Interploidy hybridization enhances floral phenotypic diversity of Iris × norrisii tetraploids

Interploidy hybridization enhances floral phenotypic diversity of Iris × norrisii tetraploids | 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 Interploidy hybridization enhances floral phenotypic diversity of Iris × norrisii tetraploids Jiacheng Tang, Liya Ding, Yike Gao, Rong Liu, Jianhua Xiao, Yi Lv, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6843341/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Oct, 2025 Read the published version in Euphytica → Version 1 posted 7 You are reading this latest preprint version Abstract Interploidy hybridization has been widely utilized in plant breeding and cultivar improvement. In ornamental species, interploidy hybridization between diverse diploid lines and synthetic tetraploids enables the transfer of rich diploid variation to polyploid backgrounds. Although previously applied in the breeding of Irises and other ornamentals, this approach remains insufficiently explored in synthetic polyploid cultivars. To address the limited floral variation observed in artificial tetraploid lines of Iris × norrisii , we performed interploidy crosses between tetraploid plants and both diploid and mixoploid lines. Hybrid progenies were evaluated based on floral traits, chromosome counts, and the meiotic behavior of microspore mother cells in parental plants. Crosses using tetraploid plants as female parents yielded tetraploid or near-tetraploid aneuploid progeny, whereas those with diploid female parents produced only diploid offspring. Cytological analysis revealed meiotic abnormalities in both diploid and tetraploid, contributing to the formation of unreduced male gametes. Interploidy hybrids displayed a broader range of perianth colors than control tetraploid crosses, whereas floral morphological variation was similar between the two groups. These findings demonstrate that diploid I. × norrisii contributes to the diversification of floral traits in tetraploid lines via the transmission of genetic variation through unreduced male gametes. Interploidy hybridization is a promising approach for enhancing phenotypic diversity in polyploid ornamental breeding. Iris × norrisii tetraploid interploidy hybridization unreduced gamete Figures Figure 1 Figure 2 Figure 3 Introduction Interploidy hybridization has been extensively applied in plant breeding programs. Crosses between diploid and tetraploid parents have been employed to develop asexual triploid fruits, such as seedless Citrullus lanatus (Thomas et al. 2003 ) and citrus varieties (Aleza et al. 2012 ). In ornamental breeding, varieties of desirable traits from diploid cultivars have been successfully introgressed into tetraploids, as demonstrated in Bearded Irises (Schreiner 1945 ; Pane-Joyce 1995 ) and Aril Irises (Warburton and Hamblen 1978 ). Additionally, the use of mixoploid Louisiana Iris cultivars has facilitated the improvement of floral diversity in tetraploid populations (Mertzweiller 1986 ). Despite these successful applications, the potential of interploidy hybridization to enrich floral trait diversity in newly induced polyploid ornamental species remains underexplored. Recently, tetraploid plants of Iris × norrisii were successfully induced through colchicine treatment (Ding et al. 2023 ). Although these tetraploids exhibited larger flowers and seeds compared to their diploid counterparts, their floral phenotypes were strikingly uniform, predominantly exhibiting purple flowers. Given that effective breeding in polyploid ornamental plants often relies on generating and selecting diverse phenotypes, expanding the floral variation of tetraploid I. × norrisii is essential for developing novel cultivars with enhanced ornamental value. To address this challenge, interploidy hybridizations were conducted between tetraploid I. × norrisii plants and both diploid and mixoploid lines, leveraging the rich floral diversity present in diploid populations. The resulting progenies were evaluated for floral traits, performed chromosome counts, and examined the meiotic behavior of parental microspore mother cells to elucidate the cytological mechanisms underlying hybrid formation. This study aims to assess the feasibility of using interploidy hybridization to diversify floral traits in tetraploid I. × norrisii , offering insights into accelerating polyploid breeding efforts in ornamental plants. Materials and methods Plant materials Tetraploid plants of the C1 generation of I. × norrisii were obtained as previously reported by Ding et al. ( 2023 ). The C2 generation was obtained through self-pollination and intercrossing among C1 individuals. Both C1 and C2 generation were served as the tetraploid parents in the hybridization. The diploid hybrid population was generated by crossing diploid I. × norrisii individuals with elite cultivars. Additionally, mixoploid plants were induced via colchicine treatment, following the method of Ding et al. ( 2023 ). All plant materials were cultivated in an open-field nursery at Beijing Forestry University (40°09′ N, 116°26′ E). Hybridization experiment Reciprocal crosses were performed between tetraploids and diploids (designated as 4X × 2X and 2X × 4X) and between tetraploids and mixoploids (designated as 4X × C0 and C0 × 4X), using mixed pollen for pollination. Self-pollination and intra-ploidy crosses within diploid and C2 populations served as controls (designated as 2X × 2X and 4X × 4X, respectively). For each cross, the numbers of pollinations, fruits, plump ovules per fruit, and seeds were recorded. Additionally, fruit set rate, germination rate, and seedling rate were calculated. The number of plump ovules per fruit was determined by dividing the total number of plump ovules produced after 14 days of pollination by the number of fruits. Seedling rate was calculated as the number of progeny obtained divided by the number of pollinations Chromosome counting in hybrid progeny Chromosome numbers were determined following the protocol of Ding et al. ( 2023 ). Root tips and developing ovaries were collected, pretreated with saturated p-dichlorobenzene in the dark, rinsed with distilled water and fixed in Carnoy's solution (ethanol: acetic acid = 3:1, v/v). After fixation, tissues were hydrolyzed in hydrochloric acid, rinsed again, and stained with Carbol fuchsin (Solarbio, Beijing, China). Chromosomes were observed using a Soptop CX40 microscope. Meiotic observation in diploid and tetraploid parents Meiosis behavior in microspore mother cells (MMCs) of diploid and tetraploid parents was examined. Developing flower buds were fixed in a fixative solution (ethanol: acetic acid: chloroform = 3: 2: 5, v/v), rinsed with distilled water, and dissected to extract anthers. The other contents were squashed on microscope slides, stained with Carbol fuchsin (Solarbio, Beijing, China), and examined under a Soptop CX40 microscope. The total number of MMCs and those showing chromosomal abnormalities were recorded at various meiotic stages, and the frequency of abnormalities was calculated for each stage. Evaluation of hybrid progeny phenotypes Floral traits of the hybrid progeny were evaluated in the outdoor nursery at Beijing Forestry University. Each individual was assessed for floral morphology, including the characteristics of perianth segments, pistils, and stamens, and classified into floral types. Perianth color was recorded, analyzed statistically, and documented through digital photography. Result Hybridization experiment The number of pollinations, fruits, plump ovules per fruit, seeds, fruit set rate, germination rate and seedling rate of each hybrid combination are summarized in Table 1 . Fruit set rates in interploidy crosses were lower than those observed in the control groups (2X × 2X and 4X × 4X). Among interploidy crosses, fruit set rates were higher when tetraploids served as the maternal parent compared to the backcrossing. Notably, the seedling rate of 4X × C0 (37.72%) was higher than that of the 4X × 4X control (19.57%). Conversely, the seedling rates of 4X × 2X, 2X × 4X, and C0 × 4X were significantly lower than that of the 4X × 4X control. Table 1 Results of hybridization experiment Crossing Types No. of Pollinations No. of Fruits Fruit Set Rate (%) No. of Plump Ovules per Fruit No. of Seeds Germination Rate (%) Seedling Rate (%) 4X × 2X 650 100 15.38 1.97 82 10.98 1.38 2X × 4X 865 108 12.49 3.59 23 34.78 0.92 4X × C0 167 26 15.57 6.54 164 38.41 37.72 C0 × 4X 290 27 9.31 1.15 28 3.57 0.34 4X × 4X 184 33 17.93 9.88 198 17.17 19.57 2X × 2X 95 38 40.00 1.89 - - - Chromosome counting of hybrid progeny Several individuals from the progenies of 4X × 2X, 2X × 4X, and 4X × C0 crosses were randomly selected for chromosome counting. Among the four offspring from the 4X × 2X crosses, three were tetraploid (2n = 4x = 64, Fig. 2 a) and one was aneuploid (2n = 4x − 2 = 62, Fig. 2 b). All five offspring from the 2X × 4X crosses were diploid (2n = 2x = 32, Fig. 2 c). Among the five samples from the 4X × C0 crosses, three were tetraploid (2n = 4x = 64, Fig. 2 d), while the remaining two were aneuploid (2n = 4x − 2 = 62, Fig. 2 f). Observation of the meiosis in diploid and tetraploid microspore mother cells A total of 609 tetraploid and 547 diploid microspore mother cells (MMCs) were examined to assess meiotic behavior. Both ploidy levels exhibited common meiotic abnormalities. During metaphase I, over 40% of MMCs at both ploidy levels exhibited chromosomes failing to congress at the equatorial plate (Table 2 , Fig. 2 c). Subsequently, unequal chromosome segregation was observed at early anaphase I (Fig. 2 d), and chromosome bridges (Fig. 2 e) were noted from early to late anaphase I, with the highest frequencies recorded during early anaphase I (29.2% in diploids; 33.3% in tetraploids). Chromosomes involved in bridges formation either separated rapidly or broke directly. Lagging chromosomes were primarily observed in anaphase I (Table 2 , Fig. 2 f), leading to micronuclei that persisted through meiosis II and into the tetrad stage. Ultimately, both diploid and tetraploid MMCs produced triads at the tetrad stage (Fig. 2 j). Despite these similarities, notable differences were evident. Tetraploid MMCs exhibited a higher frequency of meiotic abnormalities compared to diploids. At diakinesis, diploid MMCs displayed univalents (Fig. 2 a), whereas tetraploid MMCs exhibited both univalents and multivalents (Fig. 2 b). At early Anaphase I, unequal chromosome segregation was more frequent in tetraploids. Additionally, tetraploid MMCs displayed a low frequency of chromosome bridges during metaphase I and exhibited occasional perpendicular spindles during prophase II and polyads at the tetrad stage. Table 2 Frequency of meiotic anomalies in microspore mother cells in tetraploids and diploids Ploidy Level Total Abnormal Rate ( % ) Abnormal rate of different phases of meiosis of microspore mother cells Metaphase Ⅰ (%) Early Anaphase I (%) Late Anaphase Ⅰ (%) Prophase Ⅱ (%) Anaphase Ⅱ & Telophase Ⅱ (%) Tetrad Stage (%) chromosomes failing to congression Chromosome Bridge Unequal Segregation Chromosome Bridge Lagging Chromosomes Chromosome Bridge Micronucleus Perpendicular Spindle Asynchronous Separation Micronucleus Triad Micronucleus Polyad 4X 42.1 46.3 4.5 14.3 33.3 51.7 10.3 28.0 2.0 12.5 18.8 15.4 15.7 3.8 2X 26.5 41.7 0.0 0.0 29.2 35.7 7.1 18.5 0.0 9.5 14.3 8.6 5.7 0.0 Phenotypic analysis of hybrid progeny All hybrid progeny exhibited distinct floral phenotypes, which were categorized into two major types based on petal and stigma morphology: Iris dichotoma-type and Iris domestica-type. Iris domestica-type flowers displayed outer perianths without noticeable reflexing and revolute margins, inner perianths with triangular notches at the tips, and obtuse, rounded stigmatic appendages (Fig. 3 g). Conversely, Iris dichotoma-type flowers were characterized by outer perianth segments that were transversely reflexed at the middle with revolute margins, inner perianths with trapezoidal notches at the tips, and pointed stigmatic appendages (Fig. 3 h). Further phenotypic variation was observed among different crossing combinations. In the 4X × 4X control group, 20 seedlings were evaluated for perianth color: 16 exhibited purple (Fig. 3 a), two displayed light purple (Fig. 3 b), one showed white (Fig. 3 c), and one exhibited orange perianth color (Fig. 3 e). Notably, the orange-flowered individual exhibited the Iris domestica-type morphology, whereas the purple, light purple, and white flowers corresponded to the Iris dichotoma-type. Among the 4X × 2X crosses, six of nine offspring flowered, producing three purple, one white, one orange, and one pink (Fig. 3 d) perianth colors. Similarly, in the 2X × 4X crosses, five of eight offspring flowered, yielding two pink, two orange, and one pink-yellow bicolor perianths. The 4X × C0 crosses produced 59 offspring. Most individuals exhibited Iris domestica-type morphology, with 23 orange and 31 burgundy-colored flowers (Fig. 3 f). Only two individuals displayed Iris dichotoma-type morphology, bearing purple and light purple perianth colors. Additionally, perianth spotting varied considerably, ranging from strip-like to punctate patterns, with distribution areas extending from full to partial petal coverage and varying in density from sparsely to densely merged patterns. Discussion Preferential production of unreduced male gametes diploid I × norrisi A key finding of this study is the preferential production of unreduced male gametes over unreduced female gametes in diploid I × norrisii , which offers insights into reproductive barriers between different ploidy levels. In 4X × 2X crosses, tetraploid and near-tetraploid aneuploids were obtained, suggesting that unreduced (2 n ) male gametes were produced by diploid individuals. Cytological evidence further supports this inference, as triads containing 2 n pollen grains were observed in diploid MMC. When such unreduced pollen fertilizes normal female gametes from tetraploids, the resulting offspring can be tetraploid or aneuploid. Similar patterns of 2 n pollen production through triads have been reported in other species such as Populus × euramericana (Zhang et al. 2009 ) and Dimocarpus longan (Li et al. 2022 ), indicating a conserved mechanism across diverse taxa. Conversely, in 2X × 4X crosses where diploid individuals served as maternal parents, only diploid offspring were obtained. This result suggests two key inferences. First, I. × norrisii exhibits a limited capacity for producing unreduced female gametes, suggesting relatively greater stability during megaspore meiosis compared to microspore meiosis. Second, the tetraploid parent likely contributed haplotype male gametes containing 16 chromosomes, possibly due to meiotic abnormalities. This pattern differs from that observed in species such as Hieracium echioides (Peckert and Chrtek 2006 ) and Campanula rotundifolia (Sutherland and Galloway 2017 ), where interploidy crosses produced a small number of diploid progeny but commonly yielded triploid and tetraploid progeny. These differences may reflect species-specific variation in the formation of unreduced female gametes. Several meiotic irregularities observed in both diploid and tetraploid parental lines likely contribute to the production of unreduced male gametes and aneuploid offspring. These include univalents and multivalents during pachytene, lagging chromosomes, chromosome bridges, and micronuclei. Univalents and multivalents can lead to unequal chromosome segregation, while lagging chromosomes and bridges—hallmarks of chromosomal instability (Rodriguez-Muñoz et al. 2022 )—may give rise to micronuclei and trigger breakage-fusion-bridge cycles (De Marco Zompit and Stucki 2021 ) These abnormalities likely underlie the production of unreduced pollen with abnormal chromosome numbers, ultimately resulting in the observed aneuploid progeny. Diploids and mixoploids enrich floral phenotypic diversity in tetraploids via unreduced male gametes The results of this study demonstrate that diploid and mixoploid individuals can enhance the floral phenotypic diversity of I. × norrisii tetraploid progeny through interploidy hybridization. The tetraploid parental plants (C1 and C2 generations) exhibited limited perianth coloration, primarily purple and white. In contrast, novel perianth colors such as light purple and orange were observed in the 4X × 4X control crosses, while interploidy crosses (4X × 2X and 4X × C0) produced even greater diversity, including light purple, orange, burgundy, and pink flowers. Additionally, distinct floral morphologies resembling Iris dichotoma-type and Iris domestica-type were observed in both control and interploidy progeny. The diploid parental population was a genetically diverse hybrid group with highly variable perianth coloration, while the mixoploid individuals were obtained by colchicine treatment of seeds derived from this genetically diverse diploid population. As a result, both diploid and mixoploid parents carried abundant genetic variation. Combined with cytological observations, these findings suggest that diploids and mixoploids transmitted their genetic diversity to the tetraploid progeny via unreduced male gametes, thereby enriching floral phenotypic traits in the tetraploid background. Additionally, a higher seedling rate was observed in the 4X × C0 crosses than in the 4X × 2X crosses, implying that mixoploids may exhibit greater reproductive compatibility with tetraploids than diploids do. This phenomenon of generating phenotypic diversity via interploidy hybridization has significant evolutionary implications. Several recently formed polyploid species, such as Senecio cambrensis and Mimulus peregrinus (Edger et al. 2025 ), have formed multiple times in nature. The recurrent formation of allopolyploids, alongside interploidy hybridization processes as observed in I. × norrisii and other plant groups, contributes significantly to the genetic diversity that underlies the evolution of allopolyploid species. Insights from these natural events can inform and refine strategies for polyploid breeding in ornamental plants. Conclusion Interploidy hybridization has proven to be an effective strategy for accelerating polyploid breeding in I. × norrisii . Chromosome counting of hybrid progeny, along with cytological observations of meiotic behavior in parental MMCs, indicated that diploid individuals produce unreduced male gametes. These gametes transmit abundant genetic variation from diploid parents to tetraploid offspring, thereby enriching floral phenotypic diversity. However, this pathway is largely inactive diploids serve as the maternal parent. The use of mixoploid individuals increased the yield of tetraploid progeny, improving the overall efficiency of interploidy hybridization. While the ornamental traits of I. × norrisii tetraploids has been broadened, future investigations will focus on further enhancing tetraploid floral phenotypic diversity and ornamental characteristics that surpass those found in diploids. Declarations Author contributions All authors contributed to the study conception and design. Material preparation was conducted by J. T., L. D., R. L. and J. X.. Data collection and analysis were performed by J. T. and L. D.. The first draft of the manuscript was written by J. T.. Y. G., Y. L. and Z. W. commented on previous versions of the manuscript. Y. G. acquired funding. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (No. 32371943). Data availability The datasets generated and analysed during the current study are available from the corresponding author on reasonable request. Competing i nterests The authors have no relevant financial or non-financial interests to disclose. References Aleza P, Juárez J, Cuenca J, Ollitrault P, Navarro L (2012) Extensive citrus triploid hybrid production by 2x × 4x sexual hybridizations and parent-effect on the length of the juvenile phase. Plant Cell Rep 31 (9):1723–1735. doi: 10.1007/s00299-012-1286-0 De Marco Zompit M, Stucki M (2021) Mechanisms of genome stability maintenance during cell division. Dna Repair 108:103215. doi: https://doi.org/10.1016/j.dnarep.2021.103215 Ding L, Liu R, Gao Y, Xiao J, Lv Y, Zhou J, Zhang Q (2023) Effect of tetraploidization on morphological and fertility characteristics in Iris × norrisii Lenz. 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Curr Sci India 84 (6):813–816 Warburton B, Hamblen M (1978) The world of irises.The American Iris Society Zhang J, Wei Z, Li D, Li B (2009) Using SSR markers to study the mechanism of 2n pollen formation in Populus × euramericana (Dode) Guinier and P . × popularis . Ann Forest Sci 66 (5):506. doi: 10.1051/forest/2009032 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 03 Oct, 2025 Read the published version in Euphytica → Version 1 posted Editorial decision: Revision requested 11 Jul, 2025 Reviews received at journal 08 Jul, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviewers invited by journal 13 Jun, 2025 Editor assigned by journal 10 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 07 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. We do this by developing innovative software and high quality services for the global research community. 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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-6843341","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":471892380,"identity":"034bee07-e543-4a56-9a48-2cac6a585216","order_by":0,"name":"Jiacheng Tang","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Jiacheng","middleName":"","lastName":"Tang","suffix":""},{"id":471892382,"identity":"974046b8-3380-49f2-89f1-550f7b0b748c","order_by":1,"name":"Liya Ding","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Liya","middleName":"","lastName":"Ding","suffix":""},{"id":471892383,"identity":"9c47328f-c37c-43b8-b7dd-55a11672a6f3","order_by":2,"name":"Yike Gao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYJACxoaKA8zsDUAWD/Fazhxg5jlAkpbGtgMMxGuRdz98THLmvDvsPBIJjA/etjHImxPSYngmLU1y47ZnzEAtzIZz2xgMdzYQ0tKQYyb5cNthZnuJBDZp3jaGBIMDhLT0vwFqmXMYZAv7b6K0yEsAbdnYANbCxkyUFgOJZ8mWM44B/cLzsFlyzjkJww0EbelPPnizp+ZOMg978sEPb8ps5AnbcoCBRQJIJ4NiFEhLEFAPsqWBgfkDkLYjrHQUjIJRMApGLAAAi8VAim1Krj0AAAAASUVORK5CYII=","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Yike","middleName":"","lastName":"Gao","suffix":""},{"id":471892384,"identity":"a6576c70-59d6-4ac3-9f5d-b75df17992e1","order_by":3,"name":"Rong Liu","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Liu","suffix":""},{"id":471892385,"identity":"2c3173f5-ac39-4747-a2d5-51365a10400e","order_by":4,"name":"Jianhua Xiao","email":"","orcid":"","institution":"China National Botanical Garden","correspondingAuthor":false,"prefix":"","firstName":"Jianhua","middleName":"","lastName":"Xiao","suffix":""},{"id":471892387,"identity":"457c3da7-e0cf-4b98-93ed-9beef479fc2a","order_by":5,"name":"Yi Lv","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Lv","suffix":""},{"id":471892389,"identity":"6ba2983c-2e35-49af-bb37-d1be732f99b7","order_by":6,"name":"Ziyi Wang","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Ziyi","middleName":"","lastName":"Wang","suffix":""},{"id":471892390,"identity":"83eb4bfe-d89e-49d0-a2b1-33aebdadc39d","order_by":7,"name":"Qixiang Zhang","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Qixiang","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-06-07 14:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6843341/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6843341/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10681-025-03617-1","type":"published","date":"2025-10-03T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84777744,"identity":"924c6b41-2dad-443b-a4c5-28ed1ad3e0e2","added_by":"auto","created_at":"2025-06-17 09:09:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9490372,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome counting results of hybrid progeny. Bar = 10 µm. a 4X × 2X progeny, 2\u003cem\u003en\u003c/em\u003e = 4\u003cem\u003ex\u003c/em\u003e = 64; b 4X × 2X progeny, 2\u003cem\u003en\u003c/em\u003e = 4\u003cem\u003ex\u003c/em\u003e - 2 = 62; c 2X × 4X progeny, 2\u003cem\u003en\u003c/em\u003e = 4\u003cem\u003ex\u003c/em\u003e= 32; d 4X × C0 progeny, 2\u003cem\u003en\u003c/em\u003e = 4\u003cem\u003ex\u003c/em\u003e = 64; e 4X × C0 progeny, 2\u003cem\u003en\u003c/em\u003e= 4\u003cem\u003ex\u003c/em\u003e - 2 = 62\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6843341/v1/3f37bd048632fc1ff676d2c7.png"},{"id":84777748,"identity":"f514fb93-0784-4392-8921-b0f042d0a6c9","added_by":"auto","created_at":"2025-06-17 09:09:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11666623,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome abnormalities in meiotic divisions of diploid (a) and tetraploid (b-l) \u003cem\u003eI × norrisii\u003c/em\u003e microspore mother cells. Bar = 50µm. a Diakinesis, bivalents (black arrow) and univalents (white arrow); b Diakinesis, multivalents (black arrow) and univalents (white arrow); c Metaphase I, chromosomes failing to congression (black arrow); d Early anaphase Ⅰ, unequal segregation; e Late anaphase I, Chromosome bridge; f Late anaphase I, Lagging chromosomes; g Prophase Ⅱ, perpendicular spindle; h Anaphase Ⅱ, Asynchronous separation; i Telophase Ⅱ, micronucleus; j Triad; k c (black arrow); l Polyad Phenotypic analysis of hybrid progeny\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6843341/v1/6cef21467202b978debb25cf.png"},{"id":84780302,"identity":"c9fbec5e-a95f-4cea-a6ea-eef1789cda43","added_by":"auto","created_at":"2025-06-17 09:25:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3536901,"visible":true,"origin":"","legend":"\u003cp\u003eFlower colour and flower morphology of \u003cem\u003eI × norrisii \u003c/em\u003etetraploid hybrid progeny, and stigma morphologies of Iris domestica-type flower and Iris dichotoma-type flower. a-f Bar = 1 cm; g-h Bar = 2 mm. a C3 generation plants, purple, Iris dichotoma-type flower; b C3 generation plants, light purple, Iris dichotoma-type flower; c 4X × 2X progeny, white, Iris dichotoma-type flower; d 4X × 2X progeny, pink, Iris dichotoma-type flower; e 4X × C0 progeny, orange, Iris domestica-type flower; f 4X × C0 progeny, burgundy, Iris domestica-type flower; g Iris domestica-type stigma morphology; h Iris dichotoma-type stigma morphology\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6843341/v1/b8b419df0c9c1dfa49e3916c.png"},{"id":92884649,"identity":"b8caa69c-0e33-4259-8aae-fd3be9ea5180","added_by":"auto","created_at":"2025-10-06 16:13:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":25257487,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6843341/v1/1ef93d95-7083-4d59-8a4d-ad4dbe027731.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Interploidy hybridization enhances floral phenotypic diversity of Iris × norrisii tetraploids","fulltext":[{"header":"Introduction","content":"\u003cp\u003eInterploidy hybridization has been extensively applied in plant breeding programs. Crosses between diploid and tetraploid parents have been employed to develop asexual triploid fruits, such as seedless \u003cem\u003eCitrullus lanatus\u003c/em\u003e (Thomas et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and citrus varieties (Aleza et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In ornamental breeding, varieties of desirable traits from diploid cultivars have been successfully introgressed into tetraploids, as demonstrated in Bearded Irises (Schreiner \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1945\u003c/span\u003e; Pane-Joyce \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1995\u003c/span\u003e) and Aril Irises (Warburton and Hamblen \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1978\u003c/span\u003e). Additionally, the use of mixoploid Louisiana Iris cultivars has facilitated the improvement of floral diversity in tetraploid populations (Mertzweiller \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1986\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite these successful applications, the potential of interploidy hybridization to enrich floral trait diversity in newly induced polyploid ornamental species remains underexplored. Recently, tetraploid plants of \u003cem\u003eIris\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e were successfully induced through colchicine treatment (Ding et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Although these tetraploids exhibited larger flowers and seeds compared to their diploid counterparts, their floral phenotypes were strikingly uniform, predominantly exhibiting purple flowers. Given that effective breeding in polyploid ornamental plants often relies on generating and selecting diverse phenotypes, expanding the floral variation of tetraploid \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e is essential for developing novel cultivars with enhanced ornamental value.\u003c/p\u003e \u003cp\u003eTo address this challenge, interploidy hybridizations were conducted between tetraploid \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e plants and both diploid and mixoploid lines, leveraging the rich floral diversity present in diploid populations. The resulting progenies were evaluated for floral traits, performed chromosome counts, and examined the meiotic behavior of parental microspore mother cells to elucidate the cytological mechanisms underlying hybrid formation. This study aims to assess the feasibility of using interploidy hybridization to diversify floral traits in tetraploid \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e, offering insights into accelerating polyploid breeding efforts in ornamental plants.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003ePlant materials\u003c/p\u003e \u003cp\u003eTetraploid plants of the C1 generation of \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e were obtained as previously reported by Ding et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The C2 generation was obtained through self-pollination and intercrossing among C1 individuals. Both C1 and C2 generation were served as the tetraploid parents in the hybridization.\u003c/p\u003e \u003cp\u003eThe diploid hybrid population was generated by crossing diploid \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e individuals with elite cultivars. Additionally, mixoploid plants were induced via colchicine treatment, following the method of Ding et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). All plant materials were cultivated in an open-field nursery at Beijing Forestry University (40\u0026deg;09\u0026prime; N, 116\u0026deg;26\u0026prime; E).\u003c/p\u003e \u003cp\u003eHybridization experiment\u003c/p\u003e \u003cp\u003eReciprocal crosses were performed between tetraploids and diploids (designated as 4X \u0026times; 2X and 2X \u0026times; 4X) and between tetraploids and mixoploids (designated as 4X \u0026times; C0 and C0 \u0026times; 4X), using mixed pollen for pollination. Self-pollination and intra-ploidy crosses within diploid and C2 populations served as controls (designated as 2X \u0026times; 2X and 4X \u0026times; 4X, respectively).\u003c/p\u003e \u003cp\u003eFor each cross, the numbers of pollinations, fruits, plump ovules per fruit, and seeds were recorded. Additionally, fruit set rate, germination rate, and seedling rate were calculated. The number of plump ovules per fruit was determined by dividing the total number of plump ovules produced after 14 days of pollination by the number of fruits. Seedling rate was calculated as the number of progeny obtained divided by the number of pollinations\u003c/p\u003e \u003cp\u003eChromosome counting in hybrid progeny\u003c/p\u003e \u003cp\u003eChromosome numbers were determined following the protocol of Ding et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Root tips and developing ovaries were collected, pretreated with saturated p-dichlorobenzene in the dark, rinsed with distilled water and fixed in Carnoy's solution (ethanol: acetic acid\u0026thinsp;=\u0026thinsp;3:1, v/v). After fixation, tissues were hydrolyzed in hydrochloric acid, rinsed again, and stained with Carbol fuchsin (Solarbio, Beijing, China). Chromosomes were observed using a Soptop CX40 microscope.\u003c/p\u003e \u003cp\u003eMeiotic observation in diploid and tetraploid parents\u003c/p\u003e \u003cp\u003eMeiosis behavior in microspore mother cells (MMCs) of diploid and tetraploid parents was examined. Developing flower buds were fixed in a fixative solution (ethanol: acetic acid: chloroform\u0026thinsp;=\u0026thinsp;3: 2: 5, v/v), rinsed with distilled water, and dissected to extract anthers.\u003c/p\u003e \u003cp\u003eThe other contents were squashed on microscope slides, stained with Carbol fuchsin (Solarbio, Beijing, China), and examined under a Soptop CX40 microscope. The total number of MMCs and those showing chromosomal abnormalities were recorded at various meiotic stages, and the frequency of abnormalities was calculated for each stage.\u003c/p\u003e \u003cp\u003eEvaluation of hybrid progeny phenotypes\u003c/p\u003e \u003cp\u003eFloral traits of the hybrid progeny were evaluated in the outdoor nursery at Beijing Forestry University. Each individual was assessed for floral morphology, including the characteristics of perianth segments, pistils, and stamens, and classified into floral types.\u003c/p\u003e \u003cp\u003ePerianth color was recorded, analyzed statistically, and documented through digital photography.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eHybridization experiment\u003c/p\u003e \u003cp\u003eThe number of pollinations, fruits, plump ovules per fruit, seeds, fruit set rate, germination rate and seedling rate of each hybrid combination are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFruit set rates in interploidy crosses were lower than those observed in the control groups (2X \u0026times; 2X and 4X \u0026times; 4X). Among interploidy crosses, fruit set rates were higher when tetraploids served as the maternal parent compared to the backcrossing. Notably, the seedling rate of 4X \u0026times; C0 (37.72%) was higher than that of the 4X \u0026times; 4X control (19.57%). Conversely, the seedling rates of 4X \u0026times; 2X, 2X \u0026times; 4X, and C0 \u0026times; 4X were significantly lower than that of the 4X \u0026times; 4X control.\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\u003eResults of hybridization experiment\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCrossing Types\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of Pollinations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of Fruits\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFruit Set Rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNo. of Plump Ovules per Fruit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eNo. of Seeds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGermination Rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSeedling Rate (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4X \u0026times; 2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e650\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2X \u0026times; 4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e865\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e34.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4X \u0026times; C0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e15.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e38.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e37.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC0 \u0026times; 4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4X \u0026times; 4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e17.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e17.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e19.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2X \u0026times; 2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-\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\u003eChromosome counting of hybrid progeny\u003c/p\u003e \u003cp\u003eSeveral individuals from the progenies of 4X \u0026times; 2X, 2X \u0026times; 4X, and 4X \u0026times; C0 crosses were randomly selected for chromosome counting. Among the four offspring from the 4X \u0026times; 2X crosses, three were tetraploid (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;64, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea) and one was aneuploid (2n\u0026thinsp;=\u0026thinsp;4x \u0026minus;\u0026thinsp;2\u0026thinsp;=\u0026thinsp;62, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). All five offspring from the 2X \u0026times; 4X crosses were diploid (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;32, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Among the five samples from the 4X \u0026times; C0 crosses, three were tetraploid (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;64, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), while the remaining two were aneuploid (2n\u0026thinsp;=\u0026thinsp;4x \u0026minus;\u0026thinsp;2\u0026thinsp;=\u0026thinsp;62, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eObservation of the meiosis in diploid and tetraploid microspore mother cells\u003c/p\u003e \u003cp\u003eA total of 609 tetraploid and 547 diploid microspore mother cells (MMCs) were examined to assess meiotic behavior.\u003c/p\u003e \u003cp\u003eBoth ploidy levels exhibited common meiotic abnormalities. During metaphase I, over 40% of MMCs at both ploidy levels exhibited chromosomes failing to congress at the equatorial plate (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Subsequently, unequal chromosome segregation was observed at early anaphase I (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), and chromosome bridges (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) were noted from early to late anaphase I, with the highest frequencies recorded during early anaphase I (29.2% in diploids; 33.3% in tetraploids). Chromosomes involved in bridges formation either separated rapidly or broke directly. Lagging chromosomes were primarily observed in anaphase I (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef), leading to micronuclei that persisted through meiosis II and into the tetrad stage. Ultimately, both diploid and tetraploid MMCs produced triads at the tetrad stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej).\u003c/p\u003e \u003cp\u003eDespite these similarities, notable differences were evident. Tetraploid MMCs exhibited a higher frequency of meiotic abnormalities compared to diploids. At diakinesis, diploid MMCs displayed univalents (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), whereas tetraploid MMCs exhibited both univalents and multivalents (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). At early Anaphase I, unequal chromosome segregation was more frequent in tetraploids. Additionally, tetraploid MMCs displayed a low frequency of chromosome bridges during metaphase I and exhibited occasional perpendicular spindles during prophase II and polyads at the tetrad stage.\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\u003eFrequency of meiotic anomalies in microspore mother cells in tetraploids and diploids\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"15\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c15\" colnum=\"15\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003ePloidy Level\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eTotal Abnormal Rate ( % )\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"13\" nameend=\"c15\" namest=\"c3\"\u003e \u003cp\u003eAbnormal rate of different phases of meiosis of microspore mother cells\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eMetaphase Ⅰ (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eEarly Anaphase I (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eLate Anaphase Ⅰ (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003eProphase Ⅱ (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c12\" namest=\"c11\"\u003e \u003cp\u003eAnaphase Ⅱ \u0026amp; Telophase Ⅱ (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c15\" namest=\"c13\"\u003e \u003cp\u003eTetrad Stage (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003echromosomes failing to congression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChromosome Bridge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eUnequal Segregation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eChromosome Bridge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eLagging Chromosomes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChromosome Bridge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMicronucleus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003ePerpendicular Spindle\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eAsynchronous Separation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eMicronucleus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eTriad\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c14\"\u003e \u003cp\u003eMicronucleus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c15\"\u003e \u003cp\u003ePolyad\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e46.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e51.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e10.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e28.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e2.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e18.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e15.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e3.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e41.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e29.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e9.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e8.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c14\"\u003e \u003cp\u003e5.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c15\"\u003e \u003cp\u003e0.0\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\u003e \u003c/p\u003e \u003cp\u003ePhenotypic analysis of hybrid progeny\u003c/p\u003e \u003cp\u003eAll hybrid progeny exhibited distinct floral phenotypes, which were categorized into two major types based on petal and stigma morphology: Iris dichotoma-type and Iris domestica-type. Iris domestica-type flowers displayed outer perianths without noticeable reflexing and revolute margins, inner perianths with triangular notches at the tips, and obtuse, rounded stigmatic appendages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg). Conversely, Iris dichotoma-type flowers were characterized by outer perianth segments that were transversely reflexed at the middle with revolute margins, inner perianths with trapezoidal notches at the tips, and pointed stigmatic appendages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh).\u003c/p\u003e \u003cp\u003eFurther phenotypic variation was observed among different crossing combinations. In the 4X \u0026times; 4X control group, 20 seedlings were evaluated for perianth color: 16 exhibited purple (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), two displayed light purple (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), one showed white (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), and one exhibited orange perianth color (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Notably, the orange-flowered individual exhibited the Iris domestica-type morphology, whereas the purple, light purple, and white flowers corresponded to the Iris dichotoma-type.\u003c/p\u003e \u003cp\u003eAmong the 4X \u0026times; 2X crosses, six of nine offspring flowered, producing three purple, one white, one orange, and one pink (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed) perianth colors. Similarly, in the 2X \u0026times; 4X crosses, five of eight offspring flowered, yielding two pink, two orange, and one pink-yellow bicolor perianths.\u003c/p\u003e \u003cp\u003eThe 4X \u0026times; C0 crosses produced 59 offspring. Most individuals exhibited Iris domestica-type morphology, with 23 orange and 31 burgundy-colored flowers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef). Only two individuals displayed Iris dichotoma-type morphology, bearing purple and light purple perianth colors. Additionally, perianth spotting varied considerably, ranging from strip-like to punctate patterns, with distribution areas extending from full to partial petal coverage and varying in density from sparsely to densely merged patterns.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePreferential production of unreduced male gametes diploid \u003cem\u003eI\u003c/em\u003e \u0026times; \u003cem\u003enorrisi\u003c/em\u003e\u003c/p\u003e \u003cp\u003eA key finding of this study is the preferential production of unreduced male gametes over unreduced female gametes in diploid \u003cem\u003eI\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e, which offers insights into reproductive barriers between different ploidy levels.\u003c/p\u003e \u003cp\u003eIn 4X \u0026times; 2X crosses, tetraploid and near-tetraploid aneuploids were obtained, suggesting that unreduced (2\u003cem\u003en\u003c/em\u003e) male gametes were produced by diploid individuals. Cytological evidence further supports this inference, as triads containing 2\u003cem\u003en\u003c/em\u003e pollen grains were observed in diploid MMC. When such unreduced pollen fertilizes normal female gametes from tetraploids, the resulting offspring can be tetraploid or aneuploid. Similar patterns of 2\u003cem\u003en\u003c/em\u003e pollen production through triads have been reported in other species such as \u003cem\u003ePopulus \u0026times; euramericana\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and \u003cem\u003eDimocarpus longan\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), indicating a conserved mechanism across diverse taxa.\u003c/p\u003e \u003cp\u003eConversely, in 2X \u0026times; 4X crosses where diploid individuals served as maternal parents, only diploid offspring were obtained. This result suggests two key inferences. First, \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e exhibits a limited capacity for producing unreduced female gametes, suggesting relatively greater stability during megaspore meiosis compared to microspore meiosis. Second, the tetraploid parent likely contributed haplotype male gametes containing 16 chromosomes, possibly due to meiotic abnormalities. This pattern differs from that observed in species such as Hieracium echioides (Peckert and Chrtek \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and Campanula rotundifolia (Sutherland and Galloway \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), where interploidy crosses produced a small number of diploid progeny but commonly yielded triploid and tetraploid progeny. These differences may reflect species-specific variation in the formation of unreduced female gametes.\u003c/p\u003e \u003cp\u003eSeveral meiotic irregularities observed in both diploid and tetraploid parental lines likely contribute to the production of unreduced male gametes and aneuploid offspring. These include univalents and multivalents during pachytene, lagging chromosomes, chromosome bridges, and micronuclei. Univalents and multivalents can lead to unequal chromosome segregation, while lagging chromosomes and bridges\u0026mdash;hallmarks of chromosomal instability (Rodriguez-Mu\u0026ntilde;oz et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u0026mdash;may give rise to micronuclei and trigger breakage-fusion-bridge cycles (De Marco Zompit and Stucki \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) These abnormalities likely underlie the production of unreduced pollen with abnormal chromosome numbers, ultimately resulting in the observed aneuploid progeny.\u003c/p\u003e \u003cp\u003eDiploids and mixoploids enrich floral phenotypic diversity in tetraploids via unreduced male gametes\u003c/p\u003e \u003cp\u003eThe results of this study demonstrate that diploid and mixoploid individuals can enhance the floral phenotypic diversity of \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e tetraploid progeny through interploidy hybridization.\u003c/p\u003e \u003cp\u003eThe tetraploid parental plants (C1 and C2 generations) exhibited limited perianth coloration, primarily purple and white. In contrast, novel perianth colors such as light purple and orange were observed in the 4X \u0026times; 4X control crosses, while interploidy crosses (4X \u0026times; 2X and 4X \u0026times; C0) produced even greater diversity, including light purple, orange, burgundy, and pink flowers. Additionally, distinct floral morphologies resembling Iris dichotoma-type and Iris domestica-type were observed in both control and interploidy progeny.\u003c/p\u003e \u003cp\u003eThe diploid parental population was a genetically diverse hybrid group with highly variable perianth coloration, while the mixoploid individuals were obtained by colchicine treatment of seeds derived from this genetically diverse diploid population. As a result, both diploid and mixoploid parents carried abundant genetic variation. Combined with cytological observations, these findings suggest that diploids and mixoploids transmitted their genetic diversity to the tetraploid progeny via unreduced male gametes, thereby enriching floral phenotypic traits in the tetraploid background. Additionally, a higher seedling rate was observed in the 4X \u0026times; C0 crosses than in the 4X \u0026times; 2X crosses, implying that mixoploids may exhibit greater reproductive compatibility with tetraploids than diploids do.\u003c/p\u003e \u003cp\u003eThis phenomenon of generating phenotypic diversity via interploidy hybridization has significant evolutionary implications. Several recently formed polyploid species, such as \u003cem\u003eSenecio cambrensis\u003c/em\u003e and \u003cem\u003eMimulus peregrinus\u003c/em\u003e (Edger et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), have formed multiple times in nature. The recurrent formation of allopolyploids, alongside interploidy hybridization processes as observed in \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e and other plant groups, contributes significantly to the genetic diversity that underlies the evolution of allopolyploid species. Insights from these natural events can inform and refine strategies for polyploid breeding in ornamental plants.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eInterploidy hybridization has proven to be an effective strategy for accelerating polyploid breeding in \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e. Chromosome counting of hybrid progeny, along with cytological observations of meiotic behavior in parental MMCs, indicated that diploid individuals produce unreduced male gametes. These gametes transmit abundant genetic variation from diploid parents to tetraploid offspring, thereby enriching floral phenotypic diversity. However, this pathway is largely inactive diploids serve as the maternal parent. The use of mixoploid individuals increased the yield of tetraploid progeny, improving the overall efficiency of interploidy hybridization. While the ornamental traits of \u003cem\u003eI.\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e tetraploids has been broadened, future investigations will focus on further enhancing tetraploid floral phenotypic diversity and ornamental characteristics that surpass those found in diploids.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp; All authors contributed to the study conception and design. Material preparation was conducted by J. T., L. D., R. L. and J. X.. Data collection and analysis were performed by J. T. and L. D.. The first draft of the manuscript was written by J. T.. Y. G., Y. L. and Z. W. commented on previous versions of the manuscript. Y. G. acquired funding. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was supported by the National Natural Science Foundation of China (No. 32371943).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp; The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ei\u003c/strong\u003e\u003cstrong\u003enterests\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAleza P, Ju\u0026aacute;rez J, Cuenca J, Ollitrault P, Navarro L (2012) Extensive citrus triploid hybrid production by 2x \u0026times; 4x sexual hybridizations and parent-effect on the length of the juvenile phase. 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Ann Forest Sci 66 (5):506. doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1051/forest/2009032\u003c/span\u003e\u003cspan address=\"10.1051/forest/2009032\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\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":"Iris × norrisii, tetraploid, interploidy hybridization, unreduced gamete","lastPublishedDoi":"10.21203/rs.3.rs-6843341/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6843341/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInterploidy hybridization has been widely utilized in plant breeding and cultivar improvement. In ornamental species, interploidy hybridization between diverse diploid lines and synthetic tetraploids enables the transfer of rich diploid variation to polyploid backgrounds. Although previously applied in the breeding of Irises and other ornamentals, this approach remains insufficiently explored in synthetic polyploid cultivars. To address the limited floral variation observed in artificial tetraploid lines of \u003cem\u003eIris\u003c/em\u003e \u0026times; \u003cem\u003enorrisii\u003c/em\u003e, we performed interploidy crosses between tetraploid plants and both diploid and mixoploid lines. Hybrid progenies were evaluated based on floral traits, chromosome counts, and the meiotic behavior of microspore mother cells in parental plants. Crosses using tetraploid plants as female parents yielded tetraploid or near-tetraploid aneuploid progeny, whereas those with diploid female parents produced only diploid offspring. Cytological analysis revealed meiotic abnormalities in both diploid and tetraploid, contributing to the formation of unreduced male gametes. Interploidy hybrids displayed a broader range of perianth colors than control tetraploid crosses, whereas floral morphological variation was similar between the two groups. These findings demonstrate that diploid \u003cem\u003eI. \u0026times; norrisii\u003c/em\u003e contributes to the diversification of floral traits in tetraploid lines via the transmission of genetic variation through unreduced male gametes. Interploidy hybridization is a promising approach for enhancing phenotypic diversity in polyploid ornamental breeding.\u003c/p\u003e","manuscriptTitle":"Interploidy hybridization enhances floral phenotypic diversity of Iris × norrisii tetraploids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 09:09:07","doi":"10.21203/rs.3.rs-6843341/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-11T10:20:03+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-08T09:23:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286515539373447457797960312146395249316","date":"2025-06-16T10:07:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-13T08:42:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-10T14:37:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T14:35:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Euphytica","date":"2025-06-07T14:14:01+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":"b9ee24f6-277d-4d7e-88a8-db7e4bd46cb7","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T16:11:30+00:00","versionOfRecord":{"articleIdentity":"rs-6843341","link":"https://doi.org/10.1007/s10681-025-03617-1","journal":{"identity":"euphytica","isVorOnly":false,"title":"Euphytica"},"publishedOn":"2025-10-03 15:57:32","publishedOnDateReadable":"October 3rd, 2025"},"versionCreatedAt":"2025-06-17 09:09:07","video":"","vorDoi":"10.1007/s10681-025-03617-1","vorDoiUrl":"https://doi.org/10.1007/s10681-025-03617-1","workflowStages":[]},"version":"v1","identity":"rs-6843341","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6843341","identity":"rs-6843341","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}