Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier

Liao, Ben; **ang, You-Huang; Li, Yan; Yang, Kai-Yang; Shan, Jun-**ang; Ye, Wang-Wei; Dong, Nai-Qian; Kan, Yi; Yang, Yi-Bing; Zhao, Huai-Yu; Yu, Hong-**ao; Lu, Zi-Qi; Zhao, Yan; Zhao, Qiang; Guo, Dongling; Guo, Shuang-Qin; Lei, Jie-Jie; Mu, **ao-Rui; Cao, Ying-Jie; Han, Bin; Lin, Hong-Xuan

doi:10.1038/s41467-024-45377-x

Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier

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Published: 02 February 2024

Volume 15, article number 996, (2024)
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Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier

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Ben Liao^1,2^na1,
You-Huang **ang¹^na1,
Yan Li³^na1,
Kai-Yang Yang^1,4,
Jun-**ang Shan ORCID: orcid.org/0000-0002-7235-4225^1,5,
Wang-Wei Ye^1,5,
Nai-Qian Dong ORCID: orcid.org/0000-0001-7804-1417^1,5,
Yi Kan^1,5,
Yi-Bing Yang^1,4,
Huai-Yu Zhao^1,4,
Hong-**ao Yu^1,4,
Zi-Qi Lu^1,2,
Yan Zhao³,
Qiang Zhao³,
Dongling Guo³,
Shuang-Qin Guo^1,4,
Jie-Jie Lei^1,4,
**ao-Rui Mu^1,4,
Ying-Jie Cao^1,4,
Bin Han ORCID: orcid.org/0000-0001-8695-0274³ &
…
Hong-Xuan Lin ORCID: orcid.org/0000-0002-2818-023X^1,2,4,5

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Abstract

Postzygotic reproductive isolation, which results in the irreversible divergence of species, is commonly accompanied by hybrid sterility, necrosis/weakness, or lethality in the F₁ or other offspring generations. Here we show that the loss of function of HWS1 and HWS2, a couple of duplicated paralogs, together confer complete interspecific incompatibility between Asian and African rice. Both of these non-Mendelian determinants encode the putative Esa1-associated factor 6 (EAF6) protein, which functions as a characteristic subunit of the histone H4 acetyltransferase complex regulating transcriptional activation via genome-wide histone modification. The proliferating tapetum and inappropriate polar nuclei arrangement cause defective pollen and seeds in F₂ hybrid offspring due to the recombinant HWS1/2-mediated misregulation of vitamin (biotin and thiamine) metabolism and lipid synthesis. Evolutionary analysis of HWS1/2 suggests that this gene pair has undergone incomplete lineage sorting (ILS) and multiple gene duplication events during speciation. Our findings have not only uncovered a pair of speciation genes that control hybrid breakdown but also illustrate a passive mechanism that could be scaled up and used in the guidance and optimization of hybrid breeding applications for distant hybridization.

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Introduction

Reproductive isolation (RI) hinders gene flow between intraspecific or interspecific populations during speciation, therefore contributing to the maintenance of species identity^1,2. Hybrid breakdown (HB), a typical form of RI, is defined as reduced hybrid viability and/or fertility segregation in the F₂ or the later generation. Differing from other hybrid incompatibilities, HB involves intrinsic postzygotic reproductive barriers, such as hybrid inviability (including weakness³, necrosis⁴, and chlorosis⁵) and hybrid sterility (in the male, female, or both gametes). The classical Bateson-Dobzhansky-Muller (BDM) model partially addresses hybrid sterility, the most common form of postzygotic barrier that is attributed to deleterious genetic interactions between incompatible allelic variations, but fails to consider the divergent evolution process^6,7,8. A widely accepted viewpoint that has been proposed to reasonably explain the biased allele transmission and disharmonious effect conferring plant hybrid sterility is the killer-protector system^9,10. In broad terms, genomic conflicts involved in hybrid incompatibility that occurs in sporophytes and gametophytes are basically divided into two types; single or dual locus-controlled F₁/F₂ male/female sterility models^{11,Full size image}

Ultrastructural studies of the tapetal layer showed that the tapetal membrane of WYJ7 was distinctly decomposed and precipitated around Ubisch bodies at stage 10 (Fig. 2m). A typical two-layer wall structure composed of the tectum, bactum, and nexine was also clearly visible (Figs. 2n, n1). In contrast, in the chamber of sterile anthers of NIL-hws1^WYJ7/hws2^CG14 plants, there were no clear and intact Ubisch bodies observed on the inner tangential wall of the tapetum (Fig. 2o). Remarkably large vacuoles in the thickened tapetum were irregularly formed during the process of tapetal disintegration (Fig. 2o). Moreover, parts of the exine in microspores showed abnormal protuberances with uneven bactum (Figs. 2p, p1). Consistent with the above observations, the main cytological reason behind pollen abortion is considered to be the stimulated proliferation of the tapetal cells, which interferes with microspore development. Scanning electron microscopy (SEM) observations further visualized the overall appearance features of anthers and pollen grains at maturity, revealing that abortion in hws1^WY7J/hws2^CG14-type pollen grains is possibly due to their shrunken morphology and poorly-developed anther wall (Figs. 2q, q1-q8).

Dynamic examination of embryo sac development in NIL-hws1^WYJ7/hws2^CG14 plants showed no obvious defects with respect to structure and morphology prior to the eight-nucleate embryo sac stage (Fig. 2r, s). However, the two polar nuclei occasionally failed to correctly locate and form a binucleate central cell near the micropylar end during later stages of development (Fig. 2t–w). These abortive and smaller embryo sacs were also found to be developmentally arrested, either empty or degenerated, in comparison with those in the fertile control (WYJ7) plants (Fig. 2v, w). Based on these results, we concluded that inappropriate polar nuclei arrangement, stagnant development, and abnormal cellularization could result in the inability to form mature embryo sacs during female gamete development in NIL-hws1^WYJ7/hws2^CG14 plants.

Dysfunctions in HWS1/2 contribute to H4Ac loss and transcriptomic reprogramming misregulation

EAF6 is a critical component of the nucleosomal acetyltransferase of the H3/H4 (NuA3/NuA4) complex and has been reported to participate in extensive transcriptional regulation via nuclear H4 or H2A acetylation, thereby affecting plant viability^38,39,40. Subcellular localization analysis indicated that HWS1 and HWS2 are specifically targeted to the nuclei (Fig. 3a). The in vitro histone acetylation (HAT) assay showed that HWS2 is able to acetylate a histone H4 peptide (Fig. 3b). Fractionation of chromatin followed by western blotting using antibodies against various acetylation sites in the histone H2A, H3 and H4 N-terminal tail showed a pronounced decline in acetyl-H2A and acetyl-H4 in NIL-hws1^WYJ7/hws2^CG14, and this decline was most prominent at lysine 5 and 9 of histone H2A (H2AK5Ac and H2AK9Ac) and at lysine 5 of histone H4 (H4K5Ac) among all eight histone H2A and H4 lysine residues tested (Fig. 3c). To gain more insight into the correlation between the loss of H4Ac and transcriptional reprogramming, we first performed RNA deep sequencing (RNA-seq) and identified 19,806 differentially-expressed genes (DEGs) (fold-change >2) between the two corresponding NILs (Supplementary Fig. 7a). The enriched terms from combined Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis showed that the statistically significant DEGs were primarily associated with mRNA surveillance, fatty acid, lipid metabolism, RNA transport, and small molecule binding process (Supplementary Fig. 7b, c). As shown by heat maps, the expression levels of several representative male determinants related to fatty acid synthesis and lipid transport were substantially down-regulated in NIL-hws1^WYJ7/hws2^CG14 plants (Supplementary Fig. 7d)^{41,42,43,44,45}, which may account for the adverse formation of the enlarged tapetal zone, unshaped pollen exine, and unusual cuticle surface.

**Fig. 3: Involvement of HWS1/2 regulates H4Ac modification and stabilizes transcriptional reprogramming with altered histone acetylation.**

To further visualize the in vivo differences in the degree of acetylated histone H4 at the whole-genome level, a chromatin immunoprecipitation sequencing (ChIP-seq) experiment was unitedly employed to analyze the transcriptome data. Strikingly, the average H4 occupancy in NIL-hws1^WYJ7/hws2^CG14 displayed a dramatic suppression within the peak downstream of the transcriptional start site (TSS) (Fig. 3d and Supplementary Fig. 7e). A total of 2,297 H4Ac enriched peaks corresponding to 2,175 genes were identified, the vast majority of which were down-regulated and localized at the promoter (88.98%) (Supplementary Fig. 7f). The principal binding site detected in the enriched peaks was the ORB2/RRM-type motif (GGATTTGGGG) (Supplementary Fig. 7g), which basically recruits RNA-binding proteins involved in post-transcriptional regulation.

We hereupon defined the dysregulated genes with H4Ac enrichment peaks at the promoter and/or gene body regions as candidate HWS1/2 target genes (Fig. 3e). Among the categories that overlap with those of the transcriptome datasets, genes related to vitamin metabolism (biotin and thiamine), fatty acid biosynthesis, ether lipid metabolism, and carbohydrate metabolism with reduced H4Ac levels showed altered expression (Fig. 3f and Supplementary Fig. 7h). We, therefore, selected some well-characterized sterility genes belonging to these particular pathways with a strong and uniform reduction in sequencing windows to examine their mRNA and H4Ac levels as validation (Fig. 3g)⁴⁶. We examined the expression of these potential genes by qRT-PCR assays, and all of them showed greatly decreased expression in NIL-hws1^WYJ7/hws2^CG14 plants, which is in line with the results of the transcriptional profile (Fig. 3h). In parallel, chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) results also showed a relatively lower level of histone H4 acetylation in these certain genomic regions (Fig. 3i), implying that in the absence of HWS1/2, the chromatin status is not appropriately established. Besides, determined quantities of biotin were significantly decreased in NIL-hws1^WYJ7/hws2^CG14 plants in contrast with NIL-HWS1^CG14/hws2^CG14 (Fig. 3j) and NIL-hws1^WYJ7/HWS2^WYJ7 (Fig. 3k) plants. Taken together, our findings suggest that HWS1/2 is indispensable for NuA4-dependent H4 acetylation and gene transcriptional activation by proper epigenetic modifications, and is especially essential for gametophyte development through the maintenance of fatty acid, lipid, and vitamin homeostasis.

Evolutionary trajectory and EAF6 gene duplication events in Oryza species

To trace the evolutionary origins of HWS1 and HWS2 (OsEAF6), we performed a collinearity analysis around these two distinct loci among some representative Oryza species and other distantly related gramineous crop species (Fig. 4a and Supplementary Fig. 8a–d). Comparative genomics showed that there is widespread synteny in the grass family, but that EAF6 is a single-copy gene in O.punctata (BB genome), O.officinalis (CC genome), O.brachyantha (FF genome) (Fig. 4a), and the majority of AA genome wild progenitors (Supplementary Fig. 8a, b), indicating that the duplication of EAF6 pairs may have evolved de novo in the O.sativa and was distinguished from the other outgroup species Zea mays (maize), Hordeum vulgare (barley), and Zizania latifolia (Manchurian wild rice) (Fig. 4a). These sister groups to Oryza mostly showed a common block of homologous synteny around the EAF6 region retained on chromosome 2 and shared a compatible lineage with their closest wild rice relative (Leersia perrieri) aside from the non-overlap** lineage sorting in AA-genome Oryza species (Fig. 4a and Supplementary Fig. 8a, b). This suggests that the two copies of OsEAF6 in O.sativa likely originated from their ancestral EAF6 gene located in a given position of chromosome 2 through speciation, genetic duplication, or horizontal gene transfer. In addition, we made further local collinearity analysis of HWS1 between O.sativa and the other five grasses (Supplementary Fig. 8c) and HWS2 (Supplementary Fig. 8d) between O.sativa, O.officinalis, and L.perrieri. The results showed that the chromosome segment exhibited significant syntenic relationships from Zea may to Oryza sativa, but it seemed that HWS1 came out of nowhere (Supplementary Fig. 8c). On the contrary, the collinearity of the HWS2 region in related species is much worse, indicating a possibly huge chromosome structure variation in HWS2 locus (Supplementary Fig. 8d). Given that transposable elements (TEs) generally cause no synteny³², we speculated that HWS1/2 gene pair may be generated by transposon-mediated gene replication. Considering the abundant transposon insertions located nearby, we carried out genome-wide transposon annotation analysis and showed that the segmental genomic duplication carrying OsEAF6 was probably generated by DNA transposon-mediated gene replication (Supplementary Fig. 9)⁴⁷. To further clarify the evolution process of HWS1/2, we constructed the phylogenetic tree and found that the gene tree of the HWS1 locus was incongruent with the species tree⁴⁸ in the certain HWS1 allele (hws1^C) (Fig. 4b). We next generated the gene tree of HWS1 by using the 133 species (Supplementary Fig. 10a and Supplementary Data 1), and the result showed that all the African rice were more closely to nearly all the japonica and some of the O.rufipogon rice (Supplementary Fig. 10a).

**Fig. 4: Estimation of divergence time and evolutionary scenarios for *HWS1/2* homologs via multiple duplication events.**

Although these phylogenetic discrepancies potentially stem from introgression or ILS, coalescence times for regions under ILS should be earlier than the speciation event while introgression occurs later³²(Fig. 4c). To distinguish which event leads to the incongruence of gene tree and species tree, we first compared the species divergence time (t) with expected coalescence time under ILS (t_IL) which was estimated based on the gene tree of HWS1. Since O.rufipogon could be divided into three groups: Or-I/II/III and Or-I is the ancestor of indica while Or-III is the ancestor of japonica⁴⁹, we therefore selected the representative varieties and divided into two major paired-topology categories, W1970 (HWS1^T)_W1536 (hws1^C)_NH280_W1952 (Fig. 4d) and W1547 (hws1^C)_W3012 (HWS1^T)_NH280_W1952 (Fig. 4e), both of which belong to Or-I, Or-III, O.barthii, and O.meridionalis, respectively. The hws1^C gene of W1536 (Or-III) and W1547 (Or-I) was more closely related to HWS1^T of O.barthii and did not match the species tree (Fig. 4d, e). Next, we calculated the synonymous substitution rate (Ks) of 3071 single-copy gene pairs between W1970 (Or-I) and W1536 (Or-III), as well as between W1547 (Or-I) and W3012 (Or-III), and estimated the differentiation time of the two species to be 0.062 and 0.056 MYA, respectively (Fig. 4d, e and Supplementary Fig. 10b, c). Likewise, we calculated the Ks values of the HWS1 gene pairs between Or-III (hws1^C) and O.barthii (HWS1^T), along with Or-I (hws1^C) and O.barthii (HWS1^T), both of their estimated differentiation time was 0.92 MYA (Fig. 4d, e). The coalescence time of the HWS1 gene was significantly earlier than the corresponding speciation time (Fig. 4d, e). This strongly suggests that ILS, not introgression, may be probably the major reason for the pervasive signatures of incongruence in these certain HWS1 alleles (hws1^C). To further exclude the introgression event, we conducted the four-taxon D-statistic test with the ABBA-BABA method. No obvious introgression signal was observed in each of the three paired-topology categories ((Or-III, Or-I)_O.barthii_O.meridionalis, (Or-II, Or-III)_O.barthii_O.meridionalis, and (Or-II, Or-I)_O.barthii_O.meridionalis) in the window where HWS1 gene was located (Supplementary Fig. 10d).

To comprehensively investigate the evolution and duplication processes of the HWS1/2 alleles, we selectively compared the genomic sequences among a diverse group of AA-genome rice species. This analysis included wild and domesticated Asian and African rice but excluded a group of certain rice cultivars/accessions due to the presence of multiple tandemly-repeated HWS2 loci (Supplementary Fig. 9b and Supplementary Data 1). Based on the pan-genome data, we calculated the synonymous substitution (Ks) values of HWS1/2 from 133 selected varieties relative to the HWS1 of O.meridionalis. This analysis identified three evolution processes (Ks = 0.028, 0.035, and 0.053, respectively) of the most HWS1/2 alleles (Fig. 4f). The HWS1 of O.rufipogon and O.barthii were diverged from O.meridionalis (Ks = 0.028 and 0.053), and then the HWS2 (HWS2^T) gene in O.rufipogon was duplicated from HWS1 (HWS1^T) (Ks = 0.035) (Fig. 4f), while some of the HWS1 allele (hws1^C) in O.rufipogon varieties experienced the ILS event (Ks = 0.049) (Fig. 4c–e and Supplementary Fig. 10b–e). Notably, the transcriptional expression profile further revealed that the HWS1 genes all underwent gene inactivation resulting from expression failure in certain O.rufipogon and japonica (hws1^C) (Fig. 4g and Supplementary Data 2). In the three wild rice populations, nucleotide diversity (π) in adjacent regions of HWS1 gene (7.35 Mb–7.4 Mb) was significantly higher than that of cultivated rice, indicating strong positive selection in this region (Fig. 4h). Evidently, a relatively low π value for the entire 200-kb region was also detected in the japonica population (Fig. 4h), indicating the presence of a bottleneck effect shaped by demographic and selective pressure. Nevertheless, the expression failure of HWS1 in nearly all japonica might be due to the founder effect resulting from the domestication of japonica rice. Taken together, HWS1/2, the functionally redundant paralogs, are thought to be duplicated and originated from a common ancestor in each divergent lineage, and the shared genetic variation between the certain O.rufipogon and O.barthii may be likely attributed to ILS.

Variation analysis and functional classification of the HWS1/2 genes in rice

Using the nucleotide variation identified in the HWS1/2 gene bodies and the 3’ and 5’ untranslated regions (UTRs), we performed an association test with a local genomic scan and uncovered several mutations that are correlated with gene expression in the same linkage disequilibrium (LD) block (Supplementary Data 2 and Supplementary Data 3). The strongest signal was present at the SNP1_7385301 site, a T-to-C substitution located in the upstream region that is inferred to be a proposed causative variant in HWS1 (Fig. 5a and Supplementary Fig. 4c). To verify whether the T/C variant in the HWS1 promoter affects the gene expression, we generated a range of promoter mutation constructs, each fused with LUC (luciferase) reporter gene and introduced into rice protoplasts to monitor promoter activity (Fig. 5b). The quantitation of firefly luciferase expression showed that the HWS1^CG14pro: Luc activity was dramatically reduced after the point mutation (“T” to “C”) in SNP1_7385301 site. Instead, the “C” to “T” replacement in hws1^WYJ7 rescued the expression failure of HWS1 in WYJ7, indicating that this core mutation may exert a great impact on the level of promoter activity. Several Asian cultivars including four japonica and three indica varieties were crossed with SG178 to determine the responsible polymorphic site (Fig. 5c). Plants exhibiting sterility and weakness were observed in the F₂ populations derived from crosses between C-carrying varieties and SG178 (Fig. 5d–g). On the contrary, when carrying the T allele of SNP1_7385301, the functional single nucleotide polymorphism (SNP), all of the descendants with different genotypes showed full seed set (Fig. 5h–j). In summary, our experiments in which we crossed WYJ7 and SG178 with a wide range of rice varieties showed that the targeted variation in HWS1 accounts for the interspecific sterility in rice. This potentially functional allele is referred to as HWS1^T while the sterile allele lacking promoter activity or the syntenic segmental block is called hws1^C or hws2^Null, both of which represent a loss-of-function type (Fig. 4g and Supplementary Data 2). Further sequence comparisons revealed that the rice accessions/varieties could be divided into three major HWS groups; Class I (HWS1^T/HWS2), Class II (HWS1^T/hws2^Null), and Class III (hws1^C/HWS2), and all three groups contained O. rufipogon accessions (Fig. 5k).

**Fig. 5: Allelic classification of *HWS1/HWS2* combination and verification of the functional SNP in *HWS1* locus.**

On the basis of the above results, a presumptive model interpreting the duplication and ILS history of OsEAF6 in Oryza genomes was proposed as follows (Fig. 6). The ancestor EAF6 gene that originated from chromosome 2 of L. perrieri was inherited to chromosome 2 in the BB, CC, FF and GG genome species of Oryza, and then anchored on chromosome 1 in rice species with AA genome, possibly by transposon-mediated gene replication. This single-copy form of OsEAF6 gene on chromosome 1 was subsequently retained in the O.longistaminata, O.meridionalis, and O.glumipatula while an interchromosomal copy from chromosome 1 to chromosome 12 was produced in O.rufipogon followed by another ILS event occurred between O.barthii and O.rufipogon (Fig. 6a). In the primitive ancestor gene pool of O.rufipogon and O.barthii, there existed two major polymorphic HWS1 alleles: O.barthii-like (OB-like; oval) and O.rufipogon-like (OR-like; rectangle). Unlike that only OB-like HWS1 allele was inherited and directed fixed in O.barthii, these two types of HWS1 alleles were both preserved in the ancestor of O.rufipogon due to the ILS event. Then, a duplication event that generated the HWS2 allele replicated from the OR-like HWS1 and formed the Class I-type O.rufipogon. The Class I-type O.rufipogon was next crossed with the OB-like wild rice ancestor and formed the heterozygous Class I-type O.rufipogon. A random T to C mutation at the promoter region of the OB-like HWS1 allele was then initiated in the heterozygous Class I-type O.rufipogon. This T/C variation in the heterozygous Class I-type O.rufipogon eventually gave rise to the form of Class I/II/III-type O.rufipogon in the hybrid progenies (Fig. 6b). The various alleles of the O.rufipogon accessions eventually evolved to the Class I-III of O.sativa and the O.barthii, ancestor of African rice, evolved to the Class II type of O.glaberrima (Fig. 6).

**Fig. 6: Proposed evolutionary and ILS history and lineage model of the *HWS1/2* genes in *Oryza* gene pools.**

Discussion

Gene duplication, a major force that drives gene expansion, occurs spontaneously and exists broadly in RI for species differentiation in allopatric populations^50,51. Our combined results suggest that the reciprocal loss of redundant genes copied from one another brings about a selective elimination and abortion signal in the japonica-glaberrima interspecific recombinants (Fig. 1a, b and Supplementary Fig. 11a). Such biased gamete transmission is accompanied by complete seed and pollen sterility events in the hybrid offspring, which follows the two-locus interaction model but differs from the S1-mediated killer–protector system^{4a, b), suggesting that they play indispensable roles in determining cell fate. The Cas9-induced loss-of-function mutants generated in this study provide experimental evidence supporting a central hub for the two genes in both reproductive development and growth regulation (Fig. 1g, h). Previous studies in plants showed that autoimmune responses associated with NB-LRR disease-resistance genes or R gene are major and common causes of hybrid weakness and breakdown^54,55. However, we did not observe any visible lesions and typical programmed cell death (PCD) induced in the leaf and basal nodes of sterile plants compared to fertile plants (Supplementary Fig. 12a–f). No obvious upregulated or downregulated DEGs involved in autoimmune or defense response were enriched in the RNA-seq or ChIP-seq data, suggesting that this weak phenotype may not result from autoimmunity (Fig. 3f and Supplementary Fig. 12g).}

From analyses of multi-omics data (Fig. 3f and Supplementary Fig. 7c, h), it can be inferred that genes involved in the remaining metabolic processes (e.g., sugar, carotenoid, and flavonoid synthesis), Golgi-related cellular components, and endocytosis may be the other likely HWS1/2-dependent downstream regulators, leading to male/female sterility due to nutrient deficiency and autotrophic collapse. In addition, the overlap between H4Ac-enriched genes and transcriptional DEGs only occupied a small proportion, suggesting that EAF6 may also play a part in acetylating nucleosomal histone H2A or H3 as well for transcriptional activation (Fig. 3e). Overall, a comprehensive understanding of the HWS1/2-mediated reproductive isolation system not only broadens our knowledge concerning the genetic composition of Oryza species, but it also promotes the instructive utilization of gene exchange in inter-subspecific genetic improvement.

Transformation experiments demonstrated that overexpression of the HWS2 gene did not result in a weakness syndrome, and the plants possessed normal fertilization capability (Supplementary Fig. 13), indicating that there are no obvious signs of negative effects from the number of OsEAF6-specific transcripts. As an alternative approach to overcome interspecific and subspecies reproductive isolation caused by the loss of HWS gene copy number in rice, artificially engineering HWS1/2 loci provides another potential, effective target to restore pollen and spikelet fertility together that can be used in distant hybridization in rice breeding. This may require us to fully consider the practical feasibility of natural hybrid-compatible varieties as founders for use in inter-subspecific breeding other than general gene editing^{Map-based cloning of HWS1 and HWS2}

The segregating BC₅F₂ population derived from the SG178 × WYJ7 F₁ hybrid was developed for fine-map** of HWS1 and HWS2 simultaneously, and the seed fertility or segregation ratios of RI characters in all tested plants were calculated as an appraisal index for QTL linkage analysis. The HWS1 locus was roughly delimited to a genomic interval on chromosome 1 between marker loci ID1-2 and ID1-3 using 6,570 BC₅F₂ individuals. A total of 38,580 BC₅F₂ plants were then further screened using five flanking markers to detect genetic recombination. Using newly developed molecular markers, the location of HWS1 was finally narrowed down to a 35.25-kb region embodying three annotated genes. The sketchy location of HWS2 was first detected on chromosome 12 and ultimately mapped to a 337.5-kb region between marker loci ID12-8 and ID12-9 around the centromere using 60,481 BC₅F₂ plants. The query of gene fragments was searched by a web-based tool (http://ricerc.sicau.edu.cn/). All primers used for genoty** are given in Supplementary Data 4.

Evaluation of pollen and spikelet fertility

To measure the viability of male gametes, pollen grains released from broken anthers of each single spikelet at the heading stage were mixed and stained with 1% (w/v) iodine-potassium iodide (I₂-KI) solution to determine pollen fertility using a light microscope. Dark-stained pollen grains with a nearly spherical shape were scored as fertile, whereas unstained or partially stained yellow-brown and misshapen grains were deemed as sterile. The seed-setting rate on the main panicle was defined as a quantitative indicator to evaluate spikelet fertility after maturity.

Cytological analyses of anther and embryo sac development

Florets from fresh spikelets at various stages of development were excised as samples and were fixed in FAA solution (50% ethanol, 37% formalin, and 100% acetic acid). Dehydrated samples were then embedded in resin and sectioned with an EM UC7 Ultramicrotome (Leica). The semi-thin sections were subsequently stained with fast green and observed under an AxioCam MRc light microscope (Zeiss). Microspore development is roughly classified into 14 stages based on the length of the flower glume in rice and previous descriptions^57,58. For cross-section and TEM analysis, anthers from the corresponding stages were collected and then fixed in 2.5% (v/v) glutaraldehyde solution for at least 24 h and treated as described by Guo⁵⁹. Images were captured with a Hitachi H7650 electron microscope using a voltage of 80 kV. SEM was performed and modified accordingly, referring to a previous study⁶¹. Before staining, the fixed samples were washed again and transferred to 70% (v/v) ethanol. The lemma and palea were removed and discarded to take out the whole ovary. Soon afterward, the tissues were rehydrated through an ethanol series and transferred to distilled water. All ovaries were incubated in 2% (w/v) aluminum potassium sulfate dodecahydrate (Aladdin, A103914) for 10-20 min, followed by staining in 10 mg/L Eosin-B (Aladdin, E100232) for 10-12 h at room temperature. Each medium-stained ovary was repeatedly dyed in 2% (w/v) aluminum potassium sulfate dodecahydrate once again for an additional 8 h and was then processed through a graded alcohol series. Finally, the tissues were cleared in 50% (v/v) methyl salicylate (Macklin, M813577) for 1–2 h and immersed in pure methyl salicylate for 1 h. The viability of the embryo sacs was ascertained by examination using a Leica TSC SP8 STED confocal laser microscope.

Plasmid construction and plant transformation

To construct the gene overexpression construct, the full-length coding sequence of HWS2^WYJ7 was amplified using cDNA from WYJ7 leaves and was then inserted into the binary vector pHB downstream of two tandemly repeated CaMV35S promoters with an EGFP tag. The functionally inactive mutants were all generated by the CRISPR/Cas9 gene-editing system, and the selected sequences of the designed target sites were synthesized, annealed, and cloned into the pYLCRISPR/Cas9 vector using the BsaI site⁶². The primer sets and single guide RNA (sgRNA) sequences are given in Supplementary Data 4. All rice genetic transformation experiments were performed using Agrobacterium-mediated transformation of WYJ7, SG178, and/or NIL-HWS1^CG14/HWS2^WYJ7 callus tissue with the A. tumefaciens strain EHA105. Phenotypic measurements were continuously taken in each generation of transgenic plants.

RNA extraction and gene expression analysis

Anthers at various developmental stages were harvested separately based on spikelet morphology and growth position of the floret^57,58. Total RNA was extracted from the rice tissues/organs and was purified using the E.Z.N.A. Total RNA Kit (Omega), and a given mass of each RNA sample were then incubated with DnaseI for cDNA synthesis using the PrimeScript^TM RT reagent kit with gDNA Eraser (Takara) following the manufacturer’s instructions. Quantitative real-time PCR (qRT-PCR) assays were performed and analyzed using the QIAquant 96 5plex Real-Time PCR System (Qiagen) in a reaction mixture containing Fast Start Universal SYBR Green Master Mix with ROX (Roche), and there were three biological replicates for each sample. The rice Ubiquitin gene was used as the internal control to normalize the gene expression data. The relevant primer pairs used for gene amplification are given in Supplementary Data 4.

Subcellular localization assay

The 35S::HWS1/2-YFP constructs were produced separately and introduced into isolated rice protoplasts using a PEG-mediated plasmid uptake method. A fluorescently labeled organelle marker containing a nuclear localization signal (NLS) fused with DsRed was co-transfected as an indicator. Confocal imaging analysis was performed using a confocal laser scanning microscope (Zeiss LSM880) at excitation wavelengths of 514 and 563 nm. Primers used in this experiment are given in Supplementary Data 4.

Protein purification and histone acetyltransferase activity assay

The HWS2^WYJ7 coding sequence was PCR amplified and inserted between the BamHI/EcoRI restriction sites of pMAL-c5x to produce the MBP-HWS2^WYJ7 expression vector by homologous recombination. All constructs were then transformed into Escherichia coli BL21 (DE3) pLysS Rosetta-gami 2 strain, and the recombinant fusion proteins were induced by the addition of 0.1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) and prepared using the AKTA Avant protein purification system. The relevant primer sequences are given in Supplementary Data 4. For the in vitro HAT assay, the intrinsic HAT activity was detected using the fluorescent HAT Assay Kit (Active Motif) following the manufacturer’s instructions with some modifications. In brief, the purified proteins (MBP-HWS2, MBP, and positive control p300) were incubated with 20 μl histone H4 peptide in a 50 μl reaction mix that included 6 μl of 5× HAT assay buffer and 5 μl of acetyl-CoA (0.5 mM) at 28°C for 1 h. The corresponding fluorescence signals were detected using a spectrophotometer with excitation at 375 nm and emission at 460 nm (Thermo Scientific, Varioskan LUX).

High-throughput SNP genoty**

DNA samples isolated from CG14, WYJ7, and SG178 were processed and analyzed by Shanghai OE Biotech Co., Ltd. (Shanghai, China). Genotypic data for genome-wide scanning was generated using the Affymetrix SNP chip to track the DNA segments from the parental genomes. The experimental procedures and analytical processes are summarized as follows: fresh rice leaves were immediately frozen in liquid nitrogen and genomic DNA was extracted using the DNeasy Plant Mini Kit (Qiagen). PCR-amplified DNA fragments with high quality and appropriate concentration were then marked and hybridized with Axiom Rice 44k array after random interruption and purification. Non-specific binding was removed by multiple washing and each nucleotide site was finally detected to generate a raw genotype file. Among all the 42,961 SNP probes on the genoty** array, the number of markers filtered to be homozygous and polymorphic SNPs between the two parental lines, CG14 and WYJ7, was 1,233, distributed across the 12 chromosomes.

Whole-genome resequencing

The genomes of SG178 and WYJ7 were resequenced on the Illumina HiSeq4000 platform by Novogene Bioinformatics Technology Co., Ltd. (Bei**g, China). The young seedlings were first harvested to extract DNA, and sequencing libraries were prepared by sonication and ligation with adaptors. The PCR-amplified libraries were purified and sequenced to obtain the raw data with quality control. The clean sequence reads from each sample were subsequently mapped to the ‘Nipponbare’ reference genome (IRGSP-1.0) (https://rapdb.dna.affrc.go.jp/). All genetic variants were identified with functional annotation by comparison to ‘Nipponbare’ (O.sativa ssp. japonica).

RNA-seq analysis

RNA deep sequencing and analysis were performed by BGI-Tech (Shenzhen, China). Total RNA for each three biological replicates consisted of panicles (approximately 10 cm in length, 5 individual plants per biological replicate) pooled from NIL-hws1^WYJ7/hws2^CG14 and NIL-HWS1^CG14/hws2^CG14 that were isolated independently after flash freezing in liquid nitrogen. The mRNA fraction was enriched using oligo (dT) magnetic beads and was uniformly fragmented to a specified length. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand synthesis. The cDNA was next subjected to end-repair and the ends were 3’adenylated. Adapters were then ligated to the ends of 3’adenylated cDNA fragments. The cDNA fragments with adapters were then amplified and purified with Ampure XP Beads (Agencourt), and were dissolved in EB solution. The quality of the mRNA-seq libraries was determined using the Agilent Technologies 2100 bioanalyzer, and the libraries were amplified with phi29 to make DNA nanoballs (DNBs) possessing more than 300 copies per molecule. The DNBs were loaded into the patterned nanoarray and the single-end 50 (pair-end 100/150) base reads were generated using the combinatorial Probe-Anchor Synthesis (cPAS) method. RNA-seq reads were aligned to the rice reference genome (MSU-7.0) (http://rice.uga.edu/index.shtml) using Hisat2 (v2.05). Transcription levels of tested genes were quantified by FPKM (fragments per kilobase of exon per million mapped reads). The criteria of fold-change ≥2 and q-value ≤ 0.05 were established to identify the differentially expressed genes.

ChIP-seq analysis

Data acquisition and handling were performed by Jiayin BioTech Ltd. (Hangzhou, China). The tissues used for RNA-seq were harvested synchronously at the same developmental stage as the samples used in the ChIP-seq experiment with 3 individual plants but without biological repeat. The fresh immature panicles were first cross-linked in 1% formaldehyde (Sigma, 47608) under vacuum for 20 min. The nuclear chromatin was isolated by sucrose gradient centrifugation and then sonicated. The lysate was further diluted with ChIP buffer and immunoprecipitated using an anti-histone H4Ac antibody (Millipore, 06-866). The ChIP-DNA complex was then extracted and purified using the Universal DNA Purification Kit (#DP214). The ChIP-seq library was prepared using a ChIP-seq DNA sample preparation kit (NEBNext® Ultra^TMII DNA) as directed by the manufacturer. The extracted ChIP-DNA fragments were ligated to specific adaptors and sequenced on an Illumina Novaseq 6000 instrument. The sequence reads were aligned to the rice reference genome (MSU-7.0) (http://rice.uga.edu/index.shtml) and the uniquely mapped reads used for peak calling were analyzed using MACS2 (version 2.1.4) peak caller software. Motif analysis was conducted using HOMER findMotifsGenome.pl tool and the peaks were annotated using the annotatePeak function in ChIPseeker (version 1.26.2). Analyses of the differences between the two samples were performed using the ChIPDiff (version 2.3.4) program, and the detailed read count data was loaded into the IGV (version 2.8.9) genome browser for visualization.

Native ChIP-qPCR assay

It was by analogy that the young inflorescences of the two NILs (NIL-hws1^WYJ7/hws2^CG14 and NIL-HWS1^CG14/hws2^CG14) were sampled and fixed in a crosslinking solution (10 mM Tris-HCl [pH 8.0], 400 mM sucrose, 1 mM PMSF, 5 mM β-mercaptoethanol, and 1% formaldehyde) under vacuum for 20 min at room temperature. Chromatin isolation was performed using the EZ-ChIP kit (Merck) using the standard protocol with minor modifications. Briefly, the soluble chromatin was incubated with an antibody directed against Histone H4Ac and was then precipitated with Protein A/G at 4 °C overnight. The immunoprecipitated complex was retrieved and analyzed by qRT-PCR using region-specific primers, and the data were normalized by input DNA. The sequences of the primers used for ChIP-qPCR can be found in Supplementary Data 4.

Protein extraction and western blotting

Nucleoproteins were extracted from 10 cm panicles of NIL-hws1^WYJ7/hws2^CG14, NIL-HWS1^CG14/hws2^CG14, NIL-hws1^WYJ7/HWS2^WYJ7, and NIL-HWS1^CG14/HWS2^WYJ7 plants using the Nuclear isolation kit (CELLYTPN-1KT, Sigma-Aldrich), and all steps were done as indicated in the instruction and operation manual. Protein extracts were boiled and terminated in SDS sample buffer, the proteins separated by denaturing polyacrylamide gel electrophoresis (SDS-PAGE) were then transferred to polyvinylidene difluoride (PVDF) membranes (Amersham, GE Healthcare). After blocking with 5% skim milk in TBST buffer (20 mM Tris/HCl, pH 7.6, 137 mM NaCl, 0.1% Tween) at 4°C overnight, the membranes were subsequently immunoblotted with primary antibodies using recommenced dilutions, followed by washing and incubation with a similarly diluted secondary antibody (goat anti-rabbit, Agrisera). The protein bands were then imaged using a UVP ChemStudio PLUS (Analytic Jena) after incubation in prepared ECL solutions (Amersham, GE Healthcare) for 3-5 min at room temperature in the dark.

Transient expression assays of promoter activity

The ~3000-bp, promoter fragments upstream of the HWS1^CG14 and hws1^WYJ7 translation start site were amplified from CG14 and WYJ7, respectively, and the fragments with site-directed mutation at SNP1_7385301 site were synthesized by Shanghai **tubio Biotechnology. All of these fragments were inserted into pGREENII 0800-LUC vector. The rice protoplast was isolated from two-week-old rice seedling leaves after sowing. Each of the HWS1 promoter-LUC gene fusion constructs was transfected into rice protoplasts by PEG-mediated transformation. LUC and REN luciferase signals were measured using the Dual-Luciferase Reporter Assay System (Promega).

Measurement of biotin levels

The biotin levels were measured by using a commercial Enzyme-Linked Immunosorbent Assay (ELISA) kit (E-IR-R501, Elabscience). In general, 50 μl testing sample was added into each well of the ELISA plate and then incubated with 50 μl Avidin-HRP working solution for 30 min at 37°C. After 3 rounds of wash, the substrate regent (90 μl each well) was added and then incubated for 15 min at 37°C in the dark. Finally, the optical density values of samples at the 450 nm wavelength (OD450) were recorded immediately after the addition of 50 μl stop solution. The biotin levels in different samples were calculated by comparing the OD450 values of the samples to a standard four-parameter logistic curve by using the ELISACalcu (version 0.1) software.

In situ hybridization

Pollens of NIL-hws1^WYJ7/hws2^CG14 and NIL-HWS1^CG14/hws2^CG14 at different development stages were used for investigating the expression pattern of HWS1 in pollen grains. The DIG-labeled probe was used for detecting HWS1 expression and the sense probe for HWS1 was used as the negative control. All probes were synthesized by Gefan Biotechnology Company (Shanghai, China). Samples were fixed in 4% paraformaldehyde overnight and then incubated for hybridization with the detection probes at 37 °C for 24 h according to standard procedures⁶³. The DIG-labeled probe for HWS1 and the negative sense probe are listed in Supplementary Data 4.

Antibodies used in this study

The following rabbit antibodies were used for the western blotting experiment, primary antibodies: anti-Histone H4 (Abcam, ab177840, rabbit monoclonal), anti-Histone H4Ac (Millipore, 06-866, rabbit monoclonal), anti-Histone H4K5Ac (Abcam, ab51997, rabbit monoclonal), anti-Histone H4K8Ac (Abcam, ab45166, rabbit monoclonal), anti-Histone H4K16Ac (Abcam, ab109463, rabbit monoclonal), anti-Histone H4K20Ac (Abcam, ab177188, rabbit monoclonal), anti-Histone H4K77Ac (Abcam, ab241117, rabbit polyclonal), anti-Histone H3Ac (Active Motif, 39040, rabbit polyclonal), anti-Histone H2AK5Ac (Active Motif, 39108, rabbit polyclonal), and anti-Histone H2AK9Ac (Active Motif, 39110, rabbit polyclonal). Secondary antibody: goat-anti-rabbit IgG (H + L) (Proteintech, SA00001-2). A dilution of 1:2000 is used for primary and secondary antibodies in the western blotting experiment. For ChIP-seq and ChIP-qPCR experiment: anti-Histone H4Ac (Millipore, 06-866, rabbit monoclonal) is used, with a dilution of 1:200.

Identification and phylogenetic analysis of EAF6 genes in grass family

The genomes of Zea mays, Hordeum vulgare (Version TRITEX), Leersia perrieri, Oryza brachyantha (FF genome), Oryza punctata (BB genome), and Oryza barthii were all retrieved from ftp://ftp.gramene.org/pub/gramene/release-65/. The genomes of Zizania latifolia and Oryza officinalis (CC genome) were downloaded from the NCBI (GenBank accessions GCA_000418225.1 and GCA_008326285.1, respectively). The genome of Oryza granulata (GG genome) was obtained from the National Genomics Data Center under accession number GWHAAEL00000000. Publicly available data for the genomes of 20 African rice accessions from the rice super pan-genome were obtained from the Genome Warehouse (GWH) (http://bigd.big.ac.cn/gwh/) under PRJCA004295. The genomes of 71 wild rice accessions and 12 cultivars were from our recent de novo assemblies.

To retrieve the orthologs of the EAF6 gene in the representative gramineous plants, the coding sequences of HWS1 and HWS2 were inputted into the database as queries to extensively search the matched hits based on a BLAST approach (version 2.2.28)⁶⁴. All identified EAF6 genes were included in an all-versus-all alignment using MAFFT (version 7.158b) with the default parameter settings⁶⁵. A maximum likelihood phylogenetic tree was reconstructed using IQ-TREE (version 1.6.3) with 1000 bootstrap replicates⁶⁶.

The 3071 single-copy gene pairs between W1970 and W1536, as well as between W1547 and W3012 were obtained using OrthoFinder v2.3.8⁶⁷. The synonymous substitution ratio (Ks) of the gene pairs was calculated using ParaAT2.0 software⁶⁸. The peak Ks values were converted to divergence time according to the formula T = Ks/2λ (T, time; λ, average substitution rate) by using an average substitution rate of 6.5 E−09⁶⁹ for grasses to infer speciation. The Ks values for the HWS1 gene that underwent incomplete lineage sorting in W1536 and W1547 and the corresponding gene in O.barthii were also calculated by ParaAT2.0 software, and their differentiation time was calculated by the above formula.

Gene expression analysis

Data fetching and aggregate of RNA sequencing results from 33 rice varieties were downloaded from the in-house RNA-seq pipeline (BioProject number: PRJCA002103), National Genomics Data Center (http://bigd.big.ac.cn/). For transcript quantification, transcripts per million (TPM) values were determined using the Salmon method to normalize the gene expression level in the RNA pools⁷⁰. The other available transcriptomic profiles of 71 wild rice accessions and 12 African cultivars were additionally obtained from our recent de novo assemblies and the same optimization method was applied to them.

Repetitive sequence annotation

The whole-genome repetitive sequence analysis applied to all 33 rice cultivars is accessible at ‘Rice Resource Center’ (http://ricerc.sicau.edu.cn/). Transposons and tandem repeats from L. perrieri, O. officinalis, and O. punctata were annotated in detail with the Extensive de novo TE Annotator (EDTA, version1.9.6) using the curated TE library (rice 6.9.5.liban) with the parameters ‘-overwrite 0 -sensitive 1 -anno 1 -evaluate 0’⁷¹.

Genomic comparison and visualizations

MCScan software (Python version) was used to scan multiple genomes, run the pairwise synteny search program, and align putative homologous chromosomal regions with the LAST results of O. sativa versus the other selected grass species⁷². The genome pairwise synteny visualization was generated using the command ‘Python -m jcvi.graphics.karyotype’ while microsynteny visualization was produced using the command ‘Python -m jcvi.graphics.synteny’.

Gene duplication analysis

Based on the pan-genome data, we calculated the synonymous substitution (Ks) values of HWS1/2 from selected varieties relative to the HWS1 of O.meridionalis (a more ancient wild rice variety to the AA genome rice species) using ParaAT2.0 software with the parameter ‘-m clustalw2 -f axt -g -k’. The Ks distribution of paired HWS1 and HWS2 genes on orthologous data sets from 133 selected rice varieties was used as a rough indicator to determine gene duplication events and the numeric value was negatively correlated with divergence time and kinship with its ancestral copy.

Population genetic analysis

To analyze population genetics, we directly downloaded the SNP set of rice accessions from a previously published work⁴⁹ and converted the SNP coordinates of IRGSP4 to MSU RGAP 7 using BWA, which was used to process the local alignment. The SNPs covering the whole 200-kb chromosomal region upstream and downstream of HWS1 in the japonica and indica accessions of O.sativa and three types of wild rice were extracted to calculate the nucleotide diversity (π) using VCFtools in sliding windows with a 5-kb window size⁷³. The gene body and a total of 1.2-kb of DNA sequence upstream and downstream of HWS1 were extracted using BEDTools utilities, and the sequences were aligned using MAFFT software⁷⁴. The corresponding variants from 41 rice varieties in this small genomic interval were then identified using custom scripts (simple ‘shell’ command) to compute genome-wide linkage disequilibrium (LD). We next performed case-control genetic association analysis and calculated the LD to test the correlation by Pearson’s Chi-square test in PLINK version 1.90⁷⁵. The LD block was displayed using LDBlockShow software⁷⁶.

We downloaded all corresponding well-assembled genomes of 32 Asian rice varieties and 20 African rice varieties and then mapped them to the O. sativa ‘Nipponbare’ reference genome (IRGSP1.0) (https://rapdb.dna.affrc.go.jp/) using MuMmer (version 4.0) (parameters: NUCmer -l 50 -c 100 -maxmatch)⁷⁷. A delta-filter program with a one-to-one alignment model (-1) was provided to manipulate the filter of the alignment blocks and the resulting SNPs were identified accordingly using show-snps (-ClrT) in the MUMmer toolkit. Admixture among the 71O.rufipogon accessions and another 12 O. sativa cultivars from our new de novo genome assemblies were also selected to gather the SNP information.

ABBA-BABA statistic test

In the analysis of introgression, we estimated the fd statistical values using the python script ABBABABAwindows.py under topology ((X, Y), O.barthii, O.meridionalis), where X, Y are extracted from Or-III, Or-II, and Or-I, respectively. The parameters were set as “window size: 300-K bp, minimum good sites per window: 100, and minimum proportion of samples genotyped per site: 0.2. We defined a region as being introgressed if fd was in the top 0.5% of the genome-wide distribution.

Statistics and reproducibility

Statistical analysis and number of biologically independent sample (n) were indicated in the figure legends. Significant differences between two groups were determined with two-sided Student’s paired t test by using Microsoft Excel 2013 software. Differences between three or more groups were determined by using one-way ANOVA with two-sided Tukey’s HSD test with GraphPad Prism 8 software.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All the relevant data supporting the findings of this work are avaliable in this article and its Supplementary information files. The RNA-seq and ChIP-seq data generated in this study have been deposited at the National Genomics Data Center under accession numbers (ID: CRA014308, CRA014315). Source data are provided with this paper.

References

Bomblies, K. Doomed lovers: mechanisms of isolation and incompatibility in plants. Annu Rev. Plant Biol. 61, 109–124 (2010).
Article CAS PubMed Google Scholar
Westram, A. M., Stankowski, S., Surendranadh, P. & Barton, N. What is reproductive isolation? J. Evol. Biol. 35, 1143–1164 (2022).
Article PubMed PubMed Central Google Scholar
Chen, C. et al. A two-locus interaction causes interspecific hybrid weakness in rice. Nat. Commun. 5, 3357 (2014).
Article ADS PubMed Google Scholar
Chae, E. et al. Species-wide genetic incompatibility analysis identifies immune genes as hot spots of deleterious epistasis. Cell 159, 1341–1351 (2014).
Article CAS PubMed PubMed Central Google Scholar
Ichitani, K., Takemoto, Y., Iiyama, K., Taura, S. & Sato, M. Chromosomal location of HCA1 and HCA2, hybrid chlorosis genes in rice. Int. J. Plant Genomics 2012, 649081 (2012).
Article PubMed PubMed Central Google Scholar
Bateson, W. Heredity and Variation in Modern Lights. Darwin and Modern Science: Essays in Commemoration of the Centenary of the Birth of Charles Darwin and of the Fiftieth Anniversary of the Publication of The Origin of Species (ed. Seward, A. C.) 85–101 (Cambridge University Press, Cambridge, 2009).
Dobzhansky, T. Genetics and the Origin of Species (Columbia University Press, 1964)
Muller, H. J. Isolating mechanisms, evolution and temperature. Biol. Symp. 6, 71–125 (1942).
Yang, J. et al. A killer-protector system regulates both hybrid sterility and segregation distortion in rice. Science 337, 1336–1340 (2012).
Article ADS CAS PubMed Google Scholar
Yu, X. et al. A selfish genetic element confers non-Mendelian inheritance in rice. Science 360, 1130–1132 (2018).
Article ADS CAS PubMed Google Scholar
Chen, C., E, Z. & Lin, H. X. Evolution and molecular control of hybrid incompatibility in plants. Front Plant Sci. 7, 1208 (2016).
Article PubMed PubMed Central Google Scholar
**e, Y., Shen, R., Chen, L. & Liu, Y. G. Molecular mechanisms of hybrid sterility in rice. Sci. China Life Sci. 62, 737–743 (2019).
Article PubMed Google Scholar
Ouyang, Y. & Zhang, Q. Understanding reproductive isolation based on the rice model. Annu Rev. Plant Biol. 64, 111–135 (2013).
Article CAS PubMed Google Scholar
Shen, R. et al. Genomic structural variation-mediated allelic suppression causes hybrid male sterility in rice. Nat. Commun. 8, 1310 (2017).
Article ADS PubMed PubMed Central Google Scholar
Long, Y. et al. Hybrid male sterility in rice controlled by interaction between divergent alleles of two adjacent genes. Proc. Natl Acad. Sci. USA 105, 18871–18876 (2008).
Article ADS CAS PubMed PubMed Central Google Scholar
Mizuta, Y., Harushima, Y. & Kurata, N. Rice pollen hybrid incompatibility caused by reciprocal gene loss of duplicated genes. Proc. Natl Acad. Sci. USA 107, 20417–20422 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Yamagata, Y. et al. Mitochondrial gene in the nuclear genome induces reproductive barrier in rice. Proc. Natl Acad. Sci. USA 107, 1494–1499 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Hou, J. et al. ESA1 is involved in embryo sac abortion in interspecific hybrid progeny of rice. Plant Physiol. 180, 356–366 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Nguyen, G. N. et al. Duplication and Loss of Function of Genes Encoding RNA Polymerase III957 Subunit C4 Causes Hybrid Incompatibility in Rice. G3 (Bethesda) 7, 2565–2575 (2017) .
Yu, Y. et al. Hybrid sterility in rice (Oryza sativa L.) involves the tetratricopeptide repeat domain containing protein. Genetics 203, 1439–1451 (2016).
Article CAS PubMed PubMed Central Google Scholar
Wang, D. et al. Two complementary genes in a presence-absence variation contribute to indica-japonica reproductive isolation in rice. Nat. Commun. 14, 4531 (2023).
Article ADS CAS PubMed PubMed Central Google Scholar
Xu, C. et al. Spontaneous movement of a retrotransposon generated genic dominant male sterility providing a useful tool for rice breeding. Natl Sci Rev. 10, nwad210 (2023).
Lei, D. et al. Anther-specific expression of OsRIP1 causes dominant male sterility in rice. Plant Biotechnol. J. 21, 1932–1934 (2023).
Article PubMed PubMed Central Google Scholar
Wang, C. et al. A natural gene drive system confers reproductive isolation in rice. Cell 186, 3577–3592 (2023).
Article CAS PubMed Google Scholar
Li, J. et al. New Insights Into the Nature of Interspecific Hybrid Sterility in Rice. Front Plant Sci. 11, 555572 (2020).
Article PubMed PubMed Central Google Scholar
**e, Y. et al. An asymmetric allelic interaction drives allele transmission bias in interspecific rice hybrids. Nat. Commun. 10, 2501 (2019).
Article ADS PubMed PubMed Central Google Scholar
**e, Y. et al. Interspecific hybrid sterility in rice is mediated by OgTPR1 at the S1 Locus encoding a peptidase-like protein. Mol. Plant 10, 1137–1140 (2017).
Article CAS PubMed Google Scholar
Koide, Y. et al. Lineage-specific gene acquisition or loss is involved in interspecific hybrid sterility in rice. Proc. Natl Acad. Sci. USA 115, E1955–E1962 (2018).
Article CAS PubMed PubMed Central Google Scholar
Avise, J. C. & Robinson, T. J. Hemiplasy: A new term in the lexicon of phylogenetics. Syst. Biol. 57, 503–507 (2008).
Article PubMed Google Scholar
Ma, J. et al. The Chloranthus sessilifolius genome provides insight into early diversification of angiosperms. Nat. Commun. 12, 6929 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Guo, X. et al. Chloranthus genome provides insights into the early diversification of angiosperms. Nat. Commun. 12, 6930 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Feng, S. et al. Incomplete lineage sorting and phenotypic evolution in marsupials. Cell 185, 1646–1660.e18 (2022).
Article CAS PubMed PubMed Central Google Scholar
Rivas-González, I. et al. Pervasive incomplete lineage sorting illuminates speciation and selection in primates. Science 380, eabn4409 (2023).
Article PubMed Google Scholar
Edelman, N. B. et al. Genomic architecture and introgression shape a butterfly radiation. Science 366, 594–599 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
La, H. et al. Genome-wide analysis of cyclin family in rice (Oryza Sativa L.). Mol. Genet Genomics 275, 374–386 (2006).
Article CAS PubMed Google Scholar
Guo, J., Wang, F., Song, J., Sun, W. & Zhang, X. S. The expression of Orysa;CycB1;1 is essential for endosperm formation and causes embryo enlargement in rice. Planta 231, 293–303 (2010).
Article CAS PubMed Google Scholar
Presgraves, D. C. The molecular evolutionary basis of species formation. Nat. Rev. Genet 11, 175–180 (2010).
Article CAS PubMed Google Scholar
Allard, S. et al. NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex containing Esa1p and the ATM-related cofactor Tra1p. EMBO j. 18, 5108–5119 (1999).
Article CAS PubMed PubMed Central Google Scholar
Bieluszewski, T. et al. NuA4 and H2A.Z control environmental responses and autotrophic growth in Arabidopsis. Nat. Commun. 13, 277 (2022).
Article ADS CAS PubMed PubMed Central Google Scholar
Zhou, J. X. et al. The Arabidopsis NuA4 histone acetyltransferase complex is required for chlorophyll biosynthesis and photosynthesis. J. Integr. Plant Biol. 64, 901–914 (2022).
Article CAS PubMed Google Scholar
Zhang, D. et al. OsC6, encoding a lipid transfer protein, is required for postmeiotic anther development in rice. Plant Physiol. 154, 149–162 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Zheng, S. et al. OsAGO2 controls ROS production and the initiation of tapetal PCD by epigenetically regulating OsHXK1 expression in rice anthers. Proc. Natl Acad. Sci. USA 116, 7549–7558 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Yu, D. et al. Wax crystal-sparse Leaf1 encodes a beta-ketoacyl CoA synthase involved in biosynthesis of cuticular waxes on rice leaf. Planta 228, 675–685 (2008).
Article CAS PubMed Google Scholar
Li, H. et al. Cytochrome P450 family member CYP704B2 catalyzes the {omega}-hydroxylation of fatty acids and is required for anther cutin biosynthesis and pollen exine formation in rice. Plant Cell 22, 173–190 (2010).
Article CAS PubMed PubMed Central Google Scholar
Zou, T. et al. OsLAP6/OsPKS1, an orthologue of Arabidopsis PKSA/LAP6, is critical for proper pollen exine formation. Rice (N. Y) 10, 53 (2017).
Article PubMed Google Scholar
Ding, W. et al. OsKASI, a β-ketoacyl-[acyl carrier protein] synthase I, is involved in root development in rice (Oryza sativa L.). Planta 242, 203–213 (2015).
Article CAS PubMed Google Scholar
Wicker, T. et al. A unified classification system for eukaryotic transposable elements. Nat. Rev. Genet 8, 973–982 (2007).
Article CAS PubMed Google Scholar
Stein, J. C. et al. Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza. Nat. Genet. 50, 285–296 (2018).
Article CAS PubMed Google Scholar
Huang, X. et al. A map of rice genome variation reveals the origin of cultivated rice. Nature 490, 497–501 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Lynch, M. & Conery, J. S. The evolutionary fate and consequences of duplicate genes. Science 290, 1151–1155 (2000).
Article ADS CAS PubMed Google Scholar
Bikard, D. et al. Divergent evolution of duplicate genes leads to genetic incompatibilities within A. thaliana. Science 323, 623–626 (2009).
Article ADS CAS PubMed Google Scholar
Huang, X., Zhao, Q. & Han, B. Comparative population genomics reveals strong divergence and infrequent introgression between Asian and African Rice. Mol. Plant 8, 958–960 (2015).
Article CAS PubMed Google Scholar
Kubo, T., Yoshimura, A. & Kurata, N. Loss of OsEAF6, a subunit of the Histone Acetyltransferase complex, causes hybrid breakdown in intersubspecific rice crosses. Front. Plant Sci. 13 (2022).
Bomblies, K. et al. Autoimmune response as a mechanism for a Dobzhansky-Muller-type incompatibility syndrome in plants. PLOS Biol. 5, e236 (2007).
Article PubMed PubMed Central Google Scholar
Alcázar, R., García, A. V., Parker, J. E. & Reymond, M. Incremental steps toward incompatibility revealed by Arabidopsis epistatic interactions modulating salicylic acid pathway activation. Proc. Natl Acad. Sci. 106, 334–339 (2009).
Article ADS PubMed Google Scholar
**e, Y. et al. Suppression or knockout of SaF/SaM overcomes the Sa-mediated hybrid male sterility in rice. J. Integr. Plant Biol. 59, 669–679 (2017).
Article CAS PubMed Google Scholar
Itoh, J. et al. Rice plant development: from zygote to spikelet. Plant Cell Physiol. 46, 23–47 (2005).
Article CAS PubMed Google Scholar
Zhang, D., Luo, X. & Zhu, L. Cytological analysis and genetic control of rice anther development. J. Genet Genomics 38, 379–390 (2011).
Article CAS PubMed Google Scholar
Guo, T. et al. A SAC Phosphoinositide phosphatase controls rice development via hydrolyzing PI4P and PI(4,5)P(2). Plant Physiol. 182, 1346–1358 (2020).
Article CAS PubMed Google Scholar
**ang, Y. H. et al. An α/β hydrolase family member negatively regulates salt tolerance but promotes flowering through three distinct functions in rice. Mol. Plant 15, 1908–1930 (2022).
Article CAS PubMed Google Scholar
Shen, Y. et al. Fine map** of S37, a locus responsible for pollen and embryo sac sterility in hybrids between Oryza sativa L. and O. glaberrima Steud. Plant Cell Rep. 34, 1885–1897 (2015).
Article CAS PubMed Google Scholar
Ma, X. et al. A Robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol. Plant 8, 1274–1284 (2015).
Article CAS PubMed Google Scholar
Yang, M. et al. miR-71 and miR-263 jointly regulate target genes Chitin synthase and Chitinase to control locust molting. PLOS Genet. 12, e1006257 (2016).
Article PubMed PubMed Central Google Scholar
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).
Article CAS PubMed Google Scholar
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Article CAS PubMed PubMed Central Google Scholar
Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).
Article CAS PubMed Google Scholar
Emms, D. M. & Kelly, S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol. 20, 238 (2019).
Article PubMed PubMed Central Google Scholar
Zhang, Z. et al. ParaAT: a parallel tool for constructing multiple protein-coding DNA alignments. Biochem Biophys. Res Commun. 419, 779–781 (2012).
Article CAS PubMed Google Scholar
Gaut, B. S., Morton, B. R., McCaig, B. C. & Clegg, M. T. Substitution rate comparisons between grasses and palms: synonymous rate differences at the nuclear gene Adh parallel rate differences at the plastid gene rbcL. Proc. Natl Acad. Sci. 93, 10274–10279 (1996).
Article ADS CAS PubMed PubMed Central Google Scholar
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
Article CAS PubMed PubMed Central Google Scholar
Ou, S. et al. Benchmarking transposable element annotation methods for creation of a streamlined, comprehensive pipeline. Genome Biol. 20, 275 (2019).
Article CAS PubMed PubMed Central Google Scholar
Wang, Y. et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res 40, e49 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Danecek, P. et al. The variant call format and VCFtools. Bioinformatics 27, 2156–2158 (2011).
Article CAS PubMed PubMed Central Google Scholar
Quinlan, A. R. BEDTools: The Swiss-Army Tool for Genome Feature Analysis. Curr. Protoc. Bioinforma. 47, 11.12.1-34 (2014).
Article Google Scholar
Chang, C. C. et al. Second-generation PLINK: rising to the challenge of larger and richer datasets. Gigascience 4, 7 (2015).
Article PubMed PubMed Central Google Scholar
Dong, S.-S. et al. LDBlockShow: a fast and convenient tool for visualizing linkage disequilibrium and haplotype blocks based on variant call format files. Brief Bioinform. 22, bbaa227 (2021).
Marçais, G. et al. MUMmer4: A fast and versatile genome alignment system. PLoS Comput Biol. 14, e1005944 (2018).
Article PubMed PubMed Central Google Scholar

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Acknowledgements

We thank M. Shi for assistance with the transgenic assay; W. J. Cai, S. N. Yin, Z. P. Zhang, X. Y. Gao, J. Q. Li, and L. N. Xu for technical supports; Y. G. Liu for donation of CRISPR/Cas9 plasmids; L.G. Shang and Q. Qian for providing the rice super pan-genome data; S. G. Li and P. Qin for providing the geographic location of 33 O. sativa accessions. This work was supported by the grants from National Natural Science Foundation of China (32388201), the Chinese Academy of Sciences (XDB27010104, 159231KYSB20200008), Laboratory of Lingnan Modern Agriculture Project (NT2021002), CAS-Croucher Funding Scheme for Joint Laboratories.

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These authors contributed equally: Ben Liao, You-Huang **ang, Yan Li.

Authors and Affiliations

National Key Laboratory of Plant Molecular Genetics, CAS Centre for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200032, China
Ben Liao, You-Huang **ang, Kai-Yang Yang, Jun-**ang Shan, Wang-Wei Ye, Nai-Qian Dong, Yi Kan, Yi-Bing Yang, Huai-Yu Zhao, Hong-**ao Yu, Zi-Qi Lu, Shuang-Qin Guo, Jie-Jie Lei, **ao-Rui Mu, Ying-Jie Cao & Hong-Xuan Lin
School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China
Ben Liao, Zi-Qi Lu & Hong-Xuan Lin
China National Center for Gene Research, National Key Laboratory of Plant Molecular Genetics, CAS Center of Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, 200233, China
Yan Li, Yan Zhao, Qiang Zhao, Dongling Guo & Bin Han
University of the Chinese Academy of Sciences, Bei**g, 100049, China
Kai-Yang Yang, Yi-Bing Yang, Huai-Yu Zhao, Hong-**ao Yu, Shuang-Qin Guo, Jie-Jie Lei, **ao-Rui Mu, Ying-Jie Cao & Hong-Xuan Lin
Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China
Jun-**ang Shan, Wang-Wei Ye, Nai-Qian Dong, Yi Kan & Hong-Xuan Lin

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Ben Liao
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You-Huang **ang
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Yan Li
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Kai-Yang Yang
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Jun-**ang Shan
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Wang-Wei Ye
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Yi Kan
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Huai-Yu Zhao
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Hong-**ao Yu
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Zi-Qi Lu
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Yan Zhao
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Qiang Zhao
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Dongling Guo
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Shuang-Qin Guo
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Jie-Jie Lei
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**ao-Rui Mu
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Ying-Jie Cao
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Bin Han
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Hong-Xuan Lin
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Contributions

H.X.L. and B.H. conceived and supervised the project, and H.X.L., Y.H.X. and B.L. designed the experiments. B.L., Y.H.X. and Y. L. performed most of the experiments. K.Y.Y., J.X.S., W.W.Y., N.Q.D., Y.K., Y.B.Y., H.Y.Z., H.X.Y., Z.Q.L., Y.Z., Q.Z., D.G., S.Q.G., J.J.L., X.R.M., Y.J.C. and H.X.L. performed some of the experiments. Y.H.X., B.L. and H.X.L. analyzed data and wrote the manuscript.

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Correspondence to Bin Han or Hong-Xuan Lin.

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Nature Communications thanks Yao-Guang Liu, Ziqiang Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Liao, B., **ang, YH., Li, Y. et al. Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier. Nat Commun 15, 996 (2024). https://doi.org/10.1038/s41467-024-45377-x

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Received: 16 August 2023
Accepted: 21 January 2024
Published: 02 February 2024
DOI: https://doi.org/10.1038/s41467-024-45377-x
Springer Nature Limited

Dysfunction of duplicated pair rice histone acetyltransferases causes segregation distortion and an interspecific reproductive barrier

Abstract

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Introduction

Dysfunctions in HWS1/2 contribute to H4Ac loss and transcriptomic reprogramming misregulation

Evolutionary trajectory and EAF6 gene duplication events in Oryza species

Variation analysis and functional classification of the HWS1/2 genes in rice

Discussion

Evaluation of pollen and spikelet fertility

Cytological analyses of anther and embryo sac development

Plasmid construction and plant transformation

RNA extraction and gene expression analysis

Subcellular localization assay

Protein purification and histone acetyltransferase activity assay

High-throughput SNP genoty**

Whole-genome resequencing

RNA-seq analysis

ChIP-seq analysis

Native ChIP-qPCR assay

Protein extraction and western blotting

Transient expression assays of promoter activity

Measurement of biotin levels

In situ hybridization

Antibodies used in this study

Identification and phylogenetic analysis of EAF6 genes in grass family

Gene expression analysis

Repetitive sequence annotation

Genomic comparison and visualizations

Gene duplication analysis

Population genetic analysis

ABBA-BABA statistic test

Statistics and reproducibility

Reporting summary

Data availability

References

Acknowledgements

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Authors and Affiliations

Contributions

Corresponding authors

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Peer review

Peer review information

Additional information

Supplementary information

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