa physical aligned 32 linkage groups with 18 homologous groups map (LG) according the anchor SSRs. Thirty-one of 34 (XVII excluded) of the physical map. It still occurred and 36 of 38 linkage groups respectively in AD and AL that several sub-groups were aligned to the same homol- maps could be assigned to I-XIX groups (among them, ogous group of the physical map. Through BLAST, one XVIII was excluded) of poplar physical map based on 181 primer of SSR G_3766 on groups D15 and E15 had a hit Comparative genome mapping among Populus 261 in poplar genomic sequence database but another hadn’t, group E9 and LGIX. The same thing occurred on the since this marker was located on the corresponding group marker P_2818 which was linked to group ADV and ALV of the reference (Yin et al., 2004), groups D15 and E15 instead of on D2 and LGII. The primers of O_30 was regarded as the homologous group of LGXV. For the E amplified successfully in white poplar mapping popula- map, the same as Zhang et al. (2009), no linkage group tion and produced two markers which respectively were was aligned to the homologous group LGXVII of the phys- linked to group ADI, ADXIV and ALXIV non-homologous ical map. And group E21 of E map in reference (Zhang to group D3 and LGIII. The primers of O_402 amplified et al., 2009) containing only one SSR markers were found successfully in black poplar mapping population and pro- no homologous group from the physical map. duced 3 markers which respectively were linked to group Synteny and co-linearity were high conserved between D14, E6 and E13 non-homologous to group ADII and the black poplar maps and the physical map. One hun- LGII. In white poplar mapping population, the primers dred and thirty eight markers (97.2%) of 142 orthologous of O_241 produced 2 markers, one (O_241A) was linked markers in D map and 129 (92.8%) of 139 in E map were to group ADI, the homologous group of D1 and LGI, but placed onto the homologous groups in the physical map. another (O_241B) was linked to group ALIII. And the For the co-linearity, there were 134 out of 142 (94.4%) primers of P_2879 amplified successfully and produced orthologous markers between the D map and the physical three markers, P_2879A and P_2879B were distorted map, and also 131 out of 139 (94.2%) between the E map markers linked to group ADVI and P_2879C didn’t devi- and the physical map. The co-linearity between the D ated from Mendelian segregation ratios and was linked to and E linkage maps was high, with 85 allelic bridges. group ALXIII, but these two groups weren’t homologous The order of the orthologous markers was well conserved to group D3 and LGIII. between the two maps for 80 out of 85 (94.1%). Five One hundred and six markers were present on 3 maps homologous markers were placed onto deferent locations of the three sections. Among them 28 were common on between the groups D3 and E3, D4 and E4, D6 and E6, the poplar physical maps and white poplar maps, 66 were D16 and E16 (Supplementary Fig. S2). common on the poplar physical map and the black poplar maps, and 12 were common on the P. trichocarpa physical Inter-specific comparison in the genus Populus map, one of the white poplar maps and one of the black Finally, we aligned our white poplar maps and the black poplar maps. Of 12 markers existing in three section poplar maps with the P. trichocarpa physical map. The maps, the markers produced by the primers of O_465, co-alignment of these 5 maps revealed 358 common P_2861 and G_93-2 were exceptional, for example, the markers. The marker order was conserved in most of primers of P_2861 produced 3 markers which respectively the cases. The linkage group I was the largest groups in were linked to group ALII, ALIV and ALVI non-homolo- all the maps and had the greatest number of homologous gous to group E9 and LGIX. Two hundred and twelve markers (including 38 markers). The common markers markers were common to the P. trichocarpa physical map among the 5 maps are presented in detail on Fig. 1. and one of the white or black poplar maps. Of 212 mark- Only 7 of 358 markers were present on the 5 maps. ers 123 were homologous between the P. trichocarpa Five markers (P_2852, G_3269, G_2900, P_2536 and physical map and one of the white poplar maps and 89 G_2020) were on the homologous groups (linkage groups were homologous between the P. trichocarpa physical I, III, V and X) and 2 (O_40 and G_4063) were in synteny map and one of the black maps. between black poplar maps and the P. trichocarpa physical map but exceptional on the white maps. The marker DISCUSSION O_40 was on group ADVI and ALVI, instead of group D2 and E2 which were homologous group with LGII in the P. The white poplar maps We had previously constructed trichocarpa physical map. The marker G_4063 was a genetic linkage map of P. adenopoda Maxim. × P. alba mapped on 2 different groups of white poplar maps, L. based on the same markers by Joinmap4.0 (Stam, ADXIII and ALXIX, but in black poplar maps, one 1993; Wang et al., 2010). We thought the comparative (G_4063A) of those two markers produced by the primers genetic map was more valuable and reliable based on the of G_4063 in the black experimental population was same mapping strategy and software than the different. mapped on the group D19 and E19 like LGXIX of poplar In this article, two parents-specific genetic linkage maps physical map, another marker (G_4063B) was mapped on containing SSR and SRAP markers were created for the the group E12. section Populus by using a two-way pseudo-test cross Twenty-three markers were present on 4 maps, and 17 mapping strategy (Grattapaglia and Sederoff, 1994) and of them were on the homologous groups among three Mapmaker software. Two hundreds and two mapped genus maps and the rest 6 (O_23, P_2818, O_30, O_402, SSR loci allow comparisons of the maps with other genetic O_241, and P_2879) and were discrepant. The marker maps of Populus and the direct link to the P. trichocarpa O_23 was linked to group ADIII and ALIII instead of on genomic sequence. The estimated genome length (2443.2 262 Y. WANG et al.

Fig. 1. Continued Comparative genome mapping among Populus 263

Fig. 1. Continued 264 Y. WANG et al.

Fig. 1. Continued Comparative genome mapping among Populus 265

Fig. 1. Comparison of the poplar genomic maps. AD, AL, LG, D and E stand for maps of P. adenopoda, P. alba, P. trichocarpa, P. deltoides and P. euramericana. The Roman and Arabian numbers following AD, AL, LG, D and E stand for the sequence number of the linkage groups. Only homologous markers were indicated on the P. trichocarpa physical map. The distance of the markers on the linkage groups of all maps were ignored. The red markers were the orthologous markers on the homoeologous linkage groups, The green markers were paralo- gous markers on the different homoeologous groups between the white poplar map or the black map and the P. trichocarpa physical map. The blue markers of which one primer had a hit with blast but another hadn’t on the homoeologous groups of P. trichocarpa physical map were regarded as the orthologous markers. The black markers were SRAP, AFLP, RAPD, SCAR and the markers with no hit on the P. trichocarpa genome.

cM for P. adenopoda and 2719.5 cM for P. alba) fell within AL map, 19 in D map and 18 in E map respectively could the range of the values found in previous studies and was be successfully aligned to P. trichocarpa genome by at near to the original estimate of 2400–2800 cM reported by least one SSR anchor marker (Supplementary Figs. S1 Bradshaw et al. (1994), which has been verified through and S2, and Fig. 1). Many sub-groups on AD, AL and E simulation studies (Yin et al., 2004). The estimated maps were aligned to the homologous groups of P. genome length for P. adenopoda was higher than 2104 cM trichocarpa, and these sub-groups aligned to one homolo- instead of that for P. alba was little more than 2632 cM gous group couldn’t be linked because of lacking enough reported by Yin et al. ( 2001). markers to link. Therefore, enhancing the maps’ density The two white poplar maps contained two kinds of and precision was the focus of the future research aiming markers were surpassed the two parental maps based on at aligning the complete maps to P. trichocarpa genome only RAPDs reported by Yin et al. (2001). The result in and using them to identify those common QTLs that this study also verified the structure of P. alba were more affect important economic traits and fundamental biolog- complex than P. adenopoda. More segregated loci were ical process in different species genomes of Populus. identified for P. alba than for P. adenopoda, and the In the two black poplar maps, the total number of SSR observed genome length of the paternal map was 26.79% markers aligned to P. trichocarpa genome was nearly the longer than the maternal map. same as that in the white poplar maps (208 versus 204), however, owing to more fully informative loci tested in the Comparative mapping Previous simple comparison black poplar interspecific hybrid than those in the white between the genetic maps of P. deltoides and P. (Wang et al., 2010; Zhang et al., 2009), the numbers of euramericana and the composite map of P. trichocarpa × orthologous markers aligned to P. trichocarpa genome P. deltoides (Zhang et al., 2009) in our laboratory were respectively on D and E maps (142 and 139 respectively) considered as a useful starting point for this study. With were much more than AD and AL (96 and 111 respec- the help of the maps for P. alba and P. adenopoda devel- tively) (Fig. 1). Between the black poplars and P. oped based on the same set of SSR markers as Zhang et trichocarpa genome, the level of marker synteny were al. (2009) herein, additional comparisons could be made higher than that between the white poplars and P. among the genomes of the three sections in Populus, trichocarpa genome (respectively 97.2% in D map and resulting in a doubling of the number of comparison 92.8% in E map, 91.6% in AD map and 90.0% in AL map). points. The dominant markers SSR as bridges for com- At the level of genetic markers, alignment of the white parison in this study were much more than those in the poplar maps to P. trichocarpa genome showed more dis- similar previous studies (Woolbright et al., 2008; Pakull crepant than that of the black poplar maps. The most et al., 2009; Paolucci et al., 2010). likely reason for these discrepancies were that the SSR primers were mainly developed in P. trichocarpa and Genome synteny in the Populus Alignment of the therefore, SSR primer binding site sequences were not iden- genomic maps made it possible to validate a high degree tical in white poplars. P. deltoides and P. euramericana of synteny among three sections of Populus (Fig. 1). are members of the Aieiros section, the phylogenetic sister Three hundred and fifty-eight or about 31% of SSR mark- of the Tacamahaca section, and more closely related to P. ers selected from P. trichocarpa genome (more than 1140) trichocarpa than P. alba and P. adenopoda, two species of were aligned. Fifteen linkage groups in AD map, 19 in the Populus section which is the oldest monophyletic 266 Y. WANG et al. group (Cervera et al., 2005; Pakull et al., 2009). primers of O_30 were mapped on group I and II of the white poplar maps, but only one marker produced in the Duplication and reorganization of the Populus black mapping population was linked to the group D3 and genome The comparisons in the three sections genetic E3 which were homologous with group LGIII of the poplar maps revealed so many discrepancies based on the codom- physical map. The section Aigeiros was considered as inant molecular markers. They were mostly due to the phylogenetic sister of section Tacamahaca (Cervera et al., high level of duplication and reorganization of the Populus 2005) and in this study, only few translocations of mark- genome. These discrepancies were observed through the ers were found in the comparison between these two sec- comparative genomic mapping between the Populus and tions genomic maps. It should be also possible that they Tacamahaca, the oldest and the most advanced reveal species-specific chromosomal reorganization. Like monophyletic groups, and could reveal the existence of marker P_2578, it was located on D1 and E1 groups of the genomic duplication and reorganization in the history of black maps but was observed on group LGVI in the poplar the evolution of the Populus. Based on the publicly physical map. To go thoroughly into these discrepancies available EST collection, seven poplar species maybe it would be necessary to sequence some loci to confirm the share the same large-scale gene-duplication event which conservation or the reorganization of these loci among the must have occurred in the ancestor of poplar, or at least compared species. very early in the evolution of the Populus genus (Sterck Apart from these supported discrepancies there were a et al., 2005). Moreover, duplicated regions of all chromo- number of markers position and order discrepancies somes were clearly identified in the whole genome among these poplar genomes, it is however uncertain sequence of P. trichocarpa (Tuskan et al., 2006). In the whether or not there were true differences or errors in present study, the most complicated things were still pre- either the P. trichocarpa genome assembly or the white sented in the ‘pinwheel’ region which appeared after the and black poplars linkage maps. The assembly of the whole-genome duplication and reorganization (Tuskan et poplar genome is not complete and most likely the al., 2006). In the alignment of the white maps to the P. genome sequence as it is presented today contains numer- trichocarpa genome, the marker P_2818 and W_21 on ous gaps. group ADV and ALV which were homologous with group LGV were observed on group LGII of poplar physical Conclusions and prospects The genomic compara- map. And the same thing occurred on the marker O_402 tive mapping of the three sections, Populus, Aigeiros and which linked to the group ADII instead of located on Tacamahaca, revealed an interesting synteny, duplica- group LGXIV. The status resembled the set of four chro- tion and reorganization, and high conservation of the mosomes involving II, V and XIV in the genus genome in the evolutionary course. In fact, it is ‘pinwheel’. About one-third of chromosome II were the however difficult to find common markers among all the same as two-thirds of chromosome V, and more than 60% available maps published for the Populus genus due to of chromosome II corresponded to about 80% of the chro- differences about characters of different mapping popula- mosome XIV. On the first linkage groups of P. adenop- tions, amplified efficiency of SSR markers in the different oda, there were also two markers respectively observed mapping populations and mapping strategy and on LGIII and LGVI. In addition one of the markers pro- parameters. Only seven common markers were found duced by P_2861, O_241, and O_114 primers were linked among the five maps compared, but among them, two to ALVI, ALIII and ALVI instead of located on LGIX, LGI were discrepant. We also compared our white and black and LGXVI of the poplar physical map. These data sug- poplar maps with several lately reported maps from the gested a complicated course of fission or fusion in the pop- references (Gaudet et al., 2008; Pakull et al., 2009; lar lineage forming these chromosomes. Paolucci et al., 2010; Woolbright et al., 2008), and couldn’t The comparison of the white poplar maps with the find only one common marker among these maps. black poplar maps produced in the same laboratory Certainly we found more than 40 SSR markers had high (Zhang et al., 2009) was interesting because they were amplified efficiency in different mapping populations and constructed using the same mapping strategy and the were mapped on no less than 4 maps of these maps. So common set of codominant markers. In this way a rela- it might be necessary to enhance international communi- tively high number of common SSRs (42) was found. cation and cooperation among the labs in different coun- Discrepancies were observed between the two species tries. maps, and some of them could be due to the duplication To our white poplar maps, the richness should be and reorganization of the Populus genome. Like in the enhanced using SSR or EST-SSR markers and some espe- white poplar maps, the marker O_23 was mapped on the cial gene markers (for example, conserved ortholog set linkage group III, whereas, in the black poplar male par- (COS) marker) (Cabrera et al., 2009) to make comparative ent map, it was mapped on the group E9, the same as in mapping more efficient. To further investigate genome the poplar physical map. Two markers produced by the structure at a finer scale, hundreds of additional con- Comparative genome mapping among Populus 267 served orthologous markers will be necessary. The use Cervera, M., Storme, V., Soto, A., Ivens, B., Montagu, M., of single nucleotide polymorphisms (SNP) of orthologous Rajora, O. P., and Boerjan, W. (2005) Intraspecific and genes from the whole poplar genome sequence appears interspecific genetic and phylogenetic relationships in the genus Populus based on AFLP markers. Theor. Appl. promising (Berlin et al., 2010). Genet. 111, 1440–1456. 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