Dormancy Breaking Treatments in Northern Wild Rice (Zizania palustris L.) Seed Suggest a Physiological Source of Dormancy

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 scarification 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 significantly 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 scarification treatments. Hormonal treatments had no significant 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 sufficient cold storage, it is likely that NWR seed has intermediate or deep physiological dormancy.

First report of bacterial leaf streak caused by Xanthomonas translucens pv. undulosa on cultivated wild rice (Zizania palustris) in Minnesota

Known by the indigenous peoples of the Great Lakes region of North America as Manoomin, wild rice (Zizania palustris) is a native aquatic grass that is honored and central to Anishinaabe culture. Cultivated wild rice, the domesticated form of this cereal bred primarily for resistance to shattering, is grown commercially in paddies. In this study we examined four isolates (CIX303, CIX306, Xt-8, and Xt-22) of Xanthomonas translucens, the causative agent of bacterial leaf streak (BLS) on cereals and weedy grasses, in molecular and host range studies to confirm the pathovar identity of strains associated with cultivated wild rice. Two of the strains examined (CIX303 and CIX306), were isolated from cultivated wild rice in 2016 as part of a survey of the pathogen in Minnesota (Ledman 2019). Xt-8 and Xt-22 are historical strains of X. translucens isolated from symptomatic wild rice leaves collected in Minnesota in the late 1970s that were reported at the time to be X. campestris pv. cerealis (Bowden and Percich 1982). A host range assay was repeated twice in the greenhouse, where two leaves of six seedlings each of hard red spring wheat (cv. RB07), spring barley (cv. Quest), spring rye (cv. Prolific), oat (cv. Ogle), quackgrass, smooth brome grass and cultivated wild rice (cv. Itasca Cycle-12) were inoculated via leaf infiltration (Curland et al. 2020). X. translucens pv. cerealis LMG 679PT, X. translucens pv. secalis LMG 883PT, X. translucens pv. translucens LMG 876T, and X. translucens pv. undulosa LMG 892PT were included as reference strains. Host response profiles were determined for each strain by recording character states five days post inoculation. Water-soaking and necrosis were considered pathogenic reactions, whereas chlorosis was not. Three pathotype strains, LMG 679PT, LMG 876T, and LMG 892PT, caused water-soaking in cultivated wild rice, whereas LMG 883PT caused chlorosis. All four strains from cultivated wild rice produced water-soaking on wheat, barley, quackgrass, and cultivated wild rice, chlorosis or water-soaking on rye, chlorosis on oat, and a reddish water-soaking on smooth brome. The character states generated by these four isolates were identical only to the host response profile for LMG 892PT. LMG 679PT differed, causing chlorosis on wheat, no symptoms on quackgrass, and water-soaking on smooth brome. A 2645 bp concatenation of housekeeping genes (rpoD, dnaK, fyuA, gyrB) was used to perform a Bayesian analysis (GenBank accessions MW528365-MW528384) (Curland et al. 2018, Curland et al. 2020, Young et al. 2008). Subsequent phylogenies grouped all four strains from cultivated wild rice with LMG 892PT and LMG 883PT. A pairwise comparison revealed 100% identity between Xt-22 and LMG 892PT. The percentage identity of CIX303, CIX308, and Xt-8 to LMG 892PT was 99.96, 99.96, and 99.92, respectively. In contrast, when compared to LMG 679PT, the four strains from cultivated wild rice had a percent identity between 97.43 and 97.50. Based on host range studies combined with MLSA, we identified recent and historical isolates from Z. palustris as X. translucens pv. undulosa. Pathovar identity of strains causing BLS on cultivated wild rice in Minnesota is crucial when screening breeding materials for disease resistance. Furthermore, given that X. translucens pv. undulosa has been prevalent on wheat in Minnesota (Curland et al. 2018), expanding knowledge of its host range to include cultivated wild rice may inform disease management practices for both crops. References: Bowden, R., and Percich, J. 1982. Phytopath. 73:640-645. Curland, R., et al. 2018. Phytopath. 108:443–453. Curland, R., et al. 2020. Phytopath. 110:257–266. Ledman, K. 2019. M.S. Thesis, Univ. of Minnesota, St. P. Paul, USA. Young, J., et al. 2008. Syst. Appl. Microbiol. 31:366–377.

Whole Genome Assembly and Annotation of Northern Wild Rice, Zizania palustris L., Supports a Whole Genome Duplication in the Zizania Genus

ABSTRACTNorthern Wild Rice (NWR; Zizania palustris L.) 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 United States. 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 is 1.29 Gb, highly repetitive (∼76.0%), and contains 46,421 putative protein-coding genes. The expansion of retrotransposons within the genome and a whole genome duplication prior to the Zizania-Oryza speciation event have both led to an increase in genome size of NWR in comparison with O. sativa and Z. latifolia. Both events depict a genome rapidly undergoing change over a short evolutionary time. Comparative analyses revealed conservation of large syntenic blocks with Oryza sativa L., which were used to identify putative seed shattering genes. Estimates of divergence times revealed the Zizania genus diverged from Oryza ∼26-30 million years ago (MYA), while NWR and Zizania latifolia diverged from one another ∼6-8 MYA. Comparative genomics confirmed evidence of a whole genome duplication in the Zizania genus and provided support that the event was prior to the NWR-Z. latifolia speciation event. This high-quality 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.

Evaluation of a Recessive Male Floret Color in Cultivated Northern Wild Rice (Zizania palustris L.) for Pollen-Mediated Gene Flow Studies

Northern wild rice (NWR; Zizania palustris L.) is a wind-pollinated, annual, aquatic grass that grows naturally in the Great Lakes Region (GLR) 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 3200m. 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 7m from the PMF donor source with no gene flow detected past 63m. 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, auricle, and culm color. This study demonstrates that the WMF trait is an excellent candidate for use in pollen-mediated gene flow studies in NWR.

Improved Remote Sensing Methods to Detect Northern Wild Rice (Zizania palustris L.)

Declining populations of Zizania palustris L. (northern wildrice, or wildrice) during the last century drives the demand for new and innovative techniques to support monitoring of this culturally and ecologically significant crop wild relative. We trained three wildrice detection models in R and Google Earth Engine using data from annual aquatic vegetation surveys in northern Minnesota. Three different training datasets, varying in the definition of wildrice presence, were combined with Landsat 8 Operational Land Imager (OLI) and Sentinel-1 C-band synthetic aperture radar (SAR) imagery to map wildrice in 2015 using random forests. Spectral predictors were derived from phenologically important time periods of emergence (June–July) and peak harvest (August–September). The range of the Vertical Vertical (VV) polarization between the two time periods was consistently the top predictor. Model outputs were evaluated using both point and area-based validation (polygon). While all models performed well in the point validation with percent correctly classified ranging from 83.8% to 91.1%, we found polygon validation necessary to comprehensively assess wildrice detection accuracy. Our practical approach highlights a variety of applications that can be applied to guide field excursions and estimate the extent of occurrence at landscape scales. Further testing and validation of the methods we present may support multiyear monitoring which is foundational for the preservation of wildrice for future generations.