Xiangqi Zhang

Draft genome of the wheat A-genome progenitor Triticum urartu

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGAmAm), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

Molecular analysis of common wheat genes encoding three types of cytosolic heat shock protein 90 (Hsp90): functional involvement of cytosolic Hsp90s in the control of wheat seedling growth and disease resistance

Heat shock protein 90 (Hsp90) molecular chaperones play important roles in plant growth and responses to environmental stimuli. However, little is known about the genes encoding Hsp90s in common wheat. Here, we report genetic and functional analysis of the genes specifying cytosolic Hsp90s in this species. Three groups of homoeologous genes (TaHsp90.1, TaHsp90.2 and TaHsp90.3), encoding three types of cytosolic Hsp90, were isolated. The loci containing TaHsp90.1, TaHsp90.2 and TaHsp90.3 genes were assigned to groups 2, 7 and 5 chromosomes, respectively. TaHsp90.1 genes exhibited higher transcript levels in the stamen than in the leaf, root and culm. TaHsp90.2 and TaHsp90.3 genes were more ubiquitously transcribed in the vegetative and reproductive organs examined. Decreasing the expression of TaHsp90.1 genes through virus-induced gene silencing (VIGS) caused pronounced inhibition of wheat seedling growth, whereas the suppression of TaHsp90.2 or TaHsp90.3 genes via VIGS compromised the hypersensitive resistance response of the wheat variety Suwon 11 to stripe rust fungus. Our work represents the first systematic determination of wheat genes encoding cytosolic Hsp90s, and provides useful evidence for the functional involvement of cytosolic Hsp90s in the control of seedling growth and disease resistance in common wheat.

Genome-Wide Analysis of the MADS-Box Gene Family in Brachypodium distachyon

MADS-box genes are important transcription factors for plant development, especially floral organogenesis. Brachypodium distachyon is a model for biofuel plants and temperate grasses such as wheat and barley, but a comprehensive analysis of MADS-box family proteins in Brachypodium is still missing. We report here a genome-wide analysis of the MADS-box gene family in Brachypodium distachyon. We identified 57 MADS-box genes and classified them into 32 MIKCc-type, 7 MIKC*-type, 9 Mα, 7 Mβ and 2 Mγ MADS-box genes according to their phylogenetic relationships to the Arabidopsis and rice MADS-box genes. Detailed gene structure and motif distribution were then studied. Investigation of their chromosomal localizations revealed that Brachypodium MADS-box genes distributed evenly across five chromosomes. In addition, five pairs of type II MADS-box genes were found on synteny blocks derived from whole genome duplication blocks. We then performed a systematic expression analysis of Brachypodium MADS-box genes in various tissues, particular floral organs. Further detection under salt, drought, and low-temperature conditions showed that some MADS-box genes may also be involved in abiotic stress responses, including type I genes. Comparative studies of MADS-box genes among Brachypodium, rice and Arabidopsis showed that Brachypodium had fewer gene duplication events. Taken together, this work provides useful data for further functional studies of MADS-box genes in Brachypodium distachyon.

Transcriptome Analysis of H2O2-Treated Wheat Seedlings Reveals a H2O2-Responsive Fatty Acid Desaturase Gene Participating in Powdery Mildew Resistance

Hydrogen peroxide (H2O2) plays important roles in plant biotic and abiotic stress responses. However, the effect of H2O2 stress on the bread wheat transcriptome is still lacking. To investigate the cellular and metabolic responses triggered by H2O2, we performed an mRNA tag analysis of wheat seedlings under 10 mM H2O2 treatment for 6 hour in one powdery mildew (PM) resistant (PmA) and two susceptible (Cha and Han) lines. In total, 6,156, 6,875 and 3,276 transcripts were found to be differentially expressed in PmA, Han and Cha respectively. Among them, 260 genes exhibited consistent expression patterns in all three wheat lines and may represent a subset of basal H2O2 responsive genes that were associated with cell defense, signal transduction, photosynthesis, carbohydrate metabolism, lipid metabolism, redox homeostasis, and transport. Among genes specific to PmA, ‘transport’ activity was significantly enriched in Gene Ontology analysis. MapMan classification showed that, while both up- and down- regulations were observed for auxin, abscisic acid, and brassinolides signaling genes, the jasmonic acid and ethylene signaling pathway genes were all up-regulated, suggesting H2O2-enhanced JA/Et functions in PmA. To further study whether any of these genes were involved in wheat PM response, 19 H2O2-responsive putative defense related genes were assayed in wheat seedlings infected with Blumeria graminis f. sp. tritici (Bgt). Eight of these genes were found to be co-regulated by H2O2 and Bgt, among which a fatty acid desaturase gene TaFAD was then confirmed by virus induced gene silencing (VIGS) to be required for the PM resistance. Together, our data presents the first global picture of the wheat transcriptome under H2O2 stress and uncovers potential links between H2O2 and Bgt responses, hence providing important candidate genes for the PM resistance in wheat.

A New Strategy to Produce a Defensin: Stable Production of Mutated NP-1 in Nitrate Reductase-Deficient Chlorella ellipsoidea

Defensins are small cationic peptides that could be used as the potential substitute for antibiotics. However, there is no efficient method for producing defensins. In this study, we developed a new strategy to produce defensin in nitrate reductase (NR)-deficient C. ellipsoidea (nrm-4). We constructed a plant expression vector carrying mutated NP-1 gene (mNP-1), a mature α-defensin NP-1 gene from rabbit with an additional initiator codon in the 5′-terminus, in which the selection markers were NptII and NR genes. We transferred mNP-1 into nrm-4 using electroporation and obtained many transgenic lines with high efficiency under selection chemicals G418 and NaNO3. The mNP-1 was characterized using N-terminal sequencing after being isolated from transgenic lines. Excitingly, mNP-1 was produced at high levels (approximately 11.42 mg/l) even after 15 generations of continuous fermentation. In addition, mNP-1 had strong activity against Escherichia coli at 5 µg/ml. This research developed a new method for producing defensins using genetic engineering.

Microdissection and chromosome painting of the alien chromosome in an addition line of wheat--Thinopyrum intermedium.

In this study, chromosome painting was developed and used to identify alien chromosomes in TAi-27, a wheat - Thinopyrum intermedium addition line, and the chromosomes of the three different genomes of Th. Intermedium. The smallest alien chromosome of TAi-27 was microdissected and its DNA amplified by DOP-PCR was used as a probe to hybridize with metaphase chromosomes of TAi-27 and Th . intermedium . Results showed that hybridization signals were observed in all regions of a pair of the smallest alien chromosomes and the pericentromeric area of another pair of alien chromosomes in TAi-27, indicating that the probe from microdissected chromosome is species specific. In Th . intermedium , 14 chromosomes had wide and strong hybridization signals distributed mainly on the pericentromere area and 9 chromosomes with narrow and weak signals on the pericentromere area. The remaining chromosomes displayed a very weak or no signal. Sequential FISH/GISH on Th . intermedium chromosomes using the DNAs of microdissected chromosome, Pseudoroegneria spicata (St genome) and pDbH12 (a Js genome specific probe) as the probes indicated that the microdissected chromosome belonged to the St genome, three genomes (Js, J and St) in Th . intermedium could be distinguished, in which there is no hybridization signal on J genome that is similar to the genome of Th . bessarabicum . Our results showed that the smallest alien chromosomes may represent a truncated chromosome and the repetitive sequence distribution might be similar in different chromosomes within the St genome. However, the repetitive sequence distributions are different within the Js genome, within a single chromosome, and among different genomes in Th . intermedium . Our results suggested that chromosome painting could be feasible in some plants and useful in detecting chromosome variation and repetitive sequence distribution in different genomes of polyploidy plants, which is helpful for understanding the evolution of different genomes in polyploid plants.

TaRAR1 and TaSGT1 associate with TaHsp90 to function in bread wheat (Triticum aestivum L.) seedling growth and stripe rust resistance

RAR1 and SGT1 are important co-chaperones of Hsp90. We previously showed that TaHsp90.1 is required for wheat seedling growth, and that TaHsp90.2 and TaHsp90.3 are essential for resistance (R) gene mediated resistance to stripe rust fungus. Here, we report the characterization of TaRAR1 and TaSGT1 genes in bread wheat. TaRAR1 and TaSGT1 each had three homoeologs, which were located on wheat groups 2 and 3 chromosomes, respectively. Strong inhibition of seedling growth was observed after silencing TaSGT1 but not TaRAR1. In contrast, decreasing the expression of TaRAR1 or TaSGT1 could all compromise R gene mediated resistance to stripe rust fungus infection. Protein–protein interactions were found among TaRAR1, TaSGT1 and TaHsp90. The N-terminus of TaHsp90, the CHORD-I and CHORD-II domains of TaRAR1 and the CS domain of TaSGT1 may be instrumental for the interactions among the three proteins. Based on this work and our previous study on TaHsp90, we speculate that the TaSGT1–TaHsp90.1 interaction is important for maintaining bread wheat seedling growth. The TaRAR1–TaSGT1–TaHsp90.2 and TaRAR1–TaSGT1–TaHsp90.3 interactions are involved in controlling the resistance to stripe rust disease. The new information obtained here should aid further functional investigations of TaRAR1–TaSGT1–TaHsp90 complexes in regulating bread wheat growth and disease resistance.

Association of CD14 G(-1145)A and C(-159)T polymorphisms with reduced risk for tuberculosis in a Chinese Han population.

Although the role of CD14 in mediating signals from Toll-like receptors to recognize Mycobacterium tuberculosis is known, how polymorphisms in this gene affect the susceptibility to develop tuberculosis are still not clear. We examined whether single nucleotide polymorphisms at positions -1145 and -159 in the promoter region of the CD14 gene are associated with tuberculosis in a Chinese Han population in a case-control study of 432 Chinese patients with tuberculosis and 404 ethnically matched healthy controls. Genotyping was performed to identify polymorphisms of the CD14 gene by PCR-DNA sequencing. Both the frequency of allele T in the C(-159)T polymorphism (odds ratio (OR) = 1.4; 95% confidence interval (95%CI) = 1.148-1.708) and allele G in the G(-1145)A polymorphism (OR = 1.512; 95%CI = 1.236- 1.849) were significantly more frequent in cases than in controls. The frequencies of genotypes CC and CT in the C(-159)T polymorphism, as well as the frequencies of genotypes AA and AG, were lower in cases than in controls. Based on our results, we conclude that G(-1145)A and C(-159)T polymorphisms of CD14 are associated with decreased risk for the development of tuberculosis in the Chinese Han population.

Rapid EST isolation from chromosome 1R of rye

BackgroundTo obtain important expressed sequence tags (ESTs) located on specific chromosomes is currently difficult. Construction of single-chromosome EST library could be an efficient strategy to isolate important ESTs located on specific chromosomes. In this research we developed a method to rapidly isolate ESTs from chromosome 1R of rye by combining the techniques of chromosome microdissection with hybrid specific amplification (HSA).ResultsChromosome 1R was isolated by a glass needle and digested with proteinase K (PK). The DNA of chromosome 1R was amplified by two rounds of PCR using a degenerated oligonucleotide 6-MW sequence with a Sau3AI digestion site as the primer. The PCR product was digested with Sau3AI and linked with adaptor HSA1, then hybridized with the Sau3AI digested cDNA with adaptor HSA2 of rye leaves with and without salicylic acid (SA) treatment, respectively. The hybridized DNA fragments were recovered by the HSA method and cloned into pMD18-T vector. The cloned inserts were released by PCR using the partial sequences in HSA1 and HSA2 as the primers and then sequenced. Of the 94 ESTs obtained and analyzed, 6 were known sequences located on rye chromosome 1R or on homologous group 1 chromosomes of wheat; all of them were highly homologous with ESTs of wheat, barley and/or other plants in Gramineae, some of which were induced by abiotic or biotic stresses. Isolated in this research were 22 ESTs with unknown functions, probably representing some new genes on rye chromosome 1R.ConclusionWe developed a new method to rapidly clone chromosome-specific ESTs from chromosome 1R of rye. The information reported here should be useful for cloning and investigating the new genes found on chromosome 1R.

Dissecting and Enhancing the Contributions of High-Molecular-Weight Glutenin Subunits to Dough Functionality and Bread Quality

Dear Editor, Seed storage proteins (SSPs) are frequently important determinants of crop quality traits (Shewry and Casey,1999).Dissecting and enhancing the genetic contributions of individual SSPs to their target traits are essential for effectively improving crop quality attributes.However,such a task is often difficult to accomplish,because SSPs are frequently expressed from multigene families and exhibit strong allelic variation.Consequently,detailed knowledge of the function of individual SSPs in crop quality trait is still limited.This scenario is well illustrated by high-molecular-weight glutenin subunits (HMWGSs),a complex family of SSPs that are involved in wheat enduse quality through affecting dough functionality (Bekes,2012;Rasheed et al.,2014).