## 3 Discussion
#### Novel QTLs and Candidate Genes  
The GWAS-detected SNPs offer initial insights into key molecular markers and candidate genes for validation in replicated studies or marker-assisted selection (MAS). Targeted genotyping (90K-SPET) produced one of the largest SNP panels (48,445 SNP) available in faba bean. Approximately 80% of significant SNPs were located within genes, facilitating the discovery of candidate genes or causal variants. However, not all intra-genic SNPs directly impact gene function or represent the gene itself, and it is pivotal to investigate genes harboured in the LD blocks. About 30% of the intragenic SNPs were predicted to alter protein sequences (missense variants), warranting validation in functional studies to assess their effects on proteins and phenotypes. We identified intra-genic SNPs for validation as functional variants in future studies, with \(\sim\)30% predicted to alter protein sequences (missense variants). We also identified interesting genes in the LD blocks. [PERSON] et al. [29] attempted to dissect the genetic architecture of protein content via a GWAS but detected no QTLs. Herein, we report four SNPs associated with protein content, albeit with minor effects, supporting the notion that many small-effect QTLs control protein content. One SNP identified in 2021 (SNP chr5_632641479) was located near a _cationic amino acid transporter 4_ (_CAT4_) gene. Amino acid transporters contribute to nitrogen partitioning among source and sink tissues in plants [52]. Although _CAT4_ has not been directly linked to protein storage, research on another amino acid transporter subfamily in faba bean suggests that _Acid Permease_ (_AAPI1_) supplies amino acids for synthesizing storage proteins [53]. _CAT4_ may function similarly, providing amino acids used for protein storage. This is supported by studies showing that _CAT4_ mutants increased histidine levels in _Arabidopsis_ seeds [54]. Consistent with the high additive genetic correlations (type-A and type-B), the GWAS identified stable genetic signals across the years for C18:3 (SNP chr1L_1230530421, SNP chr3_1234617141, SNP chr5_505151579, and SNP contig_7845_77914). Shared genetic control was identified between oil content and C18:3 (SNP chr1S_1143102257) and between oil content and C18:1 (SNP chr4_755718730). The detected SNPs were in linkage disequilibrium with lipid biosynthesis genes, including _lipid phosphate phosphatase_ (_LPP/PAP_), _1-acyl-sn-glycerol-3-phosphate acyltransferase_ (_ATS2/LPAAT_), _3-oxoxyl-[acyl carrier protein] synthase_ (_KAS_), and a lipase gene. Interestingly, _ATS2/LPAAT_ and _LPP/PAP_ act sequentially in lipid biosynthesis. First, _ATS2/LPAAT_ converts lysophosphatidic acid (LPA) to phosphatidic acid (PA). Then, _LPP/PAP_ dephosphorylates PA to diacylglycerol (DAG). DAG is a key intermediate for triglyceride (TAG) and phospholipid synthesis [55]. Alterations in _ATS2_ and/or _LPP_ activity may affect fatty acid amounts and ratios by altering the PA/DAG balance. In _Arabidopsis_, _LPP_ overexpression or suppression alters fatty acid composition, suggesting that _LPPs_ may promote TAGs enriched with polyunsaturated fatty acids [56]. Additionally, the candidate gene _KAS_ is essential for fatty acid chain elongation by catalysing \(\beta\)-ketoacyl-ACP synthesis and providing acyl chains for lipid synthesis [57]. Our findings highlight the complex regulation required to maintain proper lipid composition in eukaryotic cells. However, these candidate genes require further validation in faba bean to confirm their roles, expression patterns, and locations. Tannins were associated with stable signals across the years, consistent with the high type-B genetic correlations. Specifically, SNP chr2_671378841 (catechin, epicatechin, procyanidin B1), SNP chr2_826275103 (epicatechin), and SNP chr2_953872991 (procyanidin B1, procyanidin B2) indicate a strong genetic control of multiple tannins on chromosome 2.