Self-incompatibility (SI) is a feature of many flowering plants, whereby self-pollen is recognized and rejected by the stigma. In grasses (Poaceae), the genes controlling this phenomenon have not been fully elucidated. Grasses have a unique two-locus system, in which two independent genetic loci (S and Z) control self-recognition. S and Z are thought to have arisen from an ancient duplication, common to all grasses. With new chromosome-scale genome data, we examined the genes present at S- and Z-loci, firstly in ryegrass (Lolium perenne), and subsequently in ~20 other grass species. We found that two DUF247 genes and a short unstructured protein (SP/ZP) were present at both S- and Z- in all SI species, while in self-compatible species these genes were often lost or mutated. Expression data suggested that DUF247 genes acted as the male components and SP/ZP were the female components. Consistent with their role in distinguishing self- from non-self, all genes were hypervariable, although key secondary structure features were conserved, including the predicted N-terminal cleavage site of SP/ZP. The evolutionary history of these genes was probed, revealing that specificity groups at the Z-locus arose before the advent of various grass subfamilies/species, while specificity groups at the S-locus arose after the split of Panicoideae, Chloridoideae, Oryzoideae and Pooideae. Finally, we propose a model explaining how the proteins encoded at the S and Z loci might function to specify self-incompatibility.

Ryegrass is the primary pasture crop in New Zealand; however, despite this importance, gains in yield through conventional breeding have been marginal. Other significant crops, particularly maize and rice, have benefited from hybrid breeding programmes whereby heterosis has been exploited. Ryegrass presents many challenges to breeders wishing to produce F1 hybrid progeny on a large scale such as self‐incompatibility, inbreeding depression, and a lack of genetic tools for creating and maintaining male‐sterile lines for crossing. Many of these challenges have been overcome in other species, providing a basis for addressing them in ryegrass. With the advent of new technologies such as CRISPR gene editing and the genome sequence for ryegrass, F1 hybrid production is now possible on a large scale. Regulations in New Zealand surrounding new genetic tools are a hurdle which must be addressed to allow the agricultural industry in New Zealand to apply these advanced technologies.

Perennial ryegrass (Lolium perenne) is a temperate grass species commonly used as pasture for livestock. Flowering (heading) of ryegrass impacts metabolizable energy content and seed yield, therefore this trait is important for both farmers and seed producers. In related grass species, theVRNgenes (VRN1-3) have been largely implicated in the determination of vernalization response and are responsible for much of the intra-species variation in this trait. Many other important flowering-time regulators have been cataloged in the model grassBrachypodium distachyon;however, in several cases, such asVRN2, their ryegrass homologs have not been well-characterized. Here, ryegrass homologs of important flowering time genes fromB. distachyonwere identified through available synteny data and sequence similarity. Phylogenetic analysis ofVRN3/FT-likeandVRN2-likegenes was performed to elucidate these families further. The expression patterns of these genes were assessed during vernalization. This confirmed the key roles played byLpVRN1andLpFT3in the promotion of flowering. Furthermore, two orthologs ofVRN2identified here, as well as an ortholog ofCO9, were expressed prior to vernalization, and were repressed in flowering plants, suggesting a role in floral repression. Significant variability in expression of these flowering pathway genes in diverse genotypes was detected and may underlie variation in flowering time and vernalization response.

Summary: Epigenetic modifications that arise during plant and animal development, such as DNA and histone modification, are mostly reset during gamete formation, but some are inherited from the germline including those marking imprinted genes ¹ . Small RNAs guide these epigenetic modifications, and some are also inherited by the next generation ^2,3 . In C. elegans , these inherited small RNAs have poly (UG) tails ⁴ , but how inherited small RNAs are distinguished in other animals and plants is unknown. Pseudouridine (Ψ) is the most abundant RNA modification but has not been explored in small RNAs. Here, we develop novel assays to detect Ψ in short RNA sequences, demonstrating its presence in mouse and Arabidopsis microRNAs and their precursors. We also detect substantial enrichment in germline small RNAs, namely epigenetically activated siRNAs (easiRNAs) in Arabidopsis pollen, and piwi-interacting piRNAs in mouse testis. In pollen, pseudouridylated easiRNAs are localized to sperm cells, and we found that PAUSED/HEN5 (PSD) , the plant homolog of Exportin-t, interacts genetically with Ψ and is required for transport of easiRNAs into sperm cells from the vegetative nucleus. We further show that Exportin-t is required for the triploid block: chromosome dosage-dependent seed lethality that is epigenetically inherited from pollen. Thus, Ψ has a conserved role in marking inherited small RNAs in the germline. One-Sentence Summary: Pseudouridine marks germline small RNAs in plants and mammals, impacting epigenetic inheritance via nuclear transport.

Premise: Advances in long‐read sequencing offer new possibilities to investigate haplotype diversity across multiple genes in plants and other taxa through multi‐locus, long‐read amplicon sequencing (multi‐locus LRAS). Despite this progress, there is a notable absence of dedicated bioinformatics pipelines for assembling diploid haplotypes of heterozygous individuals from such multi‐locus LRAS datasets, which is required for highly polymorphic populations. Methods: We first evaluated various de novo and reference‐based assembly methods, culminating in a custom pipeline (HapAsmbl) to assemble haplotypes from Oxford Nanopore Technologies (ONT) LRAS data of five flowering genes ( FT3 , FTL9 , VRN1 , VRN2A , and VRN2B ) generated from perennial ryegrass, a highly heterozygous species. After verifying the efficacy using a simulated heterozygous dataset, the HapAsmbl pipeline was used to explore haplotype diversity of CO , FT3 , and VRN1 across multiple ryegrass populations. Results: HapAsmbl outperformed existing tools by reliably reconstructing diploid haplotypes across multiple loci, enabling efficient haplotype characterization and novel allele discovery in genetically diverse populations. Discussion: HapAsmbl simplifies haplotype resolution from complex LRAS datasets from heterozygous individuals, allowing routine use of ONT long‐read sequencing for scalable haplotype analysis. HapAsmbl will enable researchers to uncover novel alleles and relate these to phenotype, supporting plant‐breeding efforts in non‐model crops.