Genetic linkage mapping in aspen (Populus tremula L. and Populus tremuloides Michx.)
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- Pakull, B., Groppe, K., Meyer, M. et al. Tree Genetics & Genomes (2009) 5: 505. doi:10.1007/s11295-009-0204-2
A large number of simple sequence repeat (SSR) marker-containing genetic maps are available for several Populus species. For aspen however, no SSR-containing map has been published so far. In this study, genetic linkage mapping was carried out with an interspecific mapping pedigree of 61 full-sib hybrids of European × quaking aspen (Populus tremula L. × Populus tremuloides Michx.), using the two-way pseudo-testcross strategy. Amplified fragment-length polymorphism (AFLP) and SSR markers were used for mapping, resulting in the first SSR-containing genetic linkage maps for aspen. The maps allow comparisons with a Populus consensus map and other published genetic maps of the genus Populus. The maps showed good collinearity to each other and to the Populus consensus map and provide a direct link to the Populus trichocarpa genomic sequence. Sex as a morphological trait was assessed in the mapping population and mapped on a non-terminal position of linkage group XIX on the male P. tremuloides map.
KeywordsSSRAFLPSexLinkage mapGenetic mapLinkage groupChromosomePopulus trichocarpa
During the last decades, the genus Populus has been established as a genetic model system for trees. The approximately 30 mostly dioecious species of the genus are classified into six sections, and all share a haploid chromosome number of 19 and a relatively small genome (about 500 Mbp for Populus trichocarpa Torr. & Gray) (Eckenwalder 1996; Tuskan et al. 2006). They are outstanding due to their fast growth, economic value, wide natural distribution and ease of genetic transformation and vegetative propagation (Bradshaw et al. 2000; Taylor 2002; Strauss and Martin 2004; Cronk 2005; Howe and Brunner 2005). Since September 2004, the assembled genome sequence of a female P. trichocarpa tree is available on the website of the Joint Genome Institute (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html, Tuskan et al. 2006).
The closely related European and quaking aspen (Populus tremula L. and Populus tremuloidesMichx.) belong to the section of aspen and white poplars (Populus spp. section Populus, syn. section LeuceDuby) and are two of the most widespread tree species of the Northern Hemisphere (Hegi 1957; Perala 1990; Tamm 2001). Like other Populus species, aspens easily hybridise inside their section, producing a strong heterosis effect in the progeny (Li and Wu 1996; Liesebach et al. 2000; Yu et al. 2001). However, their ecological behaviour diverges from that of other poplars. Unlike other Populus species, which are predominantly adapted to riparian ecosystems, aspens can tolerate a wide range of climate conditions, soil fertility and water supply. They are not sensitive to drought and wind. A large number of dense genetic linkage maps was constructed for several North American, European and Asian Populus species (Wu et al. 2000a, Cervera et al. 2001; Yin et al. 2002, Cervera et al. 2004, Yin et al. 2004a, Zhang et al. 2004, Gaudet et al. 2007, Woolbright et al. 2007). Most include microsatellite (simple sequence repeat (SSR)) markers, abundantly available on http://www.ornl.gov/sci/ipgc/ssr_resource.htm, which allow an alignment of these maps to each other and to the P. trichocarpa genomic sequence, providing information for comparative genomic studies of different species. Furthermore, these linkage maps can be used for the identification of chromosomal regions coding for monogenic or quantitative traits (QTL mapping, Frewen et al. 2000). Molecular markers, closely linked to traits of interest, can be used for marker-assisted cloning (Gibson and Sommerville 1993) or selection (Wu et al. 2000b) in plant breeding.
For aspen, just one map has been published, containing only few allozyme and restriction fragment length polymorphism markers (Liu and Furnier 1993). However, no map containing SSR markers—for comparison to other maps—has been available so far (Cervera et al. 2004).
The genetic mechanism of sex determination in poplar is largely unknown. Alstrom-Rapaport et al. (1998) suggested a multi-locus sex determination in the Salicaceae, based on the commonly observed biased sex ratios. Yin et al. (2008) reported evidence that, in P. trichocarpa, chromosome XIX seems to have sex chromosome function with the female probably being the heterogametic sex. In this paper, we report the results of a project aimed at providing genetic linkage maps for aspen (P. tremula × P. tremuloides) based on a combination of amplified fragment length polymorphism (AFLP; Vos et al. 1995) and SSR marker data. For this purpose, the two-way pseudo testcross strategy has been used (Ritter et al. 1990, Grattapaglia and Sederoff 1994). As described by Cervera et al. (2000), SSR markers allow the formation of allelic bridges between the two parental maps and, further on, an alignment to other maps of the genus Populus. Additionally, sex as a morphological trait has been mapped.
Materials and methods
Plant material and DNA isolation
The mapping pedigree consisted of 61 full-sib F1-individuals, derived from an interspecific cross between P. tremula ‘Brauna 11’ × P. tremuloides ‘Turesson 141’, produced in 1951 by the Institute for Forest Genetics and Forest Tree Breeding, Grosshansdorf, Germany. The sex of all flowering individuals was assessed visually. Genomic DNA was extracted from buds and leaves according to the protocol of Dumolin et al. (1995).
Information on the SSR markers used was gained from the SSR resource provided by the International Populus Genome Consortium IPGC (http://www.ornl.gov./sci/ipgc/ssr_resource.htm, prefix PMGC and GCPM). Primers with the prefix ORPM and WPMS were developed by the Oak Ridge National Laboratory (Tuskan et al. 2004) and the Center for Plant Breeding and Reproduction Research, respectively (van der Schoot et al. 2000, Smulders et al. 2001). Additional primers developed for P. tremuloides carrying the prefix PTR were obtained from Dayanandan et al. (1998) and Rahman et al. (2000). Further on, three additional primer pairs based on the P. trichocarpa genomic sequence were used: two primer pairs for a SSR located in a Cinnamyl-alcohol-dehydrogenase gene (CAD Ex2/Intr2, forward: 5′-CAAGGTTATCAGCTGTGG-3′, reverse: 5′-ATATTGTGTGCGTGTTCG-3′ and CAD Ex12, forward: 5′-TCAACTGGGCATCTCGCT-3′, reverse: 5′-CCACAGCTGATAACCTTG-3′), and a SSR primer pair on linkage group (LG) XIX (BPTC66, forward: 5′-GGCCGTGATTCTCATCATGATG-3′, reverse: 5′-CTTCTTCATCAACAACTGCGTCC-3′).
All primers used were tested for transferability to P. tremula/P. tremuloides and heterozygosity in the parental trees. Informative SSRs were selected for analysis in the entire mapping population.
Polymerase chain reaction (PCR) was performed in a total volume of 25 μl containing 60 ng of template DNA, 5 pmol of forward and reverse primers (synthesised by MWG, Ebersberg, Germany), 100 μM of d-ATP, d-GTP and d-TTP, 80 μM of d-CTP (Carl Roth, Karlsruhe, Germany), 0.4 μM Cy5-fluorescent labelled d-CTP (GE Healthcare, Freiburg, Germany), 1.5 mM MgCl2, 2% DMSO, 1× Taq buffer and 1 U Taq DNA-polymerase (DCSPol, DNA Cloning Service, Hamburg, Germany). Thermocycling was carried out on a TGradient cycler (Biometra, Goettingen, Germany) under the following conditions: 4 min at 94°C, 35 cycles of 94°C for 30 s, 50–58°C for 45 s, 72°C for 1 min, final extension of 10 min at 72°C.
Five to 7 μl of the PCR products were diluted in 3 μl loading buffer, 2 μl 1× TE and 1 μl of internal standard fragment solution (GE Healthcare) and separated using the automatic sequencing unit ALFexpress II (GE Healthcare) under the following conditions: running time 105–180 min, short gel plate with polyacrylamide gel ReproGel high resolution (GE Healthcare), running temperature 50°C, voltage 1,500 V.
Data analysis was carried out using the Fragment Analyser software (version 1.03; GE Healthcare).
AFLP analysis was carried out according to the protocol described by Markussen et al. (2002). Pre-amplification reactions were performed with one selective 3′-nucleotide on each primer. Main amplification was carried out using Cy5-labelled EcoRI-primers with three to four selective nucleotides and MseI-primers with three selective nucleotides. The final PCR products were separated using the automatic sequencing unit ALFexpress II (GE Healthcare) and analysed using the Fragment Analyser software (version 1.03; GE Healthcare).
AFLP markers in the genetic linkage maps were named as in Hanley et al. (2002): E for EcoRI-primer, M for MseI-primer followed by a three-letter code for the selective nucleotides and the fragment length.
The linkage maps were constructed with JoinMap 3.0® software (van Ooijen and Voorrips 2001), using the two way pseudo-testcross mapping strategy (Grattapaglia and Sederoff 1994). The maps were based on an F1 progeny, resulting in two separate maps, one for each parent.
All markers were scored as testcross markers, with alleles heterozygous in one parent and absent in the other. SSR markers segregating 1:1:1:1 (segregation types ab × cd and ef × eg) were scored by separation of their maternal and paternal segregation data. Intercross markers, with alleles heterozygous in both parents and segregating 3:1 were not used.
Markers with a high amount of missing data (>5%) were excluded from the map calculations (with the exception of GCPM_51 and GCPM_1522 on the paternal map). Markers with significant deviation from the expected Mendelian ratio (distorted markers) were detected by a χ2 test, included in JoinMap®. Highly distorted markers (P < 0.015) which were linked with no other markers or which disturbed the map calculation of their group were excluded from the map calculation due to possible spurious linkage caused by scoring mistakes. Other distorted markers remained untouched due to a possible relation with the small population size or possible linkage of these markers to genes which are subject to selection processes (Bradshaw and Stettler 1994, Cervera et al.2001). Remaining highly distorted markers (P < 0.015) were indicated on the map with underlining and italic type. Distorted markers with 0.015 < P < 0.05 were indicated on the map by italic type.
A limit of detection (LOD) threshold of 5.0 was chosen to assign markers to linkage groups. When two groups—separated at LOD 5.0—could be assigned to one linkage group by alignment to the Populus consensus map (based on a P. trichocarpa and Populus deltoides background) available on http://popgenome.ag.utk.edu/cgi-bin/cmap/map_set_info (Yin et al. 2008), those groups were—if possible—connected by choosing a LOD score of 4.0.
Maps were calculated using Kosambi’s mapping function (Kosambi 1944) and drawn using MapChart 2.1® (Voorrips 2002). Assignment to linkage groups and orientation of the linkage groups was determined by alignment of the linkage groups to the Populus consensus map or BLAST search of the primer sequences against the P. trichocarpa assembled genome sequence.
Seventy-six different AFLP primer combinations yielded 602 segregating loci, leading to an average of 7.92 informative markers per primer enzyme combination (pec). Among these AFLP markers, 278 (46.2%) were maternally informative, and 324 (53.8%) were paternally informative.
Overview of the numbers of tested and used SSR markers, subdivided according to the prefixes of the primer pairs
Final linkage analysis in the paternal parent was based on 385 AFLP and SSR markers, forming 37 linkage groups at a LOD threshold of 5.0. Twenty-eight of these linkage groups could be assigned to P. trichocarpa linkage groups I–XIX. Linkage groups I, II, VI, VII, IX, XV and XIX were split in two parts, with the two parts of linkage groups II and XIX joined at a LOD threshold of 4.0. Linkage group VIII was split into three parts, with two of them joined at a LOD threshold of 4.0. Thirty-seven of the mapped markers were distorted with P < 0.05. Remarkable clusters of distorted markers formed on linkage groups III, XI and XIX (Fig. 1).
In all cases where comparisons with the Populus consensus map (based on a P. trichocarpa and P. deltoides background) were possible or a BLAST search of the primer sequences against the P. trichocarpa genomic sequence was successful, collinearity of the mapped SSR markers with the Populus consensus map was high, with just two clear exceptions. On linkage group I of the male parent, GCPM_3502 and PMGC_2731 changed their order in relation to ORPM_20. PMGC_2515 mapped on linkage group XV on the female map instead of linkage group XIV as on the Populus consensus map. Collinearity between the male P. tremuloides and female P. tremula map was not determinable for many linkage groups because of different mapped SSR loci. If more than two identical SSR loci were present on both maps, they showed full collinearity.
Observed map length
The observed map length at LOD 5.0, including all 32 linkage groups, triplets and doublets of the maternal map and all 37 linkage groups, triplets and doublets of the paternal map was 1,681.5 cM and 1,403.2 cM, respectively.
Sex could be determined for 57 out of 61 individuals (93.4%). Twenty individuals were female (35.1%), 37 were male (64.9%). The sex trait was scored as a testcross marker in two ways, either inherited from the mother or from the father. Sex as a testcross marker inherited from the mother showed no linkage to any other marker and could not be mapped on the female map.
The sex trait scored as a testcross marker inherited from the father was mapped on a non-terminal position of linkage group XIX on the male map.
In this paper, we report the first SSR-containing genetic linkage maps for aspen (P. tremula and P. tremuloides) based on a mapping pedigree of 61 P. tremula × P. tremuloides hybrids, produced in 1951.
The maps were created by using a two-way pseudo-testcross mapping strategy (Ritter et al. 1990, Grattapaglia and Sederoff 1994). Like other forest species, aspen carries a high level of heterozygosity (Yin et al. 2001), leading to a sufficient number of mappable testcross markers, with alleles heterozygous in one parent and absent in the other in the F1 generation.
Eighty-six mapped SSR loci allow comparisons of the maps with other genetic maps of Populus, an assignment to the 19 linkage groups of P. trichocarpa and other Populus species and a direct link to the P. trichocarpa genomic sequence.
Mapping of SSR markers
At least two SSR loci are needed for a full alignment of linkage groups. For linkage groups carrying only one SSR marker, the orientation of the group remains unclear. A higher number of SSR markers on the maps would therefore be desirable. Unfortunately the number of mapped SSR markers was very low compared to the number of tested SSR primer pairs (30.7%). Furthermore, of all the SSR markers mapped, the majority (53.5%) was informative for only one parent, leading to an even lower percentage of mapped SSR markers for the individual maps (23.5% for the maternal and 21.4% for the paternal map).
The most likely cause of this low SSR yield is that the SSR primers were mainly developed in P. trichocarpa; thus, SSR primer binding site sequences are not identical in aspen.
In comparison, maps of Populus nigra L. (39.4%, Gaudet et al. 2007), P. trichocarpa × P. deltoides Marsh. (83.6%, Yin et al. 2004a) and Populus fremontii Wats × Populus angustifolia James (63.6%, Woolbright et al. 2007) showed higher percentages of informative markers. All of these species are either members of the Tacamahaca section, the section of P. trichocarpa or its phylogenetic sister section Aigeros and therefore more closely related to P. trichocarpa than the more distinct P. tremula and P. tremuloides, which are members of the Leuce section (Cervera et al. 2005). This closer relationship probably leads to a higher similarity of SSR primer binding sites. Furthermore, the tested PTR primers developed for P. tremuloides showed a higher rate of successfully mapped markers for the reported cross (66.6%) and no unamplified markers.
Map saturation and observed map length
A saturated map is defined by the correct number of linkage groups and the presence of no unlinked markers. The high number of linkage groups (32 for the maternal and 37 for the paternal map) and the presence of unlinked markers shows that the maps are not fully saturated. This is supported by the fact that many linkage groups of the male and female maps are split into two parts, compared with the Populus consensus map, available on http://popgenome.ag.utk.edu/cgi-bin/cmap/map_set_info (Yin et al. 2008).
With 1,681.5 cM, the observed map length of the female map is higher than that of the male map (1,403.2 cM). This could resemble the tendency for higher recombination rates observed in angiosperm females (Yin et al. 2004a). However, Zhang et al. (2002), Yin et al. (2004a) and Gaudet et al. (2007) all report higher observed map lengths for the male compared to the female parent. Therefore, our results could simply be an artefact based on the incomplete map saturation and the fewer and therefore larger linkage groups of the female map.
A direct comparison of genome lengths with other genetic maps of the genus is not possible due to different levels of map saturation, variations in the progeny number, varying rates of genotyping errors and the use of different mapping functions [Kosambi’s (1944) mapping algorithm leads to shorter map distances than Haldane’s (1919)].
Despite the sometimes low number of SSR markers on certain linkage groups, in all cases capable of evaluation, the maps showed a high collinearity to each other and to the Populus consensus map, with just two clear exceptions compared to the Populus consensus map.
So far, all SSR-containing maps of the genus Populus showed this high degree of collinearity, based on a common origin of the species (Tuskan et al. 2006). The two exceptions to collinearity described above could be based on genotyping errors, spurious linkage due to the relatively small population size or chromosomal rearrangements due to the—compared to other Populus species used for mapping—quite distinct species used for this mapping project.
Markers with significant deviation from the expected Mendelian ratio (distorted markers) occurred on the female and male maps. Distorted markers can disturb the mapping procedure, especially if they are based on simple scoring mistakes. On the other hand, the distortedness of markers could occur by coincidence due to the relatively small population size or by linkage of markers to genes which are the target of selective forces, such as, e.g. resistance genes (Kuang et al. 1999). The P. tremula ‘Brauna 11’ × P. tremuloides ‘Turesson 141’ pedigree was 57 years old, and the trees may therefore have been exposed to all kinds of selective forces during their lifespan, resulting in the loss of certain trees.
For this reason, distorted markers were not automatically deleted from the maps. Highly distorted markers (P < 0.015) were excluded if they were linked with no other markers or disturbed the map calculation and were therefore probably based on scoring mistakes but were kept if they formed clusters with other distorted markers. Distorted markers with 0.015 < P<0.05 were also used for mapping. A clustering of distorted markers, potentially marking regions containing genes under selective forces, appeared on certain linkage groups and has also been reported by, e.g. Cervera et al. (2001) and Yin et al. (2004a).
Two of the clusters of distorted markers reported here occur in regions of the genetic linkage map which could be assigned to distorted regions of the P. nigra map (Gaudet et al. 2007). The distorted region surrounding PMGC 2707 on linkage group II of the female P. tremula map and the distorted region on the distal end of linkage group III (including PMGC 2879) of the male P. tremuloides map also occur on the male map of the P. nigra linkage map. This could support the assumption of the influence of selective forces on these regions.
The markers closely linked to the sex trait on linkage group XIX of the male map show a certain degree of distortedness. This is probably based on the male-biased sex ratio of the population used, which could be based on coincidence but seems to be quite common in P. tremula and P. tremuloides (McLetchie and Tuskan 1994) and was, e. g., also observed for P. deltoides × P. nigra (Yin et al. 2008). Moreover, the corresponding region on the P. fremontii × P. angustifolia map (Woolbright et al. 2007) also shows a block of distortion, and Yin et al. (2004b) localised the MER-locus, involved in Melampsora rust resistance, in the same region.
Sex determination was mapped to a single locus in a non-terminal position on linkage group XIX (equal to linkage group 5 in Markussen et al. 2007) based on the analysis of the P. tremula × P. tremuloides cross reported here. The apparently terminal position of the sex trait in Markussen et al. (2007) is based on the earlier stage of development of this map, with fewer markers mapped and a lower map saturation. Furthermore, sex could only be mapped on the male P. tremuloides map. Sex remained unlinked on the female map. Not a single sex-linked marker inherited from the female parent could be mapped on linkage group XIX or any other linkage group.
Comparisons with mapped sex loci in other species of the genus Populus reveal that sex has been mapped to a single locus on linkage group XIX in all published sex-containing linkage maps so far but on different positions on this linkage group and inconsistently on either only the male or only the female map. In Populus alba L.—closely related to P. tremula and P. tremuloides—sex has been mapped to a single locus in a non-terminal position on linkage group XIX on the female map only (mentioned in Gaudet 2006, Gaudet et al. 2007). In P. nigra however, more closely related to P. trichocarpa than to P. tremula and P. tremuloides, sex has been mapped on a sub-terminal position of linkage group XIX on the male map only (Gaudet et al. 2007).
The Populus consensus map by Yin et al. (2008), which is based on a P. trichocarpa and P. deltoides background, while the sex-locus was determined in a P. deltoides × [P. deltoides × P. nigra] cross, carries the sex locus on a terminal position on the linkage group. Yin et al. (2008) reported evidence for an incipient sex chromosome in Populus with the female probably being the heterogametic sex. The latter was based on the observation that the peritelomeric region of linkage group XIX, where the sex-locus has been mapped on the Populus consensus map, showed a region of recombination suppression in the female parent and that the corresponding region in the sequenced female P. trichocarpa tree showed highly divergent haplotypes.
The consistent assignment of the sex locus to linkage group XIX shows that this linkage group definitely plays an important role in sex determination.
The exclusive mapping of sex loci in one of two parents depending on the analysed cross/species seems unusual. Sex-linked markers are inherited exclusively from the father in some species and from the mother in others. So at first sight, different species of the genus Populus seem to have an apparently different sex inheritance while some of them are crossable and even hybridise naturally (e.g. P. alba × P. tremuloides, Heimburger 1936, Peto 1938).
The different map positions of the sex loci in different species could be explained by chromosomal rearrangements. However, the collinearity of the maps of linkage group XIX based on the mapped SSR markers seems preserved, while the position of the mapped sex locus changes relative to the mapped SSR markers. So, even if the number of SSR markers mapped on linkage group XIX on the maps of P. tremuloides, P. nigra and P. alba is low and chromosomal rearrangements cannot be completely ruled out, it seems as if the mapped sex loci of the different species are located on different chromosomal positions.
A possible explanation for these inconsistent results regarding the position of the mapped sex loci on the linkage group and the mapping on male or female map could be a multi-locus system as has been suggested for the Salicaceae by Alstrom-Rapaport et al. (1998) based on the commonly observed biased sex ratios. It could be possible that several sex-determining loci with different alleles exist in different positions on linkage group XIX. The effects of individual alleles at a locus depend on genetic background with only the dominant locus being mapped in each cross.
In summary, the results obtained so far make definite conclusions about the inheritance of sex in Populus difficult, and further studies are needed.
For a deeper insight into the inheritance of sex in aspen, a more elaborate mapping of linkage group XIX with the identification of SSR sequences completely linked to sex is in progress. Primers of SSR sequences, identified with the help of the P. trichocarpa genomic sequence, are being mapped in P. tremula and P. tremuloides. Sex-linked SSR loci with defined positions may be useful as starting points for the development of probes for the screening of an existing BAC library of the male parent (Fladung et al. 2008). Impeding progress in this case could be the fact that the region surrounding the sex trait is possibly a region of high recombination suppression, a characteristic feature of sex chromosomes (Charlesworth et al. 2005, Yin et al. 2008). This would lead to large genomic regions, which may contain a high number of genes and markers, mapping in one single locus.
We thank D. Krabel (Technical University of Dresden) for helpful and supportive discussions, T. Fenning (Forest Research, Northern Research Station, Roslin, UK) for language editing and A. Tusch (vTI, Großhansdorf, Germany) for her assistance in the DNA laboratories. G. von Wuehlisch is gratefully acknowledged for providing the plant material. The research funding was provided by the DFG (German Research Foundation, ProjectFL263/15-1).