Maoqun Yu

De novo assembly and characterization of the root transcriptome of Aegilops variabilis during an interaction with the cereal cyst nematode

BackgroundAegilops variabilis No.1 is highly resistant to cereal cyst nematode (CCN). However, a lack of genomic information has restricted studies on CCN resistance genes in Ae. variabilis and has limited genetic applications in wheat breeding.ResultsUsing RNA-Seq technology, we generated a root transcriptome at a sequencing depth of 4.69 gigabases of Ae. variabilis No. 1 from a pooled RNA sample. The sample contained equal amounts of RNA extracted from CCN-infected and untreated control plants at three time-points. Using the Trinity method, nearly 52,081,238 high-quality trimmed reads were assembled into a non-redundant set of 118,064 unigenes with an average length of 500 bp and an N50 of 599 bp. The total assembly was 59.09 Mb of unique transcriptome sequences with average read-depth coverage of 33.25×. In BLAST searches of our database against public databases, 66.46% (78,467) of the unigenes were annotated with gene descriptions, conserved protein domains, or gene ontology terms. Functional categorization further revealed 7,408 individual unigenes and three pathways related to plant stress resistance.ConclusionsWe conducted high-resolution transcriptome profiling related to root development and the response to CCN infection in Ae. variabilis No.1. This research facilitates further studies on gene discovery and on the molecular mechanisms related to CCN resistance.

The draft genome of Tibetan hulless barley reveals adaptive patterns to the high stressful Tibetan Plateau

Significance The draft genome of Tibetan hulless barley provides a robust framework to better understand Poaceae evolution and a substantial basis for functional genomics of crop species with a large genome. The expansion of stress-related gene families in Tibetan hulless barley implies that it could be considered as an invaluable gene resource aiding stress tolerance improvement in Triticeae crops. Genome resequencing revealed extensive genetic diversity in Tibetan barley germplasm and divergence to sequenced barley genomes from other geographical regions. Investigation of genome-wide selection footprints demonstrated an adaptive correlation of genes under selection with extensive stressful environmental variables. These results reveal insights into the adaptation of Tibetan hulless barley to harsh environments on the highland and will facilitate future genetic improvement of crops. The Tibetan hulless barley (Hordeum vulgare L. var. nudum), also called “Qingke” in Chinese and “Ne” in Tibetan, is the staple food for Tibetans and an important livestock feed in the Tibetan Plateau. The diploid nature and adaptation to diverse environments of the highland give it unique resources for genetic research and crop improvement. Here we produced a 3.89-Gb draft assembly of Tibetan hulless barley with 36,151 predicted protein-coding genes. Comparative analyses revealed the divergence times and synteny between barley and other representative Poaceae genomes. The expansion of the gene family related to stress responses was found in Tibetan hulless barley. Resequencing of 10 barley accessions uncovered high levels of genetic variation in Tibetan wild barley and genetic divergence between Tibetan and non-Tibetan barley genomes. Selective sweep analyses demonstrate adaptive correlations of genes under selection with extensive environmental variables. Our results not only construct a genomic framework for crop improvement but also provide evolutionary insights of highland adaptation of Tibetan hulless barley.

Virus-induced silencing of genes encoding LEA protein in Tibetan hulless barley ( Hordeum vulgare ssp. vulgare ) and their relationship to drought tolerance

Expression of the late embryogenesis abundant (LEA) gene is usually associated with plant response to dehydration. In this study, a drought-tolerant genotype was screened from 48 accessions of Tibetan hulless barley (Hordeum vulgare ssp. vulgare). By using virus-induced gene silencing, the influence of two LEA genes (HVA1 and Dhn6) on drought tolerance of Tibetan hulless barley was investigated. Results of quantitative real-time PCR indicated that the relative expression levels of HVA1 and Dhn6 in silenced plants were significantly reduced compared with control plants. Both HVA1-silenced and Dhn6-silenced plants showed a consequently lower survival rate than control plants under drought stress. However, only HVA1-silenced plants exhibited a significantly higher water loss rate (WLR). These results suggested that HVA1 and Dhn6 might participate in adaptive responses to water deficit in different ways. Vegetative growth of HVA1-silenced plants was significantly retarded even under optimal growth conditions, and their biomass accumulation was also much lower than that of the controls. These results indicate that HVA1 might play a role in vegetative growth of Tibetan hulless barley.

Genotypic variability in sequence and expression of HVA1 gene in Tibetan hulless barley, Hordeum vulgare ssp. vulgare, associated with resistance to water deficit

Late embryogenesis abundant ( LEA) proteins are thought to protect against water stress in plants. Characteristics of sequence and expression of barley gene HVA1, a member of LEA group 3 protein, were investigated in hulless barley ( Hordeum vulgare ssp. vulgare), associated with phenotypically diverse drought- tolerant genotypes. Sensitive and tolerant genotypes were identified from Tibetan populations of cultivated hulless barley, based on scores of water loss rate ( WLR), maldondialdehyde ( MDA), and proline content. The results indicated that lower MDA contents, lower scores of WLR, and higher proline contents were associated with drought- tolerant genotypes in hulless barley. Notably, differential trends of expression patterns were detected among the selected contrasting genotypes, depending on the duration of dehydration stress. The HVA1 gene tended to respond earlier in the tolerance ( after 2 h) compared with sensitive genotypes ( after 4 h). Results of quantitative real-time PCR indicated that the relative level of HVA1 expression was always higher in tolerant genotypes, rapidly increasing at the earlier stages ( after 2 - 4 h of dehydration). However, HVA1 expressions of sensitive genotypes had a fast increase from 8 to 12 h of stress. Variable numbers of the 11-amino-acid-motif in LEA3 proteins were not consistent with the lines of drought resistance in hulless barley. Molecular characteristic of LEA3 protein in tolerant lines existed in the consistency of Gln32, Arg33, and Ala195 in Tibetan hulless barley. The present study may indicate that the differential HVA1 gene has a functional role in the dehydration tolerance in hulless barley. The authors suggested that the observed variability in sequence and expression of HVA1 could be related to the diverse drought- tolerant genotypes in crops.

Structural and expressional analysis of the B-hordein genes in Tibetan hull-less barley

The B-hordein gene family was analyzed from two Tibetan hull-less barley cultivars Z09 and Z26 (Hordeum vulgare subsp. vulgare). Fourteen B-hordein genes, designated BZ09-2 to BZ09-5 (from Z09) and BZ26-1 to BZ26-10 (from Z26), were sequenced. Seven of them, similar to a previously reported BZ09-1 from Z09, were predicted to encode putative active proteins each with a signal peptide, a repetitive domain, and a C-terminal region; seven of them were predicted to be pseudogenes. The B-hordein gene family was analyzed using all known representatives of B-hordein sequences and two most similar LMW-GSs of Triticum aestivum. Alignment of these seven putative proteins with known B-hordeins and two most similar LMW-GSs of T. aestivum revealed that they shared a common motif. A large variation was observed between numbers of repeat units of predicted B-hordeins of Z26 and Z09. Phylogenetic analysis revealed that all BZ26 clones were clustered in a subgroup, and BZ09-1 formed another subgroup by itself in the putative eight active genes. In addition, six 5′-upstream regulatory sequences of the B-hordein genes were isolated from the two accessions by a single oligonucleotide nested PCR, and several different mutations were identified in the cis-acting element GLM and two distinctive sequences (Z09P-2 and Z26P-3). Phylogenetic analysis of 5′-upstream regulatory regions of the B-hordein genes showed that members from the same accession were clustered together except for two distinct members. Quantitative real time PCR analysis indicated distinct expression levels of B-hordein genes in four developing stages of developing grains in two accessions. These findings suggest B-hordein genes have intrinsic differences between accessions, and this knowledge will be useful for incorporating the B-hordeins protein in barley breeding programs.

Genetic diversity of Acid-PAGE monomeric prolamins in cultivated hulless barley (Hordeum vulgare L.) from Qinghai-Tibet plateau in China

Naked barley, due to its favorable attributes such as high feed value, good human nutrition and easy processing, increasingly attracts people’s attention. Qinghai–Tibet Plateau is very rich in naked barley resources and has a long growing history. Genetic diversity of these cultivated naked barley is, however, poorly documented. This study analyzed the genetic diversity of monomeric prolamins (protein fraction corresponding to wheat gliadins) using the Acid -PAGE technique in eighty-six cultivated naked barley cultivars from Qinghai–Tibet Plateau. Extensive diversity was observed. A total of 43 different bands and 76 distinct patterns were found. Jaccard’s coefficient of similarity was calculated, and the accessions were divided into five main groups by cluster analysis using UPGMA. Differentiation among the populations from different collecting regions was investigated, based on the polymorphism of monomeric prolamins. The genetic diversity within the accessions from Tibet was slightly higher with an average diversity index of 0.29 than those from Sichuan. This study supports Tibetan Plateau is the diversity center of the cultivated naked barley and suggests that cultivated naked barley from Qinghai–Tibet Plateau is an important pool of variability that could be used in cereal breeding.

RNA-Seq Based Identification of Candidate Parasitism Genes of Cereal Cyst Nematode (Heterodera avenae) during Incompatible Infection to Aegilops variabilis.

One of the reasons for the progressive yield decline observed in cereals production is the rapid build-up of populations of the cereal cyst nematode (CCN, Heterodera avenae). These nematodes secrete so-call effectors into their host plant to suppress the plant defense responses, alter plant signaling pathways and then induce the formation of syncytium after infection. However, little is known about its molecular mechanism and parasitism during incompatible infection. To gain insight into its repertoire of parasitism genes, we investigated the transcriptome of the early parasitic second-stage (30 hours, 3 days and 9 days post infection) juveniles of the CCN as well as the CCN infected tissue of the host Aegilops variabilis by Illumina sequencing. Among all assembled unigenes, 681 putative genes of parasitic nematode were found, in which 56 putative effectors were identified, including novel pioneer genes and genes corresponding to previously reported effectors. All the 681 CCN unigenes were mapped to 229 GO terms and 200 KEGG pathways, including growth, development and several stimulus-related signaling pathways. Sixteen clusters were involved in the CCN unigene expression atlas at the early stages during infection process, and three of which were significantly gene-enriched. Besides, the protein-protein interaction network analysis revealed 35 node unigenes which may play an important role in the plant-CCN interaction. Moreover, in a comparison of differentially expressed genes between the pre-parasitic juveniles and the early parasitic juveniles, we found that hydrolase activity was up-regulated in pre J2s whereas binding activity was upregulated in infective J2s. RT-qPCR analysis on some selected genes showed detectable expression, indicating possible secretion of the proteins and putative role in infection. This study provided better insights into the incompatible interaction between H. avenae and the host plant Ae. varabilis. Moreover, RNAi targets with potential lethality were screened out and primarily validated, which provide candidates for engineering-based control of cereal cyst nematode in crops breeding.

Transcriptome assembly and analysis of Tibetan Hulless Barley (Hordeum vulgare L. var. nudum) developing grains, with emphasis on quality properties.

Background Hulless barley is attracting increasing attention due to its unique nutritional value and potential health benefits. However, the molecular biology of the barley grain development and nutrient storage are not well understood. Furthermore, the genetic potential of hulless barley has not been fully tapped for breeding. Methodology/Principal Findings In the present study, we investigated the transcriptome features during hulless barley grain development. Using Illumina paired-end RNA-Sequencing, we generated two data sets of the developing grain transcriptomes from two hulless barley landraces. A total of 13.1 and 12.9 million paired-end reads with lengths of 90 bp were generated from the two varieties and were assembled to 48,863 and 45,788 unigenes, respectively. A combined dataset of 46,485 All-Unigenes were generated from two transcriptomes with an average length of 542 bp, and 36,278 among were annotated with gene descriptions, conserved protein domains or gene ontology terms. Furthermore, sequences and expression levels of genes related to the biosynthesis of storage reserve compounds (starch, protein, and β-glucan) were analyzed, and their temporal and spatial patterns were deduced from the transcriptome data of cultivated barley Morex. Conclusions/Significance We established a sequences and functional annotation integrated database and examined the expression profiles of the developing grains of Tibetan hulless barley. The characterization of genes encoding storage proteins and enzymes of starch synthesis and (1–3;1–4)-β-D-glucan synthesis provided an overview of changes in gene expression associated with grain nutrition and health properties. Furthermore, the characterization of these genes provides a gene reservoir, which helps in quality improvement of hulless barley.

Molecular detection of rye (Secale cereale L.) chromatin in wheat line 07jian126 (Triticum aestivum L.) and its association to wheat powdery mildew resistance

Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most serious diseases for common wheat in many regions around the world. Seeking for new resistance source is urgently required to meet the challenge of the rapid loss of resistance due to the co-evolution of the pathogen’s virulence. Wheat line 07jian126 (Triticum aestivum L.) is highly resistant to the Pm disease prevailing in Sichuan province of China. Previous study showed that a SSR marker Xbarc183 was linked to the Pm resistance in 07jian126, which might be controlled by a single dominant gene, designated as Pm07J126. In this study, two additional F2 populations were used to confirm the linkage between Pm07J126 and Xbarc183. Furthermore, rye chromatin was detected in 07jian126 by molecular analysis of a rye-specific SCAR marker O5 which co-segregated with Pm07J126. This result indicated that Pm07J126 might originate from rye. The reaction patterns to 21 Bgt isolates and molecular marker analysis implied that Pm07J126 might be different from the known rye-derived Pm genes Pm7, Pm8, Pm17 and PmJZHM2RL. Chromosome observation, molecular marker, and A-PAGE analysis suggested that 07jian126 might be a rye introgression line and neither contain 1RS translocation nor secalins gene. Consequently, 07jian126 could be considered as a valuable resource for Pm resistance development of wheat. Besides, the molecular markers Xbarc183 and O5 are useful in marker-assisted selection of Pm07J126 in wheat breeding programs.

Starch granule-associated proteins of hull-less barley (Hordeum vulgare L.) from the Qinghai-Tibet Plateau in China

BACKGROUND The starch granule-associated proteins (SGAPs) are the minor components of the starch granules and a majority of them are believed to be starch biosynthetic enzymes. The Qinghai-Tibet Plateau in China, one of the centres of origin of cultivated barley, is abundant in hull-less barley resources which exhibit high polymorphism in SGAPs. RESULTS The SGAPs of hull-less barley from Qinghai-Tibet Plateau were analysed by one-dimensional (1-D) SDS-PAGE, 2-D PAGE and ESI-Q-TOF MS/MS. In the 1-D SDS-PAGE gel, four proteins including a 80 kDa starch synthase, actin, actin 4 and ATP synthase β-subunit were identified as novel SGAPs. A total of six different bands were identified as starch granule-bound starch synthase I (GBSSI) and the segregation of the novel GBSSI bands in F(1) and F(2) seeds derived from yf127 × yf70 was in accordance with Mendels law. In the 2-D PAGE gel, 92 spots were identified as 42 protein species which could be classified into 15 functional groups. Thirteen protein species were identified as SGAPs for the first time and multiple spots were identified as GBSSI. CONCLUSION This study revealed novel SGAPs in hull-less barley from the Qinghai-Tibet Plateau in China and these will be significant in further studies of starch biosynthesis in barley.