Browsing by Subject "Douglas-fir"

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    Identification and analysis of a transcriptome of Douglas-fir (Pseudotsuga menziesii) and population structure inference using different next-generation sequencing techniques
    (2015) Müller, Thomas; Schmid, Karl J.
    Predictions assume severe changes in the climatic conditions in Central Europe in the coming decades. Longer periods of drought and less precipitation during summer are expected. Plants cannot change their habitat and have to adapt to the new conditions or their offspring has to colonize new ecological niches. Due to the long generation times in trees it is important to know if and how trees can cope with the expected climatic conditions. Forest managers already give thought to the composition of future forests, because they have to choose species and populations which have no or only few problems with the changed climate. Douglas-fir (Pseudotsuga menziesii) is a promising tree species for this purpose, because it is adapted to different habitats and climate zones in its natural distribution range in North America. The two main varieties, coastal and interior Douglas-fir, differ genotypically and phenotypically, e.g. in drought tolerance. Douglas-fir trees, mainly of the coastal variety, showed good growth performances in field trials in Germany. Hence, a research project called "DougAdapt" was designed to analyze and to link genotypic and phenotypic differences in several coastal and interior Douglas-fir provenances. In this project, trees from field trials and from greenhouse experiments were sampled. To analyze the genetic diversity of the provenances we first generated reference sequences. Even with modern and cost-efficient next-generation sequencing technologies it would be very expensive to decipher the ~ 19 gigabases of the Douglas-fir genome completely. An alternative to whole genome sequencing is transcriptome sequencing, in which only the coding regions of a genome are sequenced. The transcriptome sequencing, which was performed for the first time in Douglas-fir, resulted in a large number of putative unique transcripts (PUTs). Comparisons with published transcriptomes of other plant species showed that the PUTs represented the transcriptome of Douglas-fir comprehensively. As the sampled seedlings were part of a drought stress experiment and grew under controlled conditions, we were able to identify drought related candidate PUTs, which may be part of the trees response to drought. Furthermore, more than 27,000 previously unknown single nucleotide polymorphisms (SNPs) in Douglas-fir could be identified. SNPs can influence the phenotype of individuals, and they can be used for instance as markers or to analyze genetic diversity. The analysis of genetic diversity of Douglas-fir provenances and the search for genes which may be part of local adaptation were performed with a sequence capture experiment. In sequence capture only predefined regions of a genome are sequenced. We showed that sequence capture based on PUTs as target regions is applicable in species with large and mostly unknown genomes. The polymorphic drought related candidate PUTs showed higher genetic differentiation than the remaining genes. Nevertheless, none of them was among the candidate PUTs for positive selection, which in turn are probably part of the local adaptation of the trees. Despite a high level of gene flow between coastal and interior provenances, the SNP data showed genetic differentiation between both varieties but only very low differentiation between the coastal provenances. We also investigated if genotyping-by-sequencing (GBS) is a suitable method to detect polymorphisms in Douglas-fir and compared the results of two GBS experiments with the sequence capture. The genome is digested with one or several restriction enzymes in GBS. Afterwards, only fragments with a specific length are sequenced, which considerably reduces the part of the genome that is sequenced as well as the costs. The advantage compared to sequence capture is the possibility to sample more individuals at the same time with less effort and costs. We showed that a digestion with two restriction enzymes results in more SNPs with less missing data, compared to using only one restriction enzyme. Both GBS methods returned considerably less SNPs than the sequence capture. Nevertheless, it was possible to distinguish between southern interior, northern interior, and coastal provenances using SNP data of the GBS experiments. GBS, especially with two restriction enzymes, seems to be a promising approach to genotype a large number of Douglas-fir trees and to obtain SNPs at low costs, which can be used in several tasks like genome-wide association studies. A large amount of sequence data and SNPs were analyzed in this thesis. Together with phenotypic information, these data will be crucial for the analysis of useful traits in Douglas-fir, like drought tolerance. Furthermore, the results concerning the Douglas-fir genome and the genetic diversity of different provenances will be beneficial in breeding programs and association studies, which in turn can be helpful to choose the optimal provenances for a given location.