UHD-MAP-OF FOREST SPECIES
Construction and application of a multifunctional and saturated genetic map for forest species

An ultra-high density linkage (UHD) map of Pinus pinaster will be constructed, based on 12 000 AFLP markers and 100 published microsatellites (SSR). This map will be placed online and exploited to its full extent for comparative genome analyses and QTL analysis in different genetic backgrounds. Four other published linkage maps in forest species will be aligned with the reference map. Based on reduced linkage maps derived from a common source of primer combination, comparative genome analysis will be performed in different pines and related gymnosperms. QTL analysis for important characters in different forest species will be performed and their effect and position compared. Allele-specific markers for important QTLs will be developed and their variability in local germplasm and breeding material will be analysed.

Objectives
The objectives of this project are:
1) to construct an ultra-high density AFLP linkage map (UHD map) of Pinus pinaster comprising up to 12 000 markers. This reference map will serve multiple purposes, ranging from the genetic dissection of complex phenotypes, including quantitative trait loci (QTLs), marker assisted breeding and 'BAC landing', to clone any gene of interest. Detailed marker information will be made available to the 'world community' via the Internet. The UHD map will serve as a reference map for all future forest species mapping experiments
2) to include in this reference map the positions of published microsatellites.
Simple sequence repeat (SSR) markers are codominant, highly polymorphic and widely conserved between species. They are being used extensively in many plant species, including forest species
3) to exploit this UHD map to the full extent possible for comparative genome analyses and QTL analysis. Due to the conserved linkage order and small sequence divergence, the Pinus pinaster map is also highly informative for other Pinus species and for related gymnosperms. This leads to the following specific objectives:
3a) to align other linkage maps in forest species with the reference map. Based on the position of common SSR markers and co-migrating AFLP fragments, it will be possible to align other already-published linkage maps in forest species with the reference map.
3b) to perform comparative genome analysis in different pine species and related gymnosperms. A reference collection of co-migrating markers, which can be revealed by using a standardised set of primer combinations and restriction enzymes, will facilitate switching between different genetic backgrounds and species. Therefore, reduced linkage maps will be generated in other pine and related gymnosperm species. Co-migrating AFLP markers will allow the performance of comparative genome analyses among populations
4) to perform QTL analysis for important characters in different forest species. The reduced maps are also useful for dissecting complex quantitative traits into their single genetic components through QTL analyses. These QTL analyses will be performed involving different important traits for forest breeding. Moreover, common characters for different species will allow the comparison of QTL positions in different genetic backgrounds. The information available from the reference map will provide many additional markers for specific genomic regions of interest.
5) to analyse variability of allele-specific markers for important QTLs in local germplasm and breeding material. The conversion of tightly linked AFLP markers for important QTLs into allele-specific PCR primers (CAP markers), will allow their use for marker-assisted selection or breeding. As an initial step towards their practical application, the distribution of these alleles will be analysed in local germplasm and elite breeding material.

Progress to Date
The Commission has accepted proposed changes to the third year of the work plan. Numerous new AFLP and several new EST markers were obtained during this period. Marker densities of the reference linkage map could be increased considerably.

A total of 2 193 segregating fragments derived from 316 AFLP primer combinations (PCs) and 40 EST or SSR primer pairs were available for linkage mapping. Two parental linkage maps have been constructed from the P. pinaster reference population (0024 x C803) based on AFLP, SSR and EST markers. Although segregating polymorphism was low, due to a high degree of homozygosity in the parents, 12 linkage groups with 26 to 66 markers each were obtained for each parent. The availability of 79 anchor points based on fragments common to both parents and based on co-dominant SSR and EST markers allowed the determining of homologous chromosomes for both maps and the construction of one integrated map. Total genome length of the integrated map is around 2 000 cM including 1 442 markers (120 markers per linkage group).
Partial alignments of linkage groups of the reference population with those of other published maps have been improved. This linkage map actually represents the map with the highest marker density in forest species.
Linkage maps based on AFLP and EST/SSR markers were also obtained in second partner populations (P. radiata, P. sylvestris and Pseudotsuga menziesii). Linkage maps of the first partner population have been enhanced. Further EST/SSR analyses in Pseudotsuga and the P. sylvestris populations are to be completed. QTL analysis for several characters, related to growth characteristics and physiological traits, were performed in these populations, and QTLs were detected for these characters. Previous QTL analyses in the reference population were refined and published.
Based on co-dominant EST markers mapped in different populations, previous results on comparative genome analyses were improved. Co-migrating AFLP markers seemed useless for map alignments, at least in conifer species. The analysis of several common characteristics allowed the enhancement of previous results on comparative QTL analyses in reference, partner and other published mapping populations.
CAP markers for important QTLs related to growth, wood quality and physiological characters were developed in reference and partner populations. Evaluation of these markers in local germplasm and breeding material has been initiated and will be completed in the coming months.

Results
Map alignments are crucial for comparative genome and QTL analyses. On the other hand, an unexpected low degree of polymorphism was observed in the biological research material. To improve map alignments between project maps and published maps, as well as between project maps, the Commission accepted a proposed change to the work plan. This change considered the analysis of 25 additional EST or SSR markers in the reference, as well as in both partner populations, by each partner. In order to compensate for this additional workload, the AFLP PCs to be analysed in the reference population was reduced from 50 to 25 and the number of CAP markers to be developed and analysed was reduced from 10 to 5.

Classified in FORESTRY, GENOMICS, BIOLOGICAL DIVERSITY

Scientist responsible for the project