Accessing a Russian Wheat Aphid Resistance Gene in Bread Wheat by Long-Read Technologies.

Russian wheat aphid (RWA) ( Kurdjumov) is a serious invasive pest of small-grain cereals and many grass species. An efficient strategy to defy aphid attacks is to identify sources of natural resistance and transfer resistance genes into susceptible crop cultivars. Revealing the genes helps understand plant defense mechanisms and engineer plants with durable resistance to the pest. To date, more than 15 RWA resistance genes have been identified in wheat ( L.) but none of them has been cloned. Previously, we genetically mapped the RWA resistance gene into an interval of 0.83 cM on the short arm of chromosome 7D and spanned it with five bacterial artificial chromosome (BAC) clones. Here, we used a targeted strategy combining traditional approaches toward gene cloning (genetic mapping and sequencing of BAC clones) with novel technologies, including optical mapping and long-read nanopore sequencing. The latter, with reads spanning the entire length of a BAC insert, enabled us to assemble the whole region, a task that was not achievable with short reads. Long-read optical mapping validated the DNA sequence in the interval and revealed a difference in the locus organization between resistant and susceptible genotypes. The complete and accurate sequence of the region facilitated the identification of new markers and precise annotation of the interval, revealing six high-confidence genes. Identification of as the most likely candidate opens an avenue for its validation through functional genomics approaches.

Erwinia iniecta sp. nov., isolated from Russian wheat aphid (Diuraphis noxia).

Short, Gram-negative-staining, rod-shaped bacteria were isolated from crushed bodies of Russian wheat aphid [Diuraphis noxia (Kurdjumov)] and artificial diets after Russian wheat aphid feeding. Based on multilocus sequence analysis involving the 16S rRNA, atpD, infB, gyrB and rpoB genes, these bacterial isolates constitute a novel clade in the genus Erwinia, and were most closely related to Erwinia toletana. Representative distinct strains within this clade were used for comparisons with related species of Erwinia. Phenotypic comparisons using four distinct strains and average nucleotide identity (ANI) measurements using two distinct draft genomes revealed that these strains form a novel species within the genus Erwinia. The name Erwinia iniecta sp. nov. is proposed, and strain B120T ( = CFBP 8182T = NCCB 100485T) was designated the type strain. Erwinia iniecta sp. nov. was not pathogenic to plants. However, virulence to the Russian wheat aphid was observed.

Chromosomal genomics facilitates fine mapping of a Russian wheat aphid resistance gene.

KEY MESSAGE: Making use of wheat chromosomal resources, we developed 11 gene-associated markers for the region of interest, which allowed reducing gene interval and spanning it by four BAC clones. Positional gene cloning and targeted marker development in bread wheat are hampered by high complexity and polyploidy of its nuclear genome. Aiming to clone a Russian wheat aphid resistance gene Dn2401 located on wheat chromosome arm 7DS, we have developed a strategy overcoming problems due to polyploidy and enabling efficient development of gene-associated markers from the region of interest. We employed information gathered by GenomeZipper, a synteny-based tool combining sequence data of rice, Brachypodium, sorghum and barley, and took advantage of a high-density linkage map of Aegilops tauschii. To ensure genome- and locus-specificity of markers, we made use of survey sequence assemblies of isolated wheat chromosomes 7A, 7B and 7D. Despite the low level of polymorphism of the wheat D subgenome, our approach allowed us to add in an efficient and cost-effective manner 11 new gene-associated markers in the Dn2401 region and narrow down the target interval to 0.83 cM. Screening 7DS-specific BAC library with the flanking markers revealed a contig of four BAC clones that span the Dn2401 region in wheat cultivar 'Chinese Spring'. With the availability of sequence assemblies and GenomeZippers for each of the wheat chromosome arms, the proposed strategy can be applied for focused marker development in any region of the wheat genome.

Virus-induced gene silencing suggests (1,3;1,4)-ß-glucanase is a susceptibility factor in the compatible russian wheat aphid-wheat interaction.

The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a significant insect pest of wheat (Triticum aestivum L.) and has a major economic impact worldwide, especially on winter wheat in the western United States. The continuing emergence of new RWA biotypes virulent to existing resistance genes reinforces the need for more durable resistance. Studies have indicated that resistance in previously susceptible plants can be produced by knock-down of susceptibility genes or other genes involved in host plant susceptibility. Therefore, investigation into genes involved in compatible RWA-wheat interactions could be a feasible approach to achieving durable RWA resistance. The objective of this study was to test whether silencing (1,3;1,4)-ß-glucanase, previously observed to be highly induced in susceptible compared with resistant wheat during aphid infestation, would confer resistance to a susceptible wheat genotype. Barley stripe mosaic virus-mediated virus-induced gene silencing was employed to test whether (1,3;1,4)-ß-glucanase is involved in the susceptible reaction of 'Gamtoos-S' (GS). Controlled infestation with U.S. biotype RWA2 was done to assess aphid reproduction and host symptom development. Aphids on (1,3;1,4)-ß-glucanase-silenced plants reproduced less per day and had longer prenymphipositional periods than those on control GS plants. Furthermore, the (1,3;1,4)-ß-glucanase-silenced plants exhibited less chlorosis and greater dry weight compared with GS. Aphid reproduction and host plant symptom development showed linear relationships with (1,3;1,4)-ß-glucanase transcript levels. Our results suggest that (1,3;1,4)-ß-glucanase is required for successful infestation by the RWA and may be a susceptibility factor that could be exploited as a potential target for RWA resistance breeding.

The transcriptional network of WRKY53 in cereals links oxidative responses to biotic and abiotic stress inputs.

The transcription factor WRKY53 is expressed during biotic and abiotic stress responses in cereals, but little is currently known about its regulation, structure and downstream targets. We sequenced the wheat ortholog TaWRKY53 and its promoter region, which revealed extensive similarity in gene architecture and cis-acting regulatory elements to the rice ortholog OsWRKY53, including the presence of stress-responsive abscisic acid-responsive elements (ABRE) motifs and GCC-boxes. Four proteins interacted with the WRKY53 promoter in yeast one-hybrid assays, suggesting that this gene can receive inputs from diverse stress-related pathways such as calcium signalling and senescence, and environmental cues such as drought and ultraviolet radiation. The Ser/Thr receptor kinase ORK10/LRK10 and the apoplastic peroxidase POC1 are two downstream targets for regulation by the WRKY53 transcription factor, predicted based on the presence of W-box motifs in their promoters and coregulation with WRKY53, and verified by electrophoretic mobility shift assay (EMSA). Both ORK10/LRK10 and POC1 are upregulated during cereal responses to pathogens and aphids and important components of the oxidative burst during the hypersensitive response. Taken with our yeast two-hybrid assay which identified a strong protein-protein interaction between microsomal glutathione S-transferase 3 and WRKY53, this implies that the WRKY53 transcriptional network regulates oxidative responses to a wide array of stresses.

Virus-induced gene silencing of Arabidopsis thaliana gene homologues in wheat identifies genes conferring improved drought tolerance.

In a non-model staple crop like wheat (Triticum aestivumI L.), functional validation of potential drought stress responsive genes identified in Arabidopsis could provide gene targets for breeding. Virus-induced gene silencing (VIGS) of genes of interest can overcome the inherent problems of polyploidy and limited transformation potential that hamper functional validation studies in wheat. In this study, three potential candidate genes shown to be involved in abiotic stress response pathways in Arabidopsis thaliana were selected for VIGS experiments in wheat. These include Era1 (enhanced response to abscisic acid), Cyp707a (ABA 8'-hydroxylase), and Sal1 (inositol polyphosphate 1-phosphatase). Gene homologues for these three genes were identified in wheat and cloned in the viral vector barley stripe mosaic virus (BSMV) in the antisense direction, followed by rub inoculation of BSMV viral RNA transcripts onto wheat plants. Quantitative real-time PCR showed that VIGS-treated wheat plants had significant reductions in target gene transcripts. When VIGS-treated plants generated for Era1 and Sal1 were subjected to limiting water conditions, they showed increased relative water content, improved water use efficiency, reduced gas exchange, and better vigour compared to water-stressed control plants inoculated with RNA from the empty viral vector (BSMV0). In comparison, the Cyp707a-silenced plants showed no improvement over BSMV0-inoculated plants under limited water condition. These results indicate that Era1 and Sal1 play important roles in conferring drought tolerance in wheat. Other traits affected by Era1 silencing were also studied. Delayed seed germination in Era1-silenced plants suggests this gene may be a useful target for developing resistance to pre-harvest sprouting.

BAC libraries from wheat chromosome 7D: efficient tool for positional cloning of aphid resistance genes.

Positional cloning in bread wheat is a tedious task due to its huge genome size and hexaploid character. BAC libraries represent an essential tool for positional cloning. However, wheat BAC libraries comprise more than million clones, which makes their screening very laborious. Here, we present a targeted approach based on chromosome-specific BAC libraries. Such libraries were constructed from flow-sorted arms of wheat chromosome 7D. A library from the short arm (7DS) consisting of 49,152 clones with 113 kb insert size represented 12.1 arm equivalents whereas a library from the long arm (7DL) comprised 50,304 clones of 116 kb providing 14.9x arm coverage. The 7DS library was PCR screened with markers linked to Russian wheat aphid resistance gene DnCI2401, the 7DL library was screened by hybridization with a probe linked to greenbug resistance gene Gb3. The small number of clones combined with high coverage made the screening highly efficient and cost effective.

Virus-induced gene silencing of WRKY53 and an inducible phenylalanine ammonia-lyase in wheat reduces aphid resistance.

Although several wheat genes differentially expressed during the Russian wheat aphid resistance response have recently been identified, their requirement for and specific role in resistance remain unclear. Progress in wheat-aphid interaction research is hampered by inadequate collections of mutant germplasm and difficulty in transforming hexaploid wheat. Virus-induced gene silencing (VIGS) technology is emerging as a viable reverse genetics approach in cereal crops. However, the potential of VIGS for determining aphid defence gene function in wheat has not been evaluated. We report on the use of recombinant barley stripe mosaic virus (BSMV) to target and silence a WRKY53 transcription factor and an inducible phenylalanine ammonia-lyase (PAL) gene, both predicted to contribute to aphid defence in a genetically resistant wheat line. After inoculating resistant wheat with the VIGS constructs, transcript abundance was reduced to levels similar to that observed in susceptible wheat. Notably, the level of PAL expression was also suppressed by the WKRY53 construct, suggesting that these genes operate in the same defence response network. Both knockdowns exhibited a susceptible phenotype upon aphid infestation, and aphids feeding on silenced plants exhibited a significant increase in fitness compared to aphids feeding on control plants. Altered plant phenotype and changes in aphid behaviour after silencing imply that WKRY53 and PAL play key roles in generating a successful resistance response. This study is the first report on the successful use of VIGS to investigate genes involved in wheat-insect interactions.

Transcript profiling of wheat genes expressed during feeding by two different biotypes of Diuraphis noxia.

Diuraphis noxia (Kurdjumov)(Russian wheat aphid) has severe economic impacts on wheat and barley production in the United States. The interaction between the Russian wheat aphid and its cereal hosts is poorly understood. However, the recent appearance of new biotypes in the United States showed that specific interactions exist between wheat resistance loci and Russian wheat aphid biotypes. At present, Dn7 is the only known gene in hexaploid wheat that confers resistance against all U.S. Russian wheat aphid biotypes. This study was conducted to investigate the molecular mechanism of Dn7-mediated resistance against two U.S. Russian wheat aphid biotypes (Russian wheat aphid 1 and Russian wheat aphid 2). Using GeneChip Wheat Genome Arrays, we compared transcript profiles of resistant and susceptible lines infested with either Russian wheat aphid 1 or Russian wheat aphid 2 using two time intervals (5 and 48 h after infestation). Russian wheat aphid feeding on hexaploid wheat led to the induction of groups of genes functioning in oxidative and general stress, photosynthesis, cell respiration and energy production, signal transduction, calcium-dependent signaling, pathogenesis related (PR) responses, and defense compound synthesis. The number of differentially expressed genes was higher in plants infested with Russian wheat aphid 1 compared with those infested with Russian wheat aphid 2. Although most genes involved in basic cellular functions were shared, unique genes were also obtained. This finding may indicate subtle differences in genes induced in response to different virulence proteins.

Dissimilar molecular defense responses are elicited in Triticum aestivum after infestation by different Diuraphis noxia biotypes.

For sustainable solutions to the problem of insect infestation, the study of molecular plant-insect interactions is integral to resistance breeding strategies. This also holds true in the case of wheat (Triticum aestivum), where the Russian wheat aphid (Diuraphis noxia, Kurdjumov, RWA) is responsible for significant crop losses in most major wheat producing countries around the world. Our study is focused on gaining a greater understanding of the resistance mechanisms activated by the RWA resistance gene Dn7 by comparing responses following infestation with three different aphid biotypes (RWA-SA, RWA-US1 and RWA-US2). This consisted of analyzing the resistant wheat line 94M370 (containing Dn7) and its susceptible counterpart (Gamtoos) on a transcriptional level with complementary DNA-amplified fragment length polymorphisms (cDNA-AFLPs) using 17 primer combinations, as well as quantitative reverse transcription polymerase chain reaction (qRT-PCR) of 10 differentially expressed transcripts. The results of this expression profile analysis suggest that Dn7 activates similar responses against the two US aphid biotypes, which differ noticeably from the response following infestation with a South African aphid biotype. This is consistent with recent research showing limited molecular variations between the two US aphid biotypes (approximately 0.12%), compared with a distinctly different South African biotype. We therefore conclude that Dn7 recognizes and interacts in a highly specific manner with different aphid's putative eliciting agents, which in turn activates specific defense pathways unique to that interaction.

Differentially expressed genes during malting and correlation with malting quality phenotypes in barley (Hordeum vulgare L.).

Breeding for malting quality is an important goal of malting barley breeding programs. Malting quality is a complex phenotype that combines a large number of interrelated components, each of which shows complex inheritance. Currently, only a few genes involved in determining malting quality have been characterized. We combined transcript profiling with phenotypic correlations to identify candidate genes for malting quality. The Barley1 GeneChip array containing 22,792 probe sets was used to conduct transcript profiling of genes expressed in several different stages of malting of four malting cultivars. Genes that were differentially expressed in comparisons between different malting stages relative to ungerminated seed, as well as in comparisons between malting cultivars in the same malting stage were identified. Correlation analysis of 723 differentially expressed genes with malting quality phenotypes showed that 11-102 of these genes correlated with six malting quality phenotypes. Genes involved in carbohydrate metabolism were among the positively correlated genes. Genes for protein and lipid metabolism, cell wall organization and biogenesis, and genes involved in stress and defense response also correlated with malting quality phenotypes. Expressed sequence tags (ESTs) were generated from a 'malting-gene enriched' cDNA library made by suppression subtractive hybridization between malted and ungerminated seeds of 'Morex'. Eleven percent of the ESTs had no significant homology with sequences in the databases, suggesting that there may be other malting-related genes not represented in the barley gene chip array. The results provide candidate genes for malting quality phenotypes that need to be functionally validated.

Fractionated extracts of Russian wheat aphid eliciting defense responses in wheat.

It is hypothesized that the interaction between aphids and plants follows a gene-for-gene model. The recent appearance of several new Russian wheat aphid, Diuraphis noxia (Kurdjumov) (Homoptera: Aphididae), biotypes in the United States and the differential response of wheat, Triticum aestivum L., genotypes containing different resistance genes also suggest a gene-for-gene interaction. However, aphid elicitors remain unknown. This study was conducted to identify fractionated Russian wheat aphid extracts capable of eliciting differential responses between resistant and susceptible wheat genotypes. We extracted whole soluble compounds and separated proteins and metabolites from two Russian wheat aphid biotypes (1 and 2), injected these extracts into seedlings of susceptible wheat Gamtoos (dn7) and resistant 94M370 (Dn7), and determined phenotypic and biochemical plant responses. Injections of whole extract or protein extract from both biotypes induced the typical susceptible symptom, leaf rolling, in the susceptible cultivar, but not in the resistant cultivar. Furthermore, multiple injections with protein extract from biotype 2 induced the development of chlorosis, head trapping, and stunting in susceptible wheat. Injection with metabolite, buffer, or chitin, did not produce any susceptible symptoms in either genotype. The protein extract from the two biotypes also induced significantly higher activities of three defense-response enzymes (catalase, peroxidase, and beta-glucanase) in 94M370 than in Gamtoos. These results indicate that a protein elicitor from the Russian wheat aphid is recognized by a plant receptor, and the recognition is mediated by the Dn7-gene product. The increased activities of defense-response enzymes in resistant plants after injection with the protein fraction suggest that defense response genes are induced after recognition of aphid elicitors by the plant.

Characterization of EST-derived microsatellites in the wheat genome and development of eSSR markers.

EST-derived microsatellites or simple sequence repeats (eSSR) occur in expressed sequence tags (EST). Here we report characteristics of eSSRs in the wheat genome, construction of consensus chromosome bin maps of SSR-containing ESTs ((SSR)ESTs), and development of eSSR markers for the 21 wheat chromosomes. A Perl script known as MISA was used to identify eSSRs in wheat ESTs available in the database http://wheat.pw.usda.gov/cgi-bin/ace/search/wEST ). Among 492,832 ESTs from the database, 36,520 (7.41%) contained 43,598 eSSRs. This is equivalent to 1 eSSR per 5.46 kb EST sequence. About 60% of the eSSRs were trinucleotides, 19.7% were mononucleotide, 16.7% were dinucleotides, and the remaining approximately 3% consisted of tetra-, penta-, and hexanucleotides. Among the identified eSSRs, (CCG/CGG)n is the most frequent (20.5%) followed by (A/T)n at 13.6%, (AAC/GTT)n at 11.7%, and (AG/CT)n at 8.7%. Among ESTs previously mapped to wheat chromosome bins, a total of 1,010 eSSR loci were derived from 341 (SSR)ESTs. Consensus chromosome bin maps showing the chromosome locations of (SSR)ESTs, SSR sequence motif, and cDNA library were constructed. A chi(2) test indicated that the distribution pattern of eSSR loci was generally similar to that of the original mapped ESTs in the wheat genome. Forty-eight (SSR)ESTs were converted into PCR-based eSSR markers, and 266 eSSR loci were mapped to specific chromosome arms using wheat cytogenetic stocks. The average polymorphism information content (0.45+/-0.16) of eSSR markers was lower than that reported for genomic SSRs (0.54+/-0.19), but higher than RFLPs (0.30+/-0.27). The eSSR markers were transferable among related Triticeae species, Triticum aestivum, T. durum, T. dicoccoides, Hordeum spontaneum, H. vulgare, and Secale cereale. The results confirm the presence of SSRs in expressed genes of wheat and demonstrate another application of ESTs in genomics research. eSSRs will be useful for gene tagging, gene cloning, and comparative genomics studies of cereal crops.

Synteny perturbations between wheat homoeologous chromosomes caused by locus duplications and deletions correlate with recombination rates.

Loci detected by Southern blot hybridization of 3,977 expressed sequence tag unigenes were mapped into 159 chromosome bins delineated by breakpoints of a series of overlapping deletions. These data were used to assess synteny levels along homoeologous chromosomes of the wheat A, B, and D genomes, in relation to both bin position on the centromere-telomere axis and the gradient of recombination rates along chromosome arms. Synteny level decreased with the distance of a chromosome region from the centromere. It also decreased with an increase in recombination rates along the average chromosome arm. There were twice as many unique loci in the B genome than in the A and D genomes, and synteny levels between the B genome chromosomes and the A and D genome homoeologues were lower than those between the A and D genome homoeologues. These differences among the wheat genomes were attributed to differences in the mating systems of wheat diploid ancestors. Synteny perturbations were characterized in 31 paralogous sets of loci with perturbed synteny. Both insertions and deletions of loci were detected and both preferentially occurred in high recombination regions of chromosomes.

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.

Genetic mapping of Dn7, a rye gene conferring resistance to the Russian wheat aphid in wheat.

The Russian wheat aphid is a significant pest problem in wheat and barley in North America. Genetic resistance in wheat is the most effective and economical means to control the damage caused by the aphid. Dn7 is a rye gene located on chromosome 1RS that confers resistance to the Russian wheat aphid. The gene was previously transferred from rye into a wheat background via a 1RS/1BL translocation. This study was conducted to genetically map Dn7 and to characterize the type of resistance the gene confers. The resistant line '94M370' was crossed with a susceptible wheat cultivar that also contains a pair of 1RS/1BL translocation chromosomes. The F(2) progeny from this cross segregated for resistance in a ratio of 3 resistant: 1 susceptible, indicating a single dominant gene. One-hundred and eleven RFLP markers previously mapped on wheat chromosomes 1A, 1B and 1D, barley chromosome 1H and rye chromosome 1R, were used to screen the parents for polymorphism. A genetic map containing six markers linked to Dn7, encompassing 28.2 cM, was constructed. The markers flanking Dn7 were Xbcd1434 and XksuD14, which mapped 1.4 cM and 7.4 cM from Dn7, respectively. Dn7 confers antixenosis, and provides a higher level of resistance than that provided by Dn4. The applications of Dn7 and the linked markers in wheat breeding are discussed.

The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms.

Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.