GENETIC MAPPING

Oak mapping initiatives

Genetic maps in Q. robur

Comparative mapping in the Fagaceae


Oak mapping initiatives

European white oaks France
Starting in 1995, activities in genetic mapping were implemented in European white oaks at the INRA Research Centre in Bordeaux-Cestas (France). Motivations for genetic mapping in oaks were threefold (i) the detection of genomic regions involved in species differentiation, (ii) the detection of QTLs controlling traits of adaptive significance, (ii) the comparative analysis of genomic evolution in the Fagaceae. The whole mapping project is based on 3 pedigrees: one full-sib family of Quercus robur (3PxA4), one full-sib family of Q. petraea (QS28xQS21), and one interspecific F1 full-sib family Q. robur x Q. petraea (11PxQS29). An interspecific F2 cross is currently being constructed as well. Given the objectives of the mapping experiments, the parents of the pedigrees were not selected for any particular criteria. The Q. robur parent trees originated from the Southwest of France (INRA research station of Bordeaux-Cestas, and Arcachon) and the Q. petraea parents were from the central part of France (INRA research station of Orléans-Ardon). The controlled crosses were repeatedly done over successive years until 2009. From 200 to 1000 seeds were obtained for each cross. The young seedlings were installed in a seedbed in a nursery where they are raised as stool-beds (Scotti-Saintagne et al., 2004). Starting at age 5, the full-sibs are hedged every year at the ground level at the end of winter time. Following the hedging, stump sprouts developing in spring were harvested and cut in 15 to 20 cm long cuttings. The cuttings were transferred in a greenhouse watered by a fog system for rooting. The rooted cuttings were then transplanted in the nursery where they were raised during one additional season. These cuttings were then transplanted in field tests for phenotypic observations and further QTL detection. For the time being only the Q. robur intraspecific cross has been fully exploited for genetic mapping and QTL detection. The clonal test of the full-sibs has now been planted in three different sites (2 near Bordeaux, South-West France and one near Nancy, Northeastern France). The genetic mapping of Q. robur mapping was done on a sample of 94 offspring (pedigree 3P x A4), and the QTL detection on a sample of 278 offspring (replicated on average between 5 to 10 vegetative propagules).

European white oaks Netherlands
Another mapping initiative for Q. robur was implemented in The Netherlands (ALTERRA, Wageningen). This mapping pedigree (A1xA2) consists of 101 full-sibs (Bakker 2001). The sibs were screened by paternity analysis within an open pollinated progeny set of 397 sibs collected on a single tree located in an urban area (Amsterdam). This tree was surrounded by three other oak trees within a radius of 10 m. One of these oak trees was selected to be the paternal tree. Paternity analysis revealed that 26% of the collected seeds were sired by this male parent. The selected seeds were germinated, grown individually in pots in a nursery (ALTERRA research station, Wageningen, The Netherlands) and measured for several morphological and physiological traits during the next two years (1999, 2000). The objective of this work was quite similar to the French initiative: detection of QTL controlling morphological and adaptive traits involved in species differentiation.

European white oaks Germany
A third oak mapping initiative was implemented by the University of Göttingen in 2003 (Gailing, 2008; Gailing et al., 2008). A full-sib family of 387 plants (HSL03) was obtained after crossing two adult trees of Q. robur. In contrast to the ‘French’ cross, where both crossing parents originated from southwestern France, the crossing parents of the german mapping pedigree are from different geographic regions and subspecies. The female parent (accession EF03) is situated close to Escherode (Lower Saxony, 51º19 N, 9º40 E, altitude 380 m). The male parent (SL03, ssp. slavonica) is located in the Münsterland (Hamm-Westtünnen, North Rhine-Westphalia, Abt. 161A2, altitude 36 m). This tree is characterised as a Slavonian oak by its late bud burst (from 7 to 16 days later than the mean of neighbouring indigenous oak trees), its habitus (stem straightness, stronger growth compared to indigenous oaks at the same age). Slavonian pedunculate oaks in Münsterland are characterised by relatively large leaves and late bud burst compared to indigenous oaks, but also by accelerated growth, stem straightness and a long clear bole. According to historic documents, trees of the Slavonian stand in the Münsterland were established in 1888 from plant material harvested from the lowlands between the Rivers Save and Drava in Croatia. The seedlings of the cross were further planted in the field near the University of Göttingen in the fall of 2003, for future QTL detection (Gailing, 2008; Gailing et al., 2008).

American red oaks USA
Genetic mapping in northern red oak (Quercus rubra L.) was initiated at Purdue University (Romero-Severson, 2003) and has continued at the University of Notre Dame. Using exclusion methods based on microsatellite polymorphisms (Aldrich et al., 2002, 2003a), a preliminary mapping population of 97 full-sibs was identified from the open pollinated progeny of a single tree. The most likely male parent male was the closest conspecific. Recombination patterns revealed six linkage groups of three or more markers. A second acorn harvest from the same female parent yielded 462 full-sibs. The genetic map under construction now includes 15 microsatellites, 66 AFLP markers from the first set of progeny and several hundred new AFLP markers from the second set of progeny. All of the potential pollen parents with 200 meters of the female parent are being genotyped with all fifteen microsatellite markers to eliminate any doubt over the full-sib status of the mapping population. The microsatellite markers used for genetic mapping are the same as those used for studies on interspecific gene flow (Aldrich et al. 2003b) and in northern red oak genetic diversity studies. No map has yet been published for Q. rubra. The long term goal of the red oak mapping project is the detection of QTLs and genes controlling heartwood colour and resistance to specific pests, specifically Phytophthora ramorum, the agent of sudden oak death.

Genetic maps in Q. robur

A first generation of linkage maps were constructed using the two-way pseudo test-cross strategy (Grattapaglia and Sederoff 1994), using genetic arrays obtained by randomly distributed anonymous dominant markers (RAPDs or AFLPs), isozymes, and microsatellites.

The first Quercus map was published in 1998 on Quercus robur (Barreneche et al. 1998) (pedigree 3P x A4). Using the pseudo test-cross mapping strategy, two maps were constructed comprising 307 markers (271 RAPD, 10 SCARs, 18 SSRs, 1 minisatellite, 6 isozymes and 1 ribosomal DNA marker). Both maps provide 85 to 90 % coverage of the Q. robur genome. Segregating markers could be aligned in 12 linkage groups, and the map size amounted to 893.2 cM for the paternal and 921.7 cM for the female map. This map was further upgraded by the inclusion of new SSRs (Barreneche et al. 2004) and additional AFLP and STS. The upgrading is still ongoing and to date 854 markers (271 RAPD, 457 AFLP,  10 SCAR, 59 SSR, 49 EST, 1 minisatellite, 6 isozymes and 1 ribosomal DNA marker) have been located (Table 2).

The Dutch Q. robur map (pedigree A1 x A2) was also constructed using the two-way pseudo test-cross strategy (Bakker 2001). Two parental maps were first established comprising 18 SSR and 343 AFLP markers. The total lengths of the maternal and paternal maps were respectively 496 and 566 cM. Thirteen linkage groups were obtained (for 12 chromosomes) and the two maps could be partially merged using 58 “bridge” markers (2 linkage groups could not be aligned). One of the paternal linkage groups (linkage group 13, 27 cM) was highly dissimilar to the other linkage groups in terms of marker density. This linkage group contained almost half (48%) of all paternal markers and 22% of the segregating (heterozygote) markers. This marker-dense linkage group was homologous to one of the maternal linkage groups that remarkably was composed exclusively of 13 segregating markers. The total map length of the integrated map was 659 cM map density was one marker per 2.40 cM for the map without taking the exceptionally dense linkage group 13 into account.

The German map based on 192 full sibs of the (EF03x SL03) pedigree was obtained more recently (Gailing, 2008). The construction was done with 162 AFLP markers segregating in a 1:1 ratio (92 markers from the female parent and 70 markers from the male parent) and 48 microsatellites. The resulting female map was composed of 74 markers (49 AFLPs and 25 SSRs); the male map consisted of 69 markers (48 AFLPs and 21 SSRs). Most linkage groups (apart from lg3m*, lg4m* and lg4f* that have no SSR markers) could be assigned to linkage groups of the 3PxA4 pedigree (french map). The 20 SSRs that are shared by both maps were used to align linkage groups of the male and female maps. The total length of the female map was 1135.3 cM, with an average marker distance of about 15.3 cM; the length of the male map was 938.8 cM, with an average marker spacing of about 13.6 cM.

Comparative mapping in the Fagaceae

In 1997, Steinkellner et al. showed earlier that microsatellite markers specifically developed in Quercus species could be cross-amplified in European chestnut (Castanea sativa). Hence microsatellites were considered as useful molecular markers for comparing Q. robur and C. sativa genetic linkage maps. In order to obtain orthologous markers for comparative mapping, SSR markers developed both in Quercus species and in C. sativa were therefore tested for cross-amplification and transferability between these two genera (Barreneche et al. 2004 and therein references) in a reciprocal way. Fifty three microsatellites developed in Quercus species and 30 in C. sativa were reciprocally tested on the two species. Nineteen loci, 15 from oak and 4 from chestnut, were integrated into the two previously established genetic maps allowing a first comparative mapping between linkage groups of the two species (Barreneche et al. 2004). Homeologous linkage groups identified by orthologous SSR could be assigned between the two existing genetic maps (Barreneche et al, 1998 for Q. robur; Casasoli et al., 2001 for C. sativa). These efforts were further extended to compare genetic linkage map of Q. robur and C. sativa with the C. mollissima x C. dentata, which is the north American pedigree used for mapping in American chestnut (Sisco et al., 2005).

Overall, these findings showed that microsatellite markers can be cross-transferred between Quercus and Castanea genera and can be used to recover orthologous markers for comparative mapping. SSR loci were extremely useful for comparative mapping within the same genus (Castanea) but their cross-transferability efficiency decreased between different genera. SSR loci mapped both in Q. robur and C. sativa were sequenced in order to definitely demonstrate their orthology. Sequencing results clearly showed that both orthologous and paralogous loci could be recovered among the SSR cross-transferred between the two genera. Moreover, indels were sometimes observed within the flanking regions of the repeated motif. Therefore, although SSR loci can be cross-transferred between Quercus and Castanea genera, a sequence analysis is needed to demonstrate orthology and to avoid the risk of paralogy. At this stage the transferability of SSR between Quercus and Castanea allowed to assign linkage groups, but was not sufficient to conclude on synteny or collinearity of the linkage groups (Kremer et al., 2007).

Similar attempts were also conducted to extend the comparison of genetic maps with Fagus. However the transferability rate of SSR was to low to allow assignment of linkage groups.

Genetic maps in Quercus species

References

Aldrich PR, Michler C, Sun W, Romero-Severson J (2002) Microsatellite markers for northern red oak (Fagaceae: Quercus rubra). Molecular Ecology Notes 2:472-475
Aldrich PR, Jagtap M,  Michler CH, Romero-Severson J (2003a) Amplification of North American Red Oak Microsatellite Markers in European White Oaks and Chinese Chestnut. Silvae Gene
tica 52(3-4):176-179
Aldrich PR,  Parker GR, Michler CH, Romero-Severson J (2003b) Whole-tree silvic identifications and the microsatellite genetic structure of a red oak species complex in an Indiana old-growth forest. Can J Forest Res 33:2228-2237

Bakker EG (2001) Towards molecular tools for management of oak forests, PhD, Alterra, Wageningen, 114 pages

Barreneche T, Bodenes C, Lexer C, Trontin JF, Fluch S, Streiff R, Plomion C, Roussel G, Steinkellner H, Burg K, Favre JM, Glössl J, Kremer A (1998) A genetic linkage map of Quercus robur L. (pedunculate oak) based on RAPD, SCAR, microsatellite, minisatellite, isozyme and 5S rDNA markers. Theor Appl Genet 97:1090-1103

Barreneche T, Casasoli M, Russell K, Akkak A, Meddour H, Plomion C, Villani F, Kremer A (2004) Comparative mapping between Quercus and Castanea using simple-sequence repeats (SSRs). Theor Appl Genet 108:558-566

Casasoli M, Mattioni C, Cherubini M, Villani F (2001) A genetic linkage map of European chestnut (Castanea sativa Mill.) based on RAPD, ISSR and isozyme markers. Theor Appl Genet 102:1190-1199

Gailing O. (2008) QTL analysis of leaf morphological characters in a Quercus robur full-sib family (Q-robur x Q-robur ssp slavonica). Plant Biology 10, 5: 624-634

Gailing O., Langenfeld-Heyser R., Polle A., Finkeldey R. (2008). Quantitative trait loci affecting stomatal density and growth in a Quercus robur progeny: implications for the adaptation to changing environments. Global Change Biology 14,8: 1934-1946
Grattapaglia D, Sederoff R (1994) Genetic linkage maps of Eucalyptus grandis and Eucalyptus urophylla using a pseudo test-cross mapping strategy and RAPD markers. Genetics 137:1121-1137
> Kremer, A., M. Casasoli, T. Barreneche, C. Bodénès, P. Sisco, T. Kubisiak, M. Scalfi, S. Leonardi, E.G. Bakker, J. Buiteveld, J. Romero-Severson, K. Arumuganathan, J. Derory, C. Scotti-Saintagne, G. Roussel, M.E. Bertocchi, C. Lexer, I. Porth, F. Hebard, C. Clark, J. Carlson, C. Plomion, H. Koelewijn, and F. Villani. 2007. Fagaceae: comparative Genetic Mapping in Fagaceae. P. 161-187 in “Genome Mapping & Molecular Breeding. Vol. 5: Forest Trees”, Kole, C.R. (ed.). Springer-Verlag, Berlin, Heidelberg, New York, Tokyo

Romero-Severson J, Aldrich P, Mapes E, Sun W (2003) Genetic mapping in northern red oak (Quercus rubra L.). In: Plant and Animal XI Genomes Conf, San Diego, CA, USA

Scotti-Saintagne C, Bodénès C, Barreneche T, Bertocchi E, Plomion C, Kremer A (2004) Detection of quantitative trait loci controlling bud burst and height growth in Quercus robur L. Theor Appl Genet 109:1648-1659

Sisco PH, Kubisiak TL, Casasoli M, Barreneche T, Kremer A, Clark C, Sederoff RR, Hebard FV, Villani F (2005) An improved genetic map for Castanea mollissima / Castanea dentata and its relationship to the genetic map of Castanea sativa. Acta Hortic, 693: 491-495
Steinkellner H, Lexer C, Turetschek E,  Glössl J (1997) Conservation of (GA)n microsatellite loci between Quercus species. Mol Ecol 6:1189-1194