Toepassing van meiotische plasticiteit voor ploïdieveredeling in appel (Malus x domestica Borkh.)

Apple (Malus x domestica) is a globally significant fruit crop, grown in temperate regions for both fresh consumption and industrial use. Known for its distinctive fleshy structure and health benefits, apple cultivation has been shaped by centuries of selective breeding aimed at enhancing fruit quality, appearance, and yield. However, with changing climate conditions, evolving legislation, and shifting consumer demands, plant breeding objectives are increasingly focused on resilience. As apple breeding programs adapt to these challenges, developing varieties that are resilient to biotic and abiotic stress has become a priority. (Poly)ploidy breeding, i.e. the targeted generation of offspring with three or more chromosome sets, offers a promising solution to these challenges. Recent studies across various plant species, including apple, have shown that polyploids exhibit enhanced resilience to abiotic stresses such as drought, heat, and cold, as well as increased resistance to pathogens. While most apple varieties are diploid, a significant portion of the germplasm consists of triploid individuals. The natural occurrence of triploid progeny in apple is extremely rare, suggesting that their prevalence in breeding is the result of empirical selection for advantageous agronomic and consumer traits. This is evident in the success of triploid cultivars such as 'Jonagold', 'Belle de Boskoop', and 'Mutsu'. However, the majority of these triploids have not been the result of dedicated breeding efforts towards the generation of polyploid offspring, but were generated 'on accident,' as currently, no effective tools for ploidy breeding in apple are available. While several methods, such as heteroploidy hybridization and endosperm culture, exist for producing triploid offspring, the ectopic formation of 2n gametes, i.e. gametes with the somatic chromosome number, in diploid parent provides a more straightforward and efficient pathway. Recent studies have shown that the majority of established triploids result from the ectopic formation of 2n gametes in diploid parents. However, the prevalence and mechanisms underlying 2n gamete formation in apple remain poorly understood. Furthermore, it remains unknown whether this phenomenon is driven by genetic or environmental factors, or a combination of both, and whether these can be harnessed for ploidy breeding. Therefore, this PhD project investigates the occurrence and mechanisms of 2n gamete formation in apple. In the first part of this PhD project, we developed a comprehensive framework for male reproductive development in apple. We staged male reproductive development using a standardized ontogenetic map and correlated this with phenological development in two diploid and one triploid apple cultivars. Our findings demonstrated a strong relationship between male reproductive development and flower bud phenology. Cytological analysis of male meiosis in these cultivars revealed that meiotic cell division is stable in diploids, whereas meiosis in triploids is significantly disrupted, providing a valuable baseline for future studies on apple microsporogenesis. Furthermore, this work resulted in the first complete meiotic atlas for a fruit tree species. Additionally, we assessed the timing of apple meiosis, which was found to be highly asynchronous. We also developed a pipeline for single-pollen genotyping in apple, involving nuclear extraction followed by PCR-based genotyping with molecular markers. This included the development and validation of a triplex marker set targeting the upper arm of apple chromosome 9 using haploid and diploid pollen. This method serves as an efficient tool for investigating key meiotic phenomena, such as meiotic recombination and meiotic restitution. As such, we established a reference framework of apple male reproduction to support further research, both on a fundamental or more applied scale. In the second part of this PhD project, we investigated the prevalence of spontaneous 2n pollen and ovule formation in the apple germplasm. Although the overall rate of spontaneous 2n gamete formation is rather low, our findings indicate that 2n ovule formation is the primary pathway for spontaneous sexual polyploidization in apple. We identified several 2n pollen and 2n ovule donors and explored the mechanisms underlying 2n pollen formation. Our analysis revealed that the formation of 2n pollen results from different causative mechanisms, i.e. ectopic meiotic restitution caused by altered chromosome segregation during meiosis II and pre-meiotic doubling. Additionally, we observed genotype-by-environment (GxE) effects influencing 2n gamete formation, suggesting that external factors play a role in their occurrence in apple germplasm. In the third part of this project, we examined the potential to induce 2n pollen formation through heat shock treatments. Our results showed that both a 32°C and 36°C 24h heat shock significantly affected apple male meiotic stability, with meiotic restitution increasing with temperature. Analysis of the causative mechanism revealed temperature-dependent defects in chromosome segregation at both 32°C and 36°C, particularly during meiosis II, resulting in the formation of restituted products such as dyads and triads. In addition, at 36°C, asynapsis and univalent formation were also observed. These findings suggest the formation of FDR-type 2n gametes, largely retaining parental heterozygosity. In all, our results highlight that heat shock treatments have great potential as an effective method for inducing 2n pollen formation in apple. In conclusion, this study provides valuable insights into the use of 2n gametes for ploidy breeding in apple. While the findings highlight the potential of 2n gamete formation, further research is needed to fully understand the mechanisms and factors influencing 2n gamete production. Ultimately, these insights could pave the way for more resilient and genetically diverse apple varieties, improving both agronomic traits and consumer appeal.