N 6-Methyladenosine and physiological response divergence confer autotetraploid enhanced salt tolerance compared to its diploid Hordeum bulbosum.

Polyploid species have played an essential role in plant evolution, exemplified by adaptive advantages to abiotic stress. N 6-Methyladenosine (m6A) is suggested to play an important role in stress response. However, whether genome doubling affects m6A to increase autopolyploids stress tolerance is still unclear. This study aims to compare physiological (maintaining osmoregulatory homeostasis) and m6A changes between autotetraploid and diploid wild barley (Hordeum bulbosum) in response to salt (NaCl) stress. Results showed that autotetraploids physiologically had enhanced stress tolerance based on the measured parameters of relative water content, water loss, proline, H2O2, and chlorophyll. Diploid H. bulbosum experienced an excessive abundance of proline following salt stress where tetraploids had beneficial proline accumulation and thus enhanced osmoregulation. The significantly higher level of proline and H2O2 in diploid than in autotetraploid implies that diploids suffered higher osmotic stress than autotetraploid. Autotetraploid produced enough proline to protect stress, but not so much to cause toxicity. m6A in total RNA showed no significant difference between ploidies in controls, but was significantly higher in autotetraploids than in diploids during stress and recovery. These results suggest that increased m6A might be one of molecular mechanisms that increases salt tolerance in autotetraploid H. bulbosum compared to diploids, which enhances the adaptation of autopolyploids. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01260-x.

microRNAs contribute to enhanced salt adaptation of the autopolyploid Hordeum bulbosum compared with its diploid ancestor.

Several studies have shown that autopolyploid can tolerate abiotic stresses better than its diploid ancestor. However, the underlying molecular mechanism is poorly known. microRNAs (miRNAs) are small RNAs that regulate the target gene expression post-transcriptionally and play a critical role in the response to abiotic stresses. Duplication of the whole genome can result in the expansion of miRNA families, and the innovative miRNA-target interaction is important for adaptive responses to various environments. We identified new microRNAs induced by genome duplication, that are also associated with stress response and the distinctive microRNA networks in tetraploid and diploid Hordeum bulbosum using high-throughput sequencing. Physiological results showed that autotetraploid Hordeum bulbosum tolerated salt stress better than its diploid. Comparison of miRNAs expression between diploid and tetraploid check (CK) and salt stress revealed that five miRNAs affected by genome doubling were also involved in salt stress response. Of these, miR528b-3p was only detected in the tetraploid, and downregulated under salt stress relative to that in tetraploid CK. Moreover, through target prediction, it was found that miR528b-3p was not only involved in DNA replication and repair but also participated in salt stress response. Finally, by analyzing all the differentially expressed microRNAs and their targets, we also discovered distinguished microRNAs-target regulatory networks in diploid and tetraploid, respectively. Overall, the results demonstrated the critical role of microRNAs in autopolyploid to have better tolerance salt stress.

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond.

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes.

Unlocking the secondary gene-pool of barley with next-generation sequencing.

Crop wild relatives (CWR) provide an important source of allelic diversity for any given crop plant species for counteracting the erosion of genetic diversity caused by domestication and elite breeding bottlenecks. Hordeum bulbosum L. is representing the secondary gene pool of the genus Hordeum. It has been used as a source of genetic introgressions for improving elite barley germplasm (Hordeum vulgare L.). However, genetic introgressions from H. bulbosum have yet not been broadly applied, due to a lack of suitable molecular tools for locating, characterizing, and decreasing by recombination and marker-assisted backcrossing the size of introgressed segments. We applied next-generation sequencing (NGS) based strategies for unlocking genetic diversity of three diploid introgression lines of cultivated barley containing chromosomal segments of its close relative H. bulbosum. Firstly, exome capture-based (re)-sequencing revealed large numbers of single nucleotide polymorphisms (SNPs) enabling the precise allocation of H. bulbosum introgressions. This SNP resource was further exploited by designing a custom multiplex SNP genotyping assay. Secondly, two-enzyme-based genotyping-by-sequencing (GBS) was employed to allocate the introgressed H. bulbosum segments and to genotype a mapping population. Both methods provided fast and reliable detection and mapping of the introgressed segments and enabled the identification of recombinant plants. Thus, the utilization of H. bulbosum as a resource of natural genetic diversity in barley crop improvement will be greatly facilitated by these tools in the future.

Evolutionary Conservation and Transcriptome Analyses Attribute Perenniality and Flowering to Day-Length Responsive Genes in Bulbous Barley (Hordeum bulbosum).

Rapid population growth and dramatic climatic turnovers are challenging global crop production. These challenges are spurring plant breeders to enhance adaptation and sustainability of major crops. One intriguing approach is to turn annual systems into perennial ones, yet long-term classical breeding efforts to induce perenniality have achieved limited success. Here, we report the results of our investigation of the genetic basis of bulb formation in the nonmodel organism Hordeum bulbosum, a perennial species closely related to barley. To identify candidate genes that regulate bulb formation in H. bulbosum, we applied two complementary approaches. First, we explored the evolutionary conservation of expressed genes among annual Poaceae species. Next, we assembled a reference transcriptome for H. bulbosum and conducted a differential expression (DE) analysis before and after stimulating bulb initiation. Low conservation was identified in genes related to perenniality in H. bulbosum compared with other species, including bulb development and sugar accumulation genes. We also inspected these genes using a DE analysis, which enabled identification of additional genes responsible for bulb initiation and flowering regulation. We propose a molecular model for the regulation of bulb formation involving storage organ development and starch biosynthesis genes. The high conservation observed along a major part of the pathway between H. bulbosum and barley suggests a potential for the application of biotechnological techniques to accelerate breeding toward perenniality in barley.

High Resolution Mapping of a Hordeum bulbosum-Derived Powdery Mildew Resistance Locus in Barley Using Distinct Homologous Introgression Lines.

Powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh) is one of the main foliar diseases in barley (Hordeum vulgare L.; Hv). Naturally occurring resistance genes used in barley breeding are a cost effective and environmentally sustainable strategy to minimize the impact of pathogens, however, the primary gene pool of H. vulgare contains limited diversity owing to recent domestication bottlenecks. To ensure durable resistance against this pathogen, more genes are required that could be unraveled by investigation of secondary barley gene-pool. A large set of Hordeum bulbosum (Hb) introgression lines (ILs) harboring a diverse set of desirable resistance traits have been developed and are being routinely used as source of novel diversity in gene mapping studies. Nevertheless, this strategy is often compromised by a lack of recombination between the introgressed fragment and the orthologous chromosome of the barley genome. In this study, we fine-mapped a Hb gene conferring resistance to barley powdery mildew. The initial genotyping of two Hb ILs mapping populations with differently sized 2HS introgressions revealed severely reduced interspecific recombination in the region of the introgressed segment, preventing precise localization of the gene. To overcome this difficulty, we developed an alternative strategy, exploiting intraspecific recombination by crossing two Hv/Hb ILs with collinear Hb introgressions, one of which carries a powdery mildew resistance gene, while the other doesn't. The intraspecific recombination rate in the Hb-introgressed fragment of 2HS was approximately 20 times higher than it was in the initial simple ILs mapping populations. Using high-throughput genotyping-by-sequencing (GBS), we allocated the resistance gene to a 1.4 Mb interval, based on an estimate using the Hv genome as reference, in populations of only 103 and 146 individuals, respectively, similar to what is expected at this locus in barley. The most likely candidate resistance gene within this interval is part of the coiled-coil nucleotide-binding-site leucine-rich-repeat (CC-NBS-LLR) gene family, which is over-represented among genes conferring strong dominant resistance to pathogens. The reported strategy can be applied as a general strategic approach for identifying genes underlying traits of interest in crop wild relatives.

Bulbosum to Go: A Toolbox to Utilize Hordeum vulgare/bulbosum Introgressions for Breeding and Beyond.

Hordeum bulbosum L., a wild relative of barley (Hordeum vulgare L.), has been considered as a valuable source of genetic diversity for barley improvement. Since the 1990s, a considerable number of barley/H. bulbosum introgression lines (IL)s has been generated, with segments introgressed from H. bulbosum harboring a diverse set of desirable traits. However, the efficient utilization of these ILs has been hampered, largely due to the lack of suitable molecular tools for their genetic characterization and highly reduced interspecific recombination frequencies in the region of the introgression. In the present study, we utilized genotyping-by-sequencing for the detailed molecular characterization of 145 ILs. Genotypic information allows the genetic diversity within the set of ILs to be determined and a strategy was outlined to tackle the obstacle of reduced recombination frequencies. Furthermore, we compiled exome capture re-sequencing information of barley and H. bulbosum and designed an integrated barley/H. bulbosum sequence resource with polymorphism information on interspecific and intraspecific sequence variations of both species. The integrated sequence will be valuable for marker development in barley/H. bulbosum ILs derived from any barley and H. bulbosum donors. This study provides the tools for the widespread utilization of barley/H. bulbosum ILs in applied barley breeding and academic research.