Zizania palustris L. (northern wild rice, NWR) is an aquatic grass native to North America that is notable for its nutritious grain. This is an important species with ecological, cultural and agricultural significance, specifically in the Great Lakes region of the USA. Using flow cytometry, we first estimated the NWR genome size to be 1.8 Gb. Using long-and short-range sequencing, Hi-C scaffolding and RNA-seq data from eight tissues, we generated an annotated whole-genome de novo assembly of NWR. The assembly was 1.29 Gb in length, highly repetitive (approx. 76.0%) and contained 46 421 putative protein-coding genes. The expansion of retrotransposons within the genome and a whole-genome duplication (WGD) after the Zizania-Oryza speciation event have both led to an increase in the genome size of NWR in comparison with Oryza sativa L. and Zizania latifolia. Both events depict a genome rapidly undergoing change over a short evolutionary time. Comparative analyses revealed the conservation of large syntenic blocks between NWR and O. sativa, which were used to identify putative seed-shattering genes. Estimates of divergence times revealed that the Zizania genus diverged from Oryza approximately 26-30 million years ago (26-30 MYA), whereas NWR and Z. latifolia diverged from one another approximately 6-8 MYA. Comparative genomics confirmed evidence of a WGD in the Zizania genus and provided support that the event occurred prior to the NWR-Z. latifolia speciation event. This genome assembly and annotation provides a valuable resource for comparative genomics in the Oryzeae tribe and provides an important resource for future conservation and breeding efforts of NWR.

AbstractCultivated northern wild rice (NWR; Zizania palustris L.) has been bred and studied since the 1950s. One challenge facing researchers is the lack of storage options, due to the seed's unorthodox behaviour. This study evaluated varying storage temperature and moisture conditions for the maintenance of seed viability and dormancy breaking in Minnesota-grown NWR. First, seeds were placed in non-submerged, freezing storage (NSFS) for 12–26 weeks, then in submerged, cold storage (SCS) for 2 weeks. The addition of SCS increased germination (%G) relative to NSFS alone (<0.1% NSFS, 15% NSFS and SCS), indicating that NSFS does not kill seeds but also does not break seed dormancy. Next, the required length of SCS was evaluated by placing seeds in NSFS for 12 weeks and then in SCS for 2–14 weeks. A longer SCS period increased %G from 3 to 79%, at 2 and 14 weeks of SCS, respectively. Lastly, seeds were placed in NSFS, followed by SCS, at varying intervals over a 29-week period. Across lines, germination increased from 20 to 76% between 4 and 7 weeks of SCS, respectively, then plateaued. The results of this study indicate that NSFS could be used to store NWR seeds, but at least 7 weeks in SCS is required to overcome dormancy. Additionally, while NSFS did not break seed dormancy, physiological changes related to stratification processes were occurring in non-submerged, freezing conditions. Results also suggest that the genotypic variation in NWR could be utilized for selection to improve germination and storage viability.

Northern Wild Rice (NWR; Zizania palustris L.) is an aquatic annual grass with significant ecological, cultural, and economic importance to the Great Lakes region of the United States and Canada. Understanding the genetic diversity of NWR is essential to conservation efforts. In this study, we assembled and genotyped a diverse collection of 789 NWR individuals using genotyping-by-sequencing and obtained 5,955 single-nucleotide polymorphisms (SNPs). Our collection consisted of samples from 12 wild NWR populations, some of which were collected temporally, a representative sampling of cultivated NWR germplasm, and a Zizania aquatica population (outgroup). Using these data, we characterized the genetic diversity, relatedness, and population structure of this broad collection of NWR genotypes. We found that wild populations of NWR clustered primarily by their lake or river of origin as well as larger clustering patterns based on watershed assignment. Contextualizing some clustering patterns with historical knowledge of ecosystem management helped to unravel some of the complexity of the population structure of wild populations. Cultivated materials were genetically distinct from wild populations, suggesting limited gene flow between the semi-domesticated crop and its wild counterparts. Estimates of linkage disequilibrium (LD) in NWR demonstrated that LD decays quickly across the genome and provided insights into the quality of the reference genome assembly of NWR. The first genome-wide scans of putative selection events in cultivated NWR were also evaluated. Overall, this study presents a large set of new SNP markers for use in NWR genetic studies as well as new insights into the gene flow, history, and complexity of wild and cultivated populations of NWR.

Northern wild rice (NWR; Zizania palustris L.) is a wind‐pollinated, annual, aquatic grass that grows naturally in the Great Lakes region of the United States and Canada and is also cultivated in flooded paddies, predominantly in California and Minnesota. A better understanding of pollen‐mediated gene flow is needed within the species for both conservation and breeding efforts as cultivation occurs within the species’ natural range and spatially‐isolated, paddy structures are limited within breeding programs. Widely‐cited pollen travel research in NWR demonstrated that pollen could travel at least 3200 m. However, a population segregating for male sterility was used as the pollen recipient in the study and was determined to not be adequate for NWR pollen travel studies. Here, we present the characterization of a recessive white male floret (WMF) population in contrast to the dominant, purple male floret (PMF) color of cultivated NWR along with estimates of pollen‐mediated gene flow in a cultivated paddy setting. Studies conducted in 2018 and 2019 revealed that the primary amount of pollen‐mediated gene flow occurred within the first 7 m from the PMF donor source with no gene flow detected past 63 m. These results suggest that the likelihood of pollen‐mediated gene flow between cultivated NWR and natural stands remains low. We also identified a strong linkage between male floret, collar, and culm color. This study demonstrates that the WMF trait is an excellent candidate for use in pollen‐mediated gene flow studies in NWR.

Dormancy is a limiting factor for breeding in northern wild rice (NWR; Zizania palustris L). This study developed a dormancy curve and tested a combination of scari cation and hormone treatments, across three timepoints, for their ability to break dormancy in NWR and produce viable seedlings and plants. A dormancy curve was established across 9 months post-harvest, which showed maximum germination (95 %) by 17 weeks post-harvest and high germination (≥81 %) through the rest of the testing period. Next, dormancy breaking treatments were tested. At 1 week post-harvest, few seeds germinated (≤ 15 %) across all treatment combinations. However, sulfuric acid increased germination shortly after harvest (5.8 %), compared to water (0.5 %) and NaClO (0 %) but resulted in stunted seedlings, all but one of which died shortly thereafter. At 7 weeks, sulfuric acid treated seeds did not result in signi cantly higher germination than water and maximum germination was still below 15%. By 11 weeks post-harvest, the water treatments had the highest germination and resulted in the most viable plants, indicating that dormancy had begun to break naturally and exceeded the effect of the other scari cation treatments. Hormonal treatments had no signi cant effect on germination or seed viability and no strong conclusions could be drawn about their effect on seedling or plant health. Due to the inability of early germinated seed to consistently produce viable plants and the increase in germination following su cient cold storage, it is likely that NWR seed has intermediate or deep physiological dormancy.