Analysis of small RNA-based regulatory effects on heterosis and genome-wide association study of flowering time in Brassica napus L.
by Yang Wang
Date of Examination:2025-08-19
Date of issue:2025-10-16
Advisor:Prof. Dr. Stefan Scholten
Referee:Prof. Dr. Stefan Scholten
Referee:Prof. Dr. Timothy M. Beissinger
Persistent Address:
http://dx.doi.org/10.53846/goediss-11560
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Abstract
English
Brassica napus L., commonly known as oilseed rape, is an allotetraploid that originated ~7500 years ago by natural hybridization between two diploid progenitors, Brassica rapa and Brassica oleracea. Oilseed rape is extensively cultivated providing edible oil for humans, protein-rich feed for animals, and renewable biofuels for industry. As the third largest source of vegetable oil, the yield of oilseed rape directly affects global agricultural production and food supply. Heterosis has been exploited by hybrid breeding as an important method to increase yield and yield stability, while flowering time is a key trait that affects the growth cycle, adaptability and final yield of oilseed rape. Studying the molecular mechanism of these traits will not only help to optimize breeding strategies and improve crop productivity but is also important for coping with climate change and expanding the cultivation area of oilseed rape. Heterosis is a phenomenon in which the F1 hybrid is superior to its parental inbred lines. Despite the fact that the root system, as the basis of plant growth and development, plays a crucial role in nutrient uptake, water utilization and stress tolerance, little research has been conducted on heterosis for root traits in oilseed rape. High-precision root growth rate measurements of a panel of 24 oilseed rape hybrids by a non-invasive phenotyping platform revealed remarkably high variation of mid-parent heterosis (MPH) for root growth rate ranging from -28% to 196%. Recent studies have suggested the contribution of small RNAs (sRNAs) to heterosis. Taking advantage of the high trait variation for root growth, its quantitative relationship with sRNAs expression and their regulation of genes was investigated by integrating small RNA, transcriptome, degradome and DNA methylation data. To increase statistical power, we divided 24 hybrids into 4 groups with distinct heterotic responses. sRNA expression and transcriptome analysis revealed that the number of sRNAs and genes with non-additive expression patterns increases with heterotic response. The degradome sequencing analysis confirmed targets degraded by non-additively sRNAs through post-transcriptional gene silencing (PTGS), and showed that the proportion of non-additively expressed genes that are regulated by non-additively expressed sRNAs increases with the root growth heterotic response. This regulation affects plant growth and stress/defense response genes, which have been described to contribute to heterosis formation. Additionally, 24-nt sRNAs can mediate transcriptional gene silencing (TGS) through the RNA-directed DNA methylation (RdDM) pathway. CHH methylation at loci with down-regulated 24-nt sRNA decreased in the F1 hybrid compared to the mid-parent value in the extreme high heterosis group. Non-additively expressed 24-nt sRNAs were located in genic and flanking regions of seven non-additively expressed genes. Three out of these seven genes are related to root growth. Overall, the integration of omics data illustrates that sRNA-based regulatory interactions via PTGS and TGS are quantitatively associated with root growth heterosis in oilseed rape. The timing of flowering in oilseed rape is a pivotal phenological trait that profoundly influences reproductive success, yield potential and adaptation to changing environmental conditions. Therefore, identifying genes that advance or delay flowering time will have important practical implications for sustainable agriculture under different climatic conditions, which is increasingly important in the face of global climate change. To identify quantitative trait loci (QTL) associated with flowering time, 780 accessions were used for a Genome-Wide Association Study (GWAS). Genotyping was performed using the Brassica 60K Illumina Infinium™ SNP genotyping array, and flowering time data were collected from multiple environments and locations. GWAS for flowering time was performed using five different GAPIT models: GLM, BLINK, FarmCPU, MLMM and MLM. A total of 12 quantitative trait nucleotides (QTNs) associated with flowering time were identified. Two QTNs located on chromosomes A02 and C06 were consistently identified by all models and up to 6.89% of the phenotypic variance explained (PVE). Considering flanking genomic regions of 200kb of QTNs identified the well-characterized flowering time gene of oilseed rape, BnaFT.A02, confirming the reliability of the study. Based on comparing with homologous known flowering genes in Arabidopsis thaliana, we proposed 22 promising candidate genes for flowering time in oilseed rape, involved in the regulation of photoperiodism, circadian clock and gibberellin response. In summary, this thesis reveals an important role of sRNAs in the establishment of heterosis for root growth and successfully identified QTLs related to flowering time in oilseed rape. These findings enhance our understanding of heterosis and flowering time and lay a foundation for improving the yield and yield stability of oilseed rape.
Keywords: Heterosis; non-additive; root; small RNA; flowering time; GWAS
