Rye (*Secale cereale*), a diploid species (2n=2x=14, RR), belongs to the *Secale* genus within the Triticeae tribe of the Poaceae family. Despite its phylogenetic relationship to common wheat (*Triticum aestivum*) and barley (*Hordeum vulgare*), rye possesses unique agronomic traits and genomic properties. Its notable resilience to abiotic stresses, adaptability to infertile soils, and robust resistance to fungal diseases make it a valuable crop and a significant genetic resource. The 1RS chromosome arm of rye, known for its disease-resistance genes, has been instrumental in improving wheat's resistance to powdery mildew and stripe rust. Furthermore, rye is crucial in triticale development, a synthetic crop exceeding rye in biomass and yield. The rye genome, significantly larger than those of barley and diploid wheat species (estimated at ~7.9 Gb), is characterized by a high proportion of transposon elements (approximately 90%). However, the specific contributions of these elements to genome expansion remained unclear, and a high-quality reference genome sequence was lacking. This study aimed to address this gap by sequencing and analyzing the genome of Weining rye, an early-flowering Chinese variety known for its broad-spectrum resistance to powdery mildew and stripe rust. This research sought to understand the genetic basis of Weining rye's elite traits and facilitate genomic and breeding advancements in rye and related cereals.

Previous research has highlighted the complex evolutionary history and genomic features of rye. Studies like Martis et al. (2013) explored the reticulate evolution of the rye genome, emphasizing its hybrid nature. Bauer et al. (2017) made significant progress towards a whole-genome sequence, but a high-quality reference remained elusive. Comparative genomics with other Triticeae species, including common wheat and its diploid progenitors (*Triticum urartu* and *Aegilops tauschii*), as well as barley and wild emmer wheat, provided insights into genome evolution and structure. These studies revealed significant expansion of transposable elements in rye compared to related species, contributing to its larger genome size. The role of rye in disease resistance, especially via the 1RS chromosome arm in wheat improvement, has also been extensively documented. Crespo-Herrera et al. (2017) provided a systematic review of rye's role in resistance to pathogens and pests in wheat. Similarly, the importance of rye in triticale breeding and its nutritional value have been subjects of numerous studies (e.g., Zhu, 2018). However, a detailed understanding of rye's genome structure and the genetic basis of its advantageous traits remained limited before this study.

The study employed a multi-faceted approach, integrating long-read and short-read sequencing technologies. The Weining rye genome was sequenced using both PacBio RS II (for long reads) and Illumina sequencing (for short reads). The long reads were crucial for resolving repetitive regions, while short reads enhanced accuracy. Chromatin conformation capture (Hi-C) data, genetic mapping (using a Weining x Jingzhou cross), and BioNano analysis were integrated to facilitate genome assembly and chromosome scaffolding. The genome size of Weining rye was estimated using flow cytometry. The assembly process involved several steps: read error correction using Canu, contig assembly with wtdbg, FALCON, and MECAT, and merging of assemblies with Quickmerge. Illumina reads were then used for polishing the assembly using Pilon. Hi-C data were used for scaffolding and ordering of contigs, assisted by the genetic map and BioNano data. Gene annotation involved a combination of de novo prediction, homology-based approaches (using protein sequences from other plant genomes), and transcriptome data (from various rye tissues, including developing grains). Repeat analysis utilized tools like RepeatScout, LTR-FINDER, MITE-Hunter, PILER-DF, and RepeatMasker. Gene duplications were analyzed using MCScanX, identifying different types of duplications (tandem, proximal, dispersed, and segmental). Analysis of transposable elements (TEs) focused on their contribution to genome expansion and their impact on gene duplications. Phylogenetic analysis, using single-copy orthologous genes from Weining rye and other grass genomes, was conducted using BEAST to infer divergence times. Synteny analyses, using MCScanX, were performed to compare Weining rye with rice, common wheat, and other species. To investigate seed storage proteins (SSPs), BLAST searches were conducted using SSP sequences from other Triticeae species. Transcription factors (TFs) were predicted using the iTAK pipeline. Disease-resistance-associated genes were identified by homology searches, and their distribution on rye chromosomes was analyzed. Finally, selection sweep analysis (using DRI, FST, and XP-CLR methods) was used to identify genomic regions potentially associated with rye domestication.

The researchers successfully assembled a high-quality Weining rye genome (7.74 Gb), covering 98.47% of the estimated genome size (7.86 Gb). 93.67% of the assembled genome was anchored to seven chromosomes. Transposable elements constituted a significant portion (90.31%) of the genome, with LTR retrotransposons being the most abundant. Three specific LTR-retrotransposon families (Daniela, Sumaya, and Sumana) showed remarkable expansion in rye compared to related species. The analysis revealed a higher number of tandemly and proximally duplicated genes in Weining rye than in other Triticeae species examined. A substantial number of transposed duplicated genes (TrDGs) were identified, highlighting the role of TE activity in shaping the rye genome. Analysis of starch biosynthesis-related genes (SBRGs) revealed multiple duplications and expression variations among duplicated copies, suggesting functional diversification. The study elucidated the physical organization of complex prolamin loci, providing detailed structural information on secalin genes (located in Sec-1 to Sec-4). Comparisons with wheat and barley revealed syntenic relationships and evolutionary deletions in rye. The analysis identified a higher number of transcription factors (TFs) in Weining rye compared to other grasses examined, particularly within the AP2-ERF family. The number of disease-resistance genes (DRA genes) in Weining rye was also higher than in other grasses. Investigation of the early heading trait in Weining rye revealed higher expression levels of *ScFT1* and *ScFT2* (flowering locus T genes) compared to a late-heading variety. Furthermore, functional analyses revealed that dephosphorylation of ScFT2 enhanced its ability to promote flowering. A QTL analysis identified three major QTLs associated with heading date. Selection sweep analysis identified multiple genomic regions potentially linked to rye domestication, notably including genes homologous to those involved in flowering time, grain size, and other domestication-related traits in other crops. The tandem duplication of the *ScID1* gene, homologous to the maize *ID1* gene which regulates the transition from vegetative to floral development, was noted as a potential marker of domestication selection in Weining rye.

This study's high-quality genome assembly of Weining rye provides significant advancements in rye genomics. The findings regarding TE expansion, gene duplications, and the structural organization of complex loci provide valuable insights into rye's evolutionary history and genomic architecture. The detailed characterization of SBRGs and SSPs offers promising avenues for improving rye's yield potential and nutritional qualities. The identification of genes and QTLs involved in the early heading trait provides targets for breeding programs aimed at enhancing adaptability to various growing conditions. The genomic regions identified through selection sweep analysis point to potential domestication genes and contribute to our understanding of the evolutionary processes shaping cultivated rye. The discovery of ScFT2 phosphorylation and its role in regulating flowering time represents a novel finding with broad implications for understanding flowering mechanisms in plants. This work lays the groundwork for further investigation into the functional roles of duplicated genes and their contribution to phenotypic diversity in rye.

This research produced a high-quality genome assembly of Weining rye, providing valuable resources for genetic and breeding studies. The study uncovered novel insights into rye genome characteristics, the genetic basis of important agronomic traits, and potential domestication-associated loci. Future research should focus on functional validation of the identified genes and QTLs, particularly those related to domestication, disease resistance, and stress tolerance. Further exploration of the role of TE activity in rye genome evolution and gene duplication is also warranted. This comprehensive genome resource facilitates further research into rye biology, comparative genomics among cereals, and the development of improved rye and related crops.

The study focused on a single rye variety (Weining rye), which might limit the generalizability of the findings to other rye accessions. Although the authors employed several approaches for the validation of the genome assembly, further validation and comparison with additional varieties is needed to fully capture the genomic diversity in rye. The functional characterization of many genes was not fully explored, and further investigations are needed to establish definitive causal relationships between the genomic variations and their phenotypic consequences. The selection sweep analysis relies on comparisons with a wild relative and may not fully encompass all domestication-related genetic changes.