12Despite being members of the same species, some strains of wild teosinte maintain themselves as 13 a distinct breeding population by blocking fertilization by pollen from neighboring maize plants. 14 These teosinte strains may be in the process of evolving into a separate species, since reproductive 15 barriers that block gene flow are critical components in speciation. This trait is conferred by the 16Teosinte crossing barrier1-s (Tcb1-s) haplotype, making Tcb1 a speciation gene candidate. Tcb1-17 s contains a female gene that blocks non-self-type pollen and a male function that enables self-18 type pollen to overcome that block. The Tcb1-female gene encodes a Pectin Methylesterase, 19 implying that modification of the pollen cell wall by the pistil is a key mechanism by which these 20 teosinte females reject foreign (but closely related) pollen. 21 2 22 One sentence summary: 23The Tcb1-female gene encodes a Pectin Methylesterase that in teosinte silks prevents fertilization 24 by maize pollen. 25 Short title: 26Reproductive isolation and mate rejection in Zea 27 Main text: 28Maize (Zea mays ssp mays) was domesticated from annual teosinte (Zea mays ssp 29 parviglumis) in the Balsas River valley of Mexico (1). In some locations, sympatric populations 30 of domesticated maize and annual teosinte grow in intimate associate and flower synchronously, 31 but rarely produce hybrids (2, 3). In sexually reproducing plants, reproductive barriers exist at 32 different stages, including pre-pollination, post-pollination, and post-fertilization. Post-pollination 33 barriers depend on interaction between the pollen grain and the female reproductive organs (stigma, 34 style, and ovule). In Zea mays, haplotypes at three loci, Gametophyte factor1-s (Ga1-s), 35 Gametophyte factor2-s (Ga2-s), and Teosinte crossing barrier1-s (Tcb1-s), confer Unilateral 36Cross-Incompatibility. While Ga1-s and Ga2-s are widespread in domesticated maize, Tcb1-s is 37 almost exclusively found in wild teosinte populations. The Tcb1-s haplotype, like Ga1-s and Ga2-38 s, confers unilateral cross-incompatibility against varieties carrying the tcb1 (or ga1 or ga2) 39 haplotype. Viewed otherwise, Tcb1-s provides a pollen function that overcomes the crossing 40 barrier. The latter view is preferred since pollen containing both Tcb1-s and tcb1 haplotypes 41fertilizes Tcb1-s plants, indicating that Tcb1-s compatibility is not overcome by the Tcb1-s : tcb1 42 mismatch, as is also the case for the Ga1 and Ga2 systems (4, 5). Tcb1-s was first described in 43 311The authors would like to thank Kathy Barton for the support that kept this project alive, 312 encouragement, and insight and members of the Evans and Barton labs past and present for helpful 313 discussions. We would also like to thank Beverly Oashgar for help with the screen for loss-of-314 function mutants and David Heller, Lance Cabalona, Clayton Coker, Amber Glowacki, and 315Hannah Vahldick for help growing plants and making crosses. We would also like to thank Jeffrey 316Ross-Ibarra for help i...