Acidified drinking water improves motor function, prevents tremors and changes disease trajectory in Cln2R207X mice, a model of late infantile Batten disease.

Batten disease is a group of mostly pediatric neurodegenerative lysosomal storage disorders caused by mutations in the CLN1-14 genes. We have recently shown that acidified drinking water attenuated neuropathological changes and improved motor function in the Cln1R151X and Cln3-/- mouse models of infantile CLN1 and juvenile CLN3 diseases. Here we tested if acidified drinking water has beneficial effects in Cln2R207X mice, a nonsense mutant model of late infantile CLN2 disease. Cln2R207X mice have motor deficits, muscle weakness, develop tremors, and die prematurely between 4 and 6 months of age. Acidified water administered to Cln2R207X male mice from postnatal day 21 significantly improved motor function, restored muscle strength and prevented tremors as measured at 3 months of age. Acidified drinking water also changed disease trajectory, slightly delaying the death of Cln2R207X males and females. The gut microbiota compositions of Cln2R207X and wild-type male mice were markedly different and acidified drinking water significantly altered the gut microbiota of Cln2R207X mice. This suggests that gut bacteria might contribute to the beneficial effects of acidified drinking water. Our study demonstrates that drinking water is a major environmental factor that can alter disease phenotypes and disease progression in rodent disease models.

Jurassic NLR: Conserved and dynamic evolutionary features of the atypically ancient immune receptor ZAR1.

Plant nucleotide-binding leucine-rich repeat (NLR) immune receptors generally exhibit hallmarks of rapid evolution, even at the intraspecific level. We used iterative sequence similarity searches coupled with phylogenetic analyses to reconstruct the evolutionary history of HOPZ-ACTIVATED RESISTANCE1 (ZAR1), an atypically conserved NLR that traces its origin to early flowering plant lineages ∼220 to 150 million yrs ago (Jurassic period). We discovered 120 ZAR1 orthologs in 88 species, including the monocot Colocasia esculenta, the magnoliid Cinnamomum micranthum, and most eudicots, notably the Ranunculales species Aquilegia coerulea, which is outside the core eudicots. Ortholog sequence analyses revealed highly conserved features of ZAR1, including regions for pathogen effector recognition and cell death activation. We functionally reconstructed the cell death activity of ZAR1 and its partner receptor-like cytoplasmic kinase (RLCK) from distantly related plant species, experimentally validating the hypothesis that ZAR1 evolved to partner with RLCKs early in its evolution. In addition, ZAR1 acquired novel molecular features. In cassava (Manihot esculenta) and cotton (Gossypium spp.), ZAR1 carries a C-terminal thioredoxin-like domain, and in several taxa, ZAR1 duplicated into 2 paralog families, which underwent distinct evolutionary paths. ZAR1 stands out among angiosperm NLR genes for having experienced relatively limited duplication and expansion throughout its deep evolutionary history. Nonetheless, ZAR1 also gave rise to noncanonical NLRs with integrated domains and degenerated molecular features.

Probiotic-enriched milk and dairy products increase gut microbiota diversity: a comparative study.

Targeting gut microbiota with probiotics has emerged as a promising nutritional approach for the prevention of obesity and metabolic syndrome. Cultured dairy products can be effectively employed for the delivery of probiotics to the gut as well as for the support of growth and survival of probiotic bacteria. The purpose of this study was to characterize the effects of probiotic-enriched pasteurized milk and dairy products (Greek-style yogurt and cottage cheese) of different origins (cow, goat, and camel) on taxonomic composition of the mouse gut microbiota. We hypothesized that cultured dairy products can be an effective vector for the delivery of probiotics to the gut because of its nutritional value, acidic nature, and long shelf-life. Mice were fed a standard low fat, plant polysaccharide-rich (LF/PP) diet supplemented with the probiotic-enriched milk and dairy products for 5 weeks. Next generation sequencing of DNA from mouse fecal samples was used to characterize the bacterial relative abundance. Mice fed a diet supplemented with camel milk demonstrated characteristic changes in the gut microbiota, which included an increase in relative abundance of order Clostridiales and genus Anaerostipes. Mice fed a diet supplemented with the probiotic-enriched cow cheese exhibited an increase in the relative abundance of order Clostridiales, family Ruminococcaceae, and family Lachnospiraceae. The results obtained and their bioinformatics analysis support the conclusion that camel milk and the probiotic cow cheese induce changes in the mouse gut microbiota, which can be characterized as potentially beneficial to health compared to the changes associated with a standard diet. These findings imply that probiotic-enriched milk and dairy products can be highly effective for the delivery and support of probiotic bacteria of the gut.

Single-Cell RNA Sequencing of Plant-Associated Bacterial Communities.

Plants in soil are not solitary, hence continually interact with and obtain benefits from a community of microbes ("microbiome"). The meta-functional output from the microbiome results from complex interactions among the different community members with distinct taxonomic identities and metabolic capacities. Particularly, the bacterial communities of the root surface are spatially organized structures composed of root-attached biofilms and planktonic cells arranged in complex layers. With the distinct but coordinated roles among the different member cells, bacterial communities resemble properties of a multicellular organism. High throughput sequencing technologies have allowed rapid and large-scale analysis of taxonomic composition and metabolic capacities of bacterial communities. However, these methods are generally unable to reconstruct the assembly of these communities, or how the gene expression patterns in individual cells/species are coordinated within these communities. Single-cell transcriptomes of community members can identify how gene expression patterns vary among members of the community, including differences among different cells of the same species. This information can be used to classify cells based on functional gene expression patterns, and predict the spatial organization of the community. Here we discuss strategies for the isolation of single bacterial cells, mRNA enrichment, library construction, and analysis and interpretation of the resulting single-cell RNA-Seq datasets. Unraveling regulatory and metabolic processes at the single cell level is expected to yield an unprecedented discovery of mechanisms involved in bacterial recruitment, attachment, assembly, organization of the community, or in the specific interactions among the different members of these communities.

Analysis of transcriptional responses in root tissue of bread wheat landrace (Triticum aestivum L.) reveals drought avoidance mechanisms under water scarcity.

In this study, high-throughput sequencing (RNA-Seq) was utilized to evaluate differential expression of transcripts and their related genes involved in response to terminal drought in root tissues of bread wheat landrace (L-82) and drought-sensitive genotype (Marvdasht). Subsets of 460 differentially expressed genes (DEGs) in drought-tolerant genotype and 236 in drought-sensitive genotype were distinguished and functionally annotated with 105 gene ontology (GO) terms and 77 metabolic pathways. Transcriptome profiling of drought-resistant genotype "L-82" showed up-regulation of genes mostly involved in Oxidation-reduction process, secondary metabolite biosynthesis, abiotic stress response, transferase activity and heat shock proteins. On the other hand, down-regulated genes mostly involved in signaling, oxidation-reduction process, secondary metabolite biosynthesis, auxin-responsive protein and lipid metabolism. We hypothesized that the drought tolerance in "L-82" was a result of avoidance strategies. Up-regulation of genes related to the deeper root system and adequate hydraulic characteristics to allow water uptake under water scarcity confirms our hypothesis. The transcriptomic sequences generated in this study provide information about mechanisms of acclimation to drought in the selected bread wheat landrace, "L-82", and will help us to unravel the mechanisms underlying the ability of crops to reproduce and keep its productivity even under drought stress.

Genome-Wide Association Study for Spot Blotch Resistance in Hard Winter Wheat.

Spot blotch (SB) caused by Cochliobolus sativus (anamorph: Bipolaris sorokiniana) is an economically important disease of wheat worldwide. Under a severe epidemic condition, the disease can cause yield losses up to 70%. Previous approaches like bi-parental mapping for identifying SB resistant genes/QTLs exploited only a limited portion of the available genetic diversity with a lower capacity to detect polygenic traits, and had a lower marker density. In this study, we performed genome-wide association study (GWAS) for SB resistance in hard winter wheat association mapping panel (HWWAMP) of 294 genotypes. The HWWAMP was evaluated for response to B. sorokiniana (isolate SD40), and a range of reactions was observed with 10 resistant, 38 moderately resistant, 120 moderately resistant- moderately susceptible, 111 moderately susceptible, and 15 susceptible genotypes. GWAS using 15,590 high-quality SNPs and 294 genotypes we identified six QTLs (p = <0.001) on chromosomes 2D, 3A, 4A, 4B, 5A, and 7B that collectively explained 30% of the total variation for SB resistance. Highly associated SNPs were identified for all six QTLs, QSb.sdsu-2D.1 (SNP: Kukri_c31121_1460, R2 = 4%), QSb.sdsu-3A.1 (SNP: Excalibur_c46082_440, R2 = 4%), QSb.sdsu-4A.1 (SNP: IWA8475, R2 = 5.5%), QSb.sdsu-4B.1 (SNP: Excalibur_rep_c79414_306, R2 = 4%), QSb.sdsu-5A.1 (SNP: Kukri_rep_c104877_2166, R2 = 6%), and QSb.sdsu-7B.1 (SNP: TA005844-0160, R2 = 6%). Our study not only validates three (2D, 5A, and 7B) genomic regions identified in previous studies but also provides highly associated SNP markers for marker assisted selection. In addition, we identified three novel QTLs (QSb.sdsu-3A.1, QSb.sdsu-4A.1, and QSb.sdsu-4B.1) for SB resistance in wheat. Gene annotation analysis of the candidate regions identified nine NBS-LRR and 38 other plant defense-related protein families across multiple QTLs, and these could be used for fine mapping and further characterization of SB resistance in wheat. Comparative analysis with barley indicated the SB resistance locus on wheat chromosomes 2D, 3A, 5A, and 7B identified in our study are syntenic to the previously identified SB resistance locus on chromosomes 2H, 3H, 5H, and 7H in barley. The 10 highly resistant genotypes and SNP markers identified in our study could be very useful resources for breeding of SB resistance in wheat.

Proteomic Responses of Switchgrass and Prairie Cordgrass to Senescence.

Senescence in biofuel grasses is a critical issue because early senescence decreases potential biomass production by limiting aerial growth and development. 2-Dimensional, differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometry of selected protein spots was used to evaluate differences between leaf proteomes of early (ES)- and late- senescing (LS) genotypes of Prairie cordgrass (ES/LS PCG) and switchgrass (ES/LS SG), just before and after senescence was initiated. Analysis of the manually filtered and statistically evaluated data indicated that 69 proteins were significantly differentially abundant across all comparisons, and a majority (41%) were associated with photosynthetic processes as determined by gene ontology analysis. Ten proteins were found in common between PCG and SG, and nine and 18 proteins were unique to PCG and SG respectively. Five of the 10 differentially abundant spots common to both species were increased in abundance, and five were decreased in abundance. Leaf proteomes of the LS genotypes of both grasses analyzed before senescence contained significantly higher abundances of a 14-3-3 like protein and a glutathione-S-transferase protein when compared to the ES genotypes, suggesting differential cellular metabolism in the LS vs. the ES genotypes. The higher abundance of 14-3-3 like proteins may be one factor that impacts the senescence process in both LS PCG and LS SG. Aconitase dehydratase was found in greater abundance in all four genotypes after the onset of senescence, consistent with literature reports from genetic and transcriptomic studies. A Rab protein of the Ras family of G proteins and an s-adenosylmethionine synthase were more abundant in ES PCG when compared with the LS PCG. In contrast, several proteins associated with photosynthesis and carbon assimilation were detected in greater abundance in LS PCG when compared to ES PCG, suggesting that a loss of these proteins potentially contributed to the ES phenotype in PCG. Overall, this study provides important data that can be utilized toward delaying senescence in both PCG and SG, and sets a foundational base for future improvement of perennial grass germplasm for greater aerial biomass productivity.

Validating DNA Polymorphisms Using KASP Assay in Prairie Cordgrass (Spartina pectinata Link) Populations in the U.S.

Single nucleotide polymorphisms (SNPs) are one of the most abundant DNA variants found in plant genomes and are highly efficient when comparing genome and transcriptome sequences. SNP marker analysis can be used to analyze genetic diversity, create genetic maps, and utilize marker-assisted selection breeding in many crop species. In order to utilize these technologies, one must first identify and validate putative SNPs. In this study, 121 putative SNPs, developed from a nuclear transcriptome of prairie cordgrass (Spartina pectinata Link), were analyzed using KASP technology in order to validate the SNPs. Fifty-nine SNPs were validated using a core collection of 38 natural populations and a phylogenetic tree was created with one main clade. Samples from the same population tended to cluster in the same location on the tree. Polymorphisms were identified within 52.6% of the populations, split evenly between the tetraploid and octoploid cytotypes. Twelve selected SNP markers were used to assess the fidelity of tetraploid crosses of prairie cordgrass and their resulting F2population. These markers were able to distinguish true crosses and selfs. This study provides insight into the genomic structure of prairie cordgrass, but further analysis must be done on other cytotypes to fully understand the structure of this species. This study validates putative SNPs and confirms the potential usefulness of SNP marker technology in future breeding programs of this species.

High-resolution radiation hybrid map of wheat chromosome 1D.

Physical mapping methods that do not rely on meiotic recombination are necessary for complex polyploid genomes such as wheat (Triticum aestivum L.). This need is due to the uneven distribution of recombination and significant variation in genetic to physical distance ratios. One method that has proven valuable in a number of nonplant and plant systems is radiation hybrid (RH) mapping. This work presents, for the first time, a high-resolution radiation hybrid map of wheat chromosome 1D (D genome) in a tetraploid durum wheat (T. turgidum L., AB genomes) background. An RH panel of 87 lines was used to map 378 molecular markers, which detected 2312 chromosome breaks. The total map distance ranged from approximately 3,341 cR(35,000) for five major linkage groups to 11,773 cR(35,000) for a comprehensive map. The mapping resolution was estimated to be approximately 199 kb/break and provided the starting point for BAC contig alignment. To date, this is the highest resolution that has been obtained by plant RH mapping and serves as a first step for the development of RH resources in wheat.

Comparative DNA sequence analysis of wheat and rice genomes.

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.

The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms.

Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.