Decoupled responses of above- and below-ground beta-diversity to nitrogen enrichment in a typical steppe.

Increased atmospheric nitrogen (N) deposition affects biodiversity in terrestrial ecosystems. However, we do not know whether the effects of N on above-ground plant ß-diversity are coupled with changes occurring in the soil seed bank. We conducted a long-term N-addition experiment in a typical steppe and found that above-ground ß-diversity increased and then decreased with increasing N addition, whereas below-ground ß-diversity decreased linearly. This suggests decoupled dynamics of plant communities and their soil seed bank under N enrichment. Species substitution determined above- and below-ground ß-diversity change via an increasing role of deterministic processes with N addition. These effects were mostly driven by differential responses of the above-ground vegetation and the soil seed bank ß-diversities to N-induced changes in environmental heterogeneity, increased soil inorganic N concentrations and soil acidification. Our findings highlight the importance of considering above- and below-ground processes simultaneously for effectively conserving grassland ecosystems under N enrichment.

Effect of the Seed Bank on Evolutionary Rescue in Small Populations: Univariate and Multivariate Demogenetic Dynamics.

AbstractUnder global change, the impact of seed banks on evolutionary rescue is uncertain. They buffer plant populations from demographic and genetic stochasticity but extend generation time and can become a reservoir of maladapted alleles. We built analytical and individual-based models to predict the effect of seed banks on the persistence of small annual plant populations facing an abrupt or sustained directional change in uni- or multivariate trait optima. Demogenetic dynamics predict that under most scenarios seed banks increase the lag yet enhance persistence to 200-250 years by absorbing demographic losses. Simulations indicate that the seed bank has a minimal impact on the genetic skew, although we suggest that this result could depend on the fitness component under selection. Our multivariate model reveals that by enlarging and reshaping the G matrix, seed banks can diminish the impact of mutational correlation and even accelerate adaptation under antagonistic pleiotropy relative to populations without a bank. We illustrate how the magnitude of optimum fluctuations, type and degree of optimum change, selection strength, and vital rates are weights that tip the scales determining persistence. Finally, our work highlights that migration from the past is not maladaptative when optimum fluctuations are large enough to create stepping stones to the new optimum.

Large and non-spherical seeds are less likely to form a persistent soil seed bank.

There is some evidence that seed traits can affect the long-term persistence of seeds in the soil. However, findings on this topic have differed between systems. Here, we brought together a worldwide database of seed persistence data for 1474 species to test the generality of seed mass-shape-persistence relationships. We found a significant trend for low seed persistence to be associated with larger and less spherical seeds. However, the relationship varied across different clades, growth forms and species ecological preferences. Specifically, relationships of seed mass-shape-persistence were more pronounced in Poales than in other order clades. Herbaceous species that tend to be found in sites with low soil sand content and precipitation have stronger relationships between seed shape and persistence than in sites with higher soil sand content and precipitation. For the woody plants, the relationship between persistence and seed morphology was stronger in sites with high soil sand content and low precipitation than in sites with low soil sand content and higher precipitation. Improving the ability to predict the soil seed bank formation process, including burial and persistence, could benefit the utilization of seed morphology-persistence relationships in management strategies for vegetation restoration and controlling species invasion across diverse vegetation types and environments.

A long-term experiment reveals no trade-off between seed persistence and seedling emergence.

Information on seed persistence and seedling emergence from the soil seed bank is critical for understanding species coexistence and predicting community dynamics. However, quantifying seed persistence in the soil is challenging; thus, its association with other life-history traits is poorly known on a broad scale. Using germination phenology for 349 species in a 42-yr experiment, we quantified the persistence-emergence correlations and their associations with intrinsic regeneration traits using Bayesian phylogenetic multilevel models. We showed no trade-off between seed persistence and seedling emergence. Physically dormant seeds were more persistent but exhibited lower emergence than nondormant seeds. Monocarpic species had both higher persistence and emergence than polycarpic species. Seed mass posed a marginal proxy for persistence, while emergence almost doubled from the smallest to the largest seeds. This study challenges the traditional assumption and is the first demonstration of noncorrelation between persistence and emergence, probably owing to the complexity of regenerative strategies. Species with short persistence and low emergence would be the most vulnerable for in situ conservation. Our analyses of this unique, long-term dataset provide a strong incentive for further experimental studies and a rich data resource for future syntheses.

Rodents mediate the relationship between seed rain, seed bank, and plant community with increased grazing disturbance.

Seed rain and the soil seed bank represent the dispersal of seeds in space and time, respectively, and can be important sources of recruitment of new individuals during plant community regeneration. However, the temporal dynamics of seed rain and the mechanisms by which the seed rain and soil seed bank may play a role in plant community regeneration with increased grazing disturbance remain unclear. Seed rain, soil seed bank, aboveground vegetation, and rodent density were sampled along a grazing gradient in an alpine marsh on the eastern Tibetan Plateau. We described the temporal dynamics of seed dispersal using Bayesian generalized mixed models, and nonmetric multidimensional scaling and the structural equation model were used to examine the effects of grazing disturbance on the relative role of seed rain and soil seed bank on aboveground plant community regeneration. The temporal dynamics of seed rain changed from a unimodal to a bimodal pattern with increased grazing disturbance. Both species diversity and seed density of the seed rain and seed bank increased significantly with increased grazing disturbance. Increased grazing disturbance indirectly increased the similarity of composition between seed rain, seed bank, and aboveground plant community by directly increasing species diversity and abundance of aboveground plant community. However, increased grazing disturbance also indirectly decreased the similarity of seed rain, soil seed bank, and aboveground plant community by directly increasing rodent density. The similarity between seed rain and aboveground plant community was greater than that of the soil seed bank and aboveground plant community with increased grazing disturbance. Grazing disturbance spreads the risk of seed germination and seedling establishment by changing the temporal dynamics of seed dispersal. Plants (positive) and rodents (negative) mediated the role of seed rain and soil seed bank in plant community regeneration. The role of seed rain in plant community regeneration is higher than the seed bank in disturbed alpine marshes. Our findings increase our understanding of the regeneration process of the plant community, and they provide valuable information for the conservation and restoration of alpine marsh ecosystems.

Fertility and tillage intensity affect weed community diversity and functional structure in long-term organic systems.

Knowledge of how agricultural management interacts with weed seed banks and emergent weed communities is crucial for proactive weed management. Though studies have detailed how differences in disturbance and nutrient applications between organic and conventional herbicide-based systems affect weed communities, few have focused on these same factors in contrasting organic systems. This study assessed the seed banks and emergent weed communities from the most recent crop rotation cycle (2017-2022) of a long-term experiment, which compared four organic grain and forage cropping systems differing in nutrient inputs and soil disturbance. The high fertility (HF) system received high-rate nutrient applications, low fertility (LF) received low-rate applications, enhanced weed management (EWM) focused on weed control through frequent soil disturbance, and reduced tillage (RT) prioritized soil health with less intense or frequent soil disturbance. Soil samples for greenhouse germination assays were collected at the beginning (2017) and end (2022) of the rotation to explore how these four systems influenced seed bank dynamics over time. Weed community biomass was also sampled in each crop during this time. Treatment effects on weed abundance, taxonomic diversity, and community-weighted means and functional dispersion of weed traits were analyzed with generalized mixed-effect models. The RT system had the highest weed seed bank taxonomic diversity, and EWM had the lowest. RT and LF had higher functional dispersion of traits than HF in the seed bank. Weed seed bank communities in HF and RT were characterized by short, small-seeded, and early germinating weed species. However, seed banks were also labile: Differences between systems in seed density and all other mean trait values were dependent on the crop, which preceded seed bank sampling. Likewise, differences among emergent weed communities in the four systems depended on an interaction between crop species and their planting year. Results suggest that resource availability and intensity of disturbance act as weed community assembly filters in organic cropping systems. Organic growers seeking to design systems that balance weed management and production goals can use relatively low soil disturbance and nutrient application to increase weed community taxonomic or functional diversity without necessarily increasing weed biomass or seed bank density.

Investigating seed bank potential of crustose coralline algae using DNA metabarcoding.

To examine the potential for the autogenic ecosystem engineers, crustose coralline algae (CCA), to serve as seed banks or refugia for life stages of other species, it is critical to develop sampling protocols that reflect the diversity of life present. In this pilot study on two shallow water species of CCA collected from Raoul Island (Kermadec Islands; Rangitahua) New Zealand, we investigated two preservation methods (ethanol vs. silica gel), sampled inner and outer regions of the crusts, and used DNA metabarcoding and seven genes/gene regions (16S rRNA, 18S rRNA, 23S rRNA, cox1, rbcL, and tufA genes and the ITS rRNA region) to develop a protocol for taxa identification. The results revealed immense diversity, with typically more taxa identified within the inner layers than the outer layers. As highlighted in other metabarcoding studies and in earlier work on rhodoliths (nodose coralline algae), reference databases are incomplete, and to some extent, the use of multiple markers mitigates this issue. Specifically, the 23S rRNA and rbcL genes are currently more suitable for identifying algae, while the cox1 gene fares better at capturing the diversity present inclusive of algae. Further investigation of these autogenic ecosystem engineers that likely act as marine seed banks is needed.

Rapid evolution during climate change: demographic and genetic constraints on adaptation to severe drought.

Populations often vary in their evolutionary responses to a shared environmental perturbation. A key hurdle in building more predictive models of rapid evolution is understanding this variation-why do some populations and traits evolve while others do not? We combined long-term demographic and environmental data, estimates of quantitative genetic variance components, a resurrection experiment and individual-based evolutionary simulations to gain mechanistic insights into contrasting evolutionary responses to a severe multi-year drought. We examined five traits in two populations of a native California plant, Clarkia xantiana, at three time points over 7 years. Earlier flowering phenology evolved in only one of the two populations, though both populations experienced similar drought severity and demographic declines and were estimated to have considerable additive genetic variance for flowering phenology. Pairing demographic and experimental data with evolutionary simulations suggested that while seed banks in both populations probably constrained evolutionary responses, a stronger seed bank in the non-evolving population resulted in evolutionary stasis. Gene flow through time via germ banks may be an important, underappreciated control on rapid evolution in response to extreme environmental perturbations.

Weak seed banks influence the signature and detectability of selective sweeps.

Seed banking (or dormancy) is a widespread bet-hedging strategy, generating a form of population overlap, which decreases the magnitude of genetic drift. The methodological complexity of integrating this trait implies it is ignored when developing tools to detect selective sweeps. But, as dormancy lengthens the ancestral recombination graph (ARG), increasing times to fixation, it can change the genomic signatures of selection. To detect genes under positive selection in seed banking species it is important to (1) determine whether the efficacy of selection is affected, and (2) predict the patterns of nucleotide diversity at and around positively selected alleles. We present the first tree sequence-based simulation program integrating a weak seed bank to examine the dynamics and genomic footprints of beneficial alleles in a finite population. We find that seed banking does not affect the probability of fixation and confirm expectations of increased times to fixation. We also confirm earlier findings that, for strong selection, the times to fixation are not scaled by the inbreeding effective population size in the presence of seed banks, but are shorter than would be expected. As seed banking increases the effective recombination rate, footprints of sweeps appear narrower around the selected sites and due to the scaling of the ARG are detectable for longer periods of time. The developed simulation tool can be used to predict the footprints of selection and draw statistical inference of past evolutionary events in plants, invertebrates, or fungi with seed banks.

Snow, fire and drought: How alpine and treeline soil seed banks are affected by simulated climate change.

BACKGROUND AND AIMS: Seed persistence in soil depends on environmental factors that affect seed dormancy and germination, such as temperature and water availability. In high-elevation ecosystems, rapid changes in these environmental factors due to climate change can impact future plant recruitment. To date, our knowledge on how soil seed banks from high-altitude environments will respond to climate change and extreme climate-related events is limited. Here, using the seedling emergence method, we investigated the effects of reduced snow cover, fire and drought on the density and diversity of germinants from soil seed banks of two high-altitude plant communities: a tall alpine herbfield and a treeline ecotone. METHODS: In Autumn 2020, we collected soil samples and characterized the standing vegetation of both communities at Kosciuszko National Park, Australia. Then, we employed a factorial experiment and subjected the soil samples to a series of manipulative treatments using greenhouse studies. KEY RESULTS: The treeline had a larger and more diverse soil seed bank than the herbfield. A reduction in snow had a negative effect on the number of germinants in the herbfield and increased the dissimilarity with the standing vegetation, while the treeline responses were mainly neutral. Fire did not significantly affect the number of germinants but decreased the evenness values in both communities. The drought treatment reduced the number and richness of germinants and increased the dissimilarity with the standing vegetation in both communities. Plant functional forms explained some of the detected effects but seed functional traits did not. CONCLUSIONS: Our study suggests that simulated climate change will affect plant recruitment from soil seed banks in a variety of ways. Changes in snow cover, incidences of fire and drought may be key drivers of germination from the soil seed bank and therefore the future composition of alpine plant communities.

Light controls alpine meadow community assembly during succession by affecting species recruitment from the seed bank.

Some research indicates that soil seed banks can promote species coexistence through storage effects. However, the seed bank mechanism that maintains plant assembly and its role in degraded grassland restoration are still not clear. We collected seed bank samples from early, mid and late secondary successional stages of an abandoned subalpine meadow on the Tibetan Plateau, and samples from each stage were exposed to full (i.e., natural), mid, and low light treatments in the field to represent light availability at the bottom/understory (soil surface) of a plant community in the early, mid and late stages of succession, respectively. Species richness, seed density, species composition, and community weighted mean values (CWMs) of seed mass of the species whose seeds germinated in soil samples were evaluated. In response to the light treatments, species richness increased significantly with increased light only for the late successional stage, seed density increased significantly with increased light only in the early and mid successional stages, and seed mass decreased significantly with increased light only in the mid and late successional stages. Species composition differed significantly among the light treatments only in the late successional stage. For the successional series, species richness and seed mass of the species that germinated increased significantly with succession only under mid and full light treatments. Seed density decreased significantly with succession in each light treatment. Species composition differed significantly between the early- and late stage and between the mid and late stage in each light treatment. Both the abiotic (light) and biotic (seed mass) factors influence seed bank recruitment to the plant community. Regeneration of small-seeded species in the seed bank was inhibited under low light in the late successional stage. The balance of stochastic and deterministic processes along a successional gradient was determined by regeneration from the seed bank depending on light intensity change. Differences in seed response to light intensity change largely determined plant community assembly. Our findings should help in the development of effective conservation and restoration strategies.

The 141-year period for Dr. Beal's seed viability experiment: A hybrid surprise.

PREMISE: In 1879, Dr. William Beal buried 20 glass bottles filled with seeds and sand at a single site at Michigan State University. The goal of the experiment was to understand seed longevity in the soil, a topic of general importance in ecology, restoration, conservation, and agriculture, by periodically assaying germinability of these seeds over 100 years. The interval between germination assays has been extended and the experiment will now end after 221 years, in 2100. METHODS: We dug up the 16th bottle in April 2021 and attempted to germinate the 141-year-old seeds it contained. We grew germinants to maturity and identified these to species by vegetative and reproductive phenotypes. For the first time in the history of this experiment, genomic DNA was sequenced to confirm species identities. RESULTS: Twenty seeds germinated over the 244-day assay. Eight germinated in the first 11 days. All 20 belonged to the Verbascum genus: Nineteen were V. blattaria according to phenotype and ITS2 genotype; and one had a hybrid V. blattaria × V. thapsus phenotype and ITS2 genotype. In total, 20/50 (40%) of the original Verbascum seeds in the bottle germinated in year 141. CONCLUSIONS: While most species in the Beal experiment lost all seed viability in the first 60 years, a high percentage of Verbascum seeds can still germinate after 141 years in the soil. Long-term experiments such as this one are rare and invaluable for studying seed viability in natural soil conditions.

Spatial variation of soil seed banks along a gradient of anthropogenic disturbances in tropical forests on coral islands.

Poor regeneration of natural vegetation is a major factor contributing to the degradation of tropical coral islands. Soil seed banks (SSB) are important for maintaining the resilience of plant communities. However, the community characteristics and spatial distribution of SSBs and the controlling factors along human disturbance on coral islands are unclear. To fill this gap, we measured the community structure and spatial distributions of forest SSBs on three coral islands in the South China Sea, with varying degrees of human disturbance. The results showed that strong human disturbance increased the diversity, richness, and density of SSBs, as well as increased the richness of invasive species. With increased human disturbance, the heterogeneity pattern of SSBs spatial distribution changed from difference between forest east and west to forest center and edge. The similarity between the SSBs and above-ground vegetation also increased, and the distribution of invasive species extended from the edge to the central area of the forests, demonstrating that human disturbance limited the outward dispersal of seeds of resident species but increased the inward dispersal of seeds of invasive species. Interaction between soil properties, plant characteristics, and human disturbance explained 23-45% of the spatial variation of forest SSBs on the coral islands. However, human disturbance reduced the correlations of plant communities and spatial distribution of SSBs with soil factors (i.e., available phosphorus and total nitrogen) and increased the correlations of the community characteristics of SSB with landscape heterogeneity index, road distance, and shrub and litter cover. Resident seed dispersal on tropical coral islands might be enhanced by reducing building height, constructing buildings in down-wind locations, and preserving corridors that support animal movement among forest fragments.

Seed banks alter metacommunity diversity: The interactive effects of competition, dispersal and dormancy.

Dispersal and dormancy are two common strategies allowing for species persistence and the maintenance of biodiversity in variable environments. However, theory and empirical tests of spatial diversity patterns tend to examine either mechanism in isolation. Here, we developed a stochastic, spatially explicit metacommunity model incorporating seed banks with varying germination and survival rates. We found that dormancy and dispersal had interactive, nonlinear effects on the maintenance and distribution of metacommunity diversity. Seed banks promoted local diversity when seed survival was high and maintained regional diversity through interactions with dispersal. The benefits of seed banks for regional diversity were largest when dispersal was high or intermediate, depending on whether local competition was equal or stabilising. Our study shows that classic predictions for how dispersal affects metacommunity diversity can be strongly influenced by dormancy. Together, these results emphasise the need to consider both temporal and spatial processes when predicting multi-scale patterns of diversity.

A quantitative analysis of temperature-dependent seasonal dormancy cycling in buried Arabidopsis thaliana seeds can predict seedling emergence in a global warming scenario.

Understanding how the environment regulates seed-bank dormancy changes is essential for forecasting seedling emergence in actual and future climatic scenarios, and to interpret studies of dormancy mechanisms at physiological and molecular levels. Here, we used a population threshold modelling approach to analyse dormancy changes through variations in the thermal range permissive for germination in buried seeds of Arabidopsis thaliana Cvi, a winter annual ecotype. Results showed that changes in dormancy level were mainly associated with variations in the higher limit of the thermal range permissive for germination. Changes in this limit were positively related to soil temperature during dormancy release and induction, and could be predicted using thermal time. From this, we developed a temperature-driven simulation to predict the fraction of the seed bank able to germinate in a realistic global warming scenario that approximated seedling emergence timing. Simulations predicted, in accordance with seedling emergence observed in the field, an increase in the fraction of the seed bank able to emerge as a result of global warming. In addition, our results suggest that buried seeds perceive changes in the variability of the mean daily soil temperature as the signal to change between dormancy release and induction according to the seasons.

Extinction threshold and large population limit of a plant metapopulation model with recurrent extinction events and a seed bank component.

We introduce a new model for plant metapopulations with a seed bank component, living in a fragmented environment in which local extinction events are frequent. This model is an intermediate between population dynamics models with a seed bank component, based on the classical Wright-Fisher model, and Stochastic Patch Occupancy Models (SPOMs) used in metapopulation ecology. Its main feature is the use of "ghost" individuals, which can reproduce but with a very strong selective disadvantage against "real" individuals, to artificially ensure a constant population size. We show the existence of an extinction threshold above which persistence of the subpopulation of "real" individuals is not possible, and investigate how the seed bank characteristics affect this extinction threshold. We also show the convergence of the model to a SPOM under an appropriate scaling, bridging the gap between individual-based models and occupancy models.

Long-term Spartina alterniflora invasion simplified soil seed bank and regenerated community in a coastal marsh wetland.

The coastal wetland is easily invaded by alien species due to locating in the land and sea transitional area. As a potential driving regeneration force, the soil seed bank is vital to the community restoration and species diversity protection. To reveal the long-term Spartina alterniflora invasion impact on the soil seed banks and regenerated communities, we investigated the seed banks under the different vegetation types (S. alterniflora, Phragmites australis, Scirpus mariqueter, ruderal and unvegetated site) and soil depths (0-5 and 5-10 cm) in the coastal salt marsh wetland, Chongming island, eastern China. The results showed that the soil seed bank richness and species density under different vegetation types were higher than the aboveground vegetation, and those of 0-5 cm seed banks were higher than 5-10 cm, except for the unvegetated site. The species richness and S. alterniflora seed proportion in the seed banks under S. alterniflora communities (S.AS) were lower and larger respectively than those of other sites. The species composition between S.AS and the aboveground communities showed high similarity with aggregation phylogenetic structures in two soil depths. The seed bank variations at 0-5 and 5-10 cm depths were interpreted 3.03% and 2.25% by the aboveground communities, while 4.92% and 5.55% were interpreted by the soil microbial biomass. The SEM model explained 98.1% and 91.8% of the seed banks richness at the 0-5 cm depth and 5-10 cm depth, respectively, and explained 98.8% and 46.1% of the seed banks species density at the 0-5 cm depth and 5-10 cm depth, respectively. The aboveground vegetation biomass and abundance directly affected the 0-5 cm seed banks richness and species density, while its height and biomass only affected the 5-10 cm seed banks species density. The 0-10 cm soil depth microbial biomass indirectly affected the 0-5 cm seed banks richness and species density, while affected the 5-10 cm seed banks richness. Soil physical and chemical properties only indirectly affected the 0-5 cm seed banks species density. The results provided a reference for the ecological evaluation of the impacts of S. alterniflora invasion into the coastal salt marsh wetland of eastern China, and guidance for the protection and restoration of the native plant communities.

The role of maternal age, growth, and environment in shaping offspring performance in an aerial conifer seed bank.

PREMISE: Maternal effects have been demonstrated to affect offspring performance in many organisms, and in plants, seeds are important mediators of these effects. Some woody plant species maintain long-lasting canopy seed banks as an adaptation to wildfires. Importantly, these seeds stored in serotinous cones are produced by the mother plant under varying ontogenetic and physiological conditions. METHODS: We sampled the canopy seed bank of a highly serotinous population of Pinus pinaster to test whether maternal age and growth and the environmental conditions during each crop year affected seed mass and ultimately germination and early survival. After determining retrospectively the year of each seed cohort, we followed germination and early survival in a semi-natural common garden. RESULTS: Seed mass was related to maternal age and growth at the time of seed production; i.e., slow-growing, older mothers had smaller seeds, and fast-growing, young mothers had larger seeds, which could be interpreted either as a proxy of senescence or as a maternal strategy. Seed mass had a positive effect on germination success, but aside from differences in seed mass, maternal age had a negative effect and diameter had a positive effect on germination timing and subsequent survival. CONCLUSIONS: The results highlight the importance of maternal conditions combined with seed mass in shaping seedling establishment. Our findings open new insights in the offspring performance deriving from long-term canopy seed banks, which may have high relevance for plant adaptation.

Changes in the composition of the soil seed bank of grassland after giant ragweed (Ambrosia trifida L.) invasion.

Giant ragweed (Ambrosia trifida L.), an invasive weed, has an expanding distribution area and has recently started to spread in grasslands. This unusual event threatens grasslands worldwide. In this study, we aimed to evaluate the changes in the grassland soil seed banks caused by the giant ragweed invasion in Yili Valley, Xinjiang, China. Using the space-for-time substitution approach, we compared and quantified the soil seed bank communities in a grassland over eight years following giant ragweed invasion and after its removal. The results showed that the duration of invasion determined whether giant ragweed might pose a significant threat to the native seed bank community. Four years after the invasion, the in-site seed bank density of native community significantly decreased (30.44%), while the relative coverage of giant ragweed aboveground reached 83.75%. Furthermore, the species richness in the seed bank decreased significantly (12.36%), while the relative coverage of giant ragweed reached 100% six years after the invasion. Eight years after the invasion, the seed bank density and species richness of the native community decreased by 83.28% and 39.33%, respectively, whereby the seed banks tended to be homogeneous. After the removal of giant ragweed, the potential for regeneration was limited by the residual seed bank densities of the native community. Although the native seed bank density had increased significantly after three years of restoration, new growth was dominated by weedy species, rather than by the distinctive components of the grassland habitat. Our study clarifies the process by which giant ragweed causes damage to grasslands and serves as a reference for grassland restoration and management efforts.

Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine.

BACKGROUND: We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker-free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB. RESULTS: We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy. CONCLUSIONS: All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future.