Bu-Jun Shi

A physical, genetic and functional sequence assembly of the barley genome

Barley (Hordeum vulgare L.) is among the world’s earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 ‘high-confidence’ genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement.

Boron-Toxicity Tolerance in Barley Arising from Efflux Transporter Amplification

Both limiting and toxic soil concentrations of the essential micronutrient boron represent major limitations to crop production worldwide. We identified Bot1, a BOR1 ortholog, as the gene responsible for the superior boron-toxicity tolerance of the Algerian barley landrace Sahara 3771 (Sahara). Bot1 was located at the tolerance locus by high-resolution mapping. Compared to intolerant genotypes, Sahara contains about four times as many Bot1 gene copies, produces substantially more Bot1 transcript, and encodes a Bot1 protein with a higher capacity to provide tolerance in yeast. Bot1 transcript levels identified in barley tissues are consistent with a role in limiting the net entry of boron into the root and in the disposal of boron from leaves via hydathode guttation.

Role of microRNAs in plant drought tolerance

Summary Drought is a normal and recurring climate feature in most parts of the world and plays a major role in limiting crop productivity. However, plants have their own defence systems to cope with adverse climatic conditions. One of these defence mechanisms is the reprogramming of gene expression by microRNAs (miRNAs). miRNAs are small noncoding RNAs of approximately 22 nucleotides length, which have emerged as important regulators of genes at post‐transcriptional levels in a range of organisms. Some miRNAs are functionally conserved across plant species and are regulated by drought stress. These properties suggest that miRNA‐based genetic modifications have the potential to enhance drought tolerance in cereal crops. This review summarizes the current understanding of the regulatory mechanisms of plant miRNAs, involvement of plant miRNAs in drought stress responses in barley (Hordeum vulgare L.), wheat (Triticum spp.) and other plant species, and the involvement of miRNAs in plant‐adaptive mechanisms under drought stress. Potential strategies and directions for future miRNA research and the utilization of miRNAs in the improvement of cereal crops for drought tolerance are also discussed.

Differential virulence by strains of Cucumber mosaic virus is mediated by the 2b gene

The approximately 12-kDa 2b protein, encoded by all cucumoviruses, had been shown to play an important role in viral long-distance movement, hypervirulence, and suppression of post-transcriptional gene silencing. The role of the 2b gene in the hypervirulence of Cucumber mosaic virus (CMV) and whether hypervirulence was linked to movement were analyzed using a hybrid virus (CMV-qw), generated by replacing the 2b gene in a subgroup II strain, Q-CMV, with the 2b gene from a subgroup IA strain, WAII-CMV. CMV-qw was more virulent than Q-CMV or WAII-CMV on most of the host plant species tested. Northern blot and nucleotide sequence analyses demonstrated that CMV-qw was stably maintained during the course of infection and upon passage. Kinetic studies revealed that the hypervirulence induced by the hybrid virus was associated with neither increased viral RNA accumulation nor more rapid viral movement per se, suggesting that other functions of the 2b protein are important in determining the hypervirulence.

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 Illuminas 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.

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.

Phosphate Utilization Efficiency Correlates with Expression of Low-Affinity Phosphate Transporters and Noncoding RNA, IPS1, in Barley

Genetic variation in phosphorus (P) efficiency exists among wheat (Triticum aestivum) and barley (Hordeum vulgare) genotypes, but the underlying mechanisms for the variation remain elusive. High- and low-affinity phosphate (Pi) PHT1 transporters play an indispensable role in P acquisition and remobilization. However, little is known about genetic variation in PHT1 gene expression and association with P acquisition efficiency (PAE) and P utilization efficiency (PUE). Here, we present quantitative analyses of transcript levels of high- and low-affinity PHT1 Pi transporters in four barley genotypes differing in PAE. The results showed that there was no clear pattern in the expression of four paralogs of the high-affinity Pi transporter HvPHT1;1 among the four barley genotypes, but the expression of a low-affinity Pi transporter, HvPHT1;6, and its close homolog HvHPT1;3 was correlated with the genotypes differing in PUE. Interestingly, the expression of HvPHT1;6 and HvPHT1;3 was correlated with the expression of HvIPS1 (for P starvation inducible; noncoding RNA) but not with HvIPS2, suggesting that HvIPS1 plays a distinct role in the regulation of the low-affinity Pi transporters. In addition, high PUE was found to be associated with high root-shoot ratios in low-P conditions, indicating that high carbohydrate partitioning into roots occurs simultaneously with high PUE. However, high PUE accompanying high carbon partitioning into roots could result in low PAE. Therefore, the optimization of PUE through the modification of low-affinity Pi transporter expression may assist further improvement of PAE for low-input agriculture systems.

BAC library resources for map-based cloning and physical map construction in barley (Hordeum vulgare L.).

BackgroundAlthough second generation sequencing (2GS) technologies allow re-sequencing of previously gold-standard-sequenced genomes, whole genome shotgun sequencing and de novo assembly of large and complex eukaryotic genomes is still difficult. Availability of a genome-wide physical map is therefore still a prerequisite for whole genome sequencing for genomes like barley. To start such an endeavor, large insert genomic libraries, i.e. Bacterial Artificial Chromosome (BAC) libraries, which are unbiased and representing deep haploid genome coverage, need to be ready in place.ResultFive new BAC libraries were constructed for barley (Hordeum vulgare L.) cultivar Morex. These libraries were constructed in different cloning sites (Hind III, EcoR I, Mbo I and BstX I) of the respective vectors. In order to enhance unbiased genome representation and to minimize the number of gaps between BAC contigs, which are often due to uneven distribution of restriction sites, a mechanically sheared library was also generated. The new BAC libraries were fully characterized in depth by scrutinizing the major quality parameters such as average insert size, degree of contamination (plate wide, neighboring, and chloroplast), empty wells and off-scale clones (clones with <30 or >250 fragments). Additionally a set of gene-based probes were hybridized to high density BAC filters and showed that genome coverage of each library is between 2.4 and 6.6 X.ConclusionBAC libraries representing >20 haploid genomes are available as a new resource to the barley research community. Systematic utilization of these libraries in high-throughput BAC fingerprinting should allow developing a genome-wide physical map for the barley genome, which will be instrumental for map-based gene isolation and genome sequencing.

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

BackgroundmiR399 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.ResultsIn 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.ConclusionsOur data provide an important insight into the mechanistic regulation and function of miR399, miR827 and their isomirs in barley under different P conditions.

Development of genomic resources for the narrow-leafed lupin (Lupinus angustifolius): construction of a bacterial artificial chromosome (BAC) library and BAC-end sequencing

BackgroundLupinus angustifolius L, also known as narrow-leafed lupin (NLL), is becoming an important grain legume crop that is valuable for sustainable farming and is becoming recognised as a potential human health food. Recent interest is being directed at NLL to improve grain production, disease and pest management and health benefits of the grain. However, studies have been hindered by a lack of extensive genomic resources for the species.ResultsA NLL BAC library was constructed consisting of 111,360 clones with an average insert size of 99.7 Kbp from cv Tanjil. The library has approximately 12 × genome coverage. Both ends of 9600 randomly selected BAC clones were sequenced to generate 13985 BAC end-sequences (BESs), covering approximately 1% of the NLL genome. These BESs permitted a preliminary characterisation of the NLL genome such as organisation and composition, with the BESs having approximately 39% G:C content, 16.6% repetitive DNA and 5.4% putative gene-encoding regions. From the BESs 9966 simple sequence repeat (SSR) motifs were identified and some of these are shown to be potential markers.ConclusionsThe NLL BAC library and BAC-end sequences are powerful resources for genetic and genomic research on lupin. These resources will provide a robust platform for future high-resolution mapping, map-based cloning, comparative genomics and assembly of whole-genome sequencing data for the species.