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Transcriptome reconstruction is an important component in the bioinformatical part of transcriptome studies. When a reference genome is missing, highly fragmented or incomplete, a de novo transcriptome assembly is the transcriptome reconstruction approach of choice, since in such situations, a simple alignment (or mapping) would not necessarily give all theinformation concerning splice junctions, isoforms or even the full extent of the gene. Several methods for de novo transcriptome assembly have been suggested, but many of these methods lack sufficient ability to recover isoforms or are memory intense, which requires themethods to be executed on computing clusters. One species, whose published reference genome is highly fragmented, is the Norway spruce (Picea abies (L.) H. Karst.) – a conifer, very important for Swedish forestry ande conomy, but with a long juvenile phase and irregular cone setting, the demand of cultivated seeds is larger than the supply. Thus, there is a desire to understand the molecular biology behind the cone setting in P. abies, not least regarding gene expression and its regulation. This doctoral dissertation addresses these problems by describing the biological background in general, followed by an introduction to theoretical computational problems relatedto the methods applied for transcriptome reconstruction, which then are described in depth themselves, as is P. abies. Paper I uses a novel de novo assembler to detect connections between scaffolds in the P. abies genome, and also studies P. abies var acrocona, a mutant with shorter juvenile phase and more regular cone setting than the wild type, in order to detect how cone setting is initiated. By means of allele-specific expression analysis, this study detects a SNP ina miRNA binding site on a novel gene, a mutation which is coherent with the acroconaphenotype. Paper II and paper III both introduce one novel de novo transcriptome assembler each: Paper II describes the assembly method app, Transkriptomrekonstruktion är en viktig beståndsdel i den bioinformatiska avdelningen av transkriptomstudier. När ett referensgenom saknas, är kraftigt fragmenterat eller ofullständigt, så måste sammansättningen av transkriptomet ske de novo, ty i dessa situationer skulle inte en vanlig inpassning (eller mappning) nödvändigtvis ge all information om splitsningsplatser, isoformer eller ens genens fullständiga omfattning. Flertalet metoder för sammansättning av transkriptom de novo har föreslagits, men många av dessa saknar förmåga att återskapa isoformer eller är minnesintensiva, vilket kräver att metoderna exekveras på datorkluster. En art, vars referensgenom är kraftigt fragmenterat, är rödgran (Picea abies (L.) H. Karst.) - ett barrträd som är mycket viktigt för svenskt skogsbruk och svensk ekonomi, men med en lång uppväxtsfas och oregelbunden kottsättning så är efterfrågan på förädlade fröer större än utbudet. Således finns ett intresse att förstå kottsättningens bakomliggande molekylärbiologi, inte minst med avseenede på vilka gener som uttrycks och hur de regleras. Denna doktorsavhandling behandlar dessa problem medelst beskrivande av den allmänna biologiska bakgrunden, följt av en introduktion av problem från teoretisk datalogi, vilka är relaterade till metoder för sammansättning av transkriptom, som i sin tur själva är beskrivna i detalj därefter, liksom också rödgran är. Artikel I tillämpar en ny sammasättningsmetod för att upptäcka kopplingar mellan olika fragment i grangenomet, men studerar även en mutant: P. abies var acrocona (kottegran), vilken har kortare uppväxtsfas och mer regelbunden kottsättning än vildtypen, för att avgöra hur kottsättning initieras. Med hjälp av allelspecifik uttrycksanalys hittar denna stuide en SNP i ett bindningsställe för miRNA i en tidigare ostuderad gen, vilken visar sig sammanhängande med kottegranens fenotyp. Artikel II och artikel III presenterar varsin ny metod för de novo sammansättning av transkriptom: Artikel II bes, QC 2023-11-10

Reproductive phase change is well characterized in angiosperm model species, but less studied in gymnosperms. We utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies (Norway spruce), a gymnosperm. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. We collect acrocona and wild-type samples, and RNA-sequence their messenger RNA (mRNA) and microRNA (miRNA) fractions. We establish gene expression patterns and then use allele-specific transcript assembly to identify mutations in acrocona. We genotype a segregating population of inbred acrocona trees. A member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL1, is active in reproductive meristems, whereas two putative negative regulators of PaSPL1, miRNA156 and the conifer specific miRNA529, are upregulated in vegetative and transition shoot meristems. We identify a mutation in a putative miRNA156/529 binding site of the acrocona PaSPL1 allele and show that the mutation renders the acrocona allele tolerant to these miRNAs. We show co-segregation between the early cone-setting phenotype and trees homozygous for the acrocona mutation. In conclusion, we demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating reproductive phase change in the conifer P. abies.

All seed plants go through a juvenile period before they initiate seed- and pollen-bearing organs and reproduce. Reproductive phase-change is well characterized in angiosperm model species, but much less well described in gymnosperms. Here, we utilize the early cone-setting acrocona mutant to study reproductive phase change in the conifer Picea abies; a representative of the gymnosperm lineage. The acrocona mutant frequently initiates cone-like structures, called transition shoots, in positions where wild-type P. abies always produces vegetative shoots. By sequence analysis of mRNA and microRNA transcripts, we demonstrate that orthologous components of the Age-dependent flowering pathway are active at the time of cone initiation. We show that a member of the SQUAMOSA BINDING PROTEIN-LIKE (SPL) gene family, PaSPL7, is active in reproductive meristems, whereas a putative negative regulator of PaSPL7, microRNA156 is upregulated in vegetative meristem. By allele-specific assembly, we also identify a short nucleotide polymorphism (SNP) in the miRNA156 binding of PaSPL7. By genotyping a segregating population of inbred acrocona trees, we show a clear co-segregation between the early cone-setting phenotype and trees homozygous for the SNP. Hence, the data presented demonstrate evolutionary conservation of the age-dependent flowering pathway and involvement of this pathway in regulating cone-setting in the conifer P. abies., QC 20210215

Motivation: Transcriptome assembly in species without a reference genome has to beperformed de novo, which requires a deeper sequencing than a reference based approach would require. In turn, this makes the study of alternative splicing in such species difficult,particularly for lowly expressed isoforms. Sequencing of full-length transcripts using longreads could improve this, but such techniques are still either expensive or error-prone. Result: In this paper we present the novel de novo transcript isoform assembler ClusTrast, which clusters a set of guiding contigs – e.g. long reads or a previous assembly – into(supposed) gene families. ClusTrast will then align the short reads to the guiding contigs,and thus the short reads are clustered. Finally, ClusTrast will assemble each cluster ofshort reads individually. Tested on real datasets from human, mouse, arabidopsis, rice, zebrafish and Norway spruce, ClusTrast managed to recover more of the known expressed isoforms than any of the other tested de novo assemblers, while mostly retaining precision. Therefore, we presume that ClusTrast can be a useful tool for studying alternative splicing. Contact: olofem@kth.se, QC 20210216