Effects of donor and inducer genotype and endosperm texture on haploid induction in maize assessed using R1-Navajo

Effects of donor and inducer genotype and endosperm texture on haploid induction in maize assessed using R1-Navajo | 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 Research Article Effects of donor and inducer genotype and endosperm texture on haploid induction in maize assessed using R1-Navajo Mariana Martins Marcondes, Marcos Ventura Faria, Carlos Alberto Scapim, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6978380/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Apr, 2026 Read the published version in Euphytica → Version 1 posted 9 You are reading this latest preprint version Abstract The double haploid (DH) technique is extensively utilized in maize breeding programs to accelerate the development of homozygous lines that allows the fixation of the recombinant haploid genome in a single generation. This process relies on in vivo haploid induction, in which the production of haploid seeds represents a critical step in this technique that determines the efficiency of DH line production. This study aimed to evaluate the effects of endosperm texture, donor genotype, and inducer on haploid induction rates based on the R1-navajo ( R1-nj ) marker. Eleven hybrids were assessed as donor genotypes and were pollinated with ten haploid inducers. The response was the number of putative haploid seeds based on R1-nj phenotype, evaluated across the total number of seeds, with replications obtained from individual ears. Generalized Linear Models (GLMs) with binomial distribution were fitted for the putative haploid rate (PHR). A total of 240,285 seeds were evaluated across 1,394 ears, with an observed average PHR of 6.35% in the experiment, ranging from 0.05 to 18.63% across the donors × inducers combinations. For most of the inducers evaluated, hybrids with a semi-dent endosperm texture exhibited higher PHR, whereas hybrids with flint endosperm exhibited lower rates. The three-way cross inducer (UH400 × UH401) × TAIL 9 achieved the highest PHR with five donor hybrids. These results highlight that specific combinations of donor genotypes and haploid inducers should be chosen to achieve a higher number of haploid seeds. Zea mays L. Navajo phenotype gymnogenetic haploids endosperm texture anthocyanin Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION In maize ( Zea mays L.) breeding programs focused on the production of double haploid (DH) lines, the in vivo method for obtaining gymnogenetic haploids involves four key steps: (1) haploid induction, achieved by the crossing of a gymnogenetic haploid inducer pollen donor with the donor genotype, which produces maternal haploid seeds; (2) identification and separation of haploid seeds; (3) chromosome doubling of the haploid plants using antimitotic agents; and (4) self-pollination of the doubled plants to produce double haploid lines. Compared to the conventional method that involves successive generations of self-pollination, the double haploid (DH) technique enables the development of homozygous inbred lines in a single generation, reducing the time and resources required for maize breeding (Chaikam et al. 2019 ). The haploid induction rate (HIR) is determined by the ratio of haploid seeds to the total number of seeds produced. Higher HIR values are essential for the efficiency of DH-based breeding programs and allow the production of doubled haploid lines in a large scale (Silva et al. 2023 ). The HIR characteristic is first influenced by the haploid inducer genotype. In the early use of DH in maize breeding, the Stock 6 line was widely used as inducer, with an average production rate of 2–3% haploid seeds. The later breeding for haploid induction resulted in HIR ranging from 8 to 15% in haploid inducer lines such as RWS, UH400, and TAIL (Coe 1959 , Prasanna 2012 ). In addition, the HIR is significantly influenced by the donor’s genetic background (Silva et al. 2020 ). De La Fuente et al. ( 2018 ) observed putative haploid rates ranging from 2.6–30.5% across 30 hybrids pollinated with the same inducer RWS/RWK-76. Among other potential factors, anthocyanin inhibitor alleles ( C1-I , C2-Idf , and in1-D , which act epistatically on R1-nj ) present in the donor can prevent the formation of aleurone layer pigmentation. Generally flint maize harbors anthocyanin inhibitor genes at a higher frequency than dent maize, making the identification of haploid seeds more difficult in flint or flint × dent hybrids (Eder and Chalik 2002, Chen et al. 2024 ). Despite the known occurrence of donor × inducer interactions, few studies have evaluated their effect under field conditions in tropical germplasm. Given the complex interaction that inducers and donor germplasm have on the haploid induction rate, studies are needed to determine the dependence between these factors, especially for tropical conditions (Ren et al. 2022 , Trentin et al. 2023 ). Identifying significant interactions could enable the use of specific inducer lines for the donor genotypes, thus reducing the costs associated with haploid selection. Therefore, this study aimed to evaluate the effects of endosperm textures of donors, donor and inducer genotypes on haploid induction rates, based on the R1-navajo phenotype in maize seeds. MATERIAL AND METHODS Genetic material Seeds were obtained from the pollination of eleven maize hybrids, used as donor genotypes (female parents), with ten haploid inducer maize genotypes (male parents). The donor genotypes comprised ten single hybrids and one three-way hybrid, all widely adopted in commercial maize cultivation in Brazil. These hybrids exhibited semi-dent, semi-flint and flint endosperm textures, as detailed in Table 1 . Table 1 Hybrids used as donor genotypes in the evaluation of putative haploid rate (PHR) in maize Hybrid Company Type Texture 30A68 HX Morgan SH Semi-flint AG 8780 PRO3 Bayer SH Semi-dent AS 1555 RR Bayer SH Semi-flint AS 1677 PRO3 Bayer SH Semi-flint BG 7060 Corteva TH Semi-flint Fórmula Syngenta SH Flint NS 50 PRO Syngenta SH Semi-flint NS 92 PRO Syngenta SH Semi-flint P1630 HX Corteva SH Semi-dent P4285 YHR Corteva SH Flint SYN 7205 Syngenta SH Semi-flint SH: single cross hybrid; TH: three-way cross hybrid. The evaluated haploid inducers are detailed in Table 2 . They included: a single cross hybrid derived from the temperate lines UH400 and UH401, developed by the University of Hohenheim, Germany, from the KEMS (Krasnador Embryo Marker Synthetic) inducer (Liu et al. 2016 ); tropical inbred lines adapted TAILs 7, 8, and 9 (Tropically Adapted Inducer Lines), developed by CIMMYT (International Maize and Wheat Improvement Center), Mexico, in collaboration with the University of Hohenheim, through backcrossing of the UH400 and RWS (Russian Inducer KEMS + WS14) inducers with tropical maize lines (Prasanna 2012 , Liu et al. 2016 ); inducer inbred lines developed by Nidera Seeds, originating from TAILs 1, 2, and 3; and simple and three-way cross hybrids obtained from the UH400, UH401, TAILs 7, 8, and 9 inducers. Table 2 Gymnogenetic haploid inducers used in the evaluation of putative haploid rate (PHR) in maize. Inducer Type Origin TAIL 7 IL Tropical TAIL 8 IL Tropical TAIL 9 IL Tropical TAIL 7 × TAIL 8 SH Tropical UH400 × UH401 SH Temperate (UH400 × UH401) × TAIL 7 TH Temperate/Tropical (UH400 × UH401) × TAIL 9 TH Temperate/Tropical Nidera Orig TAIL 1 IL Temperate/Tropical Nidera Orig TAIL 2 IL Temperate/Tropical Nidera Orig TAIL 3 IL Tropical IL: inbred line; SH: single cross hybrid; TH: three-way cross hybrid. Haploidy-inducing crosses and phenotypic classification The pollination field was cultivated during the 2016/2017 growing season in Abelardo Luz, Santa Catarina, Brazil. The donor and inducer genotypes were arranged in 6 m rows per combination (donors × inducers), with inter-row and intra-row spacings of 0.90 m and 0.20 m, respectively. Haploid inducers were planted in an adjacent field. To ensure synchronizing flowering periods, inducer genotypes were sown in five sequential planting dates, spaced one week apart; the first and last sowings occurred one week before and 21 days after the donor genotypes, respectively. The ears of the donor genotypes were protected prior to silk emergence. During the flowering period, pollen from each inducer was bulk-collected using paper bags, and pollinations were performed manually. The pollinated ears were harvested separately and the seeds produced were classified based on the presence of purple coloration from the expression of the R1-nj marker gene, as described by Nanda and Chase ( 1966 ). Therefore, seeds with purple endosperm and white embryos were considered putative haploids. Putative haploids classified using R1-nj are related to the true haploid induction rate and can serve as a basis for evaluating the efficiency of different inducers (Couto et al. 2015 ). Statistical analysis The response variable was the number of putative haploids ( \(\:{y}_{ijkl}\) ) out of the total number of seeds ( \(\:{\text{n}}_{\text{i}\text{j}\text{k}\text{l}}\) ) obtained from individual ears for each donor × inducer combination. Thus, \(\:{y}_{ijkl}\) follows a binomial distribution with the probability of success \(\:{\pi\:}_{ijkl}\) (Putative Haploid Rate - PHR) [ \(\:{y}_{ijkl}\:\sim\:\:Binomial({\pi\:}_{ijkl},\:{n}_{ijkl})\) ], using a logit link function, as in the Eq. (1): $$\:logit\left({\pi\:}_{ijkl}\right)\:=\:{\eta\:}_{ijkl}=\mu\:+{\alpha\:}_{i}+\:{\beta\:}_{j}+{\gamma\:}_{k\left(j\right)}+{\tau\:}_{i\left(j\right)}+{\omega\:}_{i\left(jk\right)}\left(1\right)$$ where \(\:{\eta\:}_{ijkl}\) is the linear predictor for \(\:l\) -th ear from k -th donor genotype within j -th endosperm texture and i -th inducer genotype, \(\:\mu\:\) is the constant inherent in the data, \(\:{\alpha\:}_{i}\) represents the effect of the i -th inducer, \(\:{\beta\:}_{j}\) denotes the effect of the j -th endosperm texture, \(\:{\gamma\:}_{k\left(j\right)}\) is the effect of the k -th donor genotype within the j -th endosperm texture, \(\:{\tau\:}_{i\left(j\right)}\) is the effect of the i -th inducer genotype within the j -th endosperm texture, and \(\:{\omega\:}_{i\left(jk\right)}\) is the effect of the i -th inducer genotype within the j -th endosperm texture and k -th donor genotype. The model was fitted using the ‘ glm’ function from the ‘ stats ’ package (R Core Team version 4.3.1). The model that provided a better fit to the data was subjected to residual analysis using diagnostic plots with simulated envelopes, employing the ‘ hnp ’ package (Moral et al. 2017 ). Overdispersion was detected, as the amount of variation in the data exceeded that expected by the model. Consequently, the estimated standard errors and confidence intervals were corrected using the quasibinomial model, where the variance is given by \(\:\varphi\:{\pi\:}_{ijkl}(1\:-\:{\pi\:}_{ijkl})\) , with \(\:\varphi\:\) as the dispersion parameter (Demétrio et al. 2014 ). Sequencial deviance analysis was performed, where factors of the model were assessed using the F statistic. The pairwise contrasts of the estimated means within each level of the factors were estimated, and inference was made using the Wald test. The p -values were adjusted using the Bonferroni correction to maintain the global error rate at \(\:{\alpha\:}=0.05\) . The means on the linear predictor scale were then converted to probabilities using the inverse of the link function, as in Eq. (2). $$\:{g}^{-1}\left({\pi\:}_{ijkl}\right)=\frac{{e}^{{\eta\:}_{ijkl}}}{1+{e}^{{\eta\:}_{ijkl}}}\:\left(2\right)$$ Asymptotic confidence intervals for the estimated contrasts were obtained using the ‘ emmeans ’ package in the R environment (R Core Team version 4.3.1). RESULTS A total of 240,285 seeds were evaluated across 1,394 ears. The number of ears per donor × inducer combination ranged from 0 to 30 (Supplementary Table 1), with their distribution represented in the histogram in the Fig. 1 . Eighth crosses did not produce any seeds, so their means and contrasts involving these means could not be estimated. These crosses were: BG 7060 × TAIL 7, P4285 YHR × [TAIL 9, UH400 × UH401, and (UH400 × UH401) × TAIL 9, Nidera Orig TAIL 1], and SYN 7205 × [TAIL 7, TAIL 7 × TAIL 8 and Nidera Orig TAIL 1]. A total of 15,254 putative haploid seeds were obtained, with an observed average PHR of 6.35% in the experiment, ranging from 0.05–18.63% across the donors × inducers combinations. The half-normal plots in Fig. 2 provide strong evidence in favor of the model that accounts for overdispersion, with most residuals falling within the simulated envelope. The estimated dispersion parameter for the quasibinomial overdispersion model was 2.67. The sequential deviance analysis for PHR revealed significant effects for all evaluated factors, as well as their nested effects ( p < 0.001) (Supplementary Table 2a). The interaction plot between donor endosperm textures and inducers is presented in Fig. 3 . For the semi-dent kernel texture, the inducer (UH400 × UH401) × TAIL 9 showed the highest estimated means of PHR (17.29%), differing from five lower-performing inducers (Supplementary Table 2b). For the semi-flint and flint textures, the inducers UH400 × UH401 and TAIL 7 × TAIL 8 exhibited the highest PHR (6.91% and 3.13%), respectively, differing from three and one lower-performing inducers in their respective textures. The semi-dent texture genotypes provided higher PHR in crosses with nine of the ten inducers evaluated, indicating greater inducibility of this group of donor hybrids. In all cases where contrasts between textures could be estimated, the flint endosperm texture showed a lower average PHR compared to the higher groups (Supplementary Table 2b). The interaction plot for donor genotypes and inducers is also presented in Fig. 4 . The inducer (UH400 × UH401) × TAIL 9 exhibited the highest estimated mean when used as a haploid inducer in combination with five donor hybrids, being statistically superior to the lower-performing inducers (Supplementary Table 2c). For the donor hybrid NS 50 PRO, the single hybrid inducer TAIL 7 × TAIL 8 demonstrated the highest haploid induction capacity (7.98%), outperforming the three inducers with lower haploid induction ability, including the three-way cross hybrid (UH400 × UH401) × TAIL 9 (2.80%), which was superior in other crosses. For the hybrids AS 1677 PRO3 and SYN 7205, the inducer UH400 × UH401 exhibited the highest PHR. The hybrids NS 92 PRO and P4285 YHR did not demonstrate a significant difference between the inducers. DISCUSSION Normal plots based on simulated envelopes, using deviance residuals, were employed to compare the goodness-of-fit between the binomial and quasibinomial models. The quasibinomial model, which accounts for overdispersion, provided a better fit and is more appropriate for estimating of standard errors for the estimated means. The observed overdispersion may result from heterogeneity among observations that was not explained by the model covariates (Demétrio et al. 2014 ). When the overdispersion is ignored, the standard errors of the estimated means are underestimated, inflating the Type I error rate. In this study, putative haploid rate (PHR) was significantly influenced by donor genotype, endosperm texture, and inducer. Previous studies reported a significant effect of donor genotype on haploid induction rate. Trentin et al. ( 2022 ) reported lower haploid induction rates in hybrids with flint (4.0%) and sweet (4.6%) endosperm textures compared to the dent group (9.7%). Eder and Chalyk ( 2002 ) obtained haploids from dent, flint, and flint × dent hybrids, with induction rates ranging from 2.7–8.0%. Kebede et al. ( 2011 ) evaluated the variation in HIR among tropical maize genotypes, with rates ranging from 2.9–9.7%. These results reinforce the importance of considering the genetic background of donor materials to improve haploid induction efficiency in DH-based breeding programs. The presence of anthocyanin inhibitor genes (e.g., C1-I , C2-Idf , and in1-D ) in source genotypes reduces the penetrance of R1-nj marker and can result in an increased error rate in identifying haploid seeds (Prigge et al. 2011 ). Two types of incorrect decisions can occur: (a) haploid seeds incorrectly discarded (false negatives) or (b) diploid seeds classified as haploids (false positives). The limited effectiveness of the R1-nj marker can lead to a high rate of false negatives and underestimation of haploid induction rates in the evaluated genotypes (Melchinger et al. 2014 ). Low penetrance of the R1-nj marker was reported in tropical and tropical × temperate crosses, indicating a high frequency of inhibitor alleles in the analyzed germplasm (Gain et al. 2022 ). Chaikam et al. ( 2014 ) observed that approximately 40% of the evaluated tropical maize populations showed segregation of the R1-nj marker, of which 4% exhibited complete inhibition. Similarly, Melchinger et al. ( 2014 ) and Thawarorit et al. ( 2023 ) demonstrated that flint and tropical germplasms have a higher accumulation of anthocyanin inhibitor alleles. Incomplete penetrance and variable expressivity of the R1-nj marker have also been observed in commercial sweet corn and popcorn hybrids, and to identifying haploids in these crosses cannot be reliably identified using only the R1-nj marker (Yu and Birchler 2015 , Silva et al. 2023 ). The presence of anthocyanin inhibitor genes in the donor germplasm suggests the necessity to use inducers with complementary classification methods, such as Pl-1 red root marker and kernel oil content (Melchinger et al. 2014 , Chaikam et al. 2019 , Dermail et al. 2024 , Rosa et al. 2024). Additionally, Baleroni et al. ( 2021 ) emphasize the importance to identifying haploids in germinated seedlings that may help to eliminate false positive seeds in the DH production process. The inducer (UH400 × UH401) × TAIL 9 was superior when combined with five donor hybrids. As a three-way cross hybrid, the accumulation of alleles at distinct loci responsible for haploid induction may explain the higher putative haploid rates (PHR) observed for this inducer (Chaikam et al. 2019 ). Two hypotheses, described by Meng et al. ( 2021 ), have been suggested to explain the occurrence of haploid seeds in maize: (a) single fertilization and (b) selective elimination of inducer chromosomes. The first case occurs when only the polar nuclei are fertilized by the sperm nucleus and the inducer gamete fails to fuse with the egg cell, resulting in seeds with haploid embryos. In the selective elimination process, a sperm cell of the inducer fuses with the egg cell, but the chromosomes of inducer are eliminated during subsequent zygotic divisions. The ability to induce gymnogenetic haploids is a quantitative trait controlled by multiple loci (Trentin et al. 2023 ). Two key genes have been successfully characterized: (1) a gene identified by three independent research groups, known as MATRILINEAL (MTL) , NOT LIKE DAD (NLD) , or ZmPHOSPHOLIPASE-A1 (ZmPLA1) (Gilles et al. 2017 , Liu et al. 2017 , Kelliher et al. 2017 ), located within the QTL qhir1 on chromosome 1; and (2) ZmDMP (Zhong et al. 2019 ), located within the QTL qhir8 on chromosome 9. The QTLs qhir1 and qhir8 explain 66% and 20% of the genotypic variance on the haploid induction rate, respectively (Prigge et al. 2012 ). Khammona et al. ( 2024 ) confirm the critical role of these QTLs for haploid induction rates through marker-assisted selection in segregating populations. While qhir8 alone contributes a low haploid induction rate (0.15%), it enhances the rate by two to six times when combined with the mutant allele at the MTL/NLD/ZmPLA1 locus, highlighting a synergistic effect between these genes (Zhong et al. 2019 ). Furthermore, Trentin et al. ( 2023 ) identified a major-effect QTL on chromosome 10 and several smaller-effect QTLs across six other chromosomes, reflecting the complex genetic architecture of this trait. Among the inducers tested, donor hybrids with semi-dent endosperm texture generally showed higher haploid induction rates. The flint endosperm texture typically exhibited lower induction rates, suggesting the presence of anthocyanin-inhibiting genes. In such cases, alternative methods for classifying haploid seeds should be considered. The inducer (UH400 × UH401) × TAIL 9 demonstrated the highest PHR when combined with five donor hybrids, indicating a possible accumulation of alleles responsible for haploid induction in this three-way cross inducer. For the hybrids AS 1677 PRO3 and SYN 7205, the inducer UH400 × UH401 performed better. Finally, the study highlighted the differential performance of haploid inducers when used with various donor genotypes and endosperm textures, corroborating the complex nature of induction ability. Declarations Funding This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do Paraná. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contribution Conceptualization: Marcondes MM, Faria MV, Scapim CA, Gomes R. Data curation: Faria MV, Scapim CA. Formal analysis: Rossi RM, Schuck ML. Methodology: Marcondes MM, Faria MV, Lima VA. Project administration: Faria MV. Writing-original draft: Schuck ML, Faria MV, Scapim CA. Writing-review & editing: Schuck ML, Faria MV, Scapim CA. All authors read and approved the final manuscript. Acknowledgement To Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Estado do Paraná for the financial support. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Baleroni AG, Ré F, Pelozo A, Kamphorst SH, Carneiro JWP, Rossi RM and Scapim CA (2021) Identification of haploids and diploids in maize using seedling traits and flow cytometry. Crop Breeding and Applied Biotechnology 21: e38422145. 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Supplementary Files supplementarytable1.docx supplementarytable2.docx Cite Share Download PDF Status: Published Journal Publication published 29 Apr, 2026 Read the published version in Euphytica → Version 1 posted Editorial decision: Revision requested 28 Sep, 2025 Reviews received at journal 06 Sep, 2025 Reviewers agreed at journal 22 Aug, 2025 Reviews received at journal 09 Aug, 2025 Reviewers agreed at journal 09 Jul, 2025 Reviewers invited by journal 07 Jul, 2025 Editor assigned by journal 26 Jun, 2025 Submission checks completed at journal 26 Jun, 2025 First submitted to journal 25 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6978380","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481805501,"identity":"37ec0888-0a93-46dc-84d7-1f47483cefe5","order_by":0,"name":"Mariana Martins Marcondes","email":"","orcid":"","institution":"Universidade Estadual do Centro-Oeste/UNICENTRO – Departamento de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Mariana","middleName":"Martins","lastName":"Marcondes","suffix":""},{"id":481805502,"identity":"96506b28-d7a1-454b-b7e0-5d56a7357cfe","order_by":1,"name":"Marcos Ventura Faria","email":"","orcid":"","institution":"Universidade Estadual do Centro-Oeste/UNICENTRO – Departamento de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Marcos","middleName":"Ventura","lastName":"Faria","suffix":""},{"id":481805504,"identity":"b724bccf-4237-44b9-bb7c-54622ff9a855","order_by":2,"name":"Carlos Alberto Scapim","email":"","orcid":"","institution":"Universidade Estadual de Maringá/UEM – Departamento de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"Alberto","lastName":"Scapim","suffix":""},{"id":481805506,"identity":"95771893-6a72-4e23-ac29-cbeea19409bc","order_by":3,"name":"Robson Marcelo Rossi","email":"","orcid":"","institution":"Universidade Estadual de Maringá/UEM – Departamento de Estatística","correspondingAuthor":false,"prefix":"","firstName":"Robson","middleName":"Marcelo","lastName":"Rossi","suffix":""},{"id":481805507,"identity":"2e9fb4cd-fb66-48a6-b64c-155ecee12204","order_by":4,"name":"Matheus Lucas Schuck","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYFACHiA+wMDAB+ZUADEzcwNxWtjAnDMgLYykaGFsA5P4tci3nz0m8eMMQzSbRPLDjz/n1UbztwO1/KjYhlOLwZm8NMmeGwy5bRJpxtK8247nzjjM2MDYc+Y2bi0SPGbSDB9AWnIYpBm3HcttAGphZmzDrUV+BkIL88+fc47lziekheEGSAvYYTlsErwNNbkbCGkxOJNjbNlzRiK3jeeZmTXPsQO5G4FaDuLzi3z7GcMbP47Z5PazJz+++aOmLnfe+cMHH/yowOMwCJCAMQ6DyQOE1CODOlIUj4JRMApGwQgBAKPUVzYG82b1AAAAAElFTkSuQmCC","orcid":"","institution":"Universidade Estadual de Maringá/UEM – Departamento de Agronomia","correspondingAuthor":true,"prefix":"","firstName":"Matheus","middleName":"Lucas","lastName":"Schuck","suffix":""},{"id":481805508,"identity":"59586606-e955-43bc-8120-4ab4dae81819","order_by":5,"name":"Vanderlei Aparecido de Lima","email":"","orcid":"","institution":"Universidade Tecnológica Federal do Paraná/UTFPR","correspondingAuthor":false,"prefix":"","firstName":"Vanderlei","middleName":"Aparecido","lastName":"de Lima","suffix":""},{"id":481805509,"identity":"23ddabea-abf2-458f-a4ee-cc395b17849c","order_by":6,"name":"Rodrigo Gomes","email":"","orcid":"","institution":"Universidade Estadual do Centro-Oeste/UNICENTRO – Departamento de Agronomia","correspondingAuthor":false,"prefix":"","firstName":"Rodrigo","middleName":"","lastName":"Gomes","suffix":""}],"badges":[],"createdAt":"2025-06-26 00:53:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6978380/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6978380/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10681-026-03733-6","type":"published","date":"2026-04-29T15:58:31+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86255116,"identity":"27e68a57-c01a-4258-ad6f-a756eb02f784","added_by":"auto","created_at":"2025-07-08 13:27:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":27398,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of the number of ears obtained per donor × inducer combination.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6978380/v1/8b399ac600eb5095bb08cfb0.png"},{"id":86256676,"identity":"d6fdec32-9eb0-4158-8ad1-d89a37575e0e","added_by":"auto","created_at":"2025-07-08 13:51:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90100,"visible":true,"origin":"","legend":"\u003cp\u003eDiagnostic plots with simulated envelopes of deviance residuals for (A) binomial and (B) quasibinomial logit models for the putative haploid rate (PHR) in maize.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6978380/v1/925f1b1a74b35023a796679c.png"},{"id":86255121,"identity":"0f25bcb8-91e8-4f62-8cae-f9e9f0730c05","added_by":"auto","created_at":"2025-07-08 13:27:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155303,"visible":true,"origin":"","legend":"\u003cp\u003eComplex interaction between inducer genotypes and endosperm texture on the putative haploid rate (PHR) in maize.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6978380/v1/ad7e99bb9b0364581fe96b5b.png"},{"id":86255400,"identity":"8914d6c5-3776-43cb-a9c7-f13df3f3406e","added_by":"auto","created_at":"2025-07-08 13:35:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268011,"visible":true,"origin":"","legend":"\u003cp\u003eComplex interaction between inducer genotypes and donor genotypes in the putative haploid rate in maize.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6978380/v1/27a8e79c9a0a7ce87327807d.png"},{"id":108437939,"identity":"2c7a3bbd-910e-475f-9ce1-6af33074883d","added_by":"auto","created_at":"2026-05-04 16:04:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":782492,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6978380/v1/4f62e3cd-f555-446c-a9c7-c3e9d697a12a.pdf"},{"id":86255395,"identity":"4f68a7c4-33d3-405a-a1e5-1dcf1c1d5409","added_by":"auto","created_at":"2025-07-08 13:35:30","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":25336,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6978380/v1/1127b21463406ba3900efd4c.docx"},{"id":86255117,"identity":"f4b9a07a-288d-4785-8e49-527b40a912c5","added_by":"auto","created_at":"2025-07-08 13:27:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33062,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6978380/v1/0211ed17c325431ea8c11e4c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of donor and inducer genotype and endosperm texture on haploid induction in maize assessed using R1-Navajo","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eIn maize (\u003cem\u003eZea mays\u003c/em\u003e L.) breeding programs focused on the production of double haploid (DH) lines, the \u003cem\u003ein vivo\u003c/em\u003e method for obtaining gymnogenetic haploids involves four key steps: (1) haploid induction, achieved by the crossing of a gymnogenetic haploid inducer pollen donor with the donor genotype, which produces maternal haploid seeds; (2) identification and separation of haploid seeds; (3) chromosome doubling of the haploid plants using antimitotic agents; and (4) self-pollination of the doubled plants to produce double haploid lines. Compared to the conventional method that involves successive generations of self-pollination, the double haploid (DH) technique enables the development of homozygous inbred lines in a single generation, reducing the time and resources required for maize breeding (Chaikam et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe haploid induction rate (HIR) is determined by the ratio of haploid seeds to the total number of seeds produced. Higher HIR values are essential for the efficiency of DH-based breeding programs and allow the production of doubled haploid lines in a large scale (Silva et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The HIR characteristic is first influenced by the haploid inducer genotype. In the early use of DH in maize breeding, the Stock 6 line was widely used as inducer, with an average production rate of 2\u0026ndash;3% haploid seeds. The later breeding for haploid induction resulted in HIR ranging from 8 to 15% in haploid inducer lines such as RWS, UH400, and TAIL (Coe \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1959\u003c/span\u003e, Prasanna \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition, the HIR is significantly influenced by the donor\u0026rsquo;s genetic background (Silva et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). De La Fuente et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) observed putative haploid rates ranging from 2.6\u0026ndash;30.5% across 30 hybrids pollinated with the same inducer RWS/RWK-76. Among other potential factors, anthocyanin inhibitor alleles (\u003cem\u003eC1-I\u003c/em\u003e, \u003cem\u003eC2-Idf\u003c/em\u003e, and \u003cem\u003ein1-D\u003c/em\u003e, which act epistatically on \u003cem\u003eR1-nj\u003c/em\u003e) present in the donor can prevent the formation of aleurone layer pigmentation. Generally flint maize harbors anthocyanin inhibitor genes at a higher frequency than dent maize, making the identification of haploid seeds more difficult in flint or flint \u0026times; dent hybrids (Eder and Chalik 2002, Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite the known occurrence of donor \u0026times; inducer interactions, few studies have evaluated their effect under field conditions in tropical germplasm. Given the complex interaction that inducers and donor germplasm have on the haploid induction rate, studies are needed to determine the dependence between these factors, especially for tropical conditions (Ren et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Trentin et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Identifying significant interactions could enable the use of specific inducer lines for the donor genotypes, thus reducing the costs associated with haploid selection. Therefore, this study aimed to evaluate the effects of endosperm textures of donors, donor and inducer genotypes on haploid induction rates, based on the \u003cem\u003eR1-navajo\u003c/em\u003e phenotype in maize seeds.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eGenetic material\u003c/h2\u003e\u003cp\u003eSeeds were obtained from the pollination of eleven maize hybrids, used as donor genotypes (female parents), with ten haploid inducer maize genotypes (male parents). The donor genotypes comprised ten single hybrids and one three-way hybrid, all widely adopted in commercial maize cultivation in Brazil. These hybrids exhibited semi-dent, semi-flint and flint endosperm textures, as detailed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eHybrids used as donor genotypes in the evaluation of putative haploid rate (PHR) in maize\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHybrid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCompany\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eType\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTexture\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e30A68 HX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMorgan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-flint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAG 8780 PRO3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBayer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-dent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAS 1555 RR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBayer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-flint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAS 1677 PRO3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBayer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-flint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBG 7060\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCorteva\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-flint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eF\u0026oacute;rmula\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSyngenta\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFlint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNS 50 PRO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSyngenta\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-flint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNS 92 PRO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSyngenta\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-flint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP1630 HX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCorteva\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-dent\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP4285 YHR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCorteva\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFlint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSYN 7205\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSyngenta\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSemi-flint\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eSH: single cross hybrid; TH: three-way cross hybrid.\u003c/p\u003e\u003cp\u003eThe evaluated haploid inducers are detailed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. They included: a single cross hybrid derived from the temperate lines UH400 and UH401, developed by the University of Hohenheim, Germany, from the KEMS (Krasnador Embryo Marker Synthetic) inducer (Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); tropical inbred lines adapted TAILs 7, 8, and 9 (Tropically Adapted Inducer Lines), developed by CIMMYT (International Maize and Wheat Improvement Center), Mexico, in collaboration with the University of Hohenheim, through backcrossing of the UH400 and RWS (Russian Inducer KEMS\u0026thinsp;+\u0026thinsp;WS14) inducers with tropical maize lines (Prasanna \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Liu et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); inducer inbred lines developed by Nidera Seeds, originating from TAILs 1, 2, and 3; and simple and three-way cross hybrids obtained from the UH400, UH401, TAILs 7, 8, and 9 inducers.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eGymnogenetic haploid inducers used in the evaluation of putative haploid rate (PHR) in maize.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInducer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eType\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOrigin\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTAIL 7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTAIL 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTAIL 9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTAIL 7 \u0026times; TAIL 8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUH400 \u0026times; UH401\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTemperate\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(UH400 \u0026times; UH401) \u0026times; TAIL 7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTemperate/Tropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e(UH400 \u0026times; UH401) \u0026times; TAIL 9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTemperate/Tropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNidera Orig TAIL 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTemperate/Tropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNidera Orig TAIL 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTemperate/Tropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNidera Orig TAIL 3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTropical\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIL: inbred line; SH: single cross hybrid; TH: three-way cross hybrid.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eHaploidy-inducing crosses and phenotypic classification\u003c/h3\u003e\n\u003cp\u003eThe pollination field was cultivated during the 2016/2017 growing season in Abelardo Luz, Santa Catarina, Brazil. The donor and inducer genotypes were arranged in 6 m rows per combination (donors \u0026times; inducers), with inter-row and intra-row spacings of 0.90 m and 0.20 m, respectively. Haploid inducers were planted in an adjacent field. To ensure synchronizing flowering periods, inducer genotypes were sown in five sequential planting dates, spaced one week apart; the first and last sowings occurred one week before and 21 days after the donor genotypes, respectively. The ears of the donor genotypes were protected prior to silk emergence. During the flowering period, pollen from each inducer was bulk-collected using paper bags, and pollinations were performed manually.\u003c/p\u003e\u003cp\u003eThe pollinated ears were harvested separately and the seeds produced were classified based on the presence of purple coloration from the expression of the \u003cem\u003eR1-nj\u003c/em\u003e marker gene, as described by Nanda and Chase (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1966\u003c/span\u003e). Therefore, seeds with purple endosperm and white embryos were considered putative haploids. Putative haploids classified using \u003cem\u003eR1-nj\u003c/em\u003e are related to the true haploid induction rate and can serve as a basis for evaluating the efficiency of different inducers (Couto et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe response variable was the number of putative haploids (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{y}_{ijkl}\\)\u003c/span\u003e\u003c/span\u003e) out of the total number of seeds (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\text{n}}_{\\text{i}\\text{j}\\text{k}\\text{l}}\\)\u003c/span\u003e\u003c/span\u003e) obtained from individual ears for each donor \u0026times; inducer combination. Thus, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{y}_{ijkl}\\)\u003c/span\u003e\u003c/span\u003e follows a binomial distribution with the probability of success \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\pi\\:}_{ijkl}\\)\u003c/span\u003e\u003c/span\u003e (Putative Haploid Rate - PHR) [\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{y}_{ijkl}\\:\\sim\\:\\:Binomial({\\pi\\:}_{ijkl},\\:{n}_{ijkl})\\)\u003c/span\u003e\u003c/span\u003e], using a logit link function, as in the Eq.\u0026nbsp;(1):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:logit\\left({\\pi\\:}_{ijkl}\\right)\\:=\\:{\\eta\\:}_{ijkl}=\\mu\\:+{\\alpha\\:}_{i}+\\:{\\beta\\:}_{j}+{\\gamma\\:}_{k\\left(j\\right)}+{\\tau\\:}_{i\\left(j\\right)}+{\\omega\\:}_{i\\left(jk\\right)}\\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\eta\\:}_{ijkl}\\)\u003c/span\u003e\u003c/span\u003e is the linear predictor for \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:l\\)\u003c/span\u003e\u003c/span\u003e-th ear from \u003cem\u003ek\u003c/em\u003e-th donor genotype within \u003cem\u003ej\u003c/em\u003e-th endosperm texture and \u003cem\u003ei\u003c/em\u003e-th inducer genotype, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mu\\:\\)\u003c/span\u003e\u003c/span\u003e is the constant inherent in the data, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\alpha\\:}_{i}\\)\u003c/span\u003e\u003c/span\u003e represents the effect of the \u003cem\u003ei\u003c/em\u003e-th inducer, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\beta\\:}_{j}\\)\u003c/span\u003e\u003c/span\u003e denotes the effect of the \u003cem\u003ej\u003c/em\u003e-th endosperm texture, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\gamma\\:}_{k\\left(j\\right)}\\)\u003c/span\u003e\u003c/span\u003e is the effect of the \u003cem\u003ek\u003c/em\u003e-th donor genotype within the \u003cem\u003ej\u003c/em\u003e-th endosperm texture, \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\tau\\:}_{i\\left(j\\right)}\\)\u003c/span\u003e\u003c/span\u003e is the effect of the \u003cem\u003ei\u003c/em\u003e-th inducer genotype within the \u003cem\u003ej\u003c/em\u003e-th endosperm texture, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\omega\\:}_{i\\left(jk\\right)}\\)\u003c/span\u003e\u003c/span\u003e is the effect of the \u003cem\u003ei\u003c/em\u003e-th inducer genotype within the \u003cem\u003ej\u003c/em\u003e-th endosperm texture and \u003cem\u003ek\u003c/em\u003e-th donor genotype. The model was fitted using the \u0026lsquo;\u003cem\u003eglm\u0026rsquo;\u003c/em\u003e function from the \u0026lsquo;\u003cem\u003estats\u003c/em\u003e\u0026rsquo; package (R Core Team version 4.3.1).\u003c/p\u003e\u003cp\u003eThe model that provided a better fit to the data was subjected to residual analysis using diagnostic plots with simulated envelopes, employing the \u0026lsquo;\u003cem\u003ehnp\u003c/em\u003e\u0026rsquo; package (Moral et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Overdispersion was detected, as the amount of variation in the data exceeded that expected by the model. Consequently, the estimated standard errors and confidence intervals were corrected using the quasibinomial model, where the variance is given by \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varphi\\:{\\pi\\:}_{ijkl}(1\\:-\\:{\\pi\\:}_{ijkl})\\)\u003c/span\u003e\u003c/span\u003e, with \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\varphi\\:\\)\u003c/span\u003e\u003c/span\u003e as the dispersion parameter (Dem\u0026eacute;trio et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSequencial deviance analysis was performed, where factors of the model were assessed using the F statistic. The pairwise contrasts of the estimated means within each level of the factors were estimated, and inference was made using the Wald test. The \u003cem\u003ep\u003c/em\u003e-values were adjusted using the Bonferroni correction to maintain the global error rate at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\alpha\\:}=0.05\\)\u003c/span\u003e\u003c/span\u003e. The means on the linear predictor scale were then converted to probabilities using the inverse of the link function, as in Eq.\u0026nbsp;(2).\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:{g}^{-1}\\left({\\pi\\:}_{ijkl}\\right)=\\frac{{e}^{{\\eta\\:}_{ijkl}}}{1+{e}^{{\\eta\\:}_{ijkl}}}\\:\\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eAsymptotic confidence intervals for the estimated contrasts were obtained using the \u0026lsquo;\u003cem\u003eemmeans\u003c/em\u003e\u0026rsquo; package in the R environment (R Core Team version 4.3.1).\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003eA total of 240,285 seeds were evaluated across 1,394 ears. The number of ears per donor \u0026times; inducer combination ranged from 0 to 30 (Supplementary Table\u0026nbsp;1), with their distribution represented in the histogram in the Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Eighth crosses did not produce any seeds, so their means and contrasts involving these means could not be estimated. These crosses were: BG 7060 \u0026times; TAIL 7, P4285 YHR \u0026times; [TAIL 9, UH400 \u0026times; UH401, and (UH400 \u0026times; UH401) \u0026times; TAIL 9, Nidera Orig TAIL 1], and SYN 7205 \u0026times; [TAIL 7, TAIL 7 \u0026times; TAIL 8 and Nidera Orig TAIL 1]. A total of 15,254 putative haploid seeds were obtained, with an observed average PHR of 6.35% in the experiment, ranging from 0.05\u0026ndash;18.63% across the donors \u0026times; inducers combinations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe half-normal plots in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e provide strong evidence in favor of the model that accounts for overdispersion, with most residuals falling within the simulated envelope. The estimated dispersion parameter for the quasibinomial overdispersion model was 2.67. The sequential deviance analysis for PHR revealed significant effects for all evaluated factors, as well as their nested effects (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Supplementary Table\u0026nbsp;2a).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe interaction plot between donor endosperm textures and inducers is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. For the semi-dent kernel texture, the inducer (UH400 \u0026times; UH401) \u0026times; TAIL 9 showed the highest estimated means of PHR (17.29%), differing from five lower-performing inducers (Supplementary Table\u0026nbsp;2b). For the semi-flint and flint textures, the inducers UH400 \u0026times; UH401 and TAIL 7 \u0026times; TAIL 8 exhibited the highest PHR (6.91% and 3.13%), respectively, differing from three and one lower-performing inducers in their respective textures.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe semi-dent texture genotypes provided higher PHR in crosses with nine of the ten inducers evaluated, indicating greater inducibility of this group of donor hybrids. In all cases where contrasts between textures could be estimated, the flint endosperm texture showed a lower average PHR compared to the higher groups (Supplementary Table\u0026nbsp;2b).\u003c/p\u003e\u003cp\u003eThe interaction plot for donor genotypes and inducers is also presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The inducer (UH400 \u0026times; UH401) \u0026times; TAIL 9 exhibited the highest estimated mean when used as a haploid inducer in combination with five donor hybrids, being statistically superior to the lower-performing inducers (Supplementary Table\u0026nbsp;2c). For the donor hybrid NS 50 PRO, the single hybrid inducer TAIL 7 \u0026times; TAIL 8 demonstrated the highest haploid induction capacity (7.98%), outperforming the three inducers with lower haploid induction ability, including the three-way cross hybrid (UH400 \u0026times; UH401) \u0026times; TAIL 9 (2.80%), which was superior in other crosses. For the hybrids AS 1677 PRO3 and SYN 7205, the inducer UH400 \u0026times; UH401 exhibited the highest PHR. The hybrids NS 92 PRO and P4285 YHR did not demonstrate a significant difference between the inducers.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eNormal plots based on simulated envelopes, using deviance residuals, were employed to compare the goodness-of-fit between the binomial and quasibinomial models. The quasibinomial model, which accounts for overdispersion, provided a better fit and is more appropriate for estimating of standard errors for the estimated means. The observed overdispersion may result from heterogeneity among observations that was not explained by the model covariates (Dem\u0026eacute;trio et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). When the overdispersion is ignored, the standard errors of the estimated means are underestimated, inflating the Type I error rate.\u003c/p\u003e\u003cp\u003eIn this study, putative haploid rate (PHR) was significantly influenced by donor genotype, endosperm texture, and inducer. Previous studies reported a significant effect of donor genotype on haploid induction rate. Trentin et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) reported lower haploid induction rates in hybrids with flint (4.0%) and sweet (4.6%) endosperm textures compared to the dent group (9.7%). Eder and Chalyk (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) obtained haploids from dent, flint, and flint \u0026times; dent hybrids, with induction rates ranging from 2.7\u0026ndash;8.0%. Kebede et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) evaluated the variation in HIR among tropical maize genotypes, with rates ranging from 2.9\u0026ndash;9.7%. These results reinforce the importance of considering the genetic background of donor materials to improve haploid induction efficiency in DH-based breeding programs.\u003c/p\u003e\u003cp\u003eThe presence of anthocyanin inhibitor genes (e.g., \u003cem\u003eC1-I\u003c/em\u003e, \u003cem\u003eC2-Idf\u003c/em\u003e, and \u003cem\u003ein1-D\u003c/em\u003e) in source genotypes reduces the penetrance of \u003cem\u003eR1-nj\u003c/em\u003e marker and can result in an increased error rate in identifying haploid seeds (Prigge et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Two types of incorrect decisions can occur: (a) haploid seeds incorrectly discarded (false negatives) or (b) diploid seeds classified as haploids (false positives). The limited effectiveness of the \u003cem\u003eR1-nj\u003c/em\u003e marker can lead to a high rate of false negatives and underestimation of haploid induction rates in the evaluated genotypes (Melchinger et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eLow penetrance of the \u003cem\u003eR1-nj\u003c/em\u003e marker was reported in tropical and tropical \u0026times; temperate crosses, indicating a high frequency of inhibitor alleles in the analyzed germplasm (Gain et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Chaikam et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) observed that approximately 40% of the evaluated tropical maize populations showed segregation of the \u003cem\u003eR1-nj\u003c/em\u003e marker, of which 4% exhibited complete inhibition. Similarly, Melchinger et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and Thawarorit et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrated that flint and tropical germplasms have a higher accumulation of anthocyanin inhibitor alleles. Incomplete penetrance and variable expressivity of the \u003cem\u003eR1-nj\u003c/em\u003e marker have also been observed in commercial sweet corn and popcorn hybrids, and to identifying haploids in these crosses cannot be reliably identified using only the \u003cem\u003eR1-nj\u003c/em\u003e marker (Yu and Birchler \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Silva et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe presence of anthocyanin inhibitor genes in the donor germplasm suggests the necessity to use inducers with complementary classification methods, such as \u003cem\u003ePl-1\u003c/em\u003e red root marker and kernel oil content (Melchinger et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Chaikam et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Dermail et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e, Rosa et al. 2024). Additionally, Baleroni et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) emphasize the importance to identifying haploids in germinated seedlings that may help to eliminate false positive seeds in the DH production process.\u003c/p\u003e\u003cp\u003eThe inducer (UH400 \u0026times; UH401) \u0026times; TAIL 9 was superior when combined with five donor hybrids. As a three-way cross hybrid, the accumulation of alleles at distinct loci responsible for haploid induction may explain the higher putative haploid rates (PHR) observed for this inducer (Chaikam et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Two hypotheses, described by Meng et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), have been suggested to explain the occurrence of haploid seeds in maize: (a) single fertilization and (b) selective elimination of inducer chromosomes. The first case occurs when only the polar nuclei are fertilized by the sperm nucleus and the inducer gamete fails to fuse with the egg cell, resulting in seeds with haploid embryos. In the selective elimination process, a sperm cell of the inducer fuses with the egg cell, but the chromosomes of inducer are eliminated during subsequent zygotic divisions.\u003c/p\u003e\u003cp\u003eThe ability to induce gymnogenetic haploids is a quantitative trait controlled by multiple loci (Trentin et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Two key genes have been successfully characterized: (1) a gene identified by three independent research groups, known as \u003cem\u003eMATRILINEAL (MTL)\u003c/em\u003e, \u003cem\u003eNOT LIKE DAD (NLD)\u003c/em\u003e, or \u003cem\u003eZmPHOSPHOLIPASE-A1 (ZmPLA1)\u003c/em\u003e (Gilles et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Liu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Kelliher et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), located within the QTL \u003cem\u003eqhir1\u003c/em\u003e on chromosome 1; and (2) \u003cem\u003eZmDMP\u003c/em\u003e (Zhong et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), located within the QTL \u003cem\u003eqhir8\u003c/em\u003e on chromosome 9.\u003c/p\u003e\u003cp\u003eThe QTLs \u003cem\u003eqhir1\u003c/em\u003e and \u003cem\u003eqhir8\u003c/em\u003e explain 66% and 20% of the genotypic variance on the haploid induction rate, respectively (Prigge et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Khammona et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) confirm the critical role of these QTLs for haploid induction rates through marker-assisted selection in segregating populations. While \u003cem\u003eqhir8\u003c/em\u003e alone contributes a low haploid induction rate (0.15%), it enhances the rate by two to six times when combined with the mutant allele at the MTL/NLD/ZmPLA1 locus, highlighting a synergistic effect between these genes (Zhong et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, Trentin et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) identified a major-effect QTL on chromosome 10 and several smaller-effect QTLs across six other chromosomes, reflecting the complex genetic architecture of this trait.\u003c/p\u003e\u003cp\u003eAmong the inducers tested, donor hybrids with semi-dent endosperm texture generally showed higher haploid induction rates. The flint endosperm texture typically exhibited lower induction rates, suggesting the presence of anthocyanin-inhibiting genes. In such cases, alternative methods for classifying haploid seeds should be considered. The inducer (UH400 \u0026times; UH401) \u0026times; TAIL 9 demonstrated the highest PHR when combined with five donor hybrids, indicating a possible accumulation of alleles responsible for haploid induction in this three-way cross inducer. For the hybrids AS 1677 PRO3 and SYN 7205, the inducer UH400 \u0026times; UH401 performed better. Finally, the study highlighted the differential performance of haploid inducers when used with various donor genotypes and endosperm textures, corroborating the complex nature of induction ability.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) and Funda\u0026ccedil;\u0026atilde;o Arauc\u0026aacute;ria de Apoio ao Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico do Estado do Paran\u0026aacute;.\u003c/p\u003e\u003cp\u003eCompeting Interests\u003c/p\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Marcondes MM, Faria MV, Scapim CA, Gomes R. Data curation: Faria MV, Scapim CA. Formal analysis: Rossi RM, Schuck ML. Methodology: Marcondes MM, Faria MV, Lima VA. Project administration: Faria MV. Writing-original draft: Schuck ML, Faria MV, Scapim CA. Writing-review \u0026amp; editing: Schuck ML, Faria MV, Scapim CA. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eTo Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior (CAPES) and Funda\u0026ccedil;\u0026atilde;o Arauc\u0026aacute;ria de Apoio ao Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico do Estado do Paran\u0026aacute; for the financial support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaleroni AG, R\u0026eacute; F, Pelozo A, Kamphorst SH, Carneiro JWP, Rossi RM and Scapim CA (2021) Identification of haploids and diploids in maize using seedling traits and flow cytometry. 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Biodiversitas Journal of Biological Diversity 24: 4262\u0026ndash;4268.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrentin HU, Bat\u0026icirc;ru G, Frei UK, Dutta S and L\u0026uuml;bberstedt T (2022) Investigating the Effect of the Interaction of Maize Inducer and Donor Backgrounds on Haploid Induction Rates. Plants 11: 1527\u0026ndash;1527.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTrentin HU, Krause MD, Zunjare RU, Almeida VC, Peterlini E, Rotarenco V, Frei UK, Beavis WD and L\u0026uuml;bberstedt T (2023) Genetic basis of maize maternal haploid induction beyond MATRILINEAL and ZmDMP. Frontiers in Plant Science 14: 1218042.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu W and Birchler JA (2015) A green fluorescent protein-engineered haploid inducer line facilitates haploid mutant screens and doubled haploid breeding in maize. Molecular Breeding 36: 5.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhong Y, Liu C, Qi X, Jiao Y, Wang D, Wang Y, Liu Z, Chen C, Chen B, Tian X, Li J, Chen M, Dong X, Xu X, Li L, Li W, Liu W, Jin W, Lai J and Chen S (2019) Mutation of ZmDMP enhances haploid induction in maize. Nature Plants 5: 575\u0026ndash;580.\u003c/span\u003e\u003c/li\u003e\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":"euphytica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"euph","sideBox":"Learn more about [Euphytica](https://www.springer.com/journal/10681)","snPcode":"10681","submissionUrl":"https://submission.springernature.com/new-submission/10681/3","title":"Euphytica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Zea mays L., Navajo phenotype, gymnogenetic haploids, endosperm texture, anthocyanin","lastPublishedDoi":"10.21203/rs.3.rs-6978380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6978380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe double haploid (DH) technique is extensively utilized in maize breeding programs to accelerate the development of homozygous lines that allows the fixation of the recombinant haploid genome in a single generation. This process relies on \u003cem\u003ein vivo\u003c/em\u003e haploid induction, in which the production of haploid seeds represents a critical step in this technique that determines the efficiency of DH line production. This study aimed to evaluate the effects of endosperm texture, donor genotype, and inducer on haploid induction rates based on the \u003cem\u003eR1-navajo\u003c/em\u003e (\u003cem\u003eR1-nj\u003c/em\u003e) marker. Eleven hybrids were assessed as donor genotypes and were pollinated with ten haploid inducers. The response was the number of putative haploid seeds based on \u003cem\u003eR1-nj\u003c/em\u003e phenotype, evaluated across the total number of seeds, with replications obtained from individual ears. Generalized Linear Models (GLMs) with binomial distribution were fitted for the putative haploid rate (PHR). A total of 240,285 seeds were evaluated across 1,394 ears, with an observed average PHR of 6.35% in the experiment, ranging from 0.05 to 18.63% across the donors \u0026times; inducers combinations. For most of the inducers evaluated, hybrids with a semi-dent endosperm texture exhibited higher PHR, whereas hybrids with flint endosperm exhibited lower rates. The three-way cross inducer (UH400 \u0026times; UH401) \u0026times; TAIL 9 achieved the highest PHR with five donor hybrids. These results highlight that specific combinations of donor genotypes and haploid inducers should be chosen to achieve a higher number of haploid seeds.\u003c/p\u003e","manuscriptTitle":"Effects of donor and inducer genotype and endosperm texture on haploid induction in maize assessed using R1-Navajo","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-08 13:27:26","doi":"10.21203/rs.3.rs-6978380/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-28T15:35:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-06T15:46:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"20870195392622143928056566815030749168","date":"2025-08-22T14:07:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-09T14:54:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"159553686223228103487743239865845241509","date":"2025-07-09T15:37:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-07T05:17:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-26T10:36:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-26T10:35:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Euphytica","date":"2025-06-26T00:42:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"euphytica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"euph","sideBox":"Learn more about [Euphytica](https://www.springer.com/journal/10681)","snPcode":"10681","submissionUrl":"https://submission.springernature.com/new-submission/10681/3","title":"Euphytica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a05658a5-2068-42ee-b66b-eb1908fb7f12","owner":[],"postedDate":"July 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T16:04:15+00:00","versionOfRecord":{"articleIdentity":"rs-6978380","link":"https://doi.org/10.1007/s10681-026-03733-6","journal":{"identity":"euphytica","isVorOnly":false,"title":"Euphytica"},"publishedOn":"2026-04-29 15:58:31","publishedOnDateReadable":"April 29th, 2026"},"versionCreatedAt":"2025-07-08 13:27:26","video":"","vorDoi":"10.1007/s10681-026-03733-6","vorDoiUrl":"https://doi.org/10.1007/s10681-026-03733-6","workflowStages":[]},"version":"v1","identity":"rs-6978380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6978380","identity":"rs-6978380","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}