QTL analysis uncovers the genetic architecture of resistance to chocolate spot disease caused by four Botrytis species on faba bean

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Schulman, Hamid Khazaei, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8267933/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Apr, 2026 Read the published version in BMC Plant Biology → Version 1 posted 13 You are reading this latest preprint version Abstract Background Chocolate spot (CS), caused by Botrytis species, is a major disease constraining faba bean production worldwide. In this study, we investigated the genetic basis of resistance to four Botrytis species ( B. fabae, B. cinerea , B. pseudocinerea , and B. fabiopsis ) using a recombinant inbred line (RIL) population derived from the cross Mélodie/2 × ILB 938/2, screened using a detached-leaflet assay. Results Significant variation in disease severity was observed among parental lines and RILs for all four Botrytis species. The detached-leaf assay was highly correlated with a whole-plant assay conducted with a different B. fabae isolate, validating the use of the screening tool. QTL analysis identified six loci associated with Botrytis infection across Chr1, Chr4, and Chr6, explaining 9–25% of the total phenotypic variance. The overlapping QTLs on Chr1 conferred resistance to all four Botrytis species, while QTLs on Chr4 and Chr6 were specific to B. fabae . None of the other three species showed a unique QTL. Candidate gene analysis within QTL intervals revealed several defense-related gene families, including F-box protein, WRKY, MYB, and AP2/ERF transcription factors, and peroxidases involved in oxidative stress regulation. Conclusion The consistency of response across species and the lack of unique responses from the non- fabae species indicate that selection for resistance to B. fabae should select adequately for the other species. This study provides new insights into the shared and species-specific genetic architecture of Botrytis resistance in faba bean. gene mapping quantitative trait loci candidate gene disease resistance Botrytis spp Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Faba bean ( Vicia faba L.) is an ancient grain legume crop with a large genome (2n = 2x = 12, ∼13 Gb), predominantly cultivated as a source of protein for both human and animal consumption in temperate and subtropical regions. Its seeds are rich in protein, dietary fibre, and micronutrients [ 1 ]. Faba bean cultivation enhances soil fertility through its substantial ability of biological nitrogen fixation, thereby reducing the need for synthetic fertilizers in crop production and contributing to more sustainable agricultural systems. The cultivation of faba bean has been constrained by its susceptibility to a range of abiotic and biotic stresses [ 2 ]. Among the biotic constraints, chocolate spot (CS) is one of the most important and widespread diseases affecting faba bean production worldwide, causing significant yield damage in faba bean, and in a susceptible cultivar in a favorable environment, yield loss can be complete [ 3 , 4 ]. CS is caused primarily by Botrytis fabae (Sard.), and it is now known that three other Botrytis species, namely B. cinerea , B. pseudocinerea , and B. fabiopsis , can cause the disease [ 5 ]. B. fabae has a narrow host range [ 6 ], whereas B. cinerea infects a broad range of host plants. Another distinguishing characteristic is that B. fabae is typically found on leaves, stems, and pods, while B. cinerea is primarily associated with floral parts [ 7 ]. B. fabiopsis produces symptoms similar to B. fabae [ 4 ] and can coexist with both B. fabae and B. cinerea , potentially infecting hosts in a complex manner. B. cinerea and B. pseudocinerea are morphologically identical but genetically dissimilar [ 8 ], and B. pseudocinerea produces larger lesions on detached faba bean leaves [ 9 , 10 ]. CS disease can be managed through the application of protective or preventive fungicides. While chemical control can be effective in the short term, it is costly, not environmentally friendly, labor-intensive, and may lead to the development of fungicide-resistant pathogen strains. Genetic improvement of faba bean cultivars for resistance offers a more sustainable and long-term solution [ 11 ]. Breeding for CS resistance has been challenging because it is a complex trait [ 12 ] controlled by several genes with small effects [ 13 , 14 ]. So far, only partial resistance to CS has been identified in faba bean germplasm [ 15 ]. Development of DNA markers can accelerate genomics-assisted breeding for CS resistance in this crop. The massive diploid faba bean genome has been sequenced and assembled [ 16 ], and a pan-genome is in preparation [ 17 , 18 ]. Next-generation sequencing technologies have also allowed large-scale mining of DNA markers such as single-nucleotide polymorphisms (SNPs) for this crop [ 19 – 21 ]. These recently developed, rich genomic and genetic resources need to be combined with novel phenotyping tools to identify loci, candidate genes, and pathways associated with the desired traits. These will enable understanding of the biological functions, mining of the responsible alleles, and development of molecular markers that will enable breeders to accelerate cultivar development for abiotic and biotic stressors such as CS. The first attempt at gene mapping for CS in faba bean was conducted by Gela et al. [ 13 ] using an advanced bi-parental population derived from the cross Mélodie/2 × ILB 938/2. A seven-way cross was also used for this purpose by Skovbjerg et al. [ 22 ]. In both studies, ILB 938 was used as a source of partial resistance to CS, and high-density Axiom SNP genotyping arrays were employed for genotyping [ 19 ]. Webb et al. [ 14 ] employed a multi-generation mapping population developed from the cross Maris Bead (partial resistance to CS) × IG 70726 using DArT genotyping. These studies identified several minor-effect genomic regions across all faba bean chromosomes, with loci on chromosomes 1 and 6 (Chr1, Chr6) being consistent across reports. All of these studies used B. fabae as the main Botrytis species responsible for causing CS in faba bean [ 7 ]. To date, there have been no reports on the genetic interactions and mapping of the other Botrytis species in faba bean. Thus, the main aim of this study was to study the genetic interactions of the host plant with B. fabae , B. cinerea , B. pseudocinerea , and B. fabiopsis in an extensively genotyped faba bean mapping population. To avoid questions of inconsistent plant age or developmental stage, we used a detached-leaflet assay. Materials and methods Plant material A set of 165 recombinant inbred lines (RILs) from the Mélodie/2 × ILB 938/2 mapping population [ 23 ] at the F8 generation was used for this study. Mélodie/2 is a low-vicine-convicine French cultivar with high efficiency in water use, but is susceptible to CS. ILB 938/2 (IG 12132) is a landrace originating from the Andean region of Colombia and Ecuador, and carries resistance or tolerance to several stresses and diseases, including CS [ 24 ]. The Finnish cultivar, Kontu, was included in all experiments as a susceptible check [ 9 ]. Growing conditions Five seeds from each RIL were grown in a 2.5 L pot filled with peat soil (Kekkilä Coarse potting mix WR8494, Kekkilä-BVB OY, Vantaa, Finland). Seeds were inoculated with Rhizobium leguminosarum biovar. viciae (Elomestari Oy, Tornio, Finland). Plants were maintained and grown for 6–8 weeks in a controlled-environment growth chamber (Weiss-2000, Weiss Technik GmbH, Reiskirchen-Lindsruth, Germany). The temperature was set to 22°C during the day and 20°C at night, with a 12-hour photoperiod under visible light (150 µmol/m²/s photosynthetic photon flux density, PPFD). Development of Botrytis species Virulent isolates of four Botrytis species, namely B. fabae (19B053-4), B. cinerea (19B048), B. pseudocinerea (18B11) and B. fabiopsis (19B175), were cultured on half-strength potato dextrose agar (PDA) and incubated in darkness at room temperature (20°C) for 7 days, then placed for 12h under near-ultraviolet light (NUV) and 12h in the dark for 7 days to induce sporulation. The cultured plates were flooded with sterile distilled water and scraped with a triangular steel rod to dislodge the spores. The suspension was filtered through two layers of cheesecloth into a 250 ml conical flask, then 5 ml of Tween 20 (0.03% v/v) solution was added to make a homogenous spore suspension. The suspension was evaluated using a haemocytometer and its concentration adjusted to 4×10 5 spores ml − 1 . Disease screening For disease screening, we used our recently developed detached leaflet assay [ 9 ]. Fully expanded leaves, each with four leaflets, were collected from nodes four to eight of seven- to eight-week-old plants of the RILs, two hours prior to inoculation, and kept in sterile moist tissue. Five layers of sterile paper towels were arranged in a plastic box (78 cm x 56 cm x 18 cm, 55 L), one liter of sterile water was poured on the paper towels, and a plastic net was placed so the leaf blades did not rest on the wet surface (Fig. 1 ). The leaves were arranged on the nets in a randomized complete block design (RCBD) with four replicates, with the plastic box as a replicate. Leaves of ILB 938/2, Mélodie/2, and Kontu were included as controls in each box. A 25 µl aliquot of the spore suspension of each of the four Botrytis species was inoculated using a pipette onto the leaflets in a clockwise manner, so that each leaflet received inoculum nearly simultaneously. The boxes were placed in the growth chamber and incubated for seven days at 22°C and 12 h photoperiod with 170–188 µmol/m²/s PPFD. To maintain the humidity, sterile water was sprayed 2–3 times every day inside the boxes. Visible symptoms of the fungal infection and necrosis were monitored every day. After 7 days, the symptoms on leaflets were scored following a 1–9 disease scale, where, 1: no necrotic lesion and/or chlorosis (complete resistance); 2: 1-12.5% necrotic lesion and/or chlorosis (high resistance); 3: 13-25.5% necrotic lesion and/or chlorosis (moderate resistance); 4: 26-38.5% necrotic lesion and/or chlorosis (low resistance); 5: 39-51.5% necrotic lesion and/or chlorosis (low susceptibility); 6: 52-64.5% necrotic lesion and/or chlorosis (moderate susceptibility); 7: 65-77.5% necrotic lesion and/or chlorosis (high susceptibility); 8: 78-90.5% necrotic lesion and/or chlorosis (very high susceptibility) and 9: > 91% (severe susceptibility) [ 25 ]. Map construction and QTL mapping The mapping population was genotyped using the Axiom “Vfaba_v2” 60K array [ 19 , 26 ]. The genetic map was originally produced by Gela et al. [ 13 ]. The linkage map was constructed with MapDisto v. 1.7.7.0.1 [ 27 ], applying a logarithm of odds (LOD) score of 3.0 and a recombination fraction of 0.35. The Kosambi function was used to calculate the map distance in centimorgans (cM) [ 28 ]. A final genetic map was constructed from 4,089 SNP markers, which mapped to six linkage groups (LGs) representing the six chromosomes (Chr) of faba bean [ 13 ]. The LGs were assigned to faba bean chromosomes according to the faba bean physical map [ 16 ]. The orientation of markers on the original LG1 [ 13 ] was reversed to match the orientation of the faba bean physical map, while the remaining linkage groups required no such change. QTL mapping for reactions to the four Botrytis species was performed using the composite interval mapping (CIM) method in R/qtl v.1.50 software [ 29 ]. QTL significance thresholds were determined by permutation tests (1000 permutations) at a significance level of P = 0.05 in the “scantwo” function for two-dimensional QTL scanning. The percentage of the phenotypic variance explained and effects of QTLs were obtained by fitting a mixed linear model using the “fitqtl” function. SNP marker positions were drawn by MapChart v. 2.2 [ 30 ]. The confidence intervals for each QTL were estimated using the “lodint” function that calculates the 1.5 LOD support intervals. To identify candidate genes, the coding sequences of the SNP markers in the QTL intervals were searched by BLASTn in Phytozome v13 against Vicia faba v1.1 assembly ( https://phytozome-next.jgi.doe.gov/info/Vfaba_v1_1 ). Statistical analysis The JMP Pro 14 statistical computing program was used to analyze the phenotyping data set [ 31 ]. Data were logarithmically transformed to normalize the variance and used in a two-way analysis of variance (ANOVA) to determine the effects of RILs, Botrytis species, and their interactions. Principal component analysis (PCA) was performed using RIL means with R version 4.5.1 [ 32 ] with package ggplot2 [ 33 ]. Histograms were drawn with SigmaPlot 16.0. Results Phenotypic variation RILs showed significant differences in response to CS reactions caused by the isolates of B. fabae , B. cinerea , B. pseudocinerea , and B. fabiopsis ( P < 0.0001). The interaction between RILs and Botrytis species was also significant (Table 1 ). The parental lines of the RIL population (Mélodie/2 and ILB 938/2) had significant differences in their reaction to CS across all four Botrytis species (Fig. 2 ). Mélodie/2 showed moderate susceptibility to CS caused by four Botrytis species, whereas ILB 938/2 exhibited partial resistance to CS across the Botrytis species. The pattern of disease severity in response to CS evaluation for the RILs displayed a continuous variation across different Botrytis isolates (Fig. 2 ), suggesting that the polygenic nature of CS severity and CS resistance is quantitatively inherited. PCA results showed a similar pattern for the response of RILs to B. cinerea and B. pseudocinerea , while B. fabae and B. fabiopsis showed a reverse trend. The first two principal components explained 99% of the total variation (Fig. 3 ). Table 1 Analysis of variance of infected areas of 165 RILs using four Botrytis species. Source df Mean square P- value RILs 164 0.830 0.0001 Botrytis species 3 0.270 0.0001 RILs x Botrytis species 492 0.009 0.0001 error 1980 0.006 df, degrees of freedom. RILs: Recombinant inbred lines. QTL analysis Six QTLs associated with the infection responses to the four Botrytis isolates were detected across Chr1, 4, and 6 (LG1, LG4, and LG6). The strongest signals for all four Botrytis species were located on LG1, where q-BF1 ( B. fabae ), q-BC1 ( B. cinerea ), q-BPC1( B. pseudocinerea ), and q-BFsis1 ( B. fabiopsis ) all mapped to 254–265 cM (580–663 Mb of faba bean Chr1L), with LOD scores ranging from 3.66 to 4.79 and R² values between 8.5% and 10.8%. Two additional QTLs for B. fabae response were identified on LG4 (q-BF4, 825–918 Mb of faba bean Chr4) and LG6 (q-BF6, 997–1025 Mb of faba bean Chr6) (Table 2 ; Fig. 4 ). Figure 4 indicates the location of genomic regions on Chr1L, Chr4, and Chr6 in map cM. The physical marker positions are presented in Table S1 . Positive additive values for all QTLs suggest that Mélodie/2 was the donor of susceptibility alleles (Table 2 ). Table 2 Quantitative trait loci (QTLs) for four Botrytis isolates from the cross Mélodie/2 × ILB 938/2. QTL LG QTL Peak (cM) 2-LOD interval (cM) LOD score R 2 Add q-BF1 1 259.2 254.89–263.51 4.79 10.8 0.36 q-BF4 4 70.6 65.71–75.00 3.15 7.1 0.23 q-BF6 6 110.4 107.63–111.96 3.22 7.2 0.25 q-BC1 1 259.0 255.19–264.78 4.15 10.1 0.34 q-BPC1 1 259.0 255.81–263.51 4.75 10.7 0.35 q-BFsis1 1 263.0 255.19–264.78 3.66 8.6 0.31 QTL abbreviations: BF, B. fabae (19B053-4); BC, B. cinerea (19B048); BPC, B. pseudocinerea (18B11); BFsis: B. fabiopsis (19B175). R 2 , Percentage of phenotypic variance explained by QTL; Add, Additive genetic effect. Identification of putative candidate genes Table S1 lists the candidate genes associated with each SNP marker within the QTL regions (Fig. 5 ). QTL intervals on LG1, LG4, and LG6 cover several large clusters of plant disease- and defence-related candidate genes ( Table S1 ). In LG1, the regions covered by overlapping QTLs q-BF1, q-BC1, q-BPC1, and q-BFsis1 include members of gene families for F-box proteins, DnaJ / Hsp40 chaperones, serine proteases, S-adenosylmethionine synthetases, chloride channels, SBP/MYB/FAR1 transcription factors, nodulin-like MFS transporters, lipases, and leucine-rich pentatricopeptides. Within the LG4 q-BF4 region were found genes annotated as a WRKY transcription factor, zeaxanthin epoxidase, Caffeoyl-CoA O-methyltransferase, subtilisin-like protease, RING/U-box protein, casein kinase II α1, pectin methylesterase, aspartyl protease, peroxidase, and scarecrow-like protein. LG6 q-BF6 contained members of five classical and strongly defense-related gene families, including AP2/ERF, LRR, RING/U-box E3 ligases, thioredoxin, and hydroxyproline-rich glycoproteins. Discussion This is the first study to genetically map the responses of faba bean to four Botrytis species. We found a single region on faba bean Chr1 hosting loci for resistance to B. faba , B. cinerea , B. pseudocinerea , and B. fabiopsis . B. fabae response had additional QTLs on Chr4 and Chr6. Notably, CS resistance QTLs on Chr1 and Chr6 were also reported by Gela et al. [ 13 ] using the same mapping populations and marker system but different screening methods and a different isolate of B. fabae . Putative disease- and defense-related candidate genes were identified within our identified QTL regions using faba bean genome gene annotations, some playing critical roles in resistance to Botrytis species. In our study, using the detached leaflet assay, we successfully confirmed genomic regions previously detected through whole-plant screening under different indoor growing conditions in Saskatchewan for B. fabae [ 13 ]. The phenotypic data also showed a strong positive correlation between RILs screened using the whole-plant and the detached leaflet assays (r 2 = 0.480; n = 165, data not shown). This indicates that the detached leaflet assay is an efficient alternative to whole-plant screening for Botrytis resistance under indoor climatic control conditions in this species, particularly for genetic mapping studies. It is rapid, space-efficient, and allows controlled infection with precise scoring of lesion development on excised leaves [ 34 ]. Field screening, although challenging, remains essential for evaluating the overall plant response, including plant–pathogen interactions and genotype-by-environment effects [ 35 ]. The detached leaf assay provides a valuable complementary approach to whole-plant screening. In this study, six QTLs associated with Botrytis infection were identified, five of which correspond to previously reported loci, underscoring the consistency of these genomic regions in contributing to CS resistance in faba bean. QTLs q-BF1, q-BC1, q-BPC1, and q-BFsis1 were mapped to the same region of Chr1, in the region for qBF1.2 reported by Gela et al. [ 13 ]; our qFB6 was found to be identical to qFB6.1 from the same study. These overlaps confirm the stability of these loci across different growing conditions and Botrytis isolates. Similarly, Webb et al. [ 14 ] identified a large interval for B. fabae resistance on Chr1, consistent with our findings, though their additional QTLs on Chr3 and Chr5 may reflect either environmental variation or the use of a different genetic background (cv. Maris Bead). Skovbjerg et al. [ 22 ] reported marker-trait associations for B. fabae located at the beginning of Chr1, whereas our significant peaks were near the end of this chromosome. Notably, we also detected a novel QTL on Chr4 for B. fabae , which has not been reported before. The detected minor-effect QTLs confirm the partial resistance to Botrytis infection derived from ILB 938/2, Icarus, and cv. Maris Bead, which has been used in genetic mapping efforts. Beyond these sources, additional resistance germplasm has been identified [15, Khazaei et al., unpublished data]. Further efforts are needed to study the genetic architecture and loci of these sources in order to develop the means for durable gene pyramiding and the breeding of broad-spectrum Botrytis resistance in this crop. The annotated faba bean genome facilitated the identification of candidate genes associated with the QTLs that were identified. The corresponding regions for the QTLs included gene families and candidate genes known to play a role in disease resistance or defense against various biotic stresses. Several of the candidate gene families identified in this study overlap with those reported by Webb et al. [ 14 ]. Both studies found, within the mapping intervals, genes for F-box and U-box–type E3 ubiquitin ligases, thioredoxin, and chaperone (DnaJ/Hsp40) proteins, which are central to protein turnover and stress signalling during pathogen attack. Similarly, transcription factor families including AP2/ERF, MYB, and SBP were identified as candidate genes in both studies, due to their regulatory roles in activating defence-related genes. Moreover, genes for peroxidases, lipases, and S-adenosylmethionine synthetase identified in the genome assembly for our QTL regions appear to parallel the oxidative stress and secondary metabolism genes identified in Webb et al. [ 14 ]. Consistent with this, Castillejo et al. [ 36 ] reported that resistance to B. fabae in faba bean is associated with a more efficient Photosystem II repair cycle and enhanced redox regulation, further highlighting the likely importance of oxidative balance in Botrytis resistance. Direct evidence linking specific candidate genes to Botrytis resistance in legumes remains limited. However, studies in chickpea ( Cicer arietinum L.) have reported the involvement of peroxidases, WRKY transcription factors, and antioxidant enzymes in defense responses to B. cinerea infection [ 37 – 39 ]. These genes are known to mediate oxidative stress regulation and pathogen-induced signaling pathways in legumes [ 40 ]. In Arabidopsis, functional studies have demonstrated that certain F-box proteins and WRKYs play critical roles in B. cinerea resistance by modulating jasmonate and salicylic acid pathways [ 41 , 42 ], supporting the likely conserved defense role of these gene families across species. Conclusions We confirmed previously reported minor-effect QTLs and identified a novel QTL associated with Botrytis resistance in faba bean. These loci provide valuable targets for marker-assisted and genomic selection as well as for functional verification. The detached leaf assay proved to be an efficient and reliable method for QTL mapping, offering reproducible and quantitative phenotypic data. The identified candidate genes will support further dissection of the biological mechanisms underlying Botrytis resistance. Future work on mapping additional resistance sources and combining QTLs to enhance durable resistance in breeding programs would also be highly worthwhile. Declarations a .Ethics approval and consent to participate Not applicable. b. Consent for publication Not applicable. d. Competing interest The authors declare no competing interests. Clinical trial number Not applicable. e. Funding The work was part of the projects “Fabanova: climate-ready faba beans for the Nordic and Baltic Region” funded under the NordForsk call “Sustainable agriculture and climate change” (decision 137268) and “Fabagen" funded by the Research Council of Finland, Academy projects, funding decision 363375. Author Contribution Conceptualization: FLS, HK. Methodology: M. Formal Analysis: M, HK. Investigation: M. Data Curation: M. Writing – Original Draft: M, HK. Writing – Review & Editing: all authors. Supervision: FLS, HK, MJ, AHS. Funding Acquisition: FLS, HK. Acknowledgement We thank Prof. Biruta Bankina (LBTU) for sharing the Botrytis isolates with us. We thank Dr. Pedro J. Aphalo for his kind supply of the black tube light and for measuring the NEB light intensity. 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Early wound-responsive cues regulate the expression of WRKY family genes in chickpea differently under wounded and unwounded conditions. Physiol Mol Biol Plants. 2022;28:719–35. https://doi.org/10.1007/s12298-022-01170-y . Thakur R, Sharma S, Devi R, Sirari A, Tiwari RK, Lal MK, Kumar R. Exploring the molecular basis of resistance to Botrytis cinerea in chickpea genotypes through biochemical and morphological markers. PeerJ. 2023;11:e15560. https://doi.org/10.7717/peerj.15560 . Rodríguez-Sifuentes L, Marszalek JE, Chuck-Hernández C, Serna-Saldívar SO. Legumes protease inhibitors as biopesticides and their defense mechanisms against biotic factors. Int J Mol Sci. 2020;8:3322. https://doi.org/10.3390/ijms21093322 . Jiang Y, Yu D. The WRKY57 transcription factor affects the expression of Jasmonate ZIM-domain genes transcriptionally to compromise Botrytis cinerea resistance. Plant Physiol. 2016;171:2771–82. https://doi.org/10.1104/pp.16.00747 . Zhang M, Li W, Zhang T, Liu Y, Liu L. Botrytis cinerea -induced F-box protein 1 enhances disease resistance by inhibiting JAO/JOX-mediated jasmonic acid catabolism in Arabidopsis. Mol Plant. 2024;17:297–311. https://doi.org/10.1016/j.molp.2023.12.020 . Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx Cite Share Download PDF Status: Published Journal Publication published 10 Apr, 2026 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 29 Jan, 2026 Reviews received at journal 28 Jan, 2026 Reviews received at journal 26 Jan, 2026 Reviews received at journal 11 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviewers agreed at journal 07 Jan, 2026 Reviewers agreed at journal 06 Jan, 2026 Reviewers agreed at journal 29 Dec, 2025 Reviewers invited by journal 24 Dec, 2025 Editor assigned by journal 23 Dec, 2025 Editor invited by journal 15 Dec, 2025 Submission checks completed at journal 10 Dec, 2025 First submitted to journal 10 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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13:07:58","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":132773,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8267933/v1/50fa91dec9343c1855a573a3.html"},{"id":100889554,"identity":"3e1e1e91-9deb-4f87-b2ed-12befa747079","added_by":"auto","created_at":"2026-01-22 13:07:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":858707,"visible":true,"origin":"","legend":"\u003cp\u003eSetup of the detached leaf assay for \u003cem\u003eBotrytis\u003c/em\u003e infection screening, indicating the partially resistant genotype ILB 938/2 (\u003cstrong\u003ea\u003c/strong\u003e), susceptible genotype Mélodie/2 (\u003cstrong\u003eb\u003c/strong\u003e); highly susceptible cv. Kontu (\u003cstrong\u003ec\u003c/strong\u003e); and some of the RIL population.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8267933/v1/7d21bca1c9420229c4fb2da0.png"},{"id":100950892,"identity":"4e7d492d-476e-4421-9684-442268cff8bf","added_by":"auto","created_at":"2026-01-23 07:09:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":318219,"visible":true,"origin":"","legend":"\u003cp\u003eFrequency distributions of the four \u003cem\u003eBotrytis \u003c/em\u003eisolates in 165 RILs derived from the cross Mélodie/2 × ILB 938/2 at the F8 generation. (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eB. fabae \u003c/em\u003e(19B053-4); (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eB. cinerea \u003c/em\u003e(19B048); (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eB. pseudocinerea \u003c/em\u003e(18B11); and (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eB. fabiopsis \u003c/em\u003e(19B175)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8267933/v1/af95c3bb7ff2f05179b056d7.png"},{"id":100950703,"identity":"4e53f6b2-6f0b-4b70-adf5-ea9fbfc0baeb","added_by":"auto","created_at":"2026-01-23 07:08:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":165777,"visible":true,"origin":"","legend":"\u003cp\u003ePCA-biplot graph of the first two principal components (PC1 vs PC2) for four \u003cem\u003eBotrytis \u003c/em\u003especies across 165 RILs.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8267933/v1/a6de24f21d8f6910c6eb6512.png"},{"id":100950923,"identity":"c336f62f-02ba-44c3-b3dd-262e2d56af64","added_by":"auto","created_at":"2026-01-23 07:09:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":237771,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic regions associated with \u003cem\u003eBotrytis \u003c/em\u003eresistance in faba bean showing the QTL peak locations. (\u003cstrong\u003ea\u003c/strong\u003e) \u003cem\u003eB. fabae \u003c/em\u003e(19B053-4); (\u003cstrong\u003eb\u003c/strong\u003e) \u003cem\u003eB. cinerea (\u003c/em\u003e19B048\u003cem\u003e)\u003c/em\u003e; (\u003cstrong\u003ec\u003c/strong\u003e) \u003cem\u003eB. pseudocinerea \u003c/em\u003e(18B11); and (\u003cstrong\u003ed\u003c/strong\u003e) \u003cem\u003eB. fabiopsis \u003c/em\u003e(19B175)\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8267933/v1/a4abc01f8e47ee8f392c4bf6.png"},{"id":100889555,"identity":"18c9678e-52c5-4df0-8d95-5cea56652586","added_by":"auto","created_at":"2026-01-22 13:07:58","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":732519,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of \u003cem\u003eBotrytis \u003c/em\u003eresistance genomics regions on segments of faba bean chromosomes 1L (\u003cem\u003eq-BF1\u003c/em\u003e, \u003cem\u003eq-BC1\u003c/em\u003e,\u003cem\u003e q-BPC1\u003c/em\u003e, and \u003cem\u003eq-BFsis1\u003c/em\u003e), 4 (\u003cem\u003eq-BF4\u003c/em\u003e), and 6 (\u003cem\u003eq-BF6\u003c/em\u003e).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8267933/v1/44b6a699934dc93c524801ce.png"},{"id":106809261,"identity":"38d72347-f8e0-48ca-94f7-70ad7e9a4ef9","added_by":"auto","created_at":"2026-04-13 16:08:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3120692,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8267933/v1/426c93a7-fb77-4926-9765-161cb2794813.pdf"},{"id":100889564,"identity":"889ac93f-0337-4eb5-85bb-2b653bc598a2","added_by":"auto","created_at":"2026-01-22 13:07:58","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":82086,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8267933/v1/78b9c50c53192d5060c14296.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"QTL analysis uncovers the genetic architecture of resistance to chocolate spot disease caused by four Botrytis species on faba bean","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFaba bean (\u003cem\u003eVicia faba\u003c/em\u003e L.) is an ancient grain legume crop with a large genome (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;12, \u0026sim;13 Gb), predominantly cultivated as a source of protein for both human and animal consumption in temperate and subtropical regions. Its seeds are rich in protein, dietary fibre, and micronutrients [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Faba bean cultivation enhances soil fertility through its substantial ability of biological nitrogen fixation, thereby reducing the need for synthetic fertilizers in crop production and contributing to more sustainable agricultural systems. The cultivation of faba bean has been constrained by its susceptibility to a range of abiotic and biotic stresses [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among the biotic constraints, chocolate spot (CS) is one of the most important and widespread diseases affecting faba bean production worldwide, causing significant yield damage in faba bean, and in a susceptible cultivar in a favorable environment, yield loss can be complete [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCS is caused primarily by \u003cem\u003eBotrytis fabae\u003c/em\u003e (Sard.), and it is now known that three other \u003cem\u003eBotrytis\u003c/em\u003e species, namely \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eB. pseudocinerea\u003c/em\u003e, and \u003cem\u003eB. fabiopsis\u003c/em\u003e, can cause the disease [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eB. fabae\u003c/em\u003e has a narrow host range [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], whereas \u003cem\u003eB. cinerea\u003c/em\u003e infects a broad range of host plants. Another distinguishing characteristic is that \u003cem\u003eB. fabae\u003c/em\u003e is typically found on leaves, stems, and pods, while \u003cem\u003eB. cinerea\u003c/em\u003e is primarily associated with floral parts [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eB. fabiopsis\u003c/em\u003e produces symptoms similar to \u003cem\u003eB. fabae\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and can coexist with both \u003cem\u003eB. fabae\u003c/em\u003e and \u003cem\u003eB. cinerea\u003c/em\u003e, potentially infecting hosts in a complex manner. \u003cem\u003eB. cinerea\u003c/em\u003e and \u003cem\u003eB. pseudocinerea\u003c/em\u003e are morphologically identical but genetically dissimilar [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and \u003cem\u003eB. pseudocinerea\u003c/em\u003e produces larger lesions on detached faba bean leaves [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCS disease can be managed through the application of protective or preventive fungicides. While chemical control can be effective in the short term, it is costly, not environmentally friendly, labor-intensive, and may lead to the development of fungicide-resistant pathogen strains. Genetic improvement of faba bean cultivars for resistance offers a more sustainable and long-term solution [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Breeding for CS resistance has been challenging because it is a complex trait [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] controlled by several genes with small effects [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. So far, only partial resistance to CS has been identified in faba bean germplasm [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Development of DNA markers can accelerate genomics-assisted breeding for CS resistance in this crop.\u003c/p\u003e \u003cp\u003eThe massive diploid faba bean genome has been sequenced and assembled [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and a pan-genome is in preparation [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Next-generation sequencing technologies have also allowed large-scale mining of DNA markers such as single-nucleotide polymorphisms (SNPs) for this crop [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These recently developed, rich genomic and genetic resources need to be combined with novel phenotyping tools to identify loci, candidate genes, and pathways associated with the desired traits. These will enable understanding of the biological functions, mining of the responsible alleles, and development of molecular markers that will enable breeders to accelerate cultivar development for abiotic and biotic stressors such as CS. The first attempt at gene mapping for CS in faba bean was conducted by Gela et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] using an advanced bi-parental population derived from the cross M\u0026eacute;lodie/2 \u0026times; ILB 938/2. A seven-way cross was also used for this purpose by Skovbjerg et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In both studies, ILB 938 was used as a source of partial resistance to CS, and high-density Axiom SNP genotyping arrays were employed for genotyping [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Webb et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] employed a multi-generation mapping population developed from the cross Maris Bead (partial resistance to CS) \u0026times; IG 70726 using DArT genotyping. These studies identified several minor-effect genomic regions across all faba bean chromosomes, with loci on chromosomes 1 and 6 (Chr1, Chr6) being consistent across reports. All of these studies used \u003cem\u003eB. fabae\u003c/em\u003e as the main \u003cem\u003eBotrytis\u003c/em\u003e species responsible for causing CS in faba bean [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. To date, there have been no reports on the genetic interactions and mapping of the other \u003cem\u003eBotrytis\u003c/em\u003e species in faba bean. Thus, the main aim of this study was to study the genetic interactions of the host plant with \u003cem\u003eB. fabae\u003c/em\u003e, \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eB. pseudocinerea\u003c/em\u003e, and \u003cem\u003eB. fabiopsis\u003c/em\u003e in an extensively genotyped faba bean mapping population. To avoid questions of inconsistent plant age or developmental stage, we used a detached-leaflet assay.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eA set of 165 recombinant inbred lines (RILs) from the M\u0026eacute;lodie/2 \u0026times; ILB 938/2 mapping population [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] at the F8 generation was used for this study. M\u0026eacute;lodie/2 is a low-vicine-convicine French cultivar with high efficiency in water use, but is susceptible to CS. ILB 938/2 (IG 12132) is a landrace originating from the Andean region of Colombia and Ecuador, and carries resistance or tolerance to several stresses and diseases, including CS [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The Finnish cultivar, Kontu, was included in all experiments as a susceptible check [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrowing conditions\u003c/h3\u003e\n\u003cp\u003eFive seeds from each RIL were grown in a 2.5 L pot filled with peat soil (Kekkil\u0026auml; Coarse potting mix WR8494, Kekkil\u0026auml;-BVB OY, Vantaa, Finland). Seeds were inoculated with \u003cem\u003eRhizobium leguminosarum\u003c/em\u003e biovar. \u003cem\u003eviciae\u003c/em\u003e (Elomestari Oy, Tornio, Finland). Plants were maintained and grown for 6\u0026ndash;8 weeks in a controlled-environment growth chamber (Weiss-2000, Weiss Technik GmbH, Reiskirchen-Lindsruth, Germany). The temperature was set to 22\u0026deg;C during the day and 20\u0026deg;C at night, with a 12-hour photoperiod under visible light (150 \u0026micro;mol/m\u0026sup2;/s photosynthetic photon flux density, PPFD).\u003c/p\u003e\n\u003ch3\u003eDevelopment of Botrytis species\u003c/h3\u003e\n\u003cp\u003eVirulent isolates of four \u003cem\u003eBotrytis\u003c/em\u003e species, namely \u003cem\u003eB. fabae\u003c/em\u003e (19B053-4), \u003cem\u003eB. cinerea\u003c/em\u003e (19B048), \u003cem\u003eB. pseudocinerea\u003c/em\u003e (18B11) and \u003cem\u003eB. fabiopsis\u003c/em\u003e (19B175), were cultured on half-strength potato dextrose agar (PDA) and incubated in darkness at room temperature (20\u0026deg;C) for 7 days, then placed for 12h under near-ultraviolet light (NUV) and 12h in the dark for 7 days to induce sporulation. The cultured plates were flooded with sterile distilled water and scraped with a triangular steel rod to dislodge the spores. The suspension was filtered through two layers of cheesecloth into a 250 ml conical flask, then 5 ml of Tween 20 (0.03% v/v) solution was added to make a homogenous spore suspension. The suspension was evaluated using a haemocytometer and its concentration adjusted to 4\u0026times;10\u003csup\u003e5\u003c/sup\u003e spores ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003ch3\u003eDisease screening\u003c/h3\u003e\n\u003cp\u003eFor disease screening, we used our recently developed detached leaflet assay [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Fully expanded leaves, each with four leaflets, were collected from nodes four to eight of seven- to eight-week-old plants of the RILs, two hours prior to inoculation, and kept in sterile moist tissue. Five layers of sterile paper towels were arranged in a plastic box (78 cm x 56 cm x 18 cm, 55 L), one liter of sterile water was poured on the paper towels, and a plastic net was placed so the leaf blades did not rest on the wet surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The leaves were arranged on the nets in a randomized complete block design (RCBD) with four replicates, with the plastic box as a replicate. Leaves of ILB 938/2, M\u0026eacute;lodie/2, and Kontu were included as controls in each box. A 25 \u0026micro;l aliquot of the spore suspension of each of the four \u003cem\u003eBotrytis\u003c/em\u003e species was inoculated using a pipette onto the leaflets in a clockwise manner, so that each leaflet received inoculum nearly simultaneously. The boxes were placed in the growth chamber and incubated for seven days at 22\u0026deg;C and 12 h photoperiod with 170\u0026ndash;188 \u0026micro;mol/m\u0026sup2;/s PPFD. To maintain the humidity, sterile water was sprayed 2\u0026ndash;3 times every day inside the boxes. Visible symptoms of the fungal infection and necrosis were monitored every day. After 7 days, the symptoms on leaflets were scored following a 1\u0026ndash;9 disease scale, where, 1: no necrotic lesion and/or chlorosis (complete resistance); 2: 1-12.5% necrotic lesion and/or chlorosis (high resistance); 3: 13-25.5% necrotic lesion and/or chlorosis (moderate resistance); 4: 26-38.5% necrotic lesion and/or chlorosis (low resistance); 5: 39-51.5% necrotic lesion and/or chlorosis (low susceptibility); 6: 52-64.5% necrotic lesion and/or chlorosis (moderate susceptibility); 7: 65-77.5% necrotic lesion and/or chlorosis (high susceptibility); 8: 78-90.5% necrotic lesion and/or chlorosis (very high susceptibility) and 9: \u0026gt; 91% (severe susceptibility) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMap construction and QTL mapping\u003c/h3\u003e\n\u003cp\u003eThe mapping population was genotyped using the Axiom \u0026ldquo;Vfaba_v2\u0026rdquo; 60K array [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The genetic map was originally produced by Gela et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The linkage map was constructed with MapDisto v. 1.7.7.0.1 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], applying a logarithm of odds (LOD) score of 3.0 and a recombination fraction of 0.35. The Kosambi function was used to calculate the map distance in centimorgans (cM) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A final genetic map was constructed from 4,089 SNP markers, which mapped to six linkage groups (LGs) representing the six chromosomes (Chr) of faba bean [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The LGs were assigned to faba bean chromosomes according to the faba bean physical map [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The orientation of markers on the original LG1 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] was reversed to match the orientation of the faba bean physical map, while the remaining linkage groups required no such change.\u003c/p\u003e \u003cp\u003eQTL mapping for reactions to the four \u003cem\u003eBotrytis\u003c/em\u003e species was performed using the composite interval mapping (CIM) method in R/qtl v.1.50 software [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. QTL significance thresholds were determined by permutation tests (1000 permutations) at a significance level of \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.05 in the \u0026ldquo;scantwo\u0026rdquo; function for two-dimensional QTL scanning. The percentage of the phenotypic variance explained and effects of QTLs were obtained by fitting a mixed linear model using the \u0026ldquo;fitqtl\u0026rdquo; function. SNP marker positions were drawn by MapChart v. 2.2 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The confidence intervals for each QTL were estimated using the \u0026ldquo;lodint\u0026rdquo; function that calculates the 1.5 LOD support intervals. To identify candidate genes, the coding sequences of the SNP markers in the QTL intervals were searched by BLASTn in Phytozome v13 against Vicia faba v1.1 assembly (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/info/Vfaba_v1_1\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/info/Vfaba_v1_1\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe JMP Pro 14 statistical computing program was used to analyze the phenotyping data set [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Data were logarithmically transformed to normalize the variance and used in a two-way analysis of variance (ANOVA) to determine the effects of RILs, \u003cem\u003eBotrytis\u003c/em\u003e species, and their interactions. Principal component analysis (PCA) was performed using RIL means with R version 4.5.1 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] with package ggplot2 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Histograms were drawn with SigmaPlot 16.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic variation\u003c/h2\u003e \u003cp\u003eRILs showed significant differences in response to CS reactions caused by the isolates of \u003cem\u003eB. fabae\u003c/em\u003e, \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eB. pseudocinerea\u003c/em\u003e, and \u003cem\u003eB. fabiopsis\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The interaction between RILs and \u003cem\u003eBotrytis\u003c/em\u003e species was also significant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The parental lines of the RIL population (M\u0026eacute;lodie/2 and ILB 938/2) had significant differences in their reaction to CS across all four \u003cem\u003eBotrytis\u003c/em\u003e species (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). M\u0026eacute;lodie/2 showed moderate susceptibility to CS caused by four \u003cem\u003eBotrytis\u003c/em\u003e species, whereas ILB 938/2 exhibited partial resistance to CS across the \u003cem\u003eBotrytis\u003c/em\u003e species. The pattern of disease severity in response to CS evaluation for the RILs displayed a continuous variation across different \u003cem\u003eBotrytis\u003c/em\u003e isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), suggesting that the polygenic nature of CS severity and CS resistance is quantitatively inherited. PCA results showed a similar pattern for the response of RILs to \u003cem\u003eB. cinerea\u003c/em\u003e and \u003cem\u003eB. pseudocinerea\u003c/em\u003e, while \u003cem\u003eB. fabae\u003c/em\u003e and \u003cem\u003eB. fabiopsis\u003c/em\u003e showed a reverse trend. The first two principal components explained 99% of the total variation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAnalysis of variance of infected areas of 165 RILs using four \u003cem\u003eBotrytis\u003c/em\u003e species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean square\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP-\u003c/em\u003evalue\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRILs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eBotrytis\u003c/em\u003e species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.270\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRILs x \u003cem\u003eBotrytis\u003c/em\u003e species\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e492\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eerror\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1980\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003edf, degrees of freedom. RILs: Recombinant inbred lines.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQTL analysis\u003c/h2\u003e \u003cp\u003eSix QTLs associated with the infection responses to the four \u003cem\u003eBotrytis\u003c/em\u003e isolates were detected across Chr1, 4, and 6 (LG1, LG4, and LG6). The strongest signals for all four \u003cem\u003eBotrytis\u003c/em\u003e species were located on LG1, where q-BF1 (\u003cem\u003eB. fabae\u003c/em\u003e), q-BC1 (\u003cem\u003eB. cinerea\u003c/em\u003e), q-BPC1(\u003cem\u003eB. pseudocinerea\u003c/em\u003e), and q-BFsis1 (\u003cem\u003eB. fabiopsis\u003c/em\u003e) all mapped to 254\u0026ndash;265 cM (580\u0026ndash;663 Mb of faba bean Chr1L), with LOD scores ranging from 3.66 to 4.79 and R\u0026sup2; values between 8.5% and 10.8%. Two additional QTLs for \u003cem\u003eB. fabae\u003c/em\u003e response were identified on LG4 (q-BF4, 825\u0026ndash;918 Mb of faba bean Chr4) and LG6 (q-BF6, 997\u0026ndash;1025 Mb of faba bean Chr6) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e indicates the location of genomic regions on Chr1L, Chr4, and Chr6 in map cM. The physical marker positions are presented in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Positive additive values for all QTLs suggest that M\u0026eacute;lodie/2 was the donor of susceptibility alleles (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eQuantitative trait loci (QTLs) for four \u003cem\u003eBotrytis\u003c/em\u003e isolates from the cross M\u0026eacute;lodie/2 \u0026times; ILB 938/2.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eQTL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLG\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQTL Peak (cM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2-LOD interval (cM)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLOD score\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eR\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAdd\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eq-BF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e259.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e254.89\u0026ndash;263.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eq-BF4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e70.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e65.71\u0026ndash;75.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eq-BF6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e110.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e107.63\u0026ndash;111.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eq-BC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e259.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e255.19\u0026ndash;264.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eq-BPC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e259.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e255.81\u0026ndash;263.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eq-BFsis1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e263.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e255.19\u0026ndash;264.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eQTL abbreviations: BF, \u003cem\u003eB. fabae\u003c/em\u003e (19B053-4); BC, \u003cem\u003eB. cinerea\u003c/em\u003e (19B048); BPC, \u003cem\u003eB. pseudocinerea\u003c/em\u003e (18B11); BFsis: \u003cem\u003eB. fabiopsis\u003c/em\u003e (19B175). R\u003csup\u003e2\u003c/sup\u003e, Percentage of phenotypic variance explained by QTL; Add, Additive genetic effect.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of putative candidate genes\u003c/h2\u003e \u003cp\u003e \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e lists the candidate genes associated with each SNP marker within the QTL regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). QTL intervals on LG1, LG4, and LG6 cover several large clusters of plant disease- and defence-related candidate genes (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). In LG1, the regions covered by overlapping QTLs q-BF1, q-BC1, q-BPC1, and q-BFsis1 include members of gene families for F-box proteins, DnaJ / Hsp40 chaperones, serine proteases, S-adenosylmethionine synthetases, chloride channels, SBP/MYB/FAR1 transcription factors, nodulin-like MFS transporters, lipases, and leucine-rich pentatricopeptides. Within the LG4 q-BF4 region were found genes annotated as a WRKY transcription factor, zeaxanthin epoxidase, Caffeoyl-CoA O-methyltransferase, subtilisin-like protease, RING/U-box protein, casein kinase II α1, pectin methylesterase, aspartyl protease, peroxidase, and scarecrow-like protein. LG6 q-BF6 contained members of five classical and strongly defense-related gene families, including AP2/ERF, LRR, RING/U-box E3 ligases, thioredoxin, and hydroxyproline-rich glycoproteins.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first study to genetically map the responses of faba bean to four \u003cem\u003eBotrytis\u003c/em\u003e species. We found a single region on faba bean Chr1 hosting loci for resistance to \u003cem\u003eB. faba\u003c/em\u003e, \u003cem\u003eB. cinerea\u003c/em\u003e, \u003cem\u003eB. pseudocinerea\u003c/em\u003e, and \u003cem\u003eB. fabiopsis\u003c/em\u003e. \u003cem\u003eB. fabae\u003c/em\u003e response had additional QTLs on Chr4 and Chr6. Notably, CS resistance QTLs on Chr1 and Chr6 were also reported by Gela et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] using the same mapping populations and marker system but different screening methods and a different isolate of \u003cem\u003eB. fabae\u003c/em\u003e. Putative disease- and defense-related candidate genes were identified within our identified QTL regions using faba bean genome gene annotations, some playing critical roles in resistance to \u003cem\u003eBotrytis\u003c/em\u003e species.\u003c/p\u003e \u003cp\u003eIn our study, using the detached leaflet assay, we successfully confirmed genomic regions previously detected through whole-plant screening under different indoor growing conditions in Saskatchewan for \u003cem\u003eB. fabae\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The phenotypic data also showed a strong positive correlation between RILs screened using the whole-plant and the detached leaflet assays (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.480; n\u0026thinsp;=\u0026thinsp;165, data not shown). This indicates that the detached leaflet assay is an efficient alternative to whole-plant screening for \u003cem\u003eBotrytis\u003c/em\u003e resistance under indoor climatic control conditions in this species, particularly for genetic mapping studies. It is rapid, space-efficient, and allows controlled infection with precise scoring of lesion development on excised leaves [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Field screening, although challenging, remains essential for evaluating the overall plant response, including plant\u0026ndash;pathogen interactions and genotype-by-environment effects [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The detached leaf assay provides a valuable complementary approach to whole-plant screening.\u003c/p\u003e \u003cp\u003eIn this study, six QTLs associated with \u003cem\u003eBotrytis\u003c/em\u003e infection were identified, five of which correspond to previously reported loci, underscoring the consistency of these genomic regions in contributing to CS resistance in faba bean. QTLs q-BF1, q-BC1, q-BPC1, and q-BFsis1 were mapped to the same region of Chr1, in the region for qBF1.2 reported by Gela et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; our qFB6 was found to be identical to qFB6.1 from the same study. These overlaps confirm the stability of these loci across different growing conditions and \u003cem\u003eBotrytis\u003c/em\u003e isolates. Similarly, Webb et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] identified a large interval for \u003cem\u003eB. fabae\u003c/em\u003e resistance on Chr1, consistent with our findings, though their additional QTLs on Chr3 and Chr5 may reflect either environmental variation or the use of a different genetic background (cv. Maris Bead). Skovbjerg et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] reported marker-trait associations for \u003cem\u003eB. fabae\u003c/em\u003e located at the beginning of Chr1, whereas our significant peaks were near the end of this chromosome. Notably, we also detected a novel QTL on Chr4 for \u003cem\u003eB. fabae\u003c/em\u003e, which has not been reported before. The detected minor-effect QTLs confirm the partial resistance to \u003cem\u003eBotrytis\u003c/em\u003e infection derived from ILB 938/2, Icarus, and cv. Maris Bead, which has been used in genetic mapping efforts. Beyond these sources, additional resistance germplasm has been identified [15, Khazaei et al., unpublished data]. Further efforts are needed to study the genetic architecture and loci of these sources in order to develop the means for durable gene pyramiding and the breeding of broad-spectrum \u003cem\u003eBotrytis\u003c/em\u003e resistance in this crop.\u003c/p\u003e \u003cp\u003eThe annotated faba bean genome facilitated the identification of candidate genes associated with the QTLs that were identified. The corresponding regions for the QTLs included gene families and candidate genes known to play a role in disease resistance or defense against various biotic stresses. Several of the candidate gene families identified in this study overlap with those reported by Webb et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Both studies found, within the mapping intervals, genes for F-box and U-box\u0026ndash;type E3 ubiquitin ligases, thioredoxin, and chaperone (DnaJ/Hsp40) proteins, which are central to protein turnover and stress signalling during pathogen attack. Similarly, transcription factor families including AP2/ERF, MYB, and SBP were identified as candidate genes in both studies, due to their regulatory roles in activating defence-related genes. Moreover, genes for peroxidases, lipases, and S-adenosylmethionine synthetase identified in the genome assembly for our QTL regions appear to parallel the oxidative stress and secondary metabolism genes identified in Webb et al. [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consistent with this, Castillejo et al. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] reported that resistance to \u003cem\u003eB. fabae\u003c/em\u003e in faba bean is associated with a more efficient Photosystem II repair cycle and enhanced redox regulation, further highlighting the likely importance of oxidative balance in \u003cem\u003eBotrytis\u003c/em\u003e resistance. Direct evidence linking specific candidate genes to \u003cem\u003eBotrytis\u003c/em\u003e resistance in legumes remains limited. However, studies in chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e L.) have reported the involvement of peroxidases, WRKY transcription factors, and antioxidant enzymes in defense responses to \u003cem\u003eB. cinerea\u003c/em\u003e infection [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These genes are known to mediate oxidative stress regulation and pathogen-induced signaling pathways in legumes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In Arabidopsis, functional studies have demonstrated that certain F-box proteins and WRKYs play critical roles in \u003cem\u003eB. cinerea\u003c/em\u003e resistance by modulating jasmonate and salicylic acid pathways [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], supporting the likely conserved defense role of these gene families across species.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWe confirmed previously reported minor-effect QTLs and identified a novel QTL associated with \u003cem\u003eBotrytis\u003c/em\u003e resistance in faba bean. These loci provide valuable targets for marker-assisted and genomic selection as well as for functional verification. The detached leaf assay proved to be an efficient and reliable method for QTL mapping, offering reproducible and quantitative phenotypic data. The identified candidate genes will support further dissection of the biological mechanisms underlying \u003cem\u003eBotrytis\u003c/em\u003e resistance. Future work on mapping additional resistance sources and combining QTLs to enhance durable resistance in breeding programs would also be highly worthwhile.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cb\u003ea .Ethics approval and consent to participate\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eb. Consent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003ed. Competing interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eClinical trial number\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003ee. Funding\u003c/h2\u003e \u003cp\u003eThe work was part of the projects \u0026ldquo;Fabanova: climate-ready faba beans for the Nordic and Baltic Region\u0026rdquo; funded under the NordForsk call \u0026ldquo;Sustainable agriculture and climate change\u0026rdquo; (decision 137268) and \u0026ldquo;Fabagen\" funded by the Research Council of Finland, Academy projects, funding decision 363375.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: FLS, HK. Methodology: M. Formal Analysis: M, HK. Investigation: M. Data Curation: M. Writing \u0026ndash; Original Draft: M, HK. Writing \u0026ndash; Review \u0026amp; Editing: all authors. Supervision: FLS, HK, MJ, AHS. Funding Acquisition: FLS, HK.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe thank Prof. Biruta Bankina (LBTU) for sharing the Botrytis isolates with us. We thank Dr. Pedro J. Aphalo for his kind supply of the black tube light and for measuring the NEB light intensity. We thank Marjo Kilpinen, Eija Takala, Markku Tykkyl\u0026auml;inen and Eero Kuisma, technical officers of the laboratories and glasshouses of the Department of Agricultural Sciences at the University of Helsinki, for their kind assistance during the experiments.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLabba ICM, Fr\u0026oslash;ki\u0026aelig;r H, Sandberg AS. 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Mol Plant. 2024;17:297\u0026ndash;311. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.molp.2023.12.020\u003c/span\u003e\u003cspan address=\"10.1016/j.molp.2023.12.020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"gene mapping, quantitative trait loci, candidate gene, disease resistance, Botrytis spp","lastPublishedDoi":"10.21203/rs.3.rs-8267933/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8267933/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eChocolate spot (CS), caused by \u003cem\u003eBotrytis\u003c/em\u003e species, is a major disease constraining faba bean production worldwide. In this study, we investigated the genetic basis of resistance to four \u003cem\u003eBotrytis\u003c/em\u003e species (\u003cem\u003eB. fabae, B. cinerea\u003c/em\u003e, \u003cem\u003eB. pseudocinerea\u003c/em\u003e, and \u003cem\u003eB. fabiopsis\u003c/em\u003e) using a recombinant inbred line (RIL) population derived from the cross M\u0026eacute;lodie/2 \u0026times; ILB 938/2, screened using a detached-leaflet assay.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSignificant variation in disease severity was observed among parental lines and RILs for all four \u003cem\u003eBotrytis\u003c/em\u003e species. The detached-leaf assay was highly correlated with a whole-plant assay conducted with a different \u003cem\u003eB. fabae\u003c/em\u003e isolate, validating the use of the screening tool. QTL analysis identified six loci associated with \u003cem\u003eBotrytis\u003c/em\u003e infection across Chr1, Chr4, and Chr6, explaining 9\u0026ndash;25% of the total phenotypic variance. The overlapping QTLs on Chr1 conferred resistance to all four \u003cem\u003eBotrytis\u003c/em\u003e species, while QTLs on Chr4 and Chr6 were specific to \u003cem\u003eB. fabae\u003c/em\u003e. None of the other three species showed a unique QTL. Candidate gene analysis within QTL intervals revealed several defense-related gene families, including F-box protein, WRKY, MYB, and AP2/ERF transcription factors, and peroxidases involved in oxidative stress regulation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe consistency of response across species and the lack of unique responses from the non-\u003cem\u003efabae\u003c/em\u003e species indicate that selection for resistance to \u003cem\u003eB. fabae\u003c/em\u003e should select adequately for the other species. This study provides new insights into the shared and species-specific genetic architecture of \u003cem\u003eBotrytis\u003c/em\u003e resistance in faba bean.\u003c/p\u003e","manuscriptTitle":"QTL analysis uncovers the genetic architecture of resistance to chocolate spot disease caused by four Botrytis species on faba bean","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-22 13:07:53","doi":"10.21203/rs.3.rs-8267933/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-29T22:09:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-28T06:11:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-26T17:34:35+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-11T07:55:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"30970323086113127639931885857236258953","date":"2026-01-08T06:35:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178001228568868488937002146242145953392","date":"2026-01-07T06:35:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"184496364595123074825466270121207779638","date":"2026-01-06T17:31:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"73230013126429032642570463470555152316","date":"2025-12-29T13:53:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-24T11:14:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T19:05:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-15T18:42:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-10T19:25:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-12-10T19:18:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9684b1c7-9804-43e1-b93b-bd846ba66a88","owner":[],"postedDate":"January 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T16:05:44+00:00","versionOfRecord":{"articleIdentity":"rs-8267933","link":"https://doi.org/10.1186/s12870-026-08699-0","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2026-04-10 15:58:34","publishedOnDateReadable":"April 10th, 2026"},"versionCreatedAt":"2026-01-22 13:07:53","video":"","vorDoi":"10.1186/s12870-026-08699-0","vorDoiUrl":"https://doi.org/10.1186/s12870-026-08699-0","workflowStages":[]},"version":"v1","identity":"rs-8267933","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8267933","identity":"rs-8267933","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}