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Construction and application of a multifunctional and saturaded genetic map for coniferous species

Deliverables

Based on molecular markers and QTLs (Quantitative Trait Loci) for wood quality and growth characteristics positioned on a ultra high density map of Pinus pinaster, 7 AFLP and 3 SSR fragments associated with height, diameter, wood density, cellulose, pulp yield, lignin content, and lignin extractives were amplified, isolated from agarose or acrylamide gels, cloned and sequenced. Specific primers were then designed to generate CAP markers which were evaluated in a breeding elite population of Pinus pinaster consisting in 80 fifteen years old trees each representing a full-sib family integrated in a 1000 provenance test. Complementarily, 5 other CAP markers generated from AFLPs associated with QTLs for height, diameter, vegetative and reproductive buds formation and tree form on Pinus pinaster and Pinus radiata maps, were tested in a 50 genotype breeding population of Pinus radiata. Results showed that several specific alleles of CAP markers, including CAP markers generated from Pinus radiata sequences, were informative for height, diameter, rectitude, lignin content, pulp yield, cellulose of the Pinus pinaster breeding material. Such markers can be applied conveniently for marker-assisted selection of unknown breeding material in this species. Detailed information on primer sequences and amplification protocols can be found on the web-site http://www.neiker.net/UHDfor.
QTLs for important characters related to wood quality and growth characterisitics and map locations of important qualitative charcaters in different conifer species: - QTL (Quantitative Trait Loci) -analyses of Pinus pinaster reference population: Growth characteristics and wood quality represent important criteria in forestry. Dissection of these quantitative traits into single genetic components using molecular markers and linkage maps, provide useful information for breeders about the genetic determinism of polygenic traits. In order to apply marker-assisted breeding in early selection procedures, QTL analyses for growth traits and physical and chemical wood properties were performed in a Pinus pinaster progeny. A high-density linkage map of Pinus pinaster was used for this purpose. No significant correlations were found between wood quality and growth traits. A total of 10 QTLs were detected for the growth traits height and diameter and 40 QTLs for seven wood parameters including a-cellulose content, lignin content, pulp yield, brightness and wood densities. QTLs were spread across the genome. The percentages of phenotypic variances explained by individual QTLs ranged from to 4.3% to 18.4% and the sum of trait-specific QTLs explained 20 to 53% of the total variance. - QTL-analyses of Pinus radiata: Three QTL were detected for each of the characters height and diameter located on 5 different chromosomes. As in Pinus pinaster one QTL on Lg10 influenced both traits. Individual QTL explained between 7 and 12% of the total variation. Two additional QTL were detected for the trait spring growth. They descend from either parent, are located on chromosomes 9 and 12 and explain 9 and 7% of the total variance, respectively. Quantitative characters were analysed using the interval mapping method. However, qualitative characters had been converted into presence absence data and mapped directly. In this way three different mayor genes influencing vegetative and generative bud formation and influencing the form were detected on linkage groups 12, 9 and 6 respectively. Moreover, while apparently the formation of male buds is directly influenced by the gene of generative bud formation, the appearance of female buds is triggered by the recessive action of a second independent gene located on linkage group 1. These findings can have a transcendent impact on controlling bud formation in the future. The QTL analysis for the second partner population of Pinus radiata Three QTL were detected for the character height and two QTL for diameter located on 3 different chromosomes. Two QTL on Lg11 and Lg7 influenced both traits. Individual QTL explained between 8 and 12% of the total variation. - QTL-analyses of Pinus sylvestris: Unfortunately, no QTL was detected for Lophodermium seditiosum resistance nor for breast height diameter. Several QTLs were identified for growth height (Q1-Q7, see Table 2.2.4) and are located as clusters on linkage group 10 (H78, H79b; H81b, H82b and H83b) and linkage group 4 (H79a; H80; H81a; H82 and H83a). Although QTL H79a was also located on linkage group 4, it was separately located distant to the main cluster on the same linkage group. The QTL for H77 was located distant to other QTLs on linkage group 13. The results indicate the existence of two main intervals responsible for growth height. Additionally, a second QTL analysis was performed for the second partner population of P. sylvestris for different traits such as growth height, bhd, diameter or wood density (Pilodyn), but in no case significant QTLs were detected for any of these traits. Due to missing QTL data for the second individual partner mapping population, a direct comparison of QTLs between the first and the second individual partner mapping population was not possible. No correlations between Pinus pinaster reference population and P. sylvestris partner populations were found for QTL marker or marker locations. - QTL-analyses of Pseudotsuga menziesii: Several QTLs controlling growth were detected on the male and/or the female linkage maps. Three different QTLs were detected for vegetative flushing time (D) recorded in 2002, one on female linkage group F18 and two in male linkage groups M1 and M2. Height at the end of 2001 (H1) was controlled by QTLs located on female linkage group F7 and male linkage group M1. QTLs for height at the end of 2002 (H2) were detected on female linkage group F2 and male linkage group M1 which are homologous, but not exactly in the same position than for height at the end of 2001. First flushing (f1) QTLs were located on four female linkage groups (F1, F7, F14, F17). Second flushing (f2) QTLs were suspected on female linkage groups F5 and F7, as only a part of plants showed second flushing, the sample size was very reduced. The stem elongation in 2002 (TE) was controlled by QTLs located on female linkage groups F7 and F14 and male linkage group M1. A single marker was found to be linked with polycyclism (P), located on male linkage group M.
A High density, reference linkage map for Conifer species has been developed. It contains 1697 AFLP, SSR and EST markers (141 markers per linkage group). This map correponds to a Pinus pinaster population descending from the cross 0024 Landes x C803 Corsica. Total genome length of the integrated map is around 2000 cM. Our linkage map represents actually the map with the highest marker density in forest species. Furthermore, linkage maps in six different conifer populations are available (2 populations of Pinus radiata, 2 populations of P. sylvestris, one population of Pseudotsuga menziesii and Picea abies). The individual parental and integrated linkage maps show a varying degree of saturation. They contain between 11 and 17 linkage groups and between 54 (Pseudotsuga, parental map) and over 750 markers (Picea, intgrated map). In accordance the map lengths vary between 500 and 2000 cM. Maps are partially aligned with other published maps based on co-dominant EST and SSR. 9 of the 12 LGs of the reference map could be assigned to published P. taeda linkage groups as well as to 7 Pinus elliottii, 7 P. pinaster (INRA) and 6 Pinus radiata (New Zealand) linkage groups. Moreover, several linkage groups of the P. radiata partner populations could be associated with linkage groups of the reference map and other maps. Details of maps, markers and alignments including AFLP profiles can be viewed at http://www.neiker.net/UHDfor. Results are of basic scientific character, but could be applied for marker assisted breeding and enhanced or comparative genomic research. The scientific team of this project can contribute expertise, materials and project results for collaboration in the frame of R&D projects, for providing external commercial services or for technology transfer to companies or start-ups of the Biotech sector. Potential useful applications of these project results: - Development of molecular markers and other tools for marker assisted breeding and selection. - Integration of additional markers and QTLs from external partners into the reference and sattelite maps. - Construction of sattelite maps in other genetic backgrounds of the same or related species. - Identification, characterization and mapping of cDNAs involved in response reactions to biotic and abiotic stresses or involved in wood quality. - Enhancement of the existing reference map by linking a Physical Map and a Transcriptome map, which would allow the isolation and characterization of any gene of interest in the future.

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