Cytogenetic identification and molecular mapping for the wheat-Thinopyrum ponticum introgression line with resistance to Fusarium head blight.

KEY MESSAGE: Xinong 511, a new wheat-Thinopyrum ponticum variety with excellent fusarium head blight resistance, the QTLs were mapped to the wheat chromosomes 5B and 7A with named QFhb.nwafu-5B and QFhb.nwafu-7A, respectively. Novel Fusarium head blight (FHB) resistance germplasms and genes are valuable for wheat improvement and breeding efforts. Thinopyrum ponticum, a wild relative of common wheat, is a valuable germplasm of disease resistance for wheat improvement and breeding. Xinong 511 (XN511) is a high-quality wheat variety widely cultivated in the Yellow and Huai Rivers Valley of China with stable FHB-resistance. Through analysis of pedigree materials of the wheat cultivar XN511, we found that the genetic material and FHB resistance from Th. ponticum were transmitted to the introgression line, indicating that the FHB resistance in XN511 likely originates from Th. ponticum. To further explore the genetic basis of FHB resistance in XN511, QTL mapping was conducted using the RILs population of XN511 and the susceptible line Aikang 58 (AK58). Survey with makers closely-linked to Fhb1, Fhb2, Fhb4, Fhb5, and Fhb7, indicated that both XN511 and the susceptible lines do not contain these QTL. Using bulked segregant analysis RNA-seq (BSR-Seq) and newly developed allele-specific PCR (AS-PCR) markers, QTLs in XN511 were successfully located on wheat chromosomes 5B and 7A. These findings are significant for further understanding and utilizing FHB resistance genes in wheat improvement.

GenoBaits®WheatplusEE: a targeted capture sequencing panel for quick and accurate identification of wheat-Thinopyrum derivatives.

KEY MESSAGE: A total of 90,000 capture probes derived from wheat and Thinopyrum elongatum were integrated into one chip, which served as an economical genotype for explorating Thinopyrumspecies and their derivatives. Thinopyrum species play a crucial role as a source of new genetic variations for enhancing wheat traits, including resistance to both abiotic and biotic factors. Accurate identification of exogenous chromosome(s) or chromosome segments or genes is essential following the introduction of alien genetic material into wheat, but this task remains challenging. This study aimed to develop a high-resolution wheat-Thinopyrum elongatum array, named GenoBaits®WheatplusEE, to trace alien genetic information by genotyping using a target sequencing system. This GenoBaits®WheatplusEE array included 90,000 capture probes derived from two species and integrated into one chip, with 10,000 and 80,000 originating from wheat and Th. elongatum, respectively. The capture probes were strategically positioned in genes and evenly distributed across the genome, facilitating the development of a roadmap for identifying each alien gene. The array was applied to the high-throughput identification of the alien chromosomes or segments in Thinopyrum and distantly related species and their derivatives. Our results demonstrated that the GenoBaits®WheatplusEE array could be used for direct identification of the breakpoint of alien segments, determine copy number of alien chromosomes, and reveal variations in wheat chromosomes by a single round of target sequencing of the sample. Additionally, we could efficiently and cost-effectively genotype, supporting the exploration of subgenome composition, phylogenetic relationships, and polymorphisms in essential genes (e.g., Fhb7 gene) among Thinopyrum species and their derivatives. We hope that GenoBaits®WheatplusEE will become a widely adopted tool for exporting wild germplasm for wheat improvement in the future.

Genome-Wide Comparative Analysis of the Cytochrome P450 Monooxygenase Family in 19 Aphid Species and Their Expression Analysis in 4 Cereal Crop Aphids.

Cytochrome P450 monooxygenases (CYP450s) play a variety of physiological roles, including pesticide resistance, plant allelochemical detoxification, and hormone metabolism catalysis. However, limited information is available on the classification and expression profiles of the CYP450 gene family in aphid species. This is the first study to identify the cytochrome P450 gene family in 19 aphid species at the whole genome level. A total of 1100 CYP450 genes were identified in 19 aphid species. Three hundred CYP450 genes belonged to six cereal crop aphid species, which were further classified into four subfamilies according to the phylogenetic relationship. The conserved motifs, exon-intron structures, and genomic organization of the same subfamilies were similar. Predictions of subcellular localization revealed that the endoplasmic reticulum harbored the majority of CYP450 proteins. In Sitobion avenae and Rhopalosiphum maidis, the increase in the CYP450 gene was primarily caused by segmental duplication events. However, only tandem duplication occurred in the CYP450 gene family of Diuraphis noxia, Rhopalosiphum padi, Schizaphis graminum, and Sitobion miscanthi. Synteny analysis found three continuous colinear CYP450 gene pairs among six cereal crop aphid species. Furthermore, we obtained the expression profiles of four cereal crop aphids, including R. padi, D. noxia, S. graminum, and S. avenae. Differential expression analysis provided growth stage specificity genes, tissue specificity genes, organ specificity genes and some detoxification metabolic genes among these four cereal crop aphids. Meanwhile, their expression patterns were showed. The related functions and pathways of CYP450s were revealed by GO and KEGG enrichment analysis. Above all, we picked the differentially expressed CYP450 genes from all of the differentially expressed genes (DEGs). These differentially expressed CYP450 genes provided some new potential candidates for aphid control and management. This work establishes the foundation for further investigations into the regulatory functions of the CYP450 gene family in aphid species and beyond.

Genome-wide identification and characterization of lipoxygenase genes related to the English grain aphid infestation response in wheat.

MAIN CONCLUSION: 44 wheat LOX genes were identified by silico genome-wide search method. TaLOX5, 7, 10, 24, 29, 33 were specifically expressed post aphid infestation, indicating their participation in wheat-aphid interaction. In plants, LOX genes play important roles in various biological progresses including seed germination, tuber development, plant vegetative growth and most crucially in plant signal transduction, stress response and plant defense against plant diseases and insects. Although LOX genes have been characterized in many species, the importance of the LOX family in wheat has still not been well understood, hampering further improvement of wheat under stress conditions. Here, we identified 44 LOX genes (TaLOXs) in the whole wheat genome and classified into three subfamilies (9-LOXs, Type I 13-LOXs and Type II 13-LOXs) according to phylogenetic relationships. The TaLOXs belonging to the same subgroup shared similar gene structures and motif organizations. Synteny analysis demonstrated that segmental duplication events mainly contributed to the expansion of the LOX gene family in wheat. The results of protein-protein interaction network (PPI) and miRNA-TaLOXs predictions revealed that three TaLOXs (TaLOX20, 22 and 37) interacted mostly with proteins related to methyl jasmonate (MeJA) signaling pathway. The expression patterns of TaLOXs in different tissues (root, stem, leaf, spike and grain) under diverse abiotic stresses (heat, cold, drought, drought and heat combined treatment, and salt) as well as under diverse biotic stresses (powdery mildew pathogen, Fusarium graminearum and stripe rust pathogen) were systematically analyzed using RNA-seq data. We obtained aphid-responsive candidate genes by RNA-seq data of wheat after the English grain aphid infestation. Aphid-responsive candidate genes, including TaLOX5, 7, 10, 24, 29 and 33, were up-regulated in the wheat aphid-resistant genotype (Lunxuan144), while they were little expressed in the susceptible genotype (Jimai22) during late response (48 h and 72 h) to the English grain aphid infestation. Meanwhile, qRT-PCR analysis was used to validate these aphid-responsive candidate genes. The genetic divergence and diversity of all the TaLOXs in bread wheat and its relative species were investigated by available resequencing data. Finally, the 3D structure of the TaLOX proteins was predicted based on the homology modeling method. This study not only systematically investigated the characteristics and evolutionary relationships of TaLOXs, but also provided potential candidate genes in response to the English grain aphid infestation and laid the foundation to further study the regulatory roles in the English grain aphid infestation of LOX family in wheat and beyond.

Molecular Cytological Analysis and Specific Marker Development in Wheat-Psathyrostachys huashanica Keng 3Ns Additional Line with Elongated Glume.

Psathyrostachys huashanica Keng (2n = 2x = 14, NsNs) is an excellent gene resource for wheat breeding, which is characterized by early maturity, low plant height, and disease resistance. The wheat-P. huashanica derivatives were created by the elite genes of P. huashanica and permeate into common wheat through hybridization. Among them, a long-glume material 20JH1155 was identified, with larger grains and longer spike than its parents. In the present study, the methods of cytological observation, GISH, and sequential FISH analysis showed that 20JH1155 contained 21 pairs of wheat chromosomes and a pair of P. huashanica. There were some differences in 5A and 7B chromosomes between 20JH1155 and parental wheat 7182. Molecular marker, FISH, and sequence cloning indicated 20JH1155 alien chromosomes were 3Ns of P. huashanica. In addition, differentially expressed genes during immature spikelet development of 20JH1155 and 7182 and predicted transcription factors were obtained by transcriptome sequencing. Moreover, a total of 7 makers derived from Ph#3Ns were developed from transcriptome data. Taken together, the wheat-P. huashanica derived line 20JH1155 provides a new horizon on distant hybridization of wheat and accelerates the utilization of genes of P. huashanica.

Molecular cytogenetics and development of St-chromosome-specific molecular markers of novel stripe rust resistant wheat-Thinopyrum intermedium and wheat-Thinopyrum ponticum substitution lines.

BACKGROUND: Owing to their excellent resistance to abiotic and biotic stress, Thinopyrum intermedium (2n = 6x = 42, JJJsJsStSt) and Th. ponticum (2n = 10x = 70) are both widely utilized in wheat germplasm innovation programs. Disomic substitution lines (DSLs) carrying one pair of alien chromosomes are valuable bridge materials for transmission of novel genes, fluorescence in situ hybridization (FISH) karyotype construction and specific molecular marker development. RESULTS: Six wheat-Thinopyrum DSLs derived from crosses between Abbondanza nullisomic lines (2n = 40) and two octoploid Trititrigia lines (2n = 8x = 56), were characterized by sequential FISH-genome in situ hybridization (GISH), multicolor GISH (mc-GISH), and an analysis of the wheat 15 K SNP array combined with molecular marker selection. ES-9 (DS2St (2A)) and ES-10 (DS3St (3D)) are wheat-Th. ponticum DSLs, while ES-23 (DS2St (2A)), ES-24 (DS3St (3D)), ES-25(DS2St (2B)), and ES-26 (DS2St (2D)) are wheat-Th. intermedium DSLs. ES-9, ES-23, ES-25 and ES-26 conferred high thousand-kernel weight and stripe rust resistance at adult stages, while ES-10 and ES-24 were highly resistant to stripe rust at all stages. Furthermore, cytological analysis showed that the alien chromosomes belonging to the same homoeologous group (2 or 3) derived from different donors carried the same FISH karyotype and could form a bivalent. Based on specific-locus amplified fragment sequencing (SLAF-seq), two 2St-chromosome-specific markers (PTH-005 and PTH-013) and two 3St-chromosome-specific markers (PTH-113 and PTH-135) were developed. CONCLUSIONS: The six wheat-Thinopyrum DSLs conferring stripe rust resistance can be used as bridging parents for transmission of valuable resistance genes. The utility of PTH-113 and PTH-135 in a BC1F2 population showed that the newly developed markers could be useful tools for efficient identification of St chromosomes in a common wheat background.

Identification of Differentially Expressed Genes in Resistant Tetraploid Wheat (Triticum turgidum) under Sitobion avenae (F.) Infestation.

The grain aphid Sitobion avenae (Fabricius) is one of the most destructive pests of wheat (Triticum aestivum). Deployment of resistant wheat germplasm appears as an excellent solution for this problem. Elite bread wheat cultivars only have limited resistance to this pest. The present study was carried out to investigate the potential of the tetraploid wheat (Triticum turgidum) variety Lanmai, which showed high resistance to S. avenae at both seedling and adult plant stages, as a source of resistance genes. Based on apterous adult aphids' fecundity tests and choice bioassays, Lanmai has been shown to display antixenosis and antibiosis. Suppression subtractive hybridization (SSH) was employed to identify and isolate the putative candidate defense genes in Lanmai against S. avenae infestation. A total of 134 expressed sequence tags (ESTs) were identified and categorized based on their putative functions. RT-qPCR analysis of 30 selected genes confirmed their differential expression over time between the resistant wheat variety Lanmai and susceptible wheat variety Polan305 during S. avenae infestation. There were 11 genes related to the photosynthesis process, and only 3 genes showed higher expression in Lanmai than in Polan305 after S. avenae infestation. Gene expression analysis also revealed that Lanmai played a critical role in salicylic acid and jasmonic acid pathways after S. avenae infestation. This study provided further insights into the role of defense signaling networks in wheat resistance to S. avenae and indicates that the resistant tetraploid wheat variety Lanmai may provide a valuable resource for aphid tolerance improvement in wheat.

Molecular cytogenetics for a wheat-Aegilops geniculata 3Mg alien addition line with resistance to stripe rust and powdery mildew.

BACKGROUND: Aegilops geniculata Roth is closely related to common wheat (Triticum aestivum L.) and is a valuable genetic resource for improvement of wheat. RESULTS: In this study, the W19513 line was derived from the BC1F10 progeny of a cross between wheat 'Chinese Spring' and Ae. geniculata SY159. Cytological examination showed that W19513 contained 44 chromosomes. Twenty-two bivalents were formed at the first meiotic metaphase I in the pollen mother cellsand the chromosomes were evenly distributed to opposite poles at meiotic anaphase I. Genomic in situ hybridization demonstrated that W19513 carried a pair of alien chromosomes from the M genome. Fluorescence in situ hybridization confirmed detection of variation in chromosomes 4A and 6B. Functional molecular marker analysis using expressed sequence tag-sequence-tagged site and PCR-based landmark unique gene primers revealed that the alien gene belonged to the third homologous group. The marker analysis confirmed that the alien chromosome pair was 3Mg. In addition, to further explore the molecular marker specificity of chromosome 3Mg, based on the specific locus amplified fragment sequencing technique, molecular markers specific for W19513 were developed with efficiencies of up to 47.66%. The W19513 line was inoculated with the physiological race E09 of powdery mildew (Blumeria graminis f. sp. tritici) at the seedling stage and showed moderate resistance. Field inoculation with a mixture of the races CYR31, CYR32, CYR33, and CYR34 of the stripe rust fungus (Puccinia striiformis f. sp. triticii) revealed that the line W19513 showed strong resistance. CONCLUSIONS: This study provides a foundation for use of the line W19513 in future genetic research and wheat improvement.

Chromosome karyotype and stability of new synthetic hexaploid wheat.

Synthetic hexaploid wheat offers breeders ready access to potentially novel genetic variation in wild ancestral species. In this study, we crossed MY3478 (2n = 4x = 28, AABB) as the maternal parent with the stripe rust-resistant SY41 (2n = 2x = 14, DD) as the paternal parent to construct the new hexaploid wheat line NA0928 through natural allopolyploidization. Agronomic traits and the cytology of the S8-S9 generations of NA0928 were analyzed. Abundant variation in agronomic traits was observed among each strain of NA0928 in the S8 generation. Agronomic traits were superior in strains resistant to stripe rust compared with those of highly susceptible strains. The rank order of the coefficients of variation were tiller number (55.3%) > spike length (15.3%) > number of spikelets (13.9%) > plant height (8.7). Number of tillers and spike length are important traits in wheat breeding to improve yield. Cytological observation and fluorescence in situ hybridization showed that the chromosome number and configuration showed rich variation among NA0928 strains in the S9 generation. Chromosome number ranged from 36 to 44. Variation in chromosome karyotype was detected in the A and B subgenomes. Meiotic chromosome behavior in pollen mother cells and multicolor genomic in situ hybridization revealed that two new synthetic hexaploid wheat strains showed genetic stability; one strain was resistant to stripe rust and developed multiple tillers, and the other strain was susceptible to stripe rust, but both showed improved thousand-kernel weight (TKW) weight and produced multiple tillers. The two strains will be valuable germplasm resources for use in wheat breeding.

Characterization and Evaluation of Resistance to Powdery Mildew of Wheat-Aegilops geniculata Roth 7Mg (7A) Alien Disomic Substitution Line W16998.

Aegilops geniculata Roth has been used as a donor of disease-resistance genes, to enrich the gene pool for wheat (Triticum aestivum) improvement through distant hybridization. In this study, the wheat-Ae. geniculata alien disomic substitution line W16998 was obtained from the BC1F8 progeny of a cross between the common wheat 'Chinese Spring' (CS) and Ae. geniculata Roth (serial number: SY159//CS). This line was identified using cytogenetic techniques, analysis of genomic in situ hybridization (GISH), functional molecular markers (Expressed sequence tag-sequence-tagged site (EST-STS) and PCR-based landmark unique gene (PLUG), fluorescence in situ hybridization (FISH), sequential fluorescence in situ hybridization-genomic in situ hybridization (sequential FISH-GISH), and assessment of agronomic traits and powdery mildew resistance. During the anaphase of meiosis, these were evenly distributed on both sides of the equatorial plate, and they exhibited high cytological stability during the meiotic metaphase and anaphase. GISH analysis indicated that W16998 contained a pair of Ae. geniculata alien chromosomes and 40 common wheat chromosomes. One EST-STS marker and seven PLUG marker results showed that the introduced chromosomes of Ae. geniculata belonged to homoeologous group 7. Nullisomic-tetrasomic analyses suggested that the common wheat chromosome, 7A, was absent in W16998. FISH and sequential FISH-GISH analyses confirmed that the introduced Ae. geniculata chromosome was 7Mg. Therefore, W16998 was a wheat-Ae. geniculata 7Mg (7A) alien disomic substitution line. Inoculation of isolate E09 (Blumeria graminis f. sp. tritici) in the seedling stage showed that SY159 and W16998 were resistant to powdery mildew, indeed nearly immune, whereas CS was highly susceptible. Compared to CS, W16998 exhibited increased grain weight and more spikelets, and a greater number of superior agronomic traits. Consequently, W16998 was potentially useful. Germplasms transfer new disease-resistance genes and prominent agronomic traits into common wheat, giving the latter some fine properties for breeding.

Molecular cytogenetic identification of a wheat - Thinopyrum ponticum substitution line with stripe rust resistance.

Thinopyrum ponticum (Th. ponticum) (2n = 10x = 70) is an important breeding material with excellent resistance and stress tolerance. In this study, we characterized the derivative line CH1113-B13-1-1-2-1 (CH1113-B13) through cytological, morphological, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), expressed sequence tag (EST), and PCR-based landmark unique gene (PLUG) marker analysis. The GISH analysis revealed that CH1113-B13 contained 20 pairs of common wheat chromosomes and one pair of JSt genomic chromosomes. Linkage analysis of Th. ponticum using seven EST and seven PLUG markers indicated that the pair of alien chromosomes belonged to the seventh homeologous group. Nulli-tetrasomic and FISH analysis revealed that wheat 7B chromosomes were absent in CH1113-B13; thus, CH1113-B13 was identified as a 7JSt (7B) substitution line. Finally, adult-stage CH1113-B13 exhibited immunity to wheat stripe rust. This substitution line is therefore a promising germplasm resource for wheat breeding.

Molecular cytogenetics identification of a wheat - Leymus mollis double disomic addition line with stripe rust resistance.

Leymus mollis (Trin.) Pilg. (2n = 4x = 28, NsNsXmXm) possesses a number of valuable genes against biotic and abiotic stress, which could be transferred into common wheat background for wheat improvement. In the present study, we determined the karyotypic constitution of a wheat - L. mollis double disomic addition line, M11003-4-4-1-1, selected from the F5 progeny of a stable wheat - L. mollis derivative M39 (2n = 56) × Triticum aestivum cultivar 7182, by morphological and cytogenetic identification, GISH (genomic in situ hybridization), FISH (fluorescent in situ hybridization), molecular markers analysis, and stripe rust resistance evaluation. Cytological studies demonstrated that M11003-4-4-1-1 had a chromosome karyotype of 2n = 46 with 23 bivalents, while GISH and FISH analysis indicated that this line contained 42 common wheat chromosomes and two pairs of L. mollis chromosomes. DNA markers showed that the alien chromosomes from L. mollis belonged to homoeologous groups 5 and 6. Evaluation of the agronomic traits revealed that M11003-4-4-1-1 was resistant to stripe rust at the adult stage, while the plant height was reduced and the 1000-grain weight was increased significantly. Therefore, the new line M11003-4-4-1-1 could be exploited as an important bridge material in chromosome engineering and wheat breeding.

Genome-wide identification and functional prediction of novel and fungi-responsive lincRNAs in Triticum aestivum.

BACKGROUND: Stripe rust (Puccinia striiformis f. sp. tritici; Pst) and powdery mildew (Blumeria graminis f. sp. tritici; Bgt) are important diseases of wheat (Triticum aestivum) worldwide. Increasingly evidences suggest that long intergenic ncRNAs (lincRNAs) are developmentally regulated and play important roles in development and stress responses of plants. However, identification of lincRNAs in wheat is still limited comparing with functional gene expression. RESULTS: The transcriptome of the hexaploid wheat line N9134 inoculated with the Chinese Pst race CYR31 and Bgt race E09 at 1, 2, and 3 days post-inoculation was recapitulated to detect the lincRNAs. Here, 283 differential expressed lincRNAs were identified from 58218 putative lincRNAs, which account for 31.2% of transcriptome. Of which, 254 DE-LincRNAs responded to the Bgt stress, and 52 lincRNAs in Pst. Among them, 1328 SnRNP motifs (sm sites) were detected and showed RRU4-11RR sm site element and consensus RRU1-9VU1-7RR SnRNP motifs, where the total number of uridine was more than 3 but less than 11. Additionally, 101 DE-lincRNAs were predicted as targets of miRNA by psRNATarget, while 5 target mimics were identified using target mimicry search in TAPIR. CONCLUSIONS: Taken together, our findings indicate that the lincRNA of wheat responded to Bgt and Pst stress and played important roles in splicesome and inter-regulating with miRNA. The sm site of wheat showed a more complex construction than that in mammal and model plant. The mass sequence data generated in this study provide a cue for future functional and molecular research on wheat-fungus interactions.

Molecular mapping and marker development for the Triticum dicoccoides-derived stripe rust resistance gene YrSM139-1B in bread wheat cv. Shaanmai 139.

KEY MESSAGE: YrSM139-1B maybe a new gene for effective resistance to stripe rust and useful flanking markers for marker-assisted selection were developed. ABSTRACT: Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important foliar disease of wheat. Two dominant stripe rust resistant genes YrSM139-1B and YrSM139-2D were pyramided in bread wheat cultivar Shaanmai 139; one from wild emmer and the other from Thinopyrum intermedium. Three near-isogenic F7:8 line pairs (contrasting RILs), N122-1013R/S, N122-185R/S, and N122-1812R/S, independently derived from different F2 plants and differing at the YrSM139-1B locus were generated from the cross Shaanmai 139 × Hu 901-19 through marker-assisted selection. A large F2:3 population from cross N122-1013R × N122-1013S tested for stripe rust response and subjected to analysis with markers in the 1BS10-0.5 bin region using SSR expressed sequence tags (EST) and site-specific sequence markers developed from the 90 K Illumina iSelect SNP array. Five EST-STS markers and four allele-specific PCR markers were mapped to the YrSM139-1B region. The 30.5 cM genetic map for YrSM139-1B consisted of nine markers, two of which were closer to YrSM139-1B than Xgwm273, which was used in producing the contrasting RIL pairs. Race response data and allelism tests showed that YrSM139-1B is different from Yr10, Yr15, and Yr24/26/CH42.

Molecular cytogenetic identification of a wheat-rye 1R addition line with multiple spikelets and resistance to powdery mildew.

Alien addition lines are important for transferring useful genes from alien species into common wheat. Rye is an important and valuable gene resource for improving wheat disease resistance, yield, and environment adaptation. A new wheat-rye addition line, N9436B, was developed from the progeny of the cross of common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) cultivar Shaanmai 611 and rye (Secale cereal L., 2n = 2x = 14, RR) accession Austrian rye. We characterized this new line by cytology, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), molecular markers, and disease resistance screening. N9436B was stable in morphology and cytology, with a chromosome composition of 2n = 42 + 2t = 22II. GISH investigations showed that this line contained two rye chromosomes. GISH, FISH, and molecular maker identification suggested that the introduced R chromosome and the missing wheat chromosome arms were 1R chromosome and 2DL chromosome arm, respectively. N9436B exhibited 30-37 spikelets per spike and a high level of resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) isolate E09 at the seedling stage. N9436B was cytologically stable, had the trait of multiple spikelets, and was resistant to powdery mildew; this line should thus be useful in wheat improvement.

Large-scale transcriptome comparison reveals distinct gene activations in wheat responding to stripe rust and powdery mildew.

BACKGROUND: Stripe rust (Puccinia striiformis f. sp. tritici; Pst) and powdery mildew (Blumeria graminis f. sp. tritici; Bgt) are important diseases of wheat (Triticum aestivum) worldwide. Similar mechanisms and gene transcripts are assumed to be involved in the host defense response because both pathogens are biotrophic fungi. The main objective of our study was to identify co-regulated mRNAs that show a change in expression pattern after inoculation with Pst or Bgt, and to identify mRNAs specific to the fungal stress response. RESULTS: The transcriptome of the hexaploid wheat line N9134 inoculated with the Chinese Pst race CYR 31 was compared with that of the same line inoculated with Bgt race E09 at 1, 2, and 3 days post-inoculation. Infection by Pst and Bgt affected transcription of 23.8% of all T. aestivum genes. Infection by Bgt triggered a more robust alteration in gene expression in N9134 compared with the response to Pst infection. An array of overlapping gene clusters with distinctive expression patterns provided insight into the regulatory differences in the responses to Bgt and Pst infection. The differentially expressed genes were grouped into seven enriched Kyoto Encyclopedia of Genes and Genomes pathways in Bgt-infected leaves and four pathways in Pst-infected leaves, while only two pathways overlapped. In the plant-pathogen interaction pathway, N9134 activated a higher number of genes and pathways in response to Bgt infection than in response to Pst invasion. Genomic analysis revealed that the wheat genome shared some microbial genetic fragments, which were specifically induced in response to Bgt and Pst infection. CONCLUSIONS: Taken together, our findings indicate that the responses of wheat N9134 to infection by Bgt and Pst shows differences in the pathways and genes activated. The mass sequence data for wheat-fungus interaction generated in this study provides a powerful platform for future functional and molecular research on wheat-fungus interactions.

Development and discrimination of 12 double ditelosomics in tetraploid wheat cultivar DR147.

As an important group in Triticum, tetraploid wheat plays a significant role in the research of wheat evolution. Several complete aneuploid sets of common wheat have provided valuable tools for genetic and breeding studies, while similar aneuploids of tetraploid wheat are still not well developed. Here, 12 double ditelosomics developed in Triticum turgidum L. var. durum cultivar DR147 (excluding dDT2B and dDT3A) were reported. Hybrids between DR147 and the original double-ditelosomic dDT2B of Langdon lost vigor and died prematurely after the three-leaf stage; therefore, the dDT2B line was not obtained. The cytogenetic behaviors and phenotypic characteristics of each line were detailedly described. To distinguish the entire chromosome complement of tetraploid wheat, the DR147 karyotype was established by fluorescence in situ hybridization (FISH), using the Aegilops tauschii clone pAsl and the barley clone pHvG38 as probes. FISH using a cereal-specific centromere repeat (6C6) probe suggested that all the lines possessed four telosomes, except for 4AS of double-ditelosomic dDT4A, which carried a small segment of the long arm. On the basis of the idiogram of DR147, these lines were successfully discriminated by FISH using the probes pAsl and pHvG38 and were then accurately designated.

Long non-coding genes implicated in response to stripe rust pathogen stress in wheat (Triticum aestivum L.).

The non-protein-coding genes have been reported as a critical control role in the regulation of gene expression in abiotic stress. We previously identified four expressed sequence tags numbered S18 (EL773024), S73 (EL773035), S106 (EL773041) and S108 (EL773042) from a SSH-cDNA library of bread wheat Shaanmai 139 infected with Puccinia striiformis f. sp. tritici (Pst). Here, we isolated four cDNA clones and referred them as TalncRNA18, TalncRNA73, TalncRNA106 and TalncRNA108 (GenBank: KC549675-KC549678). These cDNA separately consisted of 1,393, 667, 449 and 647 nucleotides but without any open reading frame. The alignment result showed that TalncRNA18 is a partial cDNA of E3 ubiquitin-protein ligase UPL1-like gene, TalncRNA73 is an antisense transcript of hypothetical protein, TalncRNA108 is a homolog to RRNA intron-encoded homing endonuclease, and lncRNA106 had no similarly sequence. Quantitative RT-PCR studies confirmed that these four lncRNAs were differentially expressed in three near isogenic lines. TalncRNA108 was significantly stepwise decreased at early stage of inoculation with Pst, while the others were upregulated, especially at 1 and 3 dpi (days post-inoculation). Using Chinese Spring nulli-tetrasomic lines and its ditelosomic lines, TalncRNA73 and TalncRNA108 were located to wheat chromosome 7A and the short arm of chromosome 4B, respectively, while TalncRNA18 and TalncRNA106 were located to chromosome 5B. Comparing the sequence of DNA and cDNA of four lncRNAs with polymerase chain reaction primers, the results showed that all of them have no introns. The kinetics analyses of lncRNAs expression as a result of pathogen challenge in immune resistant genotype indicated that they may play the roles of modulating or silencing the protein-coding gene into pathogen-defence response.

Construction of a Modified Clip Cage and Its Effects on the Life-History Parameters of Sitobion avenae (Fabricius) and Defense Responses of Triticum aestivum.

Clip cages are commonly used to confine aphids or other small insects to a single leaf when conducting plant-small insect interaction studies; however, clip cages are usually heavy or do not efficiently transmit light, which has an impact on leaf physiology, limiting their application. Here, simple, lightweight, and transparent modified clip cages were constructed using punched clear plastic cups, cut transparent polyvinyl chloride sheets, nylon organdy mesh, and bent duck-bill clips. These cages can be clipped directly onto dicot leaves or attached to monocot leaves with bamboo skewers and elastic bands. The weight, production time, and aphid escape rates of the modified clip cages were 3.895 ± 0.004 g, less than 3 min, and 2.154 ± 0.323%, respectively. The effects of the modified clip cage on the growth, development, and reproduction of the English grain aphid (Sitobion avenae Fabricius) in comparison with the whole cage were studied. The biochemical responses of wheat (Triticum aestivum) to the cages were also investigated. No significant differences were observed in the life table parameters, nymph mortality, and adult fecundity in S. avenae confined to clip cages and whole cages, but the clip cages were more time efficient than whole cages when conducting life table studies. Moreover, the hydrogen peroxide accumulation, callose deposition, and cell necrosis in wheat leaves covered by empty clip cages and empty whole cages were similar, and significantly lower than treatments where the aphids were inside the clip cage. The results demonstrate that the modified clip cages had negligible effects on the plant and aphid physiology, suggesting that they are effective for studying plant-small insect interactions.

Identification and expression analysis of heat-shock proteins in wheat infected with powdery mildew and stripe rust.

Heat-shock proteins (HSPs), which are encoded by conserved gene families in plants, are crucial for development and responses to diverse stresses. However, the wheat (Triticum aestivum L.) HSPs have not been systematically classified, especially those involved in protecting plants from disease. Here, we classified 119 DnaJ (Hsp40) proteins (TaDnaJs; encoded by 313 genes) and 41 Hsp70 proteins (TaHsp70s; encoded by 95 genes) into six and four groups, respectively, via a phylogenetic analysis. An examination of protein sequence alignment revealed diversity in the TaDnaJ structural organization, but a highly conserved J-domain, which was usually characterized by an HPD motif followed by DRD or DED motifs. The expression profiles of HSP-encoding homologous genes varied in response to Blumeria graminis f. sp. tritici (Bgt) and Puccinia striiformis f. sp. tritici (Pst) stress. A quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated a lack of similarity in the expression of DnaJ70b, Hsp70-30b, and Hsp90-4b in Bgt-infected resistant and susceptible wheat. Furthermore, a direct interaction between DnaJ70 and TaHsp70-30 was not detected in a yeast two-hybrid (Y2H) assay, but screening cDNA library and Y2H evidence supported that TaHsp70-30 not only interacts directly with heat-shock transcription factor (HSF) A9-like protein but also interacts with TaHsp90-4 by HSP organizing protein. This study revealed the structure and expression profiles of the HSP-encoding genes in wheat, which may be useful for future functional elucidation of wheat HSPs responses to fungal infections.