A neofunctionalized S-locus enables partial self-incompatibility in alfalfa

A neofunctionalized S-locus enables partial self-incompatibility in alfalfa | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article A neofunctionalized S-locus enables partial self-incompatibility in alfalfa Yongbiao Xue, Lizhen Song, Hong Zhao, Hongkui Zhang, Lan Zhou, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8740276/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Self-incompatibility (SI) typically enforces binary pollen rejection 1-5 , forming an all-or-none system that prevents self-pollination and promotes out-crossing. The third-largest angiosperm family, Fabaceae, lacks the canonical S-RNase-based SI and exhibits enigmatic, diversified SI 6-11 . Here, we uncover a neofunctionalized, Medicago-specific S-locus in autotetraploid alfalfa (Medicago sativa) that operates as a quantitative rheostat rather than an all-or-none switch. The locus comprises a female S determinant encoding the Fabaceous Inverted Repeat (IR)-lacking clade-specific S-RNase (FIRS-RNase) bearing an N-terminal intrinsically disordered region. This is linked to male S determinants of pollen-expressed F-box proteins carrying a DUF295 domain (S-DUF295 proteins). The FIRS-RNase undergoes liquid-liquid phase separation to form dynamic condensates; cognate S-DUF295 proteins co-condense and selectively amplify condensate size and toxicity, while the 26S proteasome rapidly clears non-self combinations. A stochastic tetraploid model, integrating allele-specific S-DUF295 expression gradients, accurately predicts the observed continuum of pollen arrest from stigma to ovary. Evolutionarily, the FIRS-RNase module likely originated in an ancestor of the IRL clade ~44 million years ago (Mya), whereas S-DUF295 emerged ~24 Mya within Medicago, following a “female-first, male-later” trajectory recurrent across angiosperm SI systems. Our quantitative mechanism elucidates how Medicago re-evolved SI following ancestral loss of the canonical S-RNase pathway, demonstrating convergent evolution of tunable reproductive barriers. Biological sciences/Plant sciences/Plant reproduction/Fertilization Biological sciences/Genetics/Gene expression Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryTables.xlsx Supplementary Tables 1-10 ExtendedDataFigures.pdf Extended Data Figures 1-20 Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8740276","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":631227705,"identity":"e48c75c1-ec35-440a-bdbc-1c3147a9d7b0","order_by":0,"name":"Yongbiao 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The third-largest angiosperm family, Fabaceae, lacks the canonical S-RNase-based SI and exhibits enigmatic, diversified SI\u003csup\u003e6-11\u003c/sup\u003e. Here, we uncover a neofunctionalized, Medicago-specific S-locus in autotetraploid alfalfa (Medicago sativa) that operates as a quantitative rheostat rather than an all-or-none switch. The locus comprises a female S determinant encoding the Fabaceous Inverted Repeat (IR)-lacking clade-specific S-RNase (FIRS-RNase) bearing an N-terminal intrinsically disordered region. This is linked to male S determinants of pollen-expressed F-box proteins carrying a DUF295 domain (S-DUF295 proteins). The FIRS-RNase undergoes liquid-liquid phase separation to form dynamic condensates; cognate S-DUF295 proteins co-condense and selectively amplify condensate size and toxicity, while the 26S proteasome rapidly clears non-self combinations. A stochastic tetraploid model, integrating allele-specific S-DUF295 expression gradients, accurately predicts the observed continuum of pollen arrest from stigma to ovary. Evolutionarily, the FIRS-RNase module likely originated in an ancestor of the IRL clade ~44 million years ago (Mya), whereas S-DUF295 emerged ~24 Mya within Medicago, following a “female-first, male-later” trajectory recurrent across angiosperm SI systems. 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