Identification and characterisation of a previously unknown drought tolerance-associated microRNA in barley.

Drought is the most serious abiotic stress, and causes crop losses on a worldwide scale. The present study identified a previously unknown microRNA (designated as hvu-miRX) of 21 nucleotides (nt) in length in barley. Its precursor (designated pre-miRX) and primary transcript (designated pri-miRX) were also identified, with lengths of 73 and 559 nt, respectively. The identified upstream sequence of pri-miRX contained both the TATA box and the CAAT box, which are both required for initiation of transcription. Transient promoter activation assays showed that the core promoter region of pri-miRX ranged 500 nt from the transcription start site. In transgenic barley overexpression of the wheat DREB3 transcription factor (TaDREB3) caused hvu-miRX to be highly expressed as compared with the same miRNA in non-transgenic barley. However, the high expression was not directly associated with TaDREB3. Genomic analysis revealed that the hvu-miRX gene was a single copy located on the short arm of chromosome 2 and appeared to be only conserved in Triticeae, but not in other plant species. Notably, transgenic barley that overexpressed hvu-miRX showed drought tolerance. Degradome library analysis and other tests showed that hvu-miRX targeted various genes including transcription factors via the cleavage mode. Our data provides an excellent opportunity to develop drought stress tolerant cereals using hvu-miRX.

Bread wheat: a role model for plant domestication and breeding.

BACKGROUND: Bread wheat is one of the most important crops in the world. Its domestication coincides with the beginning of agriculture and since then, it has been constantly under selection by humans. Its breeding has followed millennia of cultivation, sometimes with unintended selection on adaptive traits, and later by applying intentional but empirical selective pressures. For more than one century, wheat breeding has been based on science, and has been constantly evolving due to on farm agronomy and breeding program improvements. The aim of this work is to briefly review wheat breeding, with emphasis on the current advances. DISCUSSION: Improving yield potential, resistance/tolerance to biotic and abiotic stresses, and baking quality, have been priorities for breeding this cereal, however, new objectives are arising, such as biofortification enhancement. The narrow genetic diversity and complexity of its genome have hampered the breeding progress and the application of biotechnology. Old approaches, such as the introgression from relative species, mutagenesis, and hybrid breeding are strongly reappearing, motivated by an accumulation of knowledge and new technologies. A revolution has taken place regarding the use of molecular markers whereby thousands of plants can be routinely genotyped for thousands of loci. After 13 years, the wheat reference genome sequence and annotation has finally been completed, and is currently available to the scientific community. Transgenics, an unusual approach for wheat improvement, still represents a potential tool, however it is being replaced by gene editing, whose technology along with genomic selection, speed breeding, and high-throughput phenotyping make up the most recent frontiers for future wheat improvement. FINAL CONSIDERATION: Agriculture and plant breeding are constantly evolving, wheat has played a major role in these processes and will continue through decades to come.

Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome.

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

Generation of different sizes and classes of small RNAs in barley is locus, chromosome and/or cultivar-dependent.

BACKGROUND: Various small RNA (sRNA) sizes and varieties have been identified, but their relationship as well as relationship with their origins and allocations have not been well understood or investigated. RESULTS: By comparing sRNAs generated from two barley cultivars, Golden Promise (GP) and Pallas, we identified that the generation of different sizes and types of sRNAs in barley was locus-, chromosome- and/or cultivar-dependent. 20-nt sRNAs mainly comprising miRNAs and chloroplast-derived sRNAs were significantly over-expressed in Pallas vs. GP on chromosomes 3H and 6H. MiRNAs-enriched 21-nt sRNAs were significantly over-expressed in Pallas vs. GP only on chromosome 4H. On chromosome 5H this size of sRNAs was significantly under-expressed in Pallas, so were 22-nt sRNAs mainly comprising miRNAs and repeat-derived sRNAs. 24-nt sRNAs mostly derived from repeats were evenly distributed in all chromosomes and expressed similarly between GP and Pallas. Unlike other sizes of sRNAs, 24-nt sRNAs were little conserved in other plant species. Abundant sRNAs were mostly generated from 3' terminal regions of chromosome 1H and 5' terminal regions of chromosome 5H. Over-expressed miRNAs in GP vs. Pallas primarily function in stress responses and iron-binding. CONCLUSIONS: Our study indicates that 23-24-nt sRNAs may be linked to repressive chromatin modifications and function in genome stability while 20-21-nt sRNAs may be important for the cultivar specificity. This study provides a novel insight into the mechanism of sRNA expression and function in barley.

Differential expression of microRNAs and other small RNAs in barley between water and drought conditions.

Drought is a major constraint to crop production, and microRNAs (miRNAs) play an important role in plant drought tolerance. Analysis of miRNAs and other classes of small RNAs (sRNAs) in barley grown under water and drought conditions reveals that drought selectively regulates expression of miRNAs and other classes of sRNAs. Low-expressed miRNAs and all repeat-associated siRNAs (rasiRNAs) tended towards down-regulation, while tRNA-derived sRNAs (tsRNAs) had the tendency to be up-regulated, under drought. Antisense sRNAs (putative siRNAs) did not have such a tendency under drought. In drought-tolerant transgenic barley overexpressing DREB transcription factor, most of the low-expressed miRNAs were also down-regulated. In contrast, tsRNAs, rasiRNAs and other classes of sRNAs were not consistently expressed between the drought-treated and transgenic plants. The differential expression of miRNAs and siRNAs was further confirmed by Northern hybridization and quantitative real-time PCR (qRT-PCR). Targets of the drought-regulated miRNAs and siRNAs were predicted, identified by degradome libraries and confirmed by qRT-PCR. Their functions are diverse, but most are involved in transcriptional regulation. Our data provide insight into the expression profiles of miRNAs and other sRNAs, and their relationship under drought, thereby helping understand how miRNAs and sRNAs respond to drought stress in cereal crops.

Characterization of phosphorus-regulated miR399 and miR827 and their isomirs in barley under phosphorus-sufficient and phosphorus-deficient conditions.

BACKGROUND: miR399 and miR827 are both involved in conserved phosphorus (P) deficiency signalling pathways. miR399 targets the PHO2 gene encoding E2 enzyme that negatively regulates phosphate uptake and root-to-shoot allocation, while miR827 targets SPX-domain-containing genes that negatively regulate other P-responsive genes. However, the response of miR399 and miR827 to P conditions in barley has not been investigated. RESULTS: In this study, we investigated the expression profiles of miR399 and miR827 in barley (Hordeum vulagre L.) under P-deficient and P-sufficient conditions. We identified 10 members of the miR399 family and one miR827 gene in barley, all of which were significantly up-regulated under deficient P. In addition, we found many isomirs of the miR399 family and miR827, most of which were also significantly up-regulated under deficient P. Several isomirs of miR399 members were found to be able to cleave their predicted targets in vivo. Surprisingly, a few small RNAs (sRNAs) derived from the single-stranded loops of the hairpin structures of MIR399b and MIR399e-1 were also found to be able to cleave their predicted targets in vivo. Many antisense sRNAs of miR399 and a few for miR827 were also detected, but they did not seem to be regulated by P. Intriguingly, the lowest expressed member, hvu-miR399k, had four-fold more antisense sRNAs than sense sRNAs, and furthermore under P sufficiency, the antisense sRNAs are more frequent than the sense sRNAs. We identified a potential regulatory network among miR399, its target HvPHO2 and target mimics HvIPS1 and HvIPS2 in barley under P-deficient and P-sufficient conditions. CONCLUSIONS: Our data provide an important insight into the mechanistic regulation and function of miR399, miR827 and their isomirs in barley under different P conditions.

Genome merger: from sequence rearrangements in triticale to their elimination in wheat-rye addition lines.

Genetic and epigenetic modifications resulting from different genomes adjusting to a common nuclear environment have been observed in polyploids. Sequence restructuring within genomes involving retrotransposon/microsatellite-rich regions has been reported in triticale. The present study uses inter-retrotransposon amplified polymorphisms (IRAP) and retrotransposon microsatellite amplified polymorphisms (REMAP) to assess genome rearrangements in wheat-rye addition lines obtained by the controlled backcrossing of octoploid triticale to hexaploid wheat followed by self-fertilization. The comparative analysis of IRAP and REMAP banding profiles, involving a complete set of wheat-rye addition lines, and their parental species revealed in those lines the presence of wheat-origin bands absent in triticale, and the absence of rye-origin and triticale-specific bands. The presence in triticale x wheat backcrosses (BC) of rye-origin bands that were absent in the addition lines demonstrated that genomic rearrangement events were not a direct consequence of backcrossing, but resulted from further genome structural rearrangements in the BC plant progeny. PCR experiments using primers designed from different rye-origin sequences showed that the absence of a rye-origin band in wheat-rye addition lines results from sequence elimination rather than restrict changes on primer annealing sites, as noted in triticale. The level of genome restructuring events evaluated in all seven wheat-rye addition lines, compared to triticale, indicated that the unbalanced genome merger situation observed in the addition lines induced a new round of genome rearrangement, suggesting that the lesser the amount of rye chromatin introgressed into wheat the larger the outcome of genome reshuffling.

The Use of Pentaploid Crosses for the Introgression of Amblyopyrum muticum and D-Genome Chromosome Segments Into Durum Wheat.

The wild relatives of wheat provide an important source of genetic variation for wheat improvement. Much of the work in the past aimed at transferring genetic variation from wild relatives into wheat has relied on the exploitation of the ph1b mutant, located on the long arm of chromosome 5B. This mutation allows homologous recombination to occur between chromosomes from related but different genomes, e.g. between the chromosomes of wheat and related chromosomes from a wild relative resulting in the generation of interspecific recombinant chromosomes. However, the ph1b mutant also enables recombination to occur between the homologous genomes of wheat, e.g. A/B, A/D, B/D, resulting in the generation of wheat intergenomic recombinant chromosomes. In this work we report on the presence of wheat intergenomic recombinants in the genomic background of hexaploid wheat/Amblyopyrum muticum introgression lines. The transfer of genomic rearrangements involving the D-genome through pentaploid crosses provides a strategy by which the D-genome of wheat can be introgressed into durum wheat. Hence, a pentaploid crossing strategy was used to transfer D-genome segments, introgressed with either the A- and/or the B-genome, into the tetraploid background of two durum wheat genotypes Karim and Om Rabi 5 in either the presence or absence of different Am. muticum (2n = 2x = 14, TT) introgressions. Introgressions were monitored in backcross generations to the durum wheat parents via multi-color genomic in situ hybridization (mc-GISH). Tetraploid lines carrying homozygous D-genome introgressions, as well as simultaneous homozygous D- and T-genome introgressions, were developed. Introgression lines were characterized via Kompetitive Allele-Specific PCR (KASP) markers and multi-color fluorescence in situ hybridization (FISH). Results showed that new wheat sub-genomic translocations were generated at each generation in progeny that carried any Am. muticum chromosome introgression irrespective of the linkage group that the segment was derived from. The highest frequencies of homologous recombination were observed between the A- and the D-genomes. Results indicated that the genotype Karim had a higher tolerance to genomic rearrangements and T-genome introgressions compared to Om Rabi 5. This indicates the importance of the selection of the parental genotype when attempting to transfer/develop introgressions into durum wheat from pentaploid crosses.

A comprehensive expression profile of microRNAs and other classes of non-coding small RNAs in barley under phosphorous-deficient and -sufficient conditions.

Phosphorus (P) is essential for plant growth. MicroRNAs (miRNAs) play a key role in phosphate homeostasis. However, little is known about P effect on miRNA expression in barley (Hordeum vulgare L.). In this study, we used Illumina's next-generation sequencing technology to sequence small RNAs (sRNAs) in barley grown under P-deficient and P-sufficient conditions. We identified 221 conserved miRNAs and 12 novel miRNAs, of which 55 were only present in P-deficient treatment while 32 only existed in P-sufficient treatment. Total 47 miRNAs were significantly differentially expressed between the two P treatments (|log2| > 1). We also identified many other classes of sRNAs, including sense and antisense sRNAs, repeat-associated sRNAs, transfer RNA (tRNA)-derived sRNAs and chloroplast-derived sRNAs, and some of which were also significantly differentially expressed between the two P treatments. Of all the sRNAs identified, antisense sRNAs were the most abundant sRNA class in both P treatments. Surprisingly, about one-fourth of sRNAs were derived from the chloroplast genome, and a chloroplast-encoded tRNA-derived sRNA was the most abundant sRNA of all the sRNAs sequenced. Our data provide valuable clues for understanding the properties of sRNAs and new insights into the potential roles of miRNAs and other classes of sRNAs in the control of phosphate homeostasis.

A transgenic transcription factor (TaDREB3) in barley affects the expression of microRNAs and other small non-coding RNAs.

Transcription factors (TFs), microRNAs (miRNAs), small interfering RNAs (siRNAs) and other functional non-coding small RNAs (sRNAs) are important gene regulators. Comparison of sRNA expression profiles between transgenic barley over-expressing a drought tolerant TF (TaDREB3) and non-transgenic control barley revealed many group-specific sRNAs. In addition, 42% of the shared sRNAs were differentially expressed between the two groups (|log(2)| >1). Furthermore, TaDREB3-derived sRNAs were only detected in transgenic barley despite the existence of homologous genes in non-transgenic barley. These results demonstrate that the TF strongly affects the expression of sRNAs and siRNAs could in turn affect the TF stability. The TF also affects size distribution and abundance of sRNAs including miRNAs. About half of the sRNAs in each group were derived from chloroplast. A sRNA derived from tRNA-His(GUG) encoded by the chloroplast genome is the most abundant sRNA, accounting for 42.2% of the total sRNAs in transgenic barley and 28.9% in non-transgenic barley. This sRNA, which targets a gene (TC245676) involved in biological processes, was only present in barley leaves but not roots. 124 and 136 miRNAs were detected in transgenic and non-transgenic barley, respectively. miR156 was the most abundant miRNA and up-regulated in transgenic barley, while miR168 was the most abundant miRNA and up-regulated in non-transgenic barley. Eight out of 20 predicted novel miRNAs were differentially expressed between the two groups. All the predicted novel miRNA targets were validated using a degradome library. Our data provide an insight into the effect of TF on the expression of sRNAs in barley.

Polyploidization as a retraction force in plant genome evolution: sequence rearrangements in triticale.

BACKGROUND: Polyploidization is a major evolutionary process in plants where hybridization and chromosome doubling induce enormous genomic stress and can generate genetic and epigenetic modifications. However, proper evaluation of DNA sequence restructuring events and the precise characterization of sequences involved are still sparse. METHODOLOGY/PRINCIPAL FINDINGS: Inter Retrotransposons Amplified Polymorphism (IRAP), Retrotransposons Microsatellite Amplified Polymorphism (REMAP) and Inter Simple Sequence Repeat (ISSR) largely confirmed the absence of any intraspecific variation in wheat, rye and triticale. The comparative analysis of banding profiles between wheat and rye inbred lines revealed 34% of monomorphic (common to both parental species) bands for the ten different primer combinations used. The analysis of triticale plants uncovered nearly 51% of rearranged bands in the polyploid, being the majority of these modifications, due to the loss of rye bands (83%). Sequence analysis of rye fragments absent in triticale revealed for instance homology with hydroxyproline-rich glycoproteins (HRGP), a protein that belongs to a major family of inducible defence response proteins. Conversely, a wheat-specific band absent in triticale comprises a nested structure of copia-like retrotransposons elements, namely Claudia and Barbara. Sequencing of a polyploid-specific band (absent in both parents) revealed a microsatellite related sequence. Cytological studies using Fluorescent In Situ Hybridization (FISH) with REMAP products revealed a widespread distribution of retrotransposon and/or microsatellite flanking sequences on rye chromosomes, with a preferential accumulation in heterochromatic sub-telomeric domains. CONCLUSIONS/SIGNIFICANCE: Here, we used PCR-based molecular marker techniques involving retrotransposons and microsatellites to uncover polyploidization induced genetic restructuring in triticale. Sequence analysis of rearranged genomic fragments either from rye or wheat origin showed these to be retrotransposon-related as well as coding sequences. Further FISH analysis revealed possible chromosome hotspots for sequence rearrangements. The role of chromatin condensation on the origin of genomic rearrangements mediated by polyploidization in triticale is also discussed.

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.