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    A selfish genetic element confers non-Mendelian inheritance in rice Xiaowen Yu1*, Zhigang Zhao1*, Xiaoming Zheng2, Jiawu Zhou3, Weiyi Kong1, Peiran Wang1, Wenting Bai1, Hai Zheng1, Huan Zhang1, Jing Li3, Jiafan Liu2, Qiming Wang1, Long Zhang1, Kai Liu1, Yang Yu1, Xiuping Guo2, Jiulin Wang2, Qibing Lin2, Fuqing Wu2, Yulong Ren2, Shanshan Zhu2, Xin Zhang2, Zhijun Cheng2, Cailin Lei2, Shijia Liu1, Xi Liu1, Yunlu Tian1, Ling Jiang1, Song Ge4, Chuanyin Wu2†, Dayun Tao3†, Haiyang Wang2†, Jianmin Wan2†

    Selfish genetic elements are pervasive in eukaryote genomes, but their role remains controversial. We show that qHMS7, a major quantitative genetic locus for hybrid male sterility between wild rice (Oryza meridionalis) and Asian cultivated rice (O. sativa), contains two tightly linked genes [Open Reading Frame 2 (ORF2) and ORF3]. ORF2 encodes a toxic genetic element that aborts pollen in a sporophytic manner, whereas ORF3 encodes an antidote that protects pollen in a gametophytic manner. Pollens lacking ORF3 are selectively eliminated, leading to segregation distortion in the progeny. Analysis of the genetic sequence suggests that ORF3 arose first, followed by gradual functionalization of ORF2. Furthermore, this toxin-antidote system may have promoted the differentiation and/ or maintained the genome stability of wild and cultivated rice.

    I dentifying so-called speciation genes that cause reproductive isolation is a central goal in evolutionary biology. Postzygotic repro- ductive isolation (PRI), embodied by hybrid sterility, inviability, or weakness, drives specia-

    tion and maintains species identity by restricting gene flow between populations (1, 2). The Bateson- Dobzhansky-Muller model postulates that hybrid reproductive isolation results from deleterious interactions among at least two loci from evolu- tionarily divergent populations (3). Accumulating evidence suggests that selfish genetic elements (SGEs), DNA sequences that gain a transmission advantage relative to the rest of the genome, could drive genome evolution by causing hybrid incompatibilities and segregation distortion in different organisms (4–10); however, the role of SGEs in genome evolution and their underlying molecular mechanisms have remained obscure. We performed quantitative trait locus (QTL)

    analysis of a backcross F1 (BC1F1) population derived from the cross between two highly di- vergent rice species—wild rice, O. meridionalis accession 82031 (Mer), andO. sativa ssp. japonica, Dianjingyou1 (DJY1)—and detected four major QTLs controlling hybrid male sterility (fig. S1,

    A and B). Of these, qHMS7 is located near sev- eral previously identified QTLs (fig. S1C). To clone the causal gene(s) for qHMS7, we

    developed a near-isogenic line for qHMS7 (NIL- qHMS7) (fig. S1D). Intriguingly, the NIL-qHMS7 plant can only be maintained in a heterozygous status at the qHMS7 locus in theDJY1 background. Examination of the self-pollinated progeny of NIL-qHMS7 (BC6F2) revealed a bimodal distribu- tion for pollen fertility (fig. S2). Genotyping re- vealed that the semi-sterile plants were of the DJY1/Mer genotype (D/M) and the fully fertile plants were of the DJY1/DJY1 genotype (D/D)

    at the qHMS7 locus.NoMer/Mer type (M/M) plant was detected. The seed-set rate of all 50 randomly selected plants was above 85% (fig. S3), indicating that this locus did not affect female gametes and seed setting. These results suggest that qHMS7 acts as a single locus conferring male semi- sterility and that the Mer-type pollens were not transmissible to the progeny. This notion was further supported by a gametophytic transmis- sion assay using three genetic populations of DJY1 and NIL-qHMS7 (table S1). Histological and cellular examination of various stages of pollens revealed that Mer-type pollens aborted before the tricellular stage in NIL-qHMS7 (Fig. 1, A to E, and figs. S4 to S7), possibly owing to de- fects in the second mitosis that produces tri- cellular mature pollen grains. The gene(s) underlying qHMS7 locus was de-

    limited to a 31.6-kb genomic interval by the use of amap-based cloning strategy (fig. S8 and table S2). Sequencing analysis identified three genes in the qHMS7 region in DJY1 (ORF1D,ORF2D, and ORF3), but only two genes in Mer (ORF1M and ORF2M) (Fig. 1F). ORF1D, ORF1M, and ORF3 are predicted to encode homologous, grass family– specific proteins with a mitochondrial targeting signal at the N terminus, whereas ORF2D and ORF2M are predicted to encode a ribosome- inactivating protein (RIP) domain (11) contain- ing protein conserved inmonocots,with a putative bipartite nucleus localization signal and a nucleus exporting signal. The ORF2M and ORF2D proteins differ at 11 polymorphic sites (figs. S9 to S13). Quantitative reverse transcription–polymerase

    chain reaction analysis revealed that expression of both ORF2 and ORF3 was significantly higher in stages 11 to 13 of anther development (fig. S14, A and B). ORF2 expression was lower in NIL-M/M plants [near-isogenic line with M/M genotype at the qHMS7 locus inDJY1 background (see below)]


    Yu et al., Science 360, 1130–1132 (2018) 8 June 2018 1 of 3

    1National Key Laboratory for Crop Genetics and Germplasm Enhancement, Jiangsu Plant Gene Engineering Research Center, Nanjing Agricultural University, Nanjing 210095, China. 2National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China. 3Food Crops Research Institute, Yunnan Academy of Agricultural Sciences, Kunming 650200, China. 4State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China. *These authors contributed equally to this work. †Corresponding author. Email: (J.Wan); (H.W.); (D.T.); (C.W.)


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    Fig. 1. Identification of qHMS7. (A to D) Morphology (upper panels) and stained pollen (lower panels) of DJY1, Mer, hybrid F1 (Mer × DJY1), and NIL-qHMS7. Fertile pollen is stained dark and sterile pollen is not stained. Scale bars: 50 cm in the upper panels and 50 mm in the lower panels. (E) Pollen fertility of DJY1, Mer, hybrid F1, and NIL-qHMS7, shown as mean ± SD (n = 5, 5, 7, and 10 plants examined, respectively). **P < 0.01 (versus DJY1, by Student’s t test). (F) ORFs predicted in the fine-mapped region. The insertion of ORF3 in DJY1 is indicated with a gray triangle. Arrows indicate the orientation of ORFs.

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  • at mature pollen stage, compared to that in DJY1 (fig. S14C). As expected, ORF3 expression in DJY1 was about twice as high as that in NIL-qHMS7 at mature pollen stage, and no expression ofORF3 was detected in NIL-M/M plants (fig. S14D). Transient expression of green fluorescent protein fusions of ORF2D, ORF2M, and ORF3 showed that ORF2D and ORF2M are localized to both nucleus and cytoplasm (fig. S15), whereas ORF3 is localized to the mitochondria (fig. S16). Hybrid sterility is often controlled by toxin-

    antidote or killer-protector systems that subvert Mendel’s law of segregation (12). The selective abortion of Mer-type pollens in NIL-qHMS7 but not in Mer suggests that ORF3 may encode a protein that functions as an antidote to a pollen- killing toxin produced in NIL-qHMS7. To test this notion, we transformed an intact ORF3 ge- nomic fragment from DJY1 into D/M-type calli. As expected, primary (T0) transgenic plants har- boring a single-copy transgene of ORF3 (geno- typeD/M;ORF3/-) showed a partial restoration of pollen fertility (~75%), compared to the transgene- negative plants (~50% fertility). No effect on pollen fertility was observed in the T0 transformants of ORF1D or ORF2D (table S3). In addition, T1 plants (derived from single-copy T0 transformants of ORF3) of the genotype (D/M; −/−) were semi- sterile, whereas the T1 plants of the genotype (D/M; ORF3/-) showed ~75% fertility, and the T1 plants of the (D/M;ORF3/ORF3) genotype showed normal fertility (~95%). Notably, we recovered T1 plants of the (M/M; ORF3/-) and (M/M; ORF3/ ORF3) genotypes, and bothwere fully fertile (Fig. 2 and table S4). These results indicate thatORF3has a protective function for Mer-type pollen carrying it (thus acting in a gametophytic manner). The selective abortion of Mer-type pollens in

    NIL-qHMS7 but not inMer also suggests that the pollen killer is most likely encoded by the DJY1 allele(s) at the qHMS7 locus. Consistent with this notion, the T2 progeny derived from selfed (M/M; ORF3/-) type T1 plants segregated with an ex- pected ratio of 1:2:1 for the genotypes (M/M; −/−), (M/M;ORF3/-) and (M/M;ORF3/ORF3), and they were all normally fertile (fig. S17A and table S5). To identify the gene(s) encoding the predicted toxin, we individually transformed “ORF1D,” “ORF2D,” and “ORF1D+ORF2D” into calli derived from the seeds of theNIL-M/M plants. Transgenic plants harboring “ORF2D” or “ORF1D+ORF2D” were completelymale sterile, whereas the “ORF1D” transgenic plants or transgene-negative plants were normally fertile (fig. S17, B and C, and table S6). These observations suggest thatORF2D has a pollen-killing function


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