Fine-mapping of the major locus for vicine and convicine in faba bean (Vicia faba L.) and application of the findings for winter faba bean breedingDissertation
Datum der mündl. Prüfung:2023-05-09
Betreuer:Prof. Dr. Wolfgang Link
Gutachter:Prof. Dr. Wolfgang Link
Gutachter:Prof. Dr. Gunter Backes
Gutachter:Dr. Wolfgang Ecke
EnglischPresented here is a thesis on a genetic and breeding project addressing the anti-nutritional seed con-stituents vicin and convicin in faba bean. Faba bean (Vicia faba L.) is a traditional grain legume of the Old World; vicin and convicin (VC) hinder the usefulness of this crop because they are antinutritional for non-ruminant animals such as poultry, and can be dangerous to humans with a genetic deficiency of the enzyme glucose-6-phosphate dehydrogenase. One goal of this work was to identify new DNA markers that are tightly linked to the VC locus and therefore useful for breeding. Additionally, it was aimed to find the major gene for the genetic difference between normal (HVC) and genetically very low (LVC) VC levels. A further objective was to realize for the first time the genetically very low VC content of the LVC type in winter field beans. In addition, field trials were conducted over several years to test wheth-er LVC field beans might have agronomically detectable disadvantages compared to HVC types. Fungi-static effects of vicin and convicin, which suggest a higher susceptibility of LVC types to soil-borne fun-gi, have been reported in the past. Since winter faba beans have a higher yield than spring faba beans, breeding of a first winter faba bean variety with low VC content was aimed at here. Since major parts of the here-conducted research depended on the employment of near-isogenic lines, such pairs of lines were produced and their degree of isogeneity was analyzed. This was important to indicate the rele-vance of the obtained results. The central work package was the fine mapping of the major locus for vicin and convicin. For this aim, two biparental F2 families were utilized, both resulting from crosses of two near-isogenic lines, one was a HVC type, the other was a LVC type. The respective parents of these two crosses and other close-ly related pairs of close-isogenic lines were used to perform two transcriptome sequencing experiments (employing RNAseq and MACE), the results of which were used to develop 58 novel VC-associated SNP markers. The two biparental F2 families mentioned above were then genotyped with these 58 new markers and with further markers which had previously been developed by other groups. Genetic maps were generated accordingly. Phenotypic data was used for the subsequent fine mapping; the data on VC seed contents of the genotyped F2 individuals were derived from HPLC analyses and employed for this fine mapping. A comparably very small region for the possible location of the VC gene (the main VC locus mentioned above) on chromosome 1 of Vicia faba of about 0.13 cM was finally identified. In addition, based on these fine mapping results, the synteny to Medicago truncatula and Cicer ari-etinum and supported by the previous results of the Björnsdotter et al. (2021) group, the candidate gene RIBA 1 was identified as the causal gene of LVC vs. HVC. The markers most closely linked to this gene were tested on yet another F2 family, which had not yet been present in the analyses (this F2 family resulted from a cross of an HVC winter faba bean line with an LVC spring faba bean line). Eight SNP markers suitable for breeding winter faba beans for low VC content were hence identified. Using these eight markers, novel LVC winter faba bean lines were developed and grown as Syn-0 in 2020 and Syn-1 in 2021 to establish them as a LVC synthetic breeding population or cultivar candidate. LVC spring faba bean varieties have already been bred. Seeing the higher yield potential of winter beans, it seems advantageous to realize the LVC trait in winter faba beans, too. However, it is im-portant to investigate whether a LVC content could have disadvantages under field conditions, espe-cially for winter beans (which have longer growing seasons), for example higher susceptibility to soil-borne fungi. This must also lead to the consideration of VC content in roots, i.e. plant organs other than seeds. Therefore, specific studies were conducted to investigate the agronomic performance of faba beans (spring and winter) on legume-fatigued soils, to look at VC content in different plant organs, and to analyze the relationship between VC type in sown seed and VC type in harvested seed. The field trials, which were performed to study the agronomic differences between LVC and HVC faba beans, were named legume fatigue trials 1 and 2. These trials were conducted in a field plot where a faba bean monoculture had been grown prior to these trials for at least three years. This monoculture induced the accumulation of faba bean-specific soil-borne pathogens. Legume fatigue trial 1 was per-formed with diverse faba bean lines and cultivars; specifically, HVC and LVC types, as well as winter and spring faba bean cultivars. These faba bean lines and cultivars were evaluated for their agronomic per-formance, specifically for their yield and yield components. The results did not indicate any disad-vantages of the LVC lines and cultivars compared to the HVC lines and cultivars. Since the issue of agro-nomical performance of LVC faba beans compared to HVC faba beans is very important for breeding and recommending LVC winter faba bean cultivars, legume fatigue trial 2 was conducted in addition. For this, very near-isogenic line pairs, differing within one pair mainly in their VC content, were grown in the same field plot as legume fatigue trial 1. These near-isogenic line pairs were hence examined for agronomic differences in the same way as lines and cultivars in legume fatigue trial 1. Resulting from the very near-isogenic status of these pairs, detected differences in performance, if present, should now only be due to the different VC genetics and the resulting different VC status of the plants. Howev-er, the results of the legume fatigue trial 2 also showed no significant differences in yield between faba bean types with HVC and LVC. Conclusively, LVC plants seem to have no disadvantages compared to HVC plants in a field situation with high pathogen pressure from soil-borne pathogens. Therefore, breeding of LVC winter bean lines and varieties should not lead to agronomic problems caused by the LVC status of the plants. Additionally, isogenic lines were used for distribution and inheritance studies on VC, owing to the im-portance of obtaining more insights into the trait VC content. These studies revealed the highest VC content to be found in the roots of faba beans, as compared to shoots and leaves and flowers. Seeds were shown to have VC types similar to the genotype of their respective maternal plants (i.e., LVC, HVC, or intermediate), confirming seed VC content to be maternally determined. Since knowing the actual degree of isogeneity was important in assessing the validity of the results described above, one work package concentrated on this. Therefore, all near-isogenic lines utilized in the project were genotyped using the 50k Affymetrix chip for faba bean in collaboration with O'Sullivan in Reading (UK). Within the isogenic pairs, 34320 SNP markers were analyzed and used for isogeneity level analysis. The results of these calculations showed a very high level of isogeneity for all tested near-isogenic pairs (in absolute terms, and compared to the expectation of isogeneity based on the pedigree of the tested lines). Therefore, it can be deduced that the agronomic inference from of our experiments regarding the differences between HVC and LVC lines are solid. In conclusion, our research on the agronomic performance of LVC faba beans, the development of new markers closely linked to and the identification of the gene responsible for VC, and our initial approach-es to breeding LVC winter type lines paved the way for breeding LVC winter field bean varieties.
Keywords: vicine and convicine; faba bean; breeding; fine-mapping