| Edited by Yoshihiko Tsumura. Yuanxiu Wang and Li-an Xu: Corresponding author. E-mail: wangyx@mail.ahun.edu.cn. E-mail: laxu@njfu.edu.cn. Yuanxiu Wang and Bo Zhang: These authors contributed equally to this work. Note: Supplementary materials in this article are at http://www.jstage.jst.go.jp/browse/ggs |
The species of the genus Populus L. (Salicaceae) are widely distributed in the northern hemisphere from subtropical to boreal forests (Hamzeh and Dayanandan, 2004). According to a recent classification (Eckenwalder, 1996), the genus Populus is classified into 29 species in six sections, and most of the economically and ecologically important species are in the sections Populus syn. Leuce Duby (white poplar), Aigeiros Duby (black poplar) and Tacamahaca Spach (Rahman and Rajora, 2002). Phylogenetic analyses of the family Salicaceae using nuclear rDNA, molecular markers and chloroplast DNA suggest that Populus is the oldest monophyletic group, and close relationship and introgression exist between Aigeiros and Tacamahaca (Azuma et al., 2000; Cervera et al., 2005; Hamzeh and Dayanandan, 2004; Hamzeh et al., 2006; Leskinen and Alstrome-Rapaport, 1999; Rahman and Rajora, 2002).
During the last decades, Populus has been established as a favorable model system for trees and woody perennial plants due to its rapid growth, ease of vegetative and seed propagation, small genome (~480 Mbp), availability of efficient transformation systems, and conservation of chromosome number across the genus (n = 19) (Bradshaw et al., 2000; Taylor, 2002; Tuskan et al., 2004; Jansson and Douglas, 2007). These wonderful biological characters make Populus ideal for experimental researches. Many mapping populations have been produced for numerous geographically and ecologically distinct species from diverse sections within the genus. These experimental populations provide an unique opportunity for comparative mapping in the model system—an opportunity that has been further enhanced by the whole genome sequence of a female P. trichocarpa tree released (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html and http://www.phytozome.net/poplar) (Tuskan et al., 2006). Up to now, 16 mapping works have been published in Populus, leading to the construction of 30 maps relating to 10 poplar species in Populus, Aigeiros and Tacamahaca sections (Cervera et al., 2004; Gaudet et al., 2008; Woolbright et al., 2008; Zhang et al., 2009; Pakull et al., 2009; Paolucci et al., 2010). Several comparative maps with Populus consensus map (based on a P. trichocarpa and P. deltoids background) (Yin et al., 2004, 2008) or the physical map of P. trichocarpa showed high degree of synteny and provided a direct link to P. trichocarpa genome sequence (Woolbright et al., 2008; Zhang et al., 2009; Pakull et al., 2009; Paolucci et al., 2010). Most of these comparative maps are constructed for species of Populus or Aigeiros section and Tacamahaca section, few comparative mapping was actualized based on these three sections altogether.
P. adenopoda Maxim. (quaking aspen, a mountain poplar species native to China) and P. alba L. (white poplar) belong to the Populus section, and are two kinds of trees of high ecological and economic values in China. P. adenopoda is distributed in the middle and low Yangtse valley up to elevations of 1000 m and a pioneer species of ruin ecosystems, growing fast in warm and moist environmental conditions but poorly rooting. Contrarily P. alba is widely distributed across the Eurasian continent and can well adapt to dry and infertile soil. The two species are much different on their phenotype and present high genetic diversity detected using RAPD markers (Yin et al., 2001). In 2006, we obtained more than 1000 F1 hybrids of the two species through inter-specific crossing and seeds culture in green house in order to construct linkage maps, and meanwhile we expected to find some individuals combining merits of the two species in F1 population to use for environmental protection and industrial production. For these two kinds of poplar, Yin et al. (2001) once published preliminary RAPD-based linkage maps through the double pseudo-test-cross strategy. However, the subsequent research came to an end due to the gradual reduction in the size of the mapping population.
Poplar microsatellite (simple sequence repeat, SSR) markers, abundantly available on the website (http://www.ornl.gov/sci/ipgc/ssr_resources.htm) have been used for many genetic maps in both Populus and the related genus Salix (Hanley et al., 2002; Cervera et al., 2004; Yin et al., 2004; Hanley et al., 2006; Gaudet et al., 2008; Woolbright et al., 2008; Pakull et al., 2009; Zhang et al., 2009; Berlin et al., 2010; Paolucci et al., 2010; Liu et al., 2011), and allow an alignment of these maps to each other and to the P. trichocarpa genomic sequence and the consensus map, providing much information for comparative genomic studies of different species in the genus. The dominant markers (for example amplified fragment length polymorphism (AFLP) and sequence related amplified polymorphism (SRAP) (Vos et al., 1995; Li and Quiros, 2001) are unvalued for comparative mapping but can enrich the maps. Populus trees are allogamous species, with very long generation time. Natural poplar populations present a high level of heterozygosity and the possibility to obtain many full-sib progenies where markers can segregate efficiently. Consequently, classical strategies used in grass genetic mapping are not feasible for them. The pseudo-testcross strategy described by Grattapaglia and Sederoff (1994) is usually used in the mapping of forest trees. In this research the genetic maps of the white poplar were constructed using the same mapping strategy and software as two kinds of black poplar maps reported by Zhang et al. (2009) in order to compare between them (here P. deltoides and P. euramericana are regarded as deputies of the species in the section Aigeiros Duby, because the maps of P. deltoides and P. euramericana were constructed based on the common set of SSR markers in our laboratory). Therefore, the objectives of the present study were: (1) to provide genetic linkage maps for P. adenopoda and P. alba based on a combination of SSR and SRAP markers data by the two-way pseudo-testcross strategy and Mapmarker3.0 (Grattapaglia and Sederoff, 1994; Lander, 1987) (2) to align our white poplar maps and the black poplar maps to the physical map of P. trichocarpa using BLAST and the sequence of SSR primers in order to evaluate the degree of macro-synteny and macro-colinearity in Populus, Aigeiros and Tacamahaca sections genomes. Genomic comparisons of three sections poplars will allow us to detect the nuance of genome structure in the genus Populus.
The two parental species used in this study were P. adenopoda Maxim. (Chinese quaking poplar) and P. alba L. (white poplar). The female P. adenopoda parent selected for hybridization was a single tree planted at the Arboretum of Nanjing Forestry University (118°46’E, 32°03’N). However the male P. alba parent was from a natural region across a plain forestry center of Manas County (86°13’E, 44°05’N), Xinjiang, China. In spring 2006, 1100 resulting seedlings were obtained by embryo culture (culture medium: MS0, 75% alcohol 30 s + 1% HgCl 3.5 min sterilizing after 1–2 h washing by water ), and subsequently were planted in nursery garden at Nanjing Forestry University. One hundred and eighty-nine of them were randomly selected for map construction. Total DNA was extracted from frozen young leaves for the 189 progeny and the two parental clones. In this study 1186 pairs of SSRs and 163 primer pairs of SRAPs were selected for marker analysis. For each marker, a χ2 test (P < 0.01 and P < 0.05) was performed to identify alleles of each parent that deviated from Mendelian segregation ratios. Distorted markers were not excluded from linkage analysis and noted with the suffix “d” and “dd” which deviating at 0.01 < P ≤ 0.05 and P ≤ 0.01 respectively. Estimated and observed genome length and map coverage were calculated according to the previously reported method (Hulbert et al., 1988; Grattapaglia and Sederoff, 1994). All details were described in Wang et al. (2010).
The two-way pseudo-test-cross mapping strategy (Grattapaglia and Sederoff, 1994) was applied with MapMaker software version 3.0 (Lander, 1987) generating two maps, one for each parent. Therefore, two data matrices were created with SSR and SRAP markers segregating 1:1 in the progeny. To detect linkages in repulsion phase, the data set was inverted and added to the original data. The inverted markers are indicated by an “r” and represent markers in repulsion. To determine the correct genetic order, the “triple error detection” and the “error detection” features were used to recognize the cases when an event was resulted by an error rather than a recombination avoiding substantial map expansion and interference (Lincoln and Lander, 1992). Initially, markers were grouped by two-point analysis using a default LOD of 3.0 and a maximal recombination. The most likely order of markers within a linkage group was determined by multi-point analysis as follows. For linkage groups with more than five markers, the “three point” command was used to pre-compute the likelihood of all three-point crosses of each group. Then, the “order” command was used to select a subset of markers ordered at a minimum LOD of 3.0. Additional markers were added by the “try” command with a log-likelihood threshold of 2.0. The order of the marker subset was controlled with the “ripple” command. New markers were added only if the new order obtained was confirmed with this command. For the linkage groups with less than five markers, the “compare” command was used. The marker order of these groups was equally supported by a log-likelihood of 2.0. Linkage maps were generated with the “map” command using the Kosambi mapping function. Maps were drawn with the program MapChart 2.1 (Voorrips, 2002).
The female P. deltoides, I-69, from a natural population in Illinois, and P. euramericana, I-45, a natural hybrid between P. deltoides and P. nigra, were selected in the 1950s at the Research Institute of Poplars in Italy and introduced to China in 1972. Approximately 2000 seedlings were generated by interspecific cross between these two clones, from which 450 individuals were randomly selected to establish a field trial. A total of 93 genotypes randomly selected from the 450 seedlings were used for map construction (Zhang et al., 2009).
As the white poplar maps, the pseudo-test cross strategy and MapMaker 3.0 were used to construct two black poplar maps based on AFLP, ISSR, RAPD, SSR and SNP markers. A total of 329 markers (including 146 SSRs, 131 AFLPs, 47 RAPDs, 3 ISSRs, and 2 SNPs) construct a map for the maternal P. deltoids “I-69” genome. This map (denoted as D) is 2293 cM long and contains 22 linkage groups. The map for the paternal P. euramericana “I-45” genome was constructed with 300 markers (including 150 SSRs, 108 AFLPs, 39 RAPDs, 2 ISSRs, and 1SNP). This map (denoted as E) has a total length of 2345.7 cM, composed of 36 linkage groups (Zhang et al., 2009).
Comparisons among the maps of the three sections of the genus Populus were conducted with based on the SSR markers. The availability of the P. trichocarpa genome sequence allows us to align our white and black poplar maps (Zhang et al., 2009) to the physical map of P. trichocarpa. SSR markers mapped on the maps of white and black poplar were searched in the P. trichocarpa genome database (http://genome.jgi-psf.orgPoptr1_1/Poptr1_1.home.html and http://www.phytozome.net/poplar) using BLAST and the sequence of the SSR primers. When the two primers are exactly located on a scaffold (mainly refer to scaffold_1 – scaffold_19 in the P. trichocarpa genome database) and separated by about 100 to 500 bases, the sequence between the 2 primers are considered as a SSR marker. The P. trichocarpa physical map was designed with the MapChart 2.1 (Voorrips, 2002). To simplify the representation, only homologous markers are indicated in the P. trichocarpa physical map. The start base of each SSR was taken as reference. If one of two primers is found to align a homology on the physical groups and the marker was mapped on the corresponding group of the white and black maps, it was considered as a homologous marker and located it on P. trichocarpa physical map. The number of bases per cM was estimated by the ratio physical length/genetic length as described by Gerber and Rodolphe (1994).
The SSR and SRAP markers tested in this study were well defined in the reference (Wang et al., 2010). The pseudo-test cross strategy was used to construct two parent-specific maps with the segregating markers. A total of 140 markers (including 116 SSRs and 24 SRAPs) constructed a map for the maternal P. adenopoda genome. This map (denoted as AD) spanned 2168.3 cM and contained 34 linkage groups (8 triplets and 11 doublets included), with an average length of 63.8 cM, ranging from 2.1 to 211.4 cM. The average distance between adjacent markers was 20.5 cM, ranging from 2.1 to 36.2 cM. The map for the paternal P. alba genome was constructed with 175 markers (including 144 SSRs and 31 SRAPs). This map (denoted as AL) had a total length of 2749.2 cM composed of 38 linkage groups (15 triplets and 6 doublets included), with an average length of 72.3 cM ranging from 4.2 to 330.8 cM. The average distance between adjacent markers was 20.2 cM, ranging from 4.2 to 36.6 cM (Supplementary Fig. S1).
The total genome length of P. adenopoda map was estimated at 2443.2 cM (95% CI = 2154.6~2821.2 cM, p < 0.05) , and the estimate of P. alba map was 2719.5 cM (95% CI = 2436.2~3077.4 cM, p < 0.05). Using the function given by Lange and Boehnke (1992), the expected genome coverage was 93.49% for P. adenopoda map and 94.85% for P. alba map.
A total of 14 and 8 distorted markers were respectively mapped on the maternal and paternal maps. However, these markers were not distributed randomly across the whole genome. In P. adenopoda map, 4 distorted markers were clustered on linkage group ADI, and 3 on linkage groups ADVI and ADXVII. In P. alba map, there were 3 distorted marker clustered on linkage group ALXIX (Supplementary Fig. S1).
The female and male maps presented 39 allelic bridges. Nine linkage groups, with more than two common SSR loci, showed full colinearity. The discrepancies was found for SSR W_21, which mapped on linkage groups ADII and on ALV, and G_4063, which mapped on linkage groups ADXIII and ALXIX (Supplementary Fig. S1).
A physical map of SSR positions was constructed to facilitate the assignment of linkage groups and the comparison of the white and the black poplar maps with the P. trichocarpa genome. Three hundred fifty-eight SSRs were positioned on the genomic sequence resulting from a BLAST search of the primer sequence against the P. trichocarpa genome database (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html and http://www.phytozome.net/poplar).
P. adenopoda (AD) and P. alba (AL) maps were aligned to P. trichocarpa physical map (LG) according the anchor SSRs. Thirty-one of 34 and 36 of 38 linkage groups respectively in AD and AL maps could be assigned to I-XIX groups (among them, XVIII was excluded) of poplar physical map based on 181 orthologous markers of 206 common markers found in poplar genome database using BLAST. In the AD map, 96 orthologous markers aligned 31 linkage groups with 15 groups (IX, XII, XIV and XVIII excluded) of poplar physical map, and in the AL map, 111 orthologous markers aligned 36 linkage groups with 19 groups. It occurred that several sub-groups were aligned to the same homologous group of the physical map. The colinearity of SSRs between the white poplar maps and the physical map was judged to be high since 91.6% and 90.0% of syntenic markers respectively in the AD and AL maps conserved in the same order (Supplementary Fig. S1).
The alignment of the white poplar maps to poplar physical map revealed high marker synteny, however some interesting characteristics were observed for several homologous groups. SSR P_2818 located on ADII and ALII, and W_21 located on ALII and ADV were found on the homologous group LGV of P. trichocarpa. SSR O_402 on group ADV was located on the homologous group LGXIV. On the first linkage groups of white poplar, two markers (G_2955 and O_30) existed on LGIII and one marker(G_437) on LGXVII, SSR P_2861 of ALIV occurred on LGIX, and G_901 of ALXIII occurred on LGXI. The primers of O_241 amplified successfully and produced two markers, one was linked to homologous group (ADI) and another was linked to ALIII which was the homologous group of LGIII. The same things happened on the primers of O_114 and P_2020, for O_114, one was linked to ALXVI and another to ALVI, and for P_2020, one was linked to ADIV and ALIV and another to ALIX. The marker G_4063 on different groups of the white poplar maps was mapped on the group LGXIX of the physical map. The group ALXIX was regarded as the homologous group of LGXIX in poplar physical map but four markers on this group were found on other different groups, G_801 on LGXVIII, G_4067 on LGX, G_1781 on LGV and G_3376 on LGI (Supplementary Fig. S1).
P. deltoides (D) and P. euramericana (E) maps were aligned with the physical map of P. trichocarpa also based on the anchor SSR markers (detail in Supplementary Fig. S2). The result of our study was different from that of Zhang et al. (2009), and we obtained more orthologous markers between the two sections poplars. In our research all linkage groups of the D map were aligned with 19 homologous groups of the poplar physical map based on 142 orthologous SSR markers. In the E map, also 139 orthologous markers aligned 32 linkage groups with 18 homologous groups (XVII excluded) of the physical map. It still occurred that several sub-groups were aligned to the same homologous group of the physical map. Through BLAST, one primer of SSR G_3766 on groups D15 and E15 had a hit in poplar genomic sequence database but another hadn’t, since this marker was located on the corresponding group of the reference (Yin et al., 2004), groups D15 and E15 regarded as the homologous group of LGXV. For the E map, the same as Zhang et al. (2009), no linkage group was aligned to the homologous group LGXVII of the physical map. And group E21 of E map in reference (Zhang et al., 2009) containing only one SSR markers were found no homologous group from the physical map.
Synteny and co-linearity were high conserved between the black poplar maps and the physical map. One hundred and thirty eight markers (97.2%) of 142 orthologous markers in D map and 129 (92.8%) of 139 in E map were placed onto the homologous groups in the physical map. For the co-linearity, there were 134 out of 142 (94.4%) orthologous markers between the D map and the physical map, and also 131 out of 139 (94.2%) between the E map and the physical map. The co-linearity between the D and E linkage maps was high, with 85 allelic bridges. The order of the orthologous markers was well conserved between the two maps for 80 out of 85 (94.1%). Five homologous markers were placed onto deferent locations between the groups D3 and E3, D4 and E4, D6 and E6, D16 and E16 (Supplementary Fig. S2).
Finally, we aligned our white poplar maps and the black poplar maps with the P. trichocarpa physical map. The co-alignment of these 5 maps revealed 358 common markers. The marker order was conserved in most of the cases. The linkage group I was the largest groups in all the maps and had the greatest number of homologous markers (including 38 markers). The common markers among the 5 maps are presented in detail on Fig. 1.
 View Details | 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 paralogous 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. |
Only 7 of 358 markers were present on the 5 maps. Five markers (P_2852, G_3269, G_2900, P_2536 and G_2020) were on the homologous groups (linkage groups I, III, V and X) and 2 (O_40 and G_4063) were in synteny between black poplar maps and the P. trichocarpa physical map but exceptional on the white maps. The marker O_40 was on group ADVI and ALVI, instead of group D2 and E2 which were homologous group with LGII in the P. trichocarpa physical map. The marker G_4063 was mapped on 2 different groups of white poplar maps, ADXIII and ALXIX, but in black poplar maps, one (G_4063A) of those two markers produced by the primers of G_4063 in the black experimental population was mapped on the group D19 and E19 like LGXIX of poplar physical map, another marker (G_4063B) was mapped on the group E12.
Twenty-three markers were present on 4 maps, and 17 of them were on the homologous groups among three genus maps and the rest 6 (O_23, P_2818, O_30, O_402, O_241, and P_2879) and were discrepant. The marker O_23 was linked to group ADIII and ALIII instead of on group E9 and LGIX. The same thing occurred on the marker P_2818 which was linked to group ADV and ALV instead of on D2 and LGII. The primers of O_30 was amplified successfully in white poplar mapping population and produced two markers which respectively were linked to group ADI, ADXIV and ALXIV non-homologous to group D3 and LGIII. The primers of O_402 amplified successfully in black poplar mapping population and produced 3 markers which respectively were linked to group D14, E6 and E13 non-homologous to group ADII and LGII. In white poplar mapping population, the primers of O_241 produced 2 markers, one (O_241A) was linked to group ADI, the homologous group of D1 and LGI, but another (O_241B) was linked to group ALIII. And the primers of P_2879 amplified successfully and produced three markers, P_2879A and P_2879B were distorted markers linked to group ADVI and P_2879C didn’t deviated from Mendelian segregation ratios and was linked to group ALXIII, but these two groups weren’t homologous to group D3 and LGIII.
One hundred and six markers were present on 3 maps of the three sections. Among them 28 were common on the poplar physical maps and white poplar maps, 66 were common on the poplar physical map and the black poplar maps, and 12 were common on the P. trichocarpa physical map, one of the white poplar maps and one of the black poplar maps. Of 12 markers existing in three section maps, the markers produced by the primers of O_465, P_2861 and G_93-2 were exceptional, for example, the primers of P_2861 produced 3 markers which respectively were linked to group ALII, ALIV and ALVI non-homologous to group E9 and LGIX. Two hundred and twelve markers were common to the P. trichocarpa physical map and one of the white or black poplar maps. Of 212 markers 123 were homologous between the P. trichocarpa physical map and one of the white poplar maps and 89 were homologous between the P. trichocarpa physical map and one of the black maps.
We had previously constructed a genetic linkage map of P. adenopoda Maxim. × P. alba L. based on the same markers by Joinmap4.0 (Stam, 1993; Wang et al., 2010). We thought the comparative genetic map was more valuable and reliable based on the same mapping strategy and software than the different. In this article, two parents-specific genetic linkage maps containing SSR and SRAP markers were created for the section Populus by using a two-way pseudo-test cross mapping strategy (Grattapaglia and Sederoff, 1994) and Mapmaker software. Two hundreds and two mapped SSR loci allow comparisons of the maps with other genetic maps of Populus and the direct link to the P. trichocarpa genomic sequence. The estimated genome length (2443.2 cM for P. adenopoda and 2719.5 cM for P. alba) fell within the range of the values found in previous studies and was near to the original estimate of 2400–2800 cM reported by Bradshaw et al. (1994), which has been verified through simulation studies (Yin et al., 2004). The estimated genome length for P. adenopoda was higher than 2104 cM instead of that for P. alba was little more than 2632 cM reported by Yin et al. (2001).
The two white poplar maps contained two kinds of markers were surpassed the two parental maps based on only RAPDs reported by Yin et al. (2001). The result in this study also verified the structure of P. alba were more complex than P. adenopoda. More segregated loci were identified for P. alba than for P. adenopoda, and the observed genome length of the paternal map was 26.79% longer than the maternal map.
Previous simple comparison between the genetic maps of P. deltoides and P. euramericana and the composite map of P. trichocarpa × P. deltoides (Zhang et al., 2009) in our laboratory were considered as a useful starting point for this study. With the help of the maps for P. alba and P. adenopoda developed based on the same set of SSR markers as Zhang et al. (2009) herein, additional comparisons could be made among the genomes of the three sections in Populus, resulting in a doubling of the number of comparison points. The dominant markers SSR as bridges for comparison in this study were much more than those in the similar previous studies (Woolbright et al., 2008; Pakull et al., 2009; Paolucci et al., 2010).
Alignment of the genomic maps made it possible to validate a high degree of synteny among three sections of Populus (Fig. 1). Three hundred and fifty-eight or about 31% of SSR markers selected from P. trichocarpa genome (more than 1140) were aligned. Fifteen linkage groups in AD map, 19 in AL map, 19 in D map and 18 in E map respectively could be successfully aligned to P. trichocarpa genome by at least one SSR anchor marker (Supplementary Figs. S1 and S2, and Fig. 1). Many sub-groups on AD, AL and E maps were aligned to the homologous groups of P. trichocarpa, and these sub-groups aligned to one homologous group couldn’t be linked because of lacking enough markers to link. Therefore, enhancing the maps’ density and precision was the focus of the future research aiming at aligning the complete maps to P. trichocarpa genome and using them to identify those common QTLs that affect important economic traits and fundamental biological process in different species genomes of Populus.
In the two black poplar maps, the total number of SSR markers aligned to P. trichocarpa genome was nearly the same as that in the white poplar maps (208 versus 204), however, owing to more fully informative loci tested in the black poplar interspecific hybrid than those in the white (Wang et al., 2010; Zhang et al., 2009), the numbers of orthologous markers aligned to P. trichocarpa genome respectively on D and E maps (142 and 139 respectively) were much more than AD and AL (96 and 111 respectively) (Fig. 1). Between the black poplars and P. trichocarpa genome, the level of marker synteny were higher than that between the white poplars and P. trichocarpa genome (respectively 97.2% in D map and 92.8% in E map, 91.6% in AD map and 90.0% in AL map). At the level of genetic markers, alignment of the white poplar maps to P. trichocarpa genome showed more discrepant than that of the black poplar maps. The most likely reason for these discrepancies were that the SSR primers were mainly developed in P. trichocarpa and therefore, SSR primer binding site sequences were not identical in white poplars. P. deltoides and P. euramericana are members of the Aieiros section, the phylogenetic sister of the Tacamahaca section, and more closely related to P. trichocarpa than P. alba and P. adenopoda, two species of the Populus section which is the oldest monophyletic group (Cervera et al., 2005; Pakull et al., 2009).
The comparisons in the three sections genetic maps revealed so many discrepancies based on the codominant molecular markers. They were mostly due to the high level of duplication and reorganization of the Populus genome. These discrepancies were observed through the comparative genomic mapping between the Populus and Tacamahaca, the oldest and the most advanced monophyletic groups, and could reveal the existence of genomic duplication and reorganization in the history of the evolution of the Populus. Based on the publicly available EST collection, seven poplar species maybe share the same large-scale gene-duplication event which must have occurred in the ancestor of poplar, or at least very early in the evolution of the Populus genus (Sterck et al., 2005). Moreover, duplicated regions of all chromosomes were clearly identified in the whole genome sequence of P. trichocarpa (Tuskan et al., 2006). In the present study, the most complicated things were still presented in the ‘pinwheel’ region which appeared after the whole-genome duplication and reorganization (Tuskan et al., 2006). In the alignment of the white maps to the P. trichocarpa genome, the marker P_2818 and W_21 on group ADV and ALV which were homologous with group LGV were observed on group LGII of poplar physical map. And the same thing occurred on the marker O_402 which linked to the group ADII instead of located on group LGXIV. The status resembled the set of four chromosomes involving II, V and XIV in the ‘pinwheel’. About one-third of chromosome II were the same as two-thirds of chromosome V, and more than 60% of chromosome II corresponded to about 80% of the chromosome XIV. On the first linkage groups of P. adenopoda, there were also two markers respectively observed on LGIII and LGVI. In addition one of the markers produced by P_2861, O_241, and O_114 primers were linked to ALVI, ALIII and ALVI instead of located on LGIX, LGI and LGXVI of the poplar physical map. These data suggested a complicated course of fission or fusion in the poplar lineage forming these chromosomes.
The comparison of the white poplar maps with the black poplar maps produced in the same laboratory (Zhang et al., 2009) was interesting because they were constructed using the same mapping strategy and the common set of codominant markers. In this way a relatively high number of common SSRs (42) was found. Discrepancies were observed between the two species maps, and some of them could be due to the duplication and reorganization of the Populus genome. Like in the white poplar maps, the marker O_23 was mapped on the linkage group III, whereas, in the black poplar male parent map, it was mapped on the group E9, the same as in the poplar physical map. Two markers produced by the primers of O_30 were mapped on group I and II of the white poplar maps, but only one marker produced in the black mapping population was linked to the group D3 and E3 which were homologous with group LGIII of the poplar physical map. The section Aigeiros was considered as phylogenetic sister of section Tacamahaca (Cervera et al., 2005) and in this study, only few translocations of markers were found in the comparison between these two sections genomic maps. It should be also possible that they reveal species-specific chromosomal reorganization. Like marker P_2578, it was located on D1 and E1 groups of the black maps but was observed on group LGVI in the poplar physical map. To go thoroughly into these discrepancies it would be necessary to sequence some loci to confirm the conservation or the reorganization of these loci among the compared species.
Apart from these supported discrepancies there were a number of markers position and order discrepancies among these poplar genomes, it is however uncertain whether or not there were true differences or errors in either the P. trichocarpa genome assembly or the white and black poplars linkage maps. The assembly of the poplar genome is not complete and most likely the genome sequence as it is presented today contains numerous gaps.
The genomic comparative mapping of the three sections, Populus, Aigeiros and Tacamahaca, revealed an interesting synteny, duplication and reorganization, and high conservation of the genus genome in the evolutionary course. In fact, it is however difficult to find common markers among all the available maps published for the Populus genus due to differences about characters of different mapping populations, amplified efficiency of SSR markers in the different mapping populations and mapping strategy and parameters. Only seven common markers were found among the five maps compared, but among them, two were discrepant. We also compared our white and black poplar maps with several lately reported maps from the references (Gaudet et al., 2008; Pakull et al., 2009; Paolucci et al., 2010; Woolbright et al., 2008), and couldn’t find only one common marker among these maps. Certainly we found more than 40 SSR markers had high amplified efficiency in different mapping populations and were mapped on no less than 4 maps of these maps. So it might be necessary to enhance international communication and cooperation among the labs in different countries.
To our white poplar maps, the richness should be enhanced using SSR or EST-SSR markers and some especial gene markers (for example, conserved ortholog set (COS) marker) (Cabrera et al., 2009) to make comparative mapping more efficient. To further investigate genome structure at a finer scale, hundreds of additional conserved orthologous markers will be necessary. The use of single nucleotide polymorphisms (SNP) of orthologous genes from the whole poplar genome sequence appears promising (Berlin et al., 2010).
And an alignment of QTLs could be also done to identify common QTLs in different genetic backgrounds. Recently some QTLs interrelated with the characters of the white poplar leaf were located and existed on the regions of the QTL hotspots for yield in short rotation coppice bioenergy poplar (Fumin Zhang, personal communication) (Rae et al., 2009).
In this study, comparative mapping based of the co-alignment of common markers among genetic and physical maps enabled to correlate linkage information from different genetic maps and to validate the accuracy of locus ordering from the different mapping populations. Comparative mapping also allows the comparison of genome structure within the genus Populus and, thus, to study chromosomal evolution by detecting chromosome rearrangements (Cervera et al., 2004). Moreover, the alignment of the genetic map with the poplar genome sequence allows to locate large numbers of candidate genes on the genetic maps and to compare the map position with QTLs. These are our research fields in the future.
We thank Dr. Lianxin Dong of the College of Forestry at Xinjiang Agricultural University for collecting flowering branches of P. alba. This work was supported by National High Technology Research and Development Program of China (863 Program: 2006AA100109) and Key Laboratory of Forest Genetics & Biotechnology (Nanjing Forestry University), Ministry of Education of China (Grant No. FGB200802).
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