Introgression of QTL Hotspot Regions Enhances Grain Yield and Maize Lethal Necrosis Resistance in Elite Maize Lines

Introgression of QTL Hotspot Regions Enhances Grain Yield and Maize Lethal Necrosis Resistance in Elite Maize Lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Introgression of QTL Hotspot Regions Enhances Grain Yield and Maize Lethal Necrosis Resistance in Elite Maize Lines Veronica Ogugo, Vijay Chaikam, Michael S Olsen, Yoseph Beyene, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7815596/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 14 You are reading this latest preprint version Abstract Maize lethal necrosis (MLN) poses a severe threat to maize production in eastern and southern Africa, causing significant yield losses. In this study, marker-assisted backcrossing (MABC) was used to introgress major-effect MLN resistance Quantitative Trait Loci (QTL) located on chromosomes 3 and 6 into 14 elite but MLN-susceptible CIMMYT maize lines belonging to heterotic groups A and B. Ten Kompetitive Alelle Specific PCR (KASP) SNP markers closely linked to three validated QTL-hotspot regions were applied for foreground selection, with at least two hotspots polymorphic across all donor–recipient combinations. Foreground and background selection enabled fast tracking of MLN resistance alleles and recovery of near-recurrent parent genomes. The resulting BC₄F₂ introgressed lines exhibited markedly reduced MLN severity under artificial inoculation, with several lines showing a 50% reduction relative to their recurrent parents. Testcrosses of these lines demonstrated yield advantages of 2–4 t/ha under MLN pressure compared with original parental lines, while maintaining comparable performance under optimum conditions. Notably, introgressed derivatives of CML312, CML539, and CZL052 displayed both enhanced MLN resistance and superior yield performance, with CZL052-derived testcrosses achieving nearly two-fold yield gains under severe MLN stress. Importantly, equivalence trials confirmed that MLN resistance was improved without compromising resistance to gray leaf spot, turcicum leaf blight, or common rust. These findings validate the effectiveness of QTL-based conversion for enhancing MLN resistance in elite breeding lines and demonstrate the potential of these improved lines as robust parental sources for developing MLN-resilient hybrids adapted to eastern and southern Africa. Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Plant sciences maize MLN resistance disease severity grain yield MABC QTL Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Maize ( Zea mays L.) is the world’s second most cultivated cereal and, with rice and wheat, supplies over 40% of global food calories (Shiferaw et al., 2011 ; Erenstein et al., 2022 ). Demand is expected to rise by 50% by 2050 (Ignaciuk & Mason-D’Croz, 2014 ), yet production is increasingly constrained by biotic and abiotic stresses. In Sub-Saharan Africa, maize covers over 44 million hectares and feeds more than 300 million people (Goredema-Matongera et al., 2021 ) but yields average just 2.1 t/ha—less than half the global mean—due to drought, degraded soils, low inputs, and pests and diseases (Atlin et al., 2017 ; Erenstein et al., 2022 ; Cairns et al., 2021 ). Viral diseases are particularly devastating, sometimes causing complete crop failure. Major maize viruses in Africa include maize streak virus, maize chlorotic mottle virus (MCMV), sugarcane mosaic virus (SCMV), maize chlorotic dwarf virus, maize dwarf mosaic virus , and maize rough dwarf virus (Abbas et al., 2022 ; Thottappilly et al., 1992, 1993 ; Fajemisin, 2003 ). Among viral diseases in maize, Maize Lethal Necrosis (MLN) poses the greatest threat, capable of causing up to 100% yield loss in smallholder systems. MLN arises from a synergistic co-infection of MCMV and SCMV. First reported in Kenya in 2012 (Wangai et al., 2012 ), the disease has since spread across Eastern Africa, imposing an estimated annual economic burden of > $ 339 million on smallholder farmers (Marenya et al., 2018 ). MCMV, a single-stranded RNA virus in the Tombusviridae family, is transmitted primarily by thrips and beetles, but also mechanically, with rare cases of seed and soil transmission (Regassa & Dechassa, 2021 ; Bernardo et al., 2023 ). Originally identified in Peru in 1973, MCMV has since been reported in the USA, Latin America, and China (Castillo & Hebert, 1974 ; Niblett & Claflin, 1978 ; Xie et al., 2011 ). SCMV, a Potyviridae virus, is mainly aphid-transmitted but can also spread mechanically. While infections by either virus alone are usually mild, co-infection results in severe symptoms—leaf mottling, stunting, premature senescence, necrosis, sterility, and crop death before tasseling (Mahuku et al., 2015 ; Wangai et al., 2012 ). Following the MLN outbreak, surveys by International Maize and Wheat Improvement Centre (CIMMYT) and the Kenya Agriculture and Livestock Research Organization (KALRO) revealed that over 90% of commercial and pre-commercial hybrids in the region were highly susceptible to MLN (Gowda et al., 2015 , 2018 ). This triggered urgent breeding efforts to identify resistance sources. Screening of CIMMYT germplasm, commercial cultivars, and national breeding lines revealed only a few donor lines with moderate-to-high resistance to both viruses, which are now being used to introgress MLN resistance into susceptible but agronomically superior backgrounds (Boddupalli et al., 2020 ; Biswal et al., 2022 ). Genetic studies revealed that resistance to MLN is governed by few major effects and several minor effect quantitative trait loci (QTL, Gowda et al., 2015 ; Nyaga et al., 2019 ; Sitonik et al., 2019 ; Sadessa et al., 2022 ). A genome-wide association study (GWAS) identified single nucleotide polymorphisms (SNPs) explaining 8–10% of the phenotypic variance, with combined effects accounting for ~ 30% (Gowda et al., 2015 ). Subsequent biparental mapping validated these associations and revealed major QTL on chromosomes 3, 6, and 9, with effects ranging from 3.9% to 43.8% of phenotypic variation (Gowda et al., 2018 ; Awata et al., 2019 , 2020 ). Additional studies confirmed major-effect loci on chromosomes 3 and 6, consistently detected across diverse genetic backgrounds (Murithi et al., 2021 ; Sitonik et al., 2019 ). These loci represent prime candidates for introgression of MLN resistance QTL into elite maize lines. Marker-assisted backcrossing (MABC) provides a powerful tool for such trait introgression. By exploiting molecular markers tightly linked to target QTL, MABC accelerates the transfer of resistance alleles into elite cultivars while minimizing linkage drag (Mekonnen et al., 2017 ). The approach enables early and cost-effective selection, reducing breeding cycles and improving precision. MABC has been successfully applied to enhance resistance against several maize diseases—for example, introgression of qHSR1 for head smut (Zhao et al., 2012 ), ZmCCT-H5 for stalk rot, flowering regulation, and yield stability (Li et al., 2017 ; Tong et al., 2022 ), and pyramiding of major genes for maize rough dwarf disease and gray leaf spot resistance into elite inbreds (Li et al., 2024 ; Zhu et al., 2025 ). In East Africa, many commercially successful hybrids derive from parent lines that are highly susceptible to MLN. These hybrids are difficult to replace quickly because they combine high yield potential, drought tolerance, and resistance to multiple foliar diseases. A practical breeding strategy is therefore to introgress MLN resistance QTL into these susceptible parents through MABC. This approach enhances MLN resistance while retaining the superior agronomic performance of popular hybrids, extending their utility and ensuring food security as new MLN-resistant germplasm is developed. In this study, we applied MABC to introgress major-effect MLN resistance QTL into elite but MLN-susceptible CIMMYT maize lines. Using ten SNP markers tightly linked to resistance loci on chromosomes 3 and 6, we deployed five MLN-tolerant donors to transfer resistance into 14 recurrent elite lines. The specific objectives were to: (i) introgress major-effect MLN resistance QTL on chromosomes 3 and 6 into drought-tolerant but MLN-susceptible elite lines through MABC; (ii) Assess the level of MLN resistance in the improved lines relative to their recurrent parents, and (iii) Evaluate testcross hybrids derived from the introgressed lines for both agronomic performance and MLN resistance compared to the original parents. Together, these objectives aim to deliver MLN-resistant lines and hybrids without compromising yield or adaptive traits, thereby extending the utility of commercially successful germplasm and contributing to sustainable maize production and food security in Eastern Africa. Materials and Methods Germplasm used Between 2013 and 2015, CIMMYT evaluated a large panel of inbred lines and identified a subset with tolerance to MLN ( https://www.cimmyt.org/news/update-cimmyt-maize-inbred-lines-and-pre-commercial-hybrids-with-potential-resistance-to-maize-lethal-necrosis-mln-2/ ). From these, five MLN tolerant inbred lines were selected as trait donors for population development and later in resistance introgression (Table 1 ). Two and three donor lines belong to heterotic group A and B, respectively. Donor lines known for wide adaptation to the region and tolerance to MLN, while recipient lines are known for high yield potential, strong combining ability, drought tolerance, low-soil nitrogen stress tolerance and resistance to multiple foliar diseases. This combination provided an ideal foundation for introgressing MLN resistance into agronomically superior but MLN susceptible elite lines, ensuring both disease resistance and retention of desirable agronomic traits in the breeding pipeline. Genotyping Plants were tagged and leaf samples were collected after three weeks of planting. From each tagged plant, four 6 mm leaf discs were collected. The samples were dried using silica gel. Ten KASP SNPs linked to three QTL detected in multiple studies for MLN tolerance were used for parental polymorphism analyses between five donor parents and 14 recipient lines, as well as for foreground selection. Genotyping with this set of markers was conducted at LGC, UK ( https://www.lgcgroup.com ). The resulting marker data enabled the selection of plants homozygous for favorable alleles at the targeted MLN resistance QTLs (Supplementary Table S1 ). For the background selection, leaf discs from donor parent, recipient parent, and BC 4 F 3 plants were sent to Diversity Arrays Technology (DArT), Australia, for genotyping using the Maize DArTag 3.3K EiB (2.0) panel developed by the CGIAR Excellence in Breeding (EiB) platform ( https://excellenceinbreeding.org/toolbox/services/mid-density-genotyping- service). This publicly available panel comprises 3,305 DArTag markers, derived from over 10,000 genetically diverse maize inbred lines. Table 1 Donor and recipient maize inbred lines used in this study, their heterotic group classification, and SNPs linked to major-effect QTL for MLN resistance on chromosomes 3 and 6, including favorable and unfavorable alleles. Donor line HG Haplotype / QTL SNP marker Favorable allele Unfavorable allele Recipient line DTPYC9-F46-1-2-1-2-B A MLN_03.133 PZA02299_16 AA GG CML539, LaPostaSeqC7-F64-2-4-1-1 PZA00363_7 GG AA S3_133048570 CC TT CLYN261 A MLN_03.133 PZA02299_16 AA GG CML312, CML540, CML544, DTPWC9-F67-1-2-1-2, LaPostaSeqC7-F64-2-4-1-1 S3_133048570 CC TT MLN_03.140 S3_146250249 GG TT S3_146363360 TT CC S3_146602134 TT CC CML543 B MLN_03.133 PZA02299_16 AA GG CML202, CML489, CML546, CML574, CZL052, CLRCY034, CML444 PZA00363_7 GG AA S3_133048570 CC TT MLN_03.140 S3_146250249 GG TT S3_146363360 TT CC S3_146602134 TT CC MLN_06.20 PZA03047_12 GG AA S6_21007530 GG AA S6_21008211 CC TT CML574 B MLN_03.133 PZA02299_16 AA GG CML444 PZA00363_7 GG AA S3_133048570 CC TT CLRCY034 B MLN_03.133 PZA02299_16 AA GG CML507 PZA00363_7 GG AA S3_133048570 CC TT MLN_03.140 S3_146250249 GG TT S3_146363360 TT CC S3_146602134 TT CC HG – heterotic group Introgression of the QTL-hotspot genomic regions The MLN resistance QTL-hotspot region was introgressed independently into elite recipient lines using a MABC approach. In heterotic group A, donor parent CLYN261 was used to introgress MLN resistance into five elite recurrent lines: CML312, CML540, CML544, DTPWC9-F67-1-2-1-2 , and LaPostaSeqC7-F64-2-6-2-2 (Table 1 ). Additionally, DTPYC9-F46-1-2-1-2-B served as the donor for CML539 and LaPostaSeqC7-F64-2-6-2-2 . In heterotic group B, CML543 was used as the donor for seven elite recipient lines, while CML574 and CLRCY034 each contributed MLN resistance to one recipient line (Table 1 ). In the backcrossing scheme, the recipient parent was consistently used as the female and the donor parent as the male across all generations. Crosses were conducted at CIMMYT’s Kiboko Research Station, Kenya, between 2014 and 2017. To accelerate population advancement, two to three planting cycles per year were implemented. F₁ hybrids were first generated by crossing donor and recipient parents, followed by backcrossing to the recurrent parent to produce BC₁F₁ progenies in 2014. Three additional backcross generations (BC₂F₁, BC₃F₁, and BC₄F₁) were advanced during 2014–2015. Foreground selection was initiated at the BC₂F₁ generation using SNP markers tightly linked to major MLN resistance QTL. Genotyped plants carrying favorable alleles were tagged, and selected ears were advanced by planting ~ 21 seeds per row, from which 14–16 plants per row were genotyped. In 2016–2017, BC₄F₁ progenies were selfed to generate BC₄F₂ and subsequently BC₄F₃ families. Foreground selection was applied at both BC₄F₁ and BC₄F₃ stages, while background selection using genome-wide SNP markers was conducted at the BC₄F₃ stage to identify plants with the highest recurrent parent genome recovery and phenotypic similarity to their recurrent parents. From each cross, the two best BC₄F₃ lines were selected and advanced for evaluation of MLN resistance and agronomic equivalence relative to their recurrent parent. Evaluation of MLN introgressed lines and their testcrosses To assess the efficacy of introgressed QTLs and the equivalence of converted lines to their recurrent parents, testcrosses were developed at CIMMYT’s Kiboko Research Station. Inbred tester CKDHL120312 (heterotic group B) was crossed with donor lines, recurrent parents, and their BC₄F₃ introgressed derivatives from heterotic group A ( CLYN261, DTPYC9-F46-1-2-1-2, CML312, CML539, CML540, CML544, DTPWC9-F67 , and LaPostaSeqC7-F64 ). Tester CKDHL120918 (heterotic group A) was used to cross lines from heterotic group B ( CML202, CML543, CML444, CML507, CML489, CML546, CML574, CZL052 , and CLRCY034 ), along with their introgressed BC₄F₃ versions. To evaluate the effect of introgressed QTL on MLN resistance, the converted lines and their test cross hybrids were evaluated for their response to MLN under artificial infestation. The test cross hybrids were also evaluated under natural infestation at an MLN hotspot region. In all these trials, each experimental unit consisted of a two-row plot, 4 meters in length, with 0.75 meters between rows and 0.25 meters between plants within a row. Two seeds were planted per hill and later thinned to one plant per hill at three weeks after emergence, targeting a final population of 53,333 plants per hectare. Fertilizer management included a basal application of di-ammonium phosphate (DAP) at planting, providing 60 kg N and 60 kg P₂O₅ per hectare. Six weeks after emergence, all plots were top-dressed with an additional 60 kg N per hectare. Weed control was maintained through a combination of manual weeding and herbicide application. All phenotyping trials for MLN resistance under artificial infestation were conducted at CIMMYT’s MLN screening facility in Naivasha, Kenya. For inbred line trial, a total of five donor lines, 14 recurrent parent lines, and 31 selected BC₄F 3 lines (the two best lines per MABC cross) were evaluated to assess disease severity and grain yield. Testcross hybrids generated using the 31 BC₄F 3 lines along with test cross hybrids for donor and recurrent parents were evaluated under artificial MLN inoculation in 2017. Additionally, the same testcrosses were also evaluated for MLN resistance in the natural MLN hotspot region of Babati in Tanzania across two seasons, 2017 and 2018. In addition to being tested under artificial and natural MLN disease pressure, the hybrids were also evaluated under optimal conditions across multiple environments to determine both the efficacy of the introgressed QTLs under MLN pressure and the agronomic equivalence of the converted lines to the recurrent parents in the absence of MLN. These environments included Kiboko, Kitale, and Thika in Kenya under optimum management conditions in 2017. All trials conducted with an alpha-lattice experimental design with three replications per genotype. Data Collection and Statistical Analysis At each location, data were recorded on a per-plot basis for the following traits: days to 50% anthesis (AD; days from planting until 50% of plants shed pollen), days to 50% silking (SD; days from planting until 50% of plants produced silks), and anthesis–silking interval (ASI; calculated as SD – AD). Plant height (PH) was measured as the distance in centimeters from the base of the plant to the insertion of the first tassel branch, while ear height (EH) was measured as the distance from the base to the node bearing the uppermost ear; both were recorded on 10 representative plants per plot. At harvest, field weight (total weight of dehusked ears in kilograms per plot) and grain moisture (MOI; determined using a moisture meter from grains sampled at the center of five representative ears per plot) were recorded. Grain yield (GY) was computed from the field weight using a shelling percentage of 80% and adjusted to 12.5% grain moisture. Analyses of variance (ANOVA) for individual environments (under both MLN pressure and optimum conditions) were conducted using META-R software (Alvarado et al., 2020). Results Markers for foreground and background selection A total of seven recipient lines from heterotic group A and nine from heterotic group B were selected for the introgression of MLN resistance QTLs (Table 1 ). Foreground selection was carried out using a panel of ten KASP markers. Only the markers that were polymorphic between the donor and recurrent parent pairs were utilized for selection (Table 1 ; Supplementary Table S1 ). Among the ten KASP markers tested, three SNPs—PZA02299_16, PZA00363_7, and S3_133048570—were polymorphic between donor line DTPYC9-F46-1-2-1-2 and the recurrent parents CML539 and LaPostaSeqC7-F64-2-6-2-2 . In another set of crosses involving donor line CLYN261 and recipient lines ( CML312, CML540, CML544, DTPWC9-F67-1-2-1-2 , and LaPostaSeqC7-F64-2-6-2-2 ), five SNPs were identified as polymorphic (Table 1 ). For heterotic group B, nine SNPs were found polymorphic between donor line CML543 and recurrent parents ( CML202, CML489, CML546, CML574, CZL052, CLRCY034 , and CML444 ). Additionally, three SNPs were polymorphic between donor–recipient pairs CML574 × CML544 and CLRCY034 × CML507 (Table 1 ). Overall, at least two QTL-hotspot regions showed polymorphism in all donor–recipient combinations (Table 1 ; Supplementary Table S1 ). For example, in CML202, CML489 , and CML546 , SNPs linked to qMLN3_133 and qMLN6_021 were utilized for foreground selection, while in CML444 and CZL052 , all three QTL-hotspot regions were polymorphic and applied in selection. Notably, markers linked to qMLN3_133 (chromosome 3) were consistently polymorphic across all donor–recurrent parent combinations, whereas markers from the other two hotspot regions displayed polymorphism only in specific parental pairs. For background selection, among the 3305 SNPs tested on the selected donor and recipient parents, 1800 markers were used after quality check, among them, 1160 (CML312/ CLYN261), 942 (CML540/CLYN261), 1157 (CML544/CLYN261; DTPWC9-F67/CLYN261; CML539/DTPYF-46), 942 (LPSC7-F64/CLYN261; LPSC7-F64/DTPYF-46), 1119 (CML202/CML543), 1116 (CML489/CML543), 1141 (CML546/CML543), 1146 (CML574/CML543), 918 (CZL052/CML543), 1142 (CLYCR034/CML543), 1158 (CML444/CML543), and 1160 (CML507/CLYCR034) markers showed informative polymorphisms that were used for background selection (Supplementary Table S2). For the background selection, the number of polymorphic markers distributed per chromosome varied from 43 to 177. Phenotypic Evaluation of Introgression Lines for their efficacy With an objective to assess the efficacy of MABC for transferring the QTL-hotspots , 31 introgressed lines (two introgressed lines from each cross apart from LPSC7-F64 x CLYN261) were evaluated for MLN disease severity (Fig. 2 ) along with their donor and recurrent parents. Evaluation of MLN resistance showed consistent differences between donor parents, recurrent parents, and the converted lines (LNT1 and LNT2) across both heterotic groups (Fig. 2 ). In heterotic group A, recurrent parents exhibited high MLN disease severity scores (6.0–8.9), whereas donor parents consistently had lower scores (3.9–4.9). The converted lines (LNT1 and LNT2) demonstrated reduced severity compared to their recurrent parents, with scores ranging between 3.4 and 7.5, indicating a clear improvement in MLN resistance. The highest reduction of MLN disease severity was observed for introgressed lines derived from CML539 x DTPYC9-F46 (5.9 vs 3.4) and CML312 x CLYN261(6.9 vs 4.9). Similar trends were observed in heterotic group B, where recurrent parents recorded high disease severity (7.0–8.5) relative to donor parents (5.0). The converted lines exhibited lower disease severity (3.3–6.5), approaching the levels of the donor parents, particularly in CLYCR034 x CML543, CML574 × CML543 and CZL052 × CML543 combinations. Overall, the introgression of MLN resistance QTLs led to significant reductions in disease severity across both heterotic groups, confirming the effectiveness of the conversion process in improving MLN resistance. Phenotypic Evaluation of Testcrosses for their efficacy Evaluation of MLN resistance among testcross hybrids exhibited consistent differences between the testcrosses of donor parents, recurrent parents, and the converted lines (LNT1 and LNT2) across both heterotic groups (Fig. 3 ). In heterotic group A, the testcrosses of recurrent parents exhibited moderate MLN disease severity scores, whereas donor parents consistently had lower scores (2.33–3.33). The testcross of converted lines for each introgressed inbreds (LNT1 and LNT2) demonstrated reduced severity scores compared to recurrent parents, with scores ranging between 2.0 and 4.0, indicating a clear improvement in MLN resistance. The highest reduction of MLN disease severity was observed for testcrosses of introgressed lines derived from CML312. The efficacy of transferring MLN resistance associated QTL-hotspot in a cross between CLYN261 x CML312, revealed 50% reduction in MLN disease severity in introgressed lines compared to the recurrent parent CML312 (Fig. 3 ) and increase in grain yield up to 2.25 tons/ha under MLN disease pressure (Fig. 4 ). Overall, the testcross hybrids revealed considerable reduction in MLN disease severity in introgressed lines compared to their original recurrent parents, except for CLYN261 x DTPWC9-F67 cross in heterotic group A (Fig. 3 ). Similar trends were observed in heterotic group B, where testcrosses of recurrent parents recorded moderately high severity compared to test crosses of donor parents (3.00-3.67). The testcrosses of converted lines exhibited lower disease severity (2.3–4.0). Higher levels of MLN score reduction were recorded in testcrosses of converted versions of CML507, CZL052 and CML489. Overall, the introgression of MLN resistance QTLs led to significant reductions in disease severity across both heterotic groups, confirming the effectiveness of the conversion process in improving MLN resistance. Grain yield under MLN stress varied significantly among testcross hybrids of donor parents, recurrent parents, and converted lines (LNT1 and LNT2) across both heterotic groups (Fig. 4 ). In heterotic group A, testcrosses of recurrent parents consistently showed low yields under MLN (1.85–4.80 t/ha), while test crosses of donor parents recorded higher yields (4.20–5.95 t/ha). Testcrosses of Converted lines (LNT1 and LNT2) showed substantial yield advantages over recurrent parents, with hybrids yielding between 3.70 to 7.85 t/ha. Particularly testcross hybrids from converted lines of CML312 (LNT1, 7.85 t/ha) and CML539 (LNT1, 7.05 t/ha) exhibited the highest yields, clearly outperforming both donor and recurrent parents. Testcross hybrids of converted lines of CML544 (5.05 vs 1.85 t/ha), DTPWC9-F67 (6.7 vs. 3.7t/ha) showed the highest yield gains under MLN stress compared to testcross hybrids of their recurrent parents Similar trends were observed in heterotic group B, where recurrent parents yielded poorly (1.23–3.65 t/ha) compared to donor parents (4.80–5.65 t/ha) under MLN stress. The converted lines (LNT1 and LNT2) consistently outperformed recurrent parents, with yields ranging from 4.15 to 6.60 t/ha. Testcross hybrids of converted lines of CML574 (LNT2, 6.15 t/ha) and CML507 (LNT1, 6.60 t/ha) achieved the highest yields under MLN stress. Particularly, testcross hybrids of converted lines of CML543 (4.00 Vs 1.23 t/ha), CML546 (5 vs.1.65t/ha), CZL052 (4.60 vs1.40 t/ha) and CML507 (5.65 vs 1.95 t/ha) achieved the highest yields under MLN stress compared to test cross hybrids of their recurrent parents, indicating effective recovery of productivity through the introgression of MLN resistance. Overall, the converted lines significantly enhanced hybrid performance under MLN pressure in both heterotic groups, approaching or surpassing the donor parent yields. Analyses of variance on grain yield and other agronomic traits under MLN disease (Supplementary Table S3, S4 and S5) and under optimum management (Supplementary Table S6, S7 and S8) revealed significant variation and moderate to high heritability estimates for each location. Comparison of introgressed lines against their original recipient parents revealed no significant changes for agronomic traits like AD, SD, PH, EH, ER, husk cover and for foliar diseases like GLS, common rust and TLB under optimum management. Grain yield equivalency under optimum conditions in the absence of MLN disease pressure showed minimal differences among the testcrosses of donor parents, recurrent parents, and converted lines (LNT1 and LNT2) across both heterotic groups (Fig. 5 ). In heterotic group A, average yields of testcrosses of recurrent parents ranged between 4.05 and 5.25 t/ha, while donor parents yielded 4.65–4.75 t/ha. The testcrosses of converted lines performed comparably, with yields of 4.45–6.10 t/ha. LPSC7-F64 (LNT2, 6.10 t/ha) and DTPWC9-F67 (LNT2, 5.90 t/ha) and CML539 showed slightly higher yields than both donor and recurrent parents. For CML312 and CML540, the test crosses of converted lines showed higher yield compared to the recurrent parent but less than the donor parent. For CML544, the yield of testcrosses of converted lines showed less yield than the recurrent parent but same/slightly higher yield than the donor parent. Similarly, in heterotic group B, test cross hybrids of donor parents yielded 4.90 to 5.65 t/ha, while recurrent parents ranged between 4.20 and 5.20 t/ha. Testcross hybrids of converted lines matched these levels yielding 4.00–5.80 t/ha. Notably, testcross hybrids of converted lines of CML546 (LNT1, 5.80 t/ha) and CLYCR034 (LNT1, 5.65 t/ha) maintained yields equivalent to donor parents, while outperforming recurrent parents. For rest of the lines, testcross hybrids even though did not perform better than the donor parents, the conversions did slightly better or similar to the recurrent parent. These results demonstrate that the introgression of MLN resistance QTLs into elite lines did not negatively affect hybrid yield potential under optimum conditions, thereby confirming their equivalency. Discussion Climate change has intensified biotic and abiotic stresses across sub-Saharan Africa, making the development of maize hybrids with multiple stress tolerance a breeding priority (Prasanna et al., 2021 ). MLN disease, first reported in Kenya in 2011 (Wangai et al., 2012 ), has caused devastating yield losses, restricted seed movement due to transmission through seed and insect vectors, and exposed the vulnerability of many otherwise stress-tolerant and agronomically superior maize hybrids. To address this challenge, conventional breeding alone is inadequate, and the integration of modern tools such as genomics, high-throughput phenotyping, and systems modeling is essential (Prasanna et al., 2021 , 2022 ). MABC offers a strategic, quick response by introgressing MLN resistance alleles into elite, farmer-preferred hybrids backgrounds, enabling rapid genetic gain while preserving key agronomic traits. The resulting lines provide a resilient foundation for hybrid development, ensuring food security and strengthening the maize supply chain in eastern and southern Africa. MABC is particularly effective for improving disease resistance, as phenotypic selection for resistance can be unreliable due to viral mutations and strong year-to-year environmental variation. Unlike phenotypic approaches, molecular markers are environment-independent, allowing precise and consistent introgression of resistance loci into elite germplasm (Li et al. , 2018; Zhang et al., 2023 ). MLN resistance is largely quantitative, controlled by a few major-effect and several minor-effect QTL (Gowda et al., 2015 , 2018 ; Nyaga et al ., 2020; Awata et al., 2019 , 2020 ; Murithi et al., 2021 ), making it especially amenable to MABC. The present study validated seven MLN resistance linked SNPs on chromosome 3 ( PZA02299_16, PZA00363_7, S3_133048570, PZD00015_5, S3_146250249, S3_146363360 , and S3_146602134 ) and three SNPs on chromosome 6 ( PZA03047_12, S6_21007530 , and S6_21008211 ), all located within QTL-hotspot regions previously linked to MLN disease resistance in maize (Gowda et al., 2015 , 2018 ; Nyaga et al ., 2020; Awata et al., 2020 ; Lohithaswa et al., 2025 ). The strong alignment between phenotypic resistance and the presence of favorable alleles at these loci underscores their value in breeding pipelines. Indeed, 31 BC₄F 3 introgressed lines carrying these favorable alleles exhibited MLN resistance, providing direct evidence for the functional relevance of these hotspots. Similar successes have been documented in other cereals, such as the introgression of Pi-b and Pi-kh blast resistance genes into rice via MABC (Tanweer et al., 2015 ). Nevertheless, the expression of QTL can be affected by epistatic interactions or linkage drag in new genetic backgrounds (Hospital, 2005 ; Collard and Mackill, 2008 ). Therefore, the development of tightly linked or functional markers for MLN resistance remains a priority to enhance selection accuracy and reduce the number of individuals required in each backcross generation. The consistent polymorphism observed for SNP markers linked to qMLN3_133 across all donors–recipient parent combinations highlight the robustness of this QTL hotspot and its potential utility as a stable source of resistance. This finding is in line with earlier studies reporting chromosome 3 regions as critical for MLN resistance (Gowda et al., 2018 ; Nyaga et al ., 2020; Awata et al., 2020 , 2021 ). The partial polymorphism observed for the other two QTL hotspots suggests that their utility may depend on the genetic background of the parents, further emphasizing the importance of multiple resistance loci to broaden the genetic base of MLN resistance. Resistance QTL can sometimes carry negative effects on yield or plant growth (Chitwood-Brown et al., 2021 ; Klindworth et al., 2023 ) and maintaining heterosis after line conversion requires a high level of genetic background recovery. In our study, although introgressed chromosomal regions were relatively large, no yield penalties were observed in the converted lines and their testcross hybrids—because MLN resistance genes reside within the target QTL regions (Nyaga et al., 2020; Wen et al., 2024 ; Murithi et al., 2025 ). Based on marker polymorphism, 788–1160 SNPs were used to estimate recurrent parent genome (RPG) recovery. The RPG recovery in the introgressed lines ranged from 60% in CML489 to 98% in CML540, with other genetic backgrounds showing recovery rates between 83% and 90% (Supplementary Table S2). These results are consistent with findings in other crops, where, for example, 91.6% RPG recovery was achieved in rice ( Oryza sativa L.) while pyramiding blast resistance genes into elite Basmati (Singh et al., 2013 ), and > 90% recovery was reported in chickpea after just three backcross generations (Pratap et al., 2017 ; Varshney et al., 2014 ). The use of evenly distributed informative SNPs per chromosome ensured that undesired donor genome segments were minimized, a critical step to avoid linkage drag and maintain agronomic performance of elite germplasm. Similar marker-assisted breeding strategies have proven effective in other maize breeding programs targeting traits such as drought tolerance and disease resistance (Ribaut & Ragot, 2007 ; Semagn et al., 2007 ). Phenotypic evaluations of introgressed lines provided strong evidence of improved MLN resistance. Across both heterotic groups, recurrent parents were highly susceptible, whereas donor parents showed moderate resistance. Introgressed lines consistently demonstrated reduced disease severity compared to recurrent parents, with resistance levels approaching or surpassing those of the donor parents. This improvement was particularly evident in lines derived from CML539 × DTPYC9-F46 and CML312 × CLYN261 , confirming the successful transfer and expression of resistance alleles. The high level of resistance observed in converted lines of CML507, CZL052 , and CML574 in heterotic group B further demonstrates the broad effectiveness of the targeted QTLs across diverse genetic backgrounds. The introgressed lines and their testcrosses not only displayed enhanced MLN resistance but also maintained or improved yield performance under MLN stress. Testcross hybrids derived from converted lines showed yield advantages of 2–4 t/ha over their recurrent parents, underscoring the agronomic value of QTL-based conversion. For example, the widely used line CML312, known for its strong combining ability, produced BC 4 F 3 derivatives with markedly reduced MLN severity under artificial inoculation (Fig. 2 ). Testcrosses of these selections outperformed both donor and recurrent parents for MLN severity and grain yield under MLN pressure (Figs. 3 – 4 ). Similarly, CML539, an early- to medium-maturing line adapted to mid-altitudes, showed a ~ 50% reduction in MLN severity in its introgressed derivatives, with testcrosses surpassing both parents for yield under MLN stress (Fig. 4 ) as well as under optimum conditions (Fig. 5 ). In heterotic group A, CZL052—an elite, multiple-stress tolerant yet MLN-susceptible line—exhibited significant improvement following introgression, with reduced disease severity and nearly two-fold yield gains under severe MLN pressure (Fig. 4 ). Equally important, the absence of yield penalties or undesirable changes in key agronomic traits under optimum conditions suggests that the introgression process preserved the breeding value of the recurrent parents. Grain yield equivalency under disease-free conditions indicates that the incorporation of MLN resistance QTLs did not negatively impact productivity, husk cover, or resistance to other foliar diseases. This observation is critical, as it addresses a common concern of linkage drag associated with resistance gene introgression (Collard & Mackill, 2008 ; Awata et al., 2021 ). The consistent resistance across backgrounds suggests that the introgressed lines harbor favorable alleles (Supplementary Table S1 ) within three QTL-hotspot regions, reinforcing their stability and utility in breeding for durable MLN resistance. This study clearly establishes that the strategic introgression of multiple QTL-hotspots associated with MLN resistance into fourteen diverse elite genetic backgrounds significantly improved resistance levels while maintaining or enhancing grain yield performance. The enhanced resistance was evident not only under severe MLN disease pressure but also without yield penalties under optimum and disease-free conditions. The resulting superior inbred lines—each developed within distinct genetic backgrounds—represent valuable breeding resources. They hold potential both for direct substitution of the original susceptible parental lines or as a new elite line and for use in novel combinations to generate new, high-yielding hybrids with durable MLN resistance and tolerance to multiple stresses. Overall, this research delivers a dual outcome: (i) a set of elite MLN-resistant lines adapted to multiple genetic backgrounds, and (ii) comprehensive performance data validating their potential for immediate deployment in breeding programs aimed at mitigating MLN impacts while safeguarding yield potential. Conclusion The present study demonstrates the successful introgression of MLN resistance QTLs into elite maize inbred lines from both heterotic groups A and B using MABC. The integration of foreground and background selection ensured efficient recovery of the RPG while retaining favorable alleles for MLN resistance. The results not only confirmed the effectiveness of KASP-based markers in tracking resistance loci but also validated the phenotypic performance of the converted lines under artificial MLN inoculation and optimum management conditions. The improved lines developed in this study represent valuable resources for hybrid breeding programs targeting MLN-prone environments. The consistent effectiveness of qMLN3_133 across genetic backgrounds also suggests its utility as a core target in marker-assisted selection pipelines, whereas the incorporation of additional QTLs from chromosomes 3 and 6 can further broaden resistance. The introgressed lines reported here will serve as strong genetic foundations for breeding programs in sub-Saharan Africa and other MLN-affected regions, ultimately contributing to food security and the sustainability of maize production systems. Declarations Conflict of Interest Authors declare that they have no known competing personal or financial interests that could have appeared to influence the results of this study. Funding The research was supported by the Bill and Melinda Gates Foundation (B&MGF), and the United States Agency for International Development (USAID) through the Stress Tolerant Maize for Africa (STMA, B&MGF Grant # OPP1134248) Project, AGGMW (Accelerating Genetic Gains in Maize and Wheat for Improved Livelihoods, B&MGF Investment ID INV-003439) project. Author Contribution Conceptualization, funding acquisition, project & resources administration, YB, MG, PMB, VC, and MO; methodology, investigations, formal analysis and visualization, VO, SLM, VC, MG, YB, MBJ, LH, DM, and MO; supervision, MG, MO, LH, YB; Original draft preparation, VO, and MG; writing - review and editing; VO, VC, MG, PMB, MO, SLM, MBJ, LH and YB. 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09:23:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7815596/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7815596/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95229181,"identity":"fefa7563-55c1-4bd9-bbcb-13a817e5d106","added_by":"auto","created_at":"2025-11-05 16:34:33","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2074803,"visible":true,"origin":"","legend":"","description":"","filename":"OgugoetalMLNMABCMainText.docx","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/4cdcd7c181aae2be80cbd11d.docx"},{"id":95201084,"identity":"11ec8a05-ced5-4837-8dd5-62d84c01dfe7","added_by":"auto","created_at":"2025-11-05 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12:20:58","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139010,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/ad9ab48baff328591dd49f3d.html"},{"id":95201098,"identity":"47ea0a19-8f76-4388-abd5-f23949da12d3","added_by":"auto","created_at":"2025-11-05 12:20:58","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":469959,"visible":true,"origin":"","legend":"\u003cp\u003eMarker-assisted backcrossing (MABC) scheme for introgression of the MLN resistance “QTL-hotspot” genomic regions. (A) Details of the MABC scheme adopted, (B) Expression of MLN disease severity in donor parent (DP) and recurrent parent (RP) lines.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/1550aa3b99cba9954b0a2099.jpeg"},{"id":95201085,"identity":"441be254-aaab-44a0-ad5a-683390f2832c","added_by":"auto","created_at":"2025-11-05 12:20:57","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":402710,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy results on per se performance - Evaluation of donor parent, recurrent parent, and their BC₄F\u003csub\u003e3\u003c/sub\u003e lines under artificially inoculated MLN conditions. LNT1 and LNT2 represent lethal necrosis tolerant line 1 and line 2.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/a26cceaa204fdc1902551a63.jpeg"},{"id":95201099,"identity":"c8a79091-dce2-414e-9bf7-463b4ed79414","added_by":"auto","created_at":"2025-11-05 12:20:58","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":391137,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy results on testcross hybrids performance for MLN disease severity - Evaluation of testcrosses of donor parent, recurrent parent, and their BC₄F\u003csub\u003e3\u003c/sub\u003e lines under artificially inoculated MLN conditions. LNT1 and LNT2 represent lethal necrosis tolerant testcross hybrids 1 and 2.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/c6eecb6ad59156b0800772ca.jpeg"},{"id":95228759,"identity":"d27e39dd-020f-4211-b9c0-536165b4c16d","added_by":"auto","created_at":"2025-11-05 16:34:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":412603,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy results on testcross hybrids performance for grain yield under MLN disease pressure - Evaluation of testcrosses of donor parent, recurrent parent, and their BC₄F\u003csub\u003e2\u003c/sub\u003e lines under artificially inoculated MLN conditions for grain yield. LNT1 and LNT2 represent lethal necrosis tolerant testcross hybrids 1 and 2.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/a5f8ea580f3967d786c38ee8.jpeg"},{"id":95228790,"identity":"96ae848c-f2ac-4573-8ca8-33145064e8cf","added_by":"auto","created_at":"2025-11-05 16:34:08","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":331300,"visible":true,"origin":"","legend":"\u003cp\u003eEquivalency test - Evaluation of testcross hybrids of donor parent, recurrent parent, and their BC₄F\u003csub\u003e3\u003c/sub\u003e lines under optimum conditions for grain yield. LNT1 and LNT2 represent lethal necrosis tolerant testcross hybrids 1 and 2.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/baa067d16838eb46dab17c45.jpeg"},{"id":95230780,"identity":"b37b6f59-04ad-42da-88e1-a933a2f18c83","added_by":"auto","created_at":"2025-11-05 16:38:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2874584,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/365703dc-7ec1-4189-b92d-700de74d64da.pdf"},{"id":95201089,"identity":"e29e7a5a-d71a-4d9d-8679-13ec35956b68","added_by":"auto","created_at":"2025-11-05 12:20:58","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":93891,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesMS.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7815596/v1/a745f8ff8714ad63cdf918f5.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Introgression of QTL Hotspot Regions Enhances Grain Yield and Maize Lethal Necrosis Resistance in Elite Maize Lines","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMaize (\u003cem\u003eZea mays\u003c/em\u003e L.) is the world\u0026rsquo;s second most cultivated cereal and, with rice and wheat, supplies over 40% of global food calories (Shiferaw et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Erenstein et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Demand is expected to rise by 50% by 2050 (Ignaciuk \u0026amp; Mason-D\u0026rsquo;Croz, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), yet production is increasingly constrained by biotic and abiotic stresses. In Sub-Saharan Africa, maize covers over 44\u0026nbsp;million hectares and feeds more than 300\u0026nbsp;million people (Goredema-Matongera et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) but yields average just 2.1 t/ha\u0026mdash;less than half the global mean\u0026mdash;due to drought, degraded soils, low inputs, and pests and diseases (Atlin et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Erenstein et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Cairns et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Viral diseases are particularly devastating, sometimes causing complete crop failure. Major maize viruses in Africa include \u003cem\u003emaize streak virus, maize chlorotic mottle virus\u003c/em\u003e (MCMV), \u003cem\u003esugarcane mosaic virus\u003c/em\u003e (SCMV), \u003cem\u003emaize chlorotic dwarf virus, maize dwarf mosaic virus\u003c/em\u003e, and \u003cem\u003emaize rough dwarf virus\u003c/em\u003e (Abbas et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Thottappilly et al., 1992, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Fajemisin, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong viral diseases in maize, Maize Lethal Necrosis (MLN) poses the greatest threat, capable of causing up to 100% yield loss in smallholder systems. MLN arises from a synergistic co-infection of MCMV and SCMV. First reported in Kenya in 2012 (Wangai et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), the disease has since spread across Eastern Africa, imposing an estimated annual economic burden of \u0026gt;\u003cspan\u003e$\u003c/span\u003e339\u0026nbsp;million on smallholder farmers (Marenya et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). MCMV, a single-stranded RNA virus in the \u003cem\u003eTombusviridae\u003c/em\u003e family, is transmitted primarily by thrips and beetles, but also mechanically, with rare cases of seed and soil transmission (Regassa \u0026amp; Dechassa, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bernardo et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Originally identified in Peru in 1973, MCMV has since been reported in the USA, Latin America, and China (Castillo \u0026amp; Hebert, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Niblett \u0026amp; Claflin, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). SCMV, a \u003cem\u003ePotyviridae\u003c/em\u003e virus, is mainly aphid-transmitted but can also spread mechanically. While infections by either virus alone are usually mild, co-infection results in severe symptoms\u0026mdash;leaf mottling, stunting, premature senescence, necrosis, sterility, and crop death before tasseling (Mahuku et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wangai et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFollowing the MLN outbreak, surveys by International Maize and Wheat Improvement Centre (CIMMYT) and the Kenya Agriculture and Livestock Research Organization (KALRO) revealed that over 90% of commercial and pre-commercial hybrids in the region were highly susceptible to MLN (Gowda et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This triggered urgent breeding efforts to identify resistance sources. Screening of CIMMYT germplasm, commercial cultivars, and national breeding lines revealed only a few donor lines with moderate-to-high resistance to both viruses, which are now being used to introgress MLN resistance into susceptible but agronomically superior backgrounds (Boddupalli et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Biswal et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eGenetic studies revealed that resistance to MLN is governed by few major effects and several minor effect quantitative trait loci (QTL, Gowda et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nyaga et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sitonik et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sadessa et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). A genome-wide association study (GWAS) identified single nucleotide polymorphisms (SNPs) explaining 8\u0026ndash;10% of the phenotypic variance, with combined effects accounting for ~\u0026thinsp;30% (Gowda et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Subsequent biparental mapping validated these associations and revealed major QTL on chromosomes 3, 6, and 9, with effects ranging from 3.9% to 43.8% of phenotypic variation (Gowda et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Awata et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Additional studies confirmed major-effect loci on chromosomes 3 and 6, consistently detected across diverse genetic backgrounds (Murithi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sitonik et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These loci represent prime candidates for introgression of MLN resistance QTL into elite maize lines.\u003c/p\u003e\u003cp\u003eMarker-assisted backcrossing (MABC) provides a powerful tool for such trait introgression. By exploiting molecular markers tightly linked to target QTL, MABC accelerates the transfer of resistance alleles into elite cultivars while minimizing linkage drag (Mekonnen et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The approach enables early and cost-effective selection, reducing breeding cycles and improving precision. MABC has been successfully applied to enhance resistance against several maize diseases\u0026mdash;for example, introgression of \u003cem\u003eqHSR1\u003c/em\u003e for head smut (Zhao et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), \u003cem\u003eZmCCT-H5\u003c/em\u003e for stalk rot, flowering regulation, and yield stability (Li et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Tong et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and pyramiding of major genes for maize rough dwarf disease and gray leaf spot resistance into elite inbreds (Li et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhu et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn East Africa, many commercially successful hybrids derive from parent lines that are highly susceptible to MLN. These hybrids are difficult to replace quickly because they combine high yield potential, drought tolerance, and resistance to multiple foliar diseases. A practical breeding strategy is therefore to introgress MLN resistance QTL into these susceptible parents through MABC. This approach enhances MLN resistance while retaining the superior agronomic performance of popular hybrids, extending their utility and ensuring food security as new MLN-resistant germplasm is developed.\u003c/p\u003e\u003cp\u003eIn this study, we applied MABC to introgress major-effect MLN resistance QTL into elite but MLN-susceptible CIMMYT maize lines. Using ten SNP markers tightly linked to resistance loci on chromosomes 3 and 6, we deployed five MLN-tolerant donors to transfer resistance into 14 recurrent elite lines. The specific objectives were to: (i) introgress major-effect MLN resistance QTL on chromosomes 3 and 6 into drought-tolerant but MLN-susceptible elite lines through MABC; (ii) Assess the level of MLN resistance in the improved lines relative to their recurrent parents, and (iii) Evaluate testcross hybrids derived from the introgressed lines for both agronomic performance and MLN resistance compared to the original parents. Together, these objectives aim to deliver MLN-resistant lines and hybrids without compromising yield or adaptive traits, thereby extending the utility of commercially successful germplasm and contributing to sustainable maize production and food security in Eastern Africa.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eGermplasm used\u003c/h2\u003e\u003cp\u003eBetween 2013 and 2015, CIMMYT evaluated a large panel of inbred lines and identified a subset with tolerance to MLN (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cimmyt.org/news/update-cimmyt-maize-inbred-lines-and-pre-commercial-hybrids-with-potential-resistance-to-maize-lethal-necrosis-mln-2/\u003c/span\u003e\u003cspan address=\"https://www.cimmyt.org/news/update-cimmyt-maize-inbred-lines-and-pre-commercial-hybrids-with-potential-resistance-to-maize-lethal-necrosis-mln-2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). From these, five MLN tolerant inbred lines were selected as trait donors for population development and later in resistance introgression (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two and three donor lines belong to heterotic group A and B, respectively. Donor lines known for wide adaptation to the region and tolerance to MLN, while recipient lines are known for high yield potential, strong combining ability, drought tolerance, low-soil nitrogen stress tolerance and resistance to multiple foliar diseases. This combination provided an ideal foundation for introgressing MLN resistance into agronomically superior but MLN susceptible elite lines, ensuring both disease resistance and retention of desirable agronomic traits in the breeding pipeline.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGenotyping\u003c/h3\u003e\n\u003cp\u003ePlants were tagged and leaf samples were collected after three weeks of planting. From each tagged plant, four 6 mm leaf discs were collected. The samples were dried using silica gel. Ten KASP SNPs linked to three QTL detected in multiple studies for MLN tolerance were used for parental polymorphism analyses between five donor parents and 14 recipient lines, as well as for foreground selection. Genotyping with this set of markers was conducted at LGC, UK (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.lgcgroup.com\u003c/span\u003e\u003cspan address=\"https://www.lgcgroup.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The resulting marker data enabled the selection of plants homozygous for favorable alleles at the targeted MLN resistance QTLs (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For the background selection, leaf discs from donor parent, recipient parent, and BC\u003csub\u003e4\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e plants were sent to Diversity Arrays Technology (DArT), Australia, for genotyping using the Maize DArTag 3.3K EiB (2.0) panel developed by the CGIAR Excellence in Breeding (EiB) platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://excellenceinbreeding.org/toolbox/services/mid-density-genotyping-\u003c/span\u003e\u003cspan address=\"https://excellenceinbreeding.org/toolbox/services/mid-density-genotyping-\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e service). This publicly available panel comprises 3,305 DArTag markers, derived from over 10,000 genetically diverse maize inbred lines.\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\u003eDonor and recipient maize inbred lines used in this study, their heterotic group classification, and SNPs linked to major-effect QTL for MLN resistance on chromosomes 3 and 6, including favorable and unfavorable alleles.\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=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDonor line\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHG\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHaplotype / QTL\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSNP marker\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFavorable allele\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eUnfavorable allele\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eRecipient line\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDTPYC9-F46-1-2-1-2-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eMLN_03.133\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA02299_16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eCML539, LaPostaSeqC7-F64-2-4-1-1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA00363_7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_133048570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eCLYN261\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eMLN_03.133\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA02299_16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"4\" rowspan=\"5\"\u003e\u003cp\u003eCML312, CML540, CML544, DTPWC9-F67-1-2-1-2, LaPostaSeqC7-F64-2-4-1-1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_133048570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eMLN_03.140\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146250249\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146363360\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146602134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003eCML543\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eMLN_03.133\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA02299_16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"8\" rowspan=\"9\"\u003e\u003cp\u003eCML202, CML489, CML546, CML574, CZL052, CLRCY034, CML444\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA00363_7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_133048570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eMLN_03.140\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146250249\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146363360\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146602134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eMLN_06.20\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA03047_12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS6_21007530\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS6_21008211\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eCML574\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eMLN_03.133\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA02299_16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eCML444\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA00363_7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_133048570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eCLRCY034\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eMLN_03.133\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA02299_16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\" morerows=\"5\" rowspan=\"6\"\u003e\u003cp\u003eCML507\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePZA00363_7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_133048570\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cem\u003eMLN_03.140\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146250249\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146363360\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eS3_146602134\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eCC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eHG \u0026ndash; heterotic group\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\n\u003ch3\u003eIntrogression of the QTL-hotspot genomic regions\u003c/h3\u003e\n\u003cp\u003eThe MLN resistance QTL-hotspot region was introgressed independently into elite recipient lines using a MABC approach. In heterotic group A, donor parent \u003cem\u003eCLYN261\u003c/em\u003e was used to introgress MLN resistance into five elite recurrent lines: \u003cem\u003eCML312, CML540, CML544, DTPWC9-F67-1-2-1-2\u003c/em\u003e, and \u003cem\u003eLaPostaSeqC7-F64-2-6-2-2\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, \u003cem\u003eDTPYC9-F46-1-2-1-2-B\u003c/em\u003e served as the donor for \u003cem\u003eCML539\u003c/em\u003e and \u003cem\u003eLaPostaSeqC7-F64-2-6-2-2\u003c/em\u003e. In heterotic group B, \u003cem\u003eCML543\u003c/em\u003e was used as the donor for seven elite recipient lines, while \u003cem\u003eCML574\u003c/em\u003e and \u003cem\u003eCLRCY034\u003c/em\u003e each contributed MLN resistance to one recipient line (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn the backcrossing scheme, the recipient parent was consistently used as the female and the donor parent as the male across all generations. Crosses were conducted at CIMMYT\u0026rsquo;s Kiboko Research Station, Kenya, between 2014 and 2017. To accelerate population advancement, two to three planting cycles per year were implemented. F₁ hybrids were first generated by crossing donor and recipient parents, followed by backcrossing to the recurrent parent to produce BC₁F₁ progenies in 2014. Three additional backcross generations (BC₂F₁, BC₃F₁, and BC₄F₁) were advanced during 2014\u0026ndash;2015. Foreground selection was initiated at the BC₂F₁ generation using SNP markers tightly linked to major MLN resistance QTL. Genotyped plants carrying favorable alleles were tagged, and selected ears were advanced by planting\u0026thinsp;~\u0026thinsp;21 seeds per row, from which 14\u0026ndash;16 plants per row were genotyped.\u003c/p\u003e\u003cp\u003eIn 2016\u0026ndash;2017, BC₄F₁ progenies were selfed to generate BC₄F₂ and subsequently BC₄F₃ families. Foreground selection was applied at both BC₄F₁ and BC₄F₃ stages, while background selection using genome-wide SNP markers was conducted at the BC₄F₃ stage to identify plants with the highest recurrent parent genome recovery and phenotypic similarity to their recurrent parents. From each cross, the two best BC₄F₃ lines were selected and advanced for evaluation of MLN resistance and agronomic equivalence relative to their recurrent parent.\u003c/p\u003e\n\u003ch3\u003eEvaluation of MLN introgressed lines and their testcrosses\u003c/h3\u003e\n\u003cp\u003eTo assess the efficacy of introgressed QTLs and the equivalence of converted lines to their recurrent parents, testcrosses were developed at CIMMYT\u0026rsquo;s Kiboko Research Station. Inbred tester CKDHL120312 (heterotic group B) was crossed with donor lines, recurrent parents, and their BC₄F₃ introgressed derivatives from heterotic group A (\u003cem\u003eCLYN261, DTPYC9-F46-1-2-1-2, CML312, CML539, CML540, CML544, DTPWC9-F67\u003c/em\u003e, and \u003cem\u003eLaPostaSeqC7-F64\u003c/em\u003e). Tester CKDHL120918 (heterotic group A) was used to cross lines from heterotic group B (\u003cem\u003eCML202, CML543, CML444, CML507, CML489, CML546, CML574, CZL052\u003c/em\u003e, and \u003cem\u003eCLRCY034\u003c/em\u003e), along with their introgressed BC₄F₃ versions.\u003c/p\u003e\u003cp\u003eTo evaluate the effect of introgressed QTL on MLN resistance, the converted lines and their test cross hybrids were evaluated for their response to MLN under artificial infestation. The test cross hybrids were also evaluated under natural infestation at an MLN hotspot region. In all these trials, each experimental unit consisted of a two-row plot, 4 meters in length, with 0.75 meters between rows and 0.25 meters between plants within a row. Two seeds were planted per hill and later thinned to one plant per hill at three weeks after emergence, targeting a final population of 53,333 plants per hectare. Fertilizer management included a basal application of di-ammonium phosphate (DAP) at planting, providing 60 kg N and 60 kg P₂O₅ per hectare. Six weeks after emergence, all plots were top-dressed with an additional 60 kg N per hectare. Weed control was maintained through a combination of manual weeding and herbicide application.\u003c/p\u003e\u003cp\u003eAll phenotyping trials for MLN resistance under artificial infestation were conducted at CIMMYT\u0026rsquo;s MLN screening facility in Naivasha, Kenya. For inbred line trial, a total of five donor lines, 14 recurrent parent lines, and 31 selected BC₄F\u003csub\u003e3\u003c/sub\u003e lines (the two best lines per MABC cross) were evaluated to assess disease severity and grain yield. Testcross hybrids generated using the 31 BC₄F\u003csub\u003e3\u003c/sub\u003e lines along with test cross hybrids for donor and recurrent parents were evaluated under artificial MLN inoculation in 2017. Additionally, the same testcrosses were also evaluated for MLN resistance in the natural MLN hotspot region of Babati in Tanzania across two seasons, 2017 and 2018.\u003c/p\u003e\u003cp\u003eIn addition to being tested under artificial and natural MLN disease pressure, the hybrids were also evaluated under optimal conditions across multiple environments to determine both the efficacy of the introgressed QTLs under MLN pressure and the agronomic equivalence of the converted lines to the recurrent parents in the absence of MLN. These environments included Kiboko, Kitale, and Thika in Kenya under optimum management conditions in 2017. All trials conducted with an alpha-lattice experimental design with three replications per genotype.\u003c/p\u003e\n\u003ch3\u003eData Collection and Statistical Analysis\u003c/h3\u003e\n\u003cp\u003eAt each location, data were recorded on a per-plot basis for the following traits: days to 50% anthesis (AD; days from planting until 50% of plants shed pollen), days to 50% silking (SD; days from planting until 50% of plants produced silks), and anthesis\u0026ndash;silking interval (ASI; calculated as SD \u0026ndash; AD). Plant height (PH) was measured as the distance in centimeters from the base of the plant to the insertion of the first tassel branch, while ear height (EH) was measured as the distance from the base to the node bearing the uppermost ear; both were recorded on 10 representative plants per plot. At harvest, field weight (total weight of dehusked ears in kilograms per plot) and grain moisture (MOI; determined using a moisture meter from grains sampled at the center of five representative ears per plot) were recorded. Grain yield (GY) was computed from the field weight using a shelling percentage of 80% and adjusted to 12.5% grain moisture. Analyses of variance (ANOVA) for individual environments (under both MLN pressure and optimum conditions) were conducted using META-R software (Alvarado et al., 2020).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003eMarkers for foreground and background selection\u003c/h2\u003e\u003cp\u003eA total of seven recipient lines from heterotic group A and nine from heterotic group B were selected for the introgression of MLN resistance QTLs (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Foreground selection was carried out using a panel of ten KASP markers. Only the markers that were polymorphic between the donor and recurrent parent pairs were utilized for selection (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Among the ten KASP markers tested, three SNPs\u0026mdash;PZA02299_16, PZA00363_7, and S3_133048570\u0026mdash;were polymorphic between donor line \u003cem\u003eDTPYC9-F46-1-2-1-2\u003c/em\u003e and the recurrent parents \u003cem\u003eCML539\u003c/em\u003e and \u003cem\u003eLaPostaSeqC7-F64-2-6-2-2\u003c/em\u003e. In another set of crosses involving donor line \u003cem\u003eCLYN261\u003c/em\u003e and recipient lines (\u003cem\u003eCML312, CML540, CML544, DTPWC9-F67-1-2-1-2\u003c/em\u003e, and \u003cem\u003eLaPostaSeqC7-F64-2-6-2-2\u003c/em\u003e), five SNPs were identified as polymorphic (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For heterotic group B, nine SNPs were found polymorphic between donor line \u003cem\u003eCML543\u003c/em\u003e and recurrent parents (\u003cem\u003eCML202, CML489, CML546, CML574, CZL052, CLRCY034\u003c/em\u003e, and \u003cem\u003eCML444\u003c/em\u003e). Additionally, three SNPs were polymorphic between donor\u0026ndash;recipient pairs \u003cem\u003eCML574 \u0026times; CML544\u003c/em\u003e and \u003cem\u003eCLRCY034 \u0026times; CML507\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, at least two QTL-hotspot regions showed polymorphism in all donor\u0026ndash;recipient combinations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). For example, in \u003cem\u003eCML202, CML489\u003c/em\u003e, and \u003cem\u003eCML546\u003c/em\u003e, SNPs linked to \u003cem\u003eqMLN3_133\u003c/em\u003e and \u003cem\u003eqMLN6_021\u003c/em\u003e were utilized for foreground selection, while in \u003cem\u003eCML444\u003c/em\u003e and \u003cem\u003eCZL052\u003c/em\u003e, all three QTL-hotspot regions were polymorphic and applied in selection. Notably, markers linked to \u003cem\u003eqMLN3_133\u003c/em\u003e (chromosome 3) were consistently polymorphic across all donor\u0026ndash;recurrent parent combinations, whereas markers from the other two hotspot regions displayed polymorphism only in specific parental pairs.\u003c/p\u003e\u003cp\u003eFor background selection, among the 3305 SNPs tested on the selected donor and recipient parents, 1800 markers were used after quality check, among them, 1160 (CML312/ CLYN261), 942 (CML540/CLYN261), 1157 (CML544/CLYN261; DTPWC9-F67/CLYN261; CML539/DTPYF-46), 942 (LPSC7-F64/CLYN261; LPSC7-F64/DTPYF-46), 1119 (CML202/CML543), 1116 (CML489/CML543), 1141 (CML546/CML543), 1146 (CML574/CML543), 918 (CZL052/CML543), 1142 (CLYCR034/CML543), 1158 (CML444/CML543), and 1160 (CML507/CLYCR034) markers showed informative polymorphisms that were used for background selection (Supplementary Table S2). For the background selection, the number of polymorphic markers distributed per chromosome varied from 43 to 177.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhenotypic Evaluation of Introgression Lines for their efficacy\u003c/h3\u003e\n\u003cp\u003eWith an objective to assess the efficacy of MABC for transferring the \u003cem\u003eQTL-hotspots\u003c/em\u003e, 31 introgressed lines (two introgressed lines from each cross apart from LPSC7-F64 x CLYN261) were evaluated for MLN disease severity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) along with their donor and recurrent parents. Evaluation of MLN resistance showed consistent differences between donor parents, recurrent parents, and the converted lines (LNT1 and LNT2) across both heterotic groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In heterotic group A, recurrent parents exhibited high MLN disease severity scores (6.0\u0026ndash;8.9), whereas donor parents consistently had lower scores (3.9\u0026ndash;4.9). The converted lines (LNT1 and LNT2) demonstrated reduced severity compared to their recurrent parents, with scores ranging between 3.4 and 7.5, indicating a clear improvement in MLN resistance. The highest reduction of MLN disease severity was observed for introgressed lines derived from CML539 x DTPYC9-F46 (5.9 vs 3.4) and CML312 x CLYN261(6.9 vs 4.9). Similar trends were observed in heterotic group B, where recurrent parents recorded high disease severity (7.0\u0026ndash;8.5) relative to donor parents (5.0). The converted lines exhibited lower disease severity (3.3\u0026ndash;6.5), approaching the levels of the donor parents, particularly in CLYCR034 x CML543, CML574 \u0026times; CML543 and CZL052 \u0026times; CML543 combinations. Overall, the introgression of MLN resistance QTLs led to significant reductions in disease severity across both heterotic groups, confirming the effectiveness of the conversion process in improving MLN resistance.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ePhenotypic Evaluation of Testcrosses for their efficacy\u003c/h2\u003e\u003cp\u003eEvaluation of MLN resistance among testcross hybrids exhibited consistent differences between the testcrosses of donor parents, recurrent parents, and the converted lines (LNT1 and LNT2) across both heterotic groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In heterotic group A, the testcrosses of recurrent parents exhibited moderate MLN disease severity scores, whereas donor parents consistently had lower scores (2.33\u0026ndash;3.33). The testcross of converted lines for each introgressed inbreds (LNT1 and LNT2) demonstrated reduced severity scores compared to recurrent parents, with scores ranging between 2.0 and 4.0, indicating a clear improvement in MLN resistance. The highest reduction of MLN disease severity was observed for testcrosses of introgressed lines derived from CML312. The efficacy of transferring MLN resistance associated \u003cem\u003eQTL-hotspot\u003c/em\u003e in a cross between CLYN261 x CML312, revealed 50% reduction in MLN disease severity in introgressed lines compared to the recurrent parent CML312 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and increase in grain yield up to 2.25 tons/ha under MLN disease pressure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Overall, the testcross hybrids revealed considerable reduction in MLN disease severity in introgressed lines compared to their original recurrent parents, except for CLYN261 x DTPWC9-F67 cross in heterotic group A (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSimilar trends were observed in heterotic group B, where testcrosses of recurrent parents recorded moderately high severity compared to test crosses of donor parents (3.00-3.67). The testcrosses of converted lines exhibited lower disease severity (2.3\u0026ndash;4.0). Higher levels of MLN score reduction were recorded in testcrosses of converted versions of CML507, CZL052 and CML489. Overall, the introgression of MLN resistance QTLs led to significant reductions in disease severity across both heterotic groups, confirming the effectiveness of the conversion process in improving MLN resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGrain yield under MLN stress varied significantly among testcross hybrids of donor parents, recurrent parents, and converted lines (LNT1 and LNT2) across both heterotic groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In heterotic group A, testcrosses of recurrent parents consistently showed low yields under MLN (1.85\u0026ndash;4.80 t/ha), while test crosses of donor parents recorded higher yields (4.20\u0026ndash;5.95 t/ha). Testcrosses of Converted lines (LNT1 and LNT2) showed substantial yield advantages over recurrent parents, with hybrids yielding between 3.70 to 7.85 t/ha. Particularly testcross hybrids from converted lines of CML312 (LNT1, 7.85 t/ha) and CML539 (LNT1, 7.05 t/ha) exhibited the highest yields, clearly outperforming both donor and recurrent parents. Testcross hybrids of converted lines of CML544 (5.05 vs 1.85 t/ha), DTPWC9-F67 (6.7 vs. 3.7t/ha) showed the highest yield gains under MLN stress compared to testcross hybrids of their recurrent parents\u003c/p\u003e\u003cp\u003eSimilar trends were observed in heterotic group B, where recurrent parents yielded poorly (1.23\u0026ndash;3.65 t/ha) compared to donor parents (4.80\u0026ndash;5.65 t/ha) under MLN stress. The converted lines (LNT1 and LNT2) consistently outperformed recurrent parents, with yields ranging from 4.15 to 6.60 t/ha. Testcross hybrids of converted lines of CML574 (LNT2, 6.15 t/ha) and CML507 (LNT1, 6.60 t/ha) achieved the highest yields under MLN stress. Particularly, testcross hybrids of converted lines of CML543 (4.00 Vs 1.23 t/ha), CML546 (5 vs.1.65t/ha), CZL052 (4.60 vs1.40 t/ha) and CML507 (5.65 vs 1.95 t/ha) achieved the highest yields under MLN stress compared to test cross hybrids of their recurrent parents, indicating effective recovery of productivity through the introgression of MLN resistance. Overall, the converted lines significantly enhanced hybrid performance under MLN pressure in both heterotic groups, approaching or surpassing the donor parent yields.\u003c/p\u003e\u003cp\u003eAnalyses of variance on grain yield and other agronomic traits under MLN disease (Supplementary Table S3, S4 and S5) and under optimum management (Supplementary Table S6, S7 and S8) revealed significant variation and moderate to high heritability estimates for each location. Comparison of introgressed lines against their original recipient parents revealed no significant changes for agronomic traits like AD, SD, PH, EH, ER, husk cover and for foliar diseases like GLS, common rust and TLB under optimum management.\u003c/p\u003e\u003cp\u003eGrain yield equivalency under optimum conditions in the absence of MLN disease pressure showed minimal differences among the testcrosses of donor parents, recurrent parents, and converted lines (LNT1 and LNT2) across both heterotic groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In heterotic group A, average yields of testcrosses of recurrent parents ranged between 4.05 and 5.25 t/ha, while donor parents yielded 4.65\u0026ndash;4.75 t/ha. The testcrosses of converted lines performed comparably, with yields of 4.45\u0026ndash;6.10 t/ha. LPSC7-F64 (LNT2, 6.10 t/ha) and DTPWC9-F67 (LNT2, 5.90 t/ha) and CML539 showed slightly higher yields than both donor and recurrent parents. For CML312 and CML540, the test crosses of converted lines showed higher yield compared to the recurrent parent but less than the donor parent. For CML544, the yield of testcrosses of converted lines showed less yield than the recurrent parent but same/slightly higher yield than the donor parent.\u003c/p\u003e\u003cp\u003eSimilarly, in heterotic group B, test cross hybrids of donor parents yielded 4.90 to 5.65 t/ha, while recurrent parents ranged between 4.20 and 5.20 t/ha. Testcross hybrids of converted lines matched these levels yielding 4.00\u0026ndash;5.80 t/ha. Notably, testcross hybrids of converted lines of CML546 (LNT1, 5.80 t/ha) and CLYCR034 (LNT1, 5.65 t/ha) maintained yields equivalent to donor parents, while outperforming recurrent parents. For rest of the lines, testcross hybrids even though did not perform better than the donor parents, the conversions did slightly better or similar to the recurrent parent. These results demonstrate that the introgression of MLN resistance QTLs into elite lines did not negatively affect hybrid yield potential under optimum conditions, thereby confirming their equivalency.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eClimate change has intensified biotic and abiotic stresses across sub-Saharan Africa, making the development of maize hybrids with multiple stress tolerance a breeding priority (Prasanna et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). MLN disease, first reported in Kenya in 2011 (Wangai et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), has caused devastating yield losses, restricted seed movement due to transmission through seed and insect vectors, and exposed the vulnerability of many otherwise stress-tolerant and agronomically superior maize hybrids. To address this challenge, conventional breeding alone is inadequate, and the integration of modern tools such as genomics, high-throughput phenotyping, and systems modeling is essential (Prasanna et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). MABC offers a strategic, quick response by introgressing MLN resistance alleles into elite, farmer-preferred hybrids backgrounds, enabling rapid genetic gain while preserving key agronomic traits. The resulting lines provide a resilient foundation for hybrid development, ensuring food security and strengthening the maize supply chain in eastern and southern Africa.\u003c/p\u003e\u003cp\u003eMABC is particularly effective for improving disease resistance, as phenotypic selection for resistance can be unreliable due to viral mutations and strong year-to-year environmental variation. Unlike phenotypic approaches, molecular markers are environment-independent, allowing precise and consistent introgression of resistance loci into elite germplasm (Li \u003cem\u003eet al.\u003c/em\u003e, 2018; Zhang et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). MLN resistance is largely quantitative, controlled by a few major-effect and several minor-effect QTL (Gowda et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nyaga \u003cem\u003eet al\u003c/em\u003e., 2020; Awata et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Murithi et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), making it especially amenable to MABC. The present study validated seven MLN resistance linked SNPs on chromosome 3 (\u003cem\u003ePZA02299_16, PZA00363_7, S3_133048570, PZD00015_5, S3_146250249, S3_146363360\u003c/em\u003e, and \u003cem\u003eS3_146602134\u003c/em\u003e) and three SNPs on chromosome 6 (\u003cem\u003ePZA03047_12, S6_21007530\u003c/em\u003e, and \u003cem\u003eS6_21008211\u003c/em\u003e), all located within \u003cem\u003eQTL-hotspot\u003c/em\u003e regions previously linked to MLN disease resistance in maize (Gowda et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nyaga \u003cem\u003eet al\u003c/em\u003e., 2020; Awata et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lohithaswa et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The strong alignment between phenotypic resistance and the presence of favorable alleles at these loci underscores their value in breeding pipelines. Indeed, 31 BC₄F\u003csub\u003e3\u003c/sub\u003e introgressed lines carrying these favorable alleles exhibited MLN resistance, providing direct evidence for the functional relevance of these hotspots. Similar successes have been documented in other cereals, such as the introgression of \u003cem\u003ePi-b\u003c/em\u003e and \u003cem\u003ePi-kh\u003c/em\u003e blast resistance genes into rice via MABC (Tanweer et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Nevertheless, the expression of QTL can be affected by epistatic interactions or linkage drag in new genetic backgrounds (Hospital, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Collard and Mackill, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, the development of tightly linked or functional markers for MLN resistance remains a priority to enhance selection accuracy and reduce the number of individuals required in each backcross generation.\u003c/p\u003e\u003cp\u003eThe consistent polymorphism observed for SNP markers linked to \u003cem\u003eqMLN3_133\u003c/em\u003e across all donors\u0026ndash;recipient parent combinations highlight the robustness of this \u003cem\u003eQTL hotspot\u003c/em\u003e and its potential utility as a stable source of resistance. This finding is in line with earlier studies reporting chromosome 3 regions as critical for MLN resistance (Gowda et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nyaga \u003cem\u003eet al\u003c/em\u003e., 2020; Awata et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The partial polymorphism observed for the other two QTL hotspots suggests that their utility may depend on the genetic background of the parents, further emphasizing the importance of multiple resistance loci to broaden the genetic base of MLN resistance.\u003c/p\u003e\u003cp\u003eResistance QTL can sometimes carry negative effects on yield or plant growth (Chitwood-Brown et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Klindworth et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and maintaining heterosis after line conversion requires a high level of genetic background recovery. In our study, although introgressed chromosomal regions were relatively large, no yield penalties were observed in the converted lines and their testcross hybrids\u0026mdash;because MLN resistance genes reside within the target QTL regions (Nyaga et al., 2020; Wen et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Murithi et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Based on marker polymorphism, 788\u0026ndash;1160 SNPs were used to estimate recurrent parent genome (RPG) recovery. The RPG recovery in the introgressed lines ranged from 60% in CML489 to 98% in CML540, with other genetic backgrounds showing recovery rates between 83% and 90% (Supplementary Table S2). These results are consistent with findings in other crops, where, for example, 91.6% RPG recovery was achieved in rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.) while pyramiding blast resistance genes into elite Basmati (Singh et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and \u0026gt;\u0026thinsp;90% recovery was reported in chickpea after just three backcross generations (Pratap et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Varshney et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The use of evenly distributed informative SNPs per chromosome ensured that undesired donor genome segments were minimized, a critical step to avoid linkage drag and maintain agronomic performance of elite germplasm. Similar marker-assisted breeding strategies have proven effective in other maize breeding programs targeting traits such as drought tolerance and disease resistance (Ribaut \u0026amp; Ragot, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Semagn et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePhenotypic evaluations of introgressed lines provided strong evidence of improved MLN resistance. Across both heterotic groups, recurrent parents were highly susceptible, whereas donor parents showed moderate resistance. Introgressed lines consistently demonstrated reduced disease severity compared to recurrent parents, with resistance levels approaching or surpassing those of the donor parents. This improvement was particularly evident in lines derived from \u003cem\u003eCML539 \u0026times; DTPYC9-F46\u003c/em\u003e and \u003cem\u003eCML312 \u0026times; CLYN261\u003c/em\u003e, confirming the successful transfer and expression of resistance alleles. The high level of resistance observed in converted lines of \u003cem\u003eCML507, CZL052\u003c/em\u003e, and \u003cem\u003eCML574\u003c/em\u003e in heterotic group B further demonstrates the broad effectiveness of the targeted QTLs across diverse genetic backgrounds.\u003c/p\u003e\u003cp\u003eThe introgressed lines and their testcrosses not only displayed enhanced MLN resistance but also maintained or improved yield performance under MLN stress. Testcross hybrids derived from converted lines showed yield advantages of 2\u0026ndash;4 t/ha over their recurrent parents, underscoring the agronomic value of QTL-based conversion. For example, the widely used line CML312, known for its strong combining ability, produced BC\u003csub\u003e4\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e derivatives with markedly reduced MLN severity under artificial inoculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Testcrosses of these selections outperformed both donor and recurrent parents for MLN severity and grain yield under MLN pressure (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, CML539, an early- to medium-maturing line adapted to mid-altitudes, showed a\u0026thinsp;~\u0026thinsp;50% reduction in MLN severity in its introgressed derivatives, with testcrosses surpassing both parents for yield under MLN stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) as well as under optimum conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In heterotic group A, CZL052\u0026mdash;an elite, multiple-stress tolerant yet MLN-susceptible line\u0026mdash;exhibited significant improvement following introgression, with reduced disease severity and nearly two-fold yield gains under severe MLN pressure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Equally important, the absence of yield penalties or undesirable changes in key agronomic traits under optimum conditions suggests that the introgression process preserved the breeding value of the recurrent parents. Grain yield equivalency under disease-free conditions indicates that the incorporation of MLN resistance QTLs did not negatively impact productivity, husk cover, or resistance to other foliar diseases. This observation is critical, as it addresses a common concern of linkage drag associated with resistance gene introgression (Collard \u0026amp; Mackill, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Awata et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The consistent resistance across backgrounds suggests that the introgressed lines harbor favorable alleles (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) within three \u003cem\u003eQTL-hotspot\u003c/em\u003e regions, reinforcing their stability and utility in breeding for durable MLN resistance.\u003c/p\u003e\u003cp\u003eThis study clearly establishes that the strategic introgression of multiple \u003cem\u003eQTL-hotspots\u003c/em\u003e associated with MLN resistance into fourteen diverse elite genetic backgrounds significantly improved resistance levels while maintaining or enhancing grain yield performance. The enhanced resistance was evident not only under severe MLN disease pressure but also without yield penalties under optimum and disease-free conditions. The resulting superior inbred lines\u0026mdash;each developed within distinct genetic backgrounds\u0026mdash;represent valuable breeding resources. They hold potential both for direct substitution of the original susceptible parental lines or as a new elite line and for use in novel combinations to generate new, high-yielding hybrids with durable MLN resistance and tolerance to multiple stresses. Overall, this research delivers a dual outcome: (i) a set of elite MLN-resistant lines adapted to multiple genetic backgrounds, and (ii) comprehensive performance data validating their potential for immediate deployment in breeding programs aimed at mitigating MLN impacts while safeguarding yield potential.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study demonstrates the successful introgression of MLN resistance QTLs into elite maize inbred lines from both heterotic groups A and B using MABC. The integration of foreground and background selection ensured efficient recovery of the RPG while retaining favorable alleles for MLN resistance. The results not only confirmed the effectiveness of KASP-based markers in tracking resistance loci but also validated the phenotypic performance of the converted lines under artificial MLN inoculation and optimum management conditions. The improved lines developed in this study represent valuable resources for hybrid breeding programs targeting MLN-prone environments. The consistent effectiveness of \u003cem\u003eqMLN3_133\u003c/em\u003e across genetic backgrounds also suggests its utility as a core target in marker-assisted selection pipelines, whereas the incorporation of additional QTLs from chromosomes 3 and 6 can further broaden resistance. The introgressed lines reported here will serve as strong genetic foundations for breeding programs in sub-Saharan Africa and other MLN-affected regions, ultimately contributing to food security and the sustainability of maize production systems.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e\u003cp\u003eAuthors declare that they have no known competing personal or financial interests that could have appeared to influence the results of this study.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe research was supported by the Bill and Melinda Gates Foundation (B\u0026amp;MGF), and the United States Agency for International Development (USAID) through the Stress Tolerant Maize for Africa (STMA, B\u0026amp;MGF Grant # OPP1134248) Project, AGGMW (Accelerating Genetic Gains in Maize and Wheat for Improved Livelihoods, B\u0026amp;MGF Investment ID INV-003439) project.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, funding acquisition, project \u0026amp; resources administration, YB, MG, PMB, VC, and MO; methodology, investigations, formal analysis and visualization, VO, SLM, VC, MG, YB, MBJ, LH, DM, and MO; supervision, MG, MO, LH, YB; Original draft preparation, VO, and MG; writing - review and editing; VO, VC, MG, PMB, MO, SLM, MBJ, LH and YB.\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThe authors are grateful to the International Maize and Wheat Improvement Center (CIMMYT) scientists and technicians who generated the germplasm, and highly appreciate the technical support received from the staff members affiliated to CIMMYT maize research station in Naivasha and Kiboko, Kenya.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll the generated data in this study is included in the supplementary Tables.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbbas, M. 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Marker-assisted introgression of qHSR1 to improve maize resistance to head smut. \u003cem\u003eMolecular Breeding\u003c/em\u003e, \u003cem\u003e30\u003c/em\u003e(2), 1077\u0026ndash;1088.\u003c/li\u003e\n\u003cli\u003eZhu, M., Song, H., Xu, J., Jiang, X., Zhang, Y., Ma, J., Jiang, M., Li, Y., Xie, Z., \u0026amp; Liu, T. (2025). Introgression of ZmCPK39 in maize hybrids enhances resistance to gray leaf spot disease without compromising yield. \u003cem\u003eMolecular Breeding\u003c/em\u003e, \u003cem\u003e45\u003c/em\u003e(3), 28.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"maize, MLN resistance, disease severity, grain yield, MABC, QTL","lastPublishedDoi":"10.21203/rs.3.rs-7815596/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7815596/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMaize lethal necrosis (MLN) poses a severe threat to maize production in eastern and southern Africa, causing significant yield losses. In this study, marker-assisted backcrossing (MABC) was used to introgress major-effect MLN resistance Quantitative Trait Loci (QTL) located on chromosomes 3 and 6 into 14 elite but MLN-susceptible CIMMYT maize lines belonging to heterotic groups A and B. Ten Kompetitive Alelle Specific PCR (KASP) SNP markers closely linked to three validated \u003cem\u003eQTL-hotspot\u003c/em\u003e regions were applied for foreground selection, with at least two hotspots polymorphic across all donor\u0026ndash;recipient combinations. Foreground and background selection enabled fast tracking of MLN resistance alleles and recovery of near-recurrent parent genomes. The resulting BC₄F₂ introgressed lines exhibited markedly reduced MLN severity under artificial inoculation, with several lines showing a 50% reduction relative to their recurrent parents. Testcrosses of these lines demonstrated yield advantages of 2\u0026ndash;4 t/ha under MLN pressure compared with original parental lines, while maintaining comparable performance under optimum conditions. Notably, introgressed derivatives of CML312, CML539, and CZL052 displayed both enhanced MLN resistance and superior yield performance, with CZL052-derived testcrosses achieving nearly two-fold yield gains under severe MLN stress. Importantly, equivalence trials confirmed that MLN resistance was improved without compromising resistance to gray leaf spot, turcicum leaf blight, or common rust. These findings validate the effectiveness of QTL-based conversion for enhancing MLN resistance in elite breeding lines and demonstrate the potential of these improved lines as robust parental sources for developing MLN-resilient hybrids adapted to eastern and southern Africa.\u003c/p\u003e","manuscriptTitle":"Introgression of QTL Hotspot Regions Enhances Grain Yield and Maize Lethal Necrosis Resistance in Elite Maize Lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-05 12:20:53","doi":"10.21203/rs.3.rs-7815596/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-19T10:33:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-18T01:34:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-10T13:50:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162672755849647807285088762577941588539","date":"2025-11-01T14:40:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"161325232634220399417304262943178923670","date":"2025-11-01T07:31:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"217430831950219088985910464374401971735","date":"2025-10-28T04:28:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"177188877223861369627835061780062987659","date":"2025-10-28T04:14:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185114398851715858110464236231156233492","date":"2025-10-27T13:47:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159553686223228103487743239865845241509","date":"2025-10-27T08:07:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-27T07:07:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-15T12:06:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-10T14:56:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-10T14:54:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-09T09:12:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2ebf148c-f11b-4a9e-b803-02d52c3270d9","owner":[],"postedDate":"November 5th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57274825,"name":"Biological sciences/Genetics"},{"id":57274826,"name":"Biological sciences/Molecular biology"},{"id":57274827,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-05-13T16:08:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-05 12:20:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7815596","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7815596","identity":"rs-7815596","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}