Linyi Qiao

Chromosomal location and comparative genomics analysis of powdery mildew resistance gene Pm51 in a putative wheat-Thinopyrum ponticum introgression line.

Powdery mildew (PM) is a very destructive disease of wheat (Triticum aestivum L.). Wheat-Thinopyrum ponticum introgression line CH7086 was shown to possess powdery mildew resistance possibly originating from Th. ponticum. Genomic in situ hybridization and molecular characterization of the alien introgression failed to identify alien chromatin. To study the genetics of resistance, CH7086 was crossed with susceptible genotypes. Segregation in F2 populations and F2:3 lines tested with Chinese Bgt race E09 under controlled conditions indicated that CH7086 carries a single dominant gene for powdery mildew resistance. Fourteen SSR and EST-PCR markers linked with the locus were identified. The genetic distances between the locus and the two flanking markers were 1.5 and 3.2 cM, respectively. Based on the locations of the markers by nullisomic-tetrasomic and deletion lines of ‘Chinese Spring’, the resistance gene was located in deletion bin 2BL-0.89-1.00. Conserved orthologous marker analysis indicated that the genomic region flanking the resistance gene has a high level of collinearity to that of rice chromosome 4 and Brachypodium chromosome 5. Both resistance specificities and tests of allelism suggested the resistance gene in CH7086 was different from previously reported powdery mildew resistance genes on 2BL, and the gene was provisionally designated PmCH86. Molecular analysis of PmCH86 compared with other genes for resistance to Bgt in the 2BL-0.89-1.00 region suggested that PmCH86 may be a new PM resistance gene, and it was therefore designated as Pm51. The closely linked flanking markers could be useful in exploiting this putative wheat-Thinopyrum translocation line for rapid transfer of Pm51 to wheat breeding programs.

A genome-wide analysis of the auxin/indole-3-acetic acid gene family in hexaploid bread wheat (Triticum aestivum L.)

The Auxin/indole-3-acetic acid (Aux/IAA) gene family plays key roles in the primary auxin-response process and controls a number of important traits in plants. However, the characteristics of the Aux/IAA gene family in hexaploid bread wheat (Triticum aestivum L.) have long been unknown. In this study, a comprehensive identification of the Aux/IAA gene family was performed using the latest draft genome sequence of the bread wheat “Chinese Spring.” Thirty-four Aux/IAA genes were identified, 30 of which have duplicated genes on the A, B or D sub-genome, with a total of 84 Aux/IAA sequences. These predicted Aux/IAA genes were non-randomly distributed in all the wheat chromosomes except for chromosome 2D. The information of wheat Aux/IAA proteins is also described. Based on an analysis of phylogeny, expression and adaptive evolution, we prove that the Aux/IAA family in wheat has been replicated twice in the two allopolyploidization events of bread wheat, when the tandem duplication also occurred. The duplicated genes have undergone an evolutionary process of purifying selection, resulting in the high conservation of copy genes among sub-genomes and functional redundancy among several members of the TaIAA family. However, functional divergence probably existed in most TaIAA members due to the diversity of the functional domain and expression pattern. Our research provides useful information for further research into the function of Aux/IAA genes in wheat.

Genome-wide identification and analysis of the MADS-box gene family in bread wheat (Triticum aestivum L.)

The MADS-box genes encode transcription factors with key roles in plant growth and development. A comprehensive analysis of the MADS-box gene family in bread wheat (Triticum aestivum) has not yet been conducted, and our understanding of their roles in stress is rather limited. Here, we report the identification and characterization of the MADS-box gene family in wheat. A total of 180 MADS-box genes classified as 32 Mα, 5 Mγ, 5 Mδ, and 138 MIKC types were identified. Evolutionary analysis of the orthologs among T. urartu, Aegilops tauschii and wheat as well as homeologous sequences analysis among the three sub-genomes in wheat revealed that gene loss and chromosomal rearrangements occurred during and/or after the origin of bread wheat. Forty wheat MADS-box genes that were expressed throughout the investigated tissues and development stages were identified. The genes that were regulated in response to both abiotic stresses (i.e., phosphorus deficiency, drought, heat, and combined drought and heat) and biotic stresses (i.e., Fusarium graminearum, Septoria tritici, stripe rust and powdery mildew) were detected as well. A few notable MADS-box genes were specifically expressed in a single tissue and those showed relatively higher expression differences between the stress and control treatment. The expression patterns of considerable MADS-box genes differed from those of their orthologs in Brachypodium, rice, and Arabidopsis. Collectively, the present study provides new insights into the possible roles of MADS-box genes in response to stresses and will be valuable for further functional studies of important candidate MADS-box genes.

Molecular characterization of a new wheat-Thinopyrum intermedium translocation line with resistance to powdery mildew and stripe rust.

A new wheat-Thinopyrum translocation line CH13-21 was selected from the progenies derived from a cross between wheat-Th. intermedium partial amphiploid TAI7047 and wheat line Mianyang11. CH13-21 was characterized by using genomic in situ hybridization (GISH), multicolor-GISH (mc-GISH), multicolor-fluorescence in situ hybridization (mc-FISH) and chromosome-specific molecular markers. When inoculated with stripe rust and powdery mildew isolates, CH13-21 displayed novel resistance to powdery mildew and stripe rust which inherited from its Thinopyrum parent. The chromosomal counting analyses indicated that CH13-21 has 42 chromosomes, with normal bivalent pairing at metaphase I of meiosis. GISH probed by Th. intermedium genomic DNA showed that CH13-21 contained a pair of wheat-Th. intermedium translocated chromosomes. Sequential mc-FISH analyses probed by pSc119.2 and pAs1 clearly revealed that chromosome arm 6BS of CH13-21 was replaced by Thinopyrum chromatin in the translocation chromosome. The molecular markers analysis further confirmed that the introduced Th. intermedium chromatin in CH13-21 belonged to the long arm of homoeologous group 6 chromosome. Therefore, CH13-21 was a new T6BS.6Ai#1L compensating Robertsonian translocation line. It concludes that CH13-21 is a new genetic resource for wheat breeding programs providing novel variation for disease resistances.

Genome-wide identification and resistance expression analysis of the NBS gene family in Triticum urartu

As the largest class of resistant genes, the nucleotide binding site (NBS) has been studied extensively at a genome-wide level in rice, sorghum, maize, barley and hexaploid wheat. However, no such comprehensive analysis has been conducted of the NBS gene family in Triticum urartu, the donor of the A genome to the common wheat. Using a bioinformatics method, 463 NBS genes were isolated from the whole genome of T. urartu, of which 461 had location information. The expansion pattern and evolution of the 461 NBS candidate proteins were analyzed, and 118 of them were duplicated. By calculating the lengths of the copies, it was inferred that the NBS resistance gene family of T. urartu has experienced at least two duplication events. Expression analysis based on RNA-seq data found that 6 genes were differentially expressed among Tu38, Tu138 and Tu158 in response to Blumeria graminis f.sp.tritici (Bgt). Following Bgt infection, the expression levels of these genes were up-regulated. These results provide critical references for further identification and analysis of NBS family genes with important functions.

Mapping of Powdery Mildew Resistance Gene pmCH89 in a Putative Wheat-Thinopyrum intermedium Introgression Line.

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a globally serious disease adversely affecting wheat production. The Bgt-resistant wheat breeding line CH09W89 was derived after backcrossing a Bgt resistant wheat-Thinopyrum intermedium partial amphiploid TAI7045 with susceptible wheat cultivars. At the seedling stage, CH09W89 exhibited immunity or high resistance to Bgt pathotypes E09, E20, E21, E23, E26, Bg1, and Bg2, similar to its donor line TAI7045 and Th. intermedium. No Th. intermedium chromatin was detected based on genomic in situ hybridization of mitotic chromosomes. To determine the mode of inheritance of the Bgt resistance and the chromosomal location of the resistance gene, CH09W89 was crossed with two susceptible wheat cultivars. The results of the genetic analysis showed that the adult resistance to Bgt E09 in CH09W89 was controlled by a single recessive gene, which was tentatively designated as pmCH89. Two polymorphic SSR markers, Xwmc310 and Xwmc125, were linked to the resistance gene with genetic distances 3.1 and 2.7 cM, respectively. Using the Chinese Spring aneuploid and deletion lines, the resistance gene and its linked markers were assigned to chromosome arm 4BL in the bin 0.68–0.78. Due to its unique position on chromosome 4BL, pmCH89 appears to be a new locus for resistance to powdery mildew. These results will be of benefit for improving powdery mildew resistance in wheat breeding programs.

Characterization and Expression Patterns of Auxin Response Factors in Wheat

Auxin response factors (ARFs) are important transcription factors involved in both the auxin signaling pathway and the regulatory development of various plant organs. In this study, 23 TaARF members encoded by a total of 68 homeoalleles were isolated from 18 wheat chromosomes (excluding chromosome 4). The TaARFs, including their conserved domains, exon/intron structures, related microRNAs, and alternative splicing (AS) variants, were then characterized. Phylogenetic analysis revealed that members of the TaARF family share close homology with ARFs in other grass species. qRT-PCR analyses revealed that 20 TaARF members were expressed in different organs and tissues and that the expression of some members significantly differed in the roots, stems, and leaves of wheat seedlings in response to exogenous auxin treatment. Moreover, protein network analyses and co-expression results showed that TaTIR1–TaARF15/18/19–TaIAA13 may interact at both the protein and genetic levels. The results of subsequent evolutionary analyses showed that three transcripts of TaARF15 in the A subgenome of wheat exhibited high evolutionary rate and underwent positive selection. Transgenic analyses indicated that TaARF15-A.1 promoted the growth of roots and leaves of Arabidopsis thaliana and was upregulated in the overexpression plants after auxin treatment. Our results will provide reference information for subsequent research and utilization of the TaARF gene family.

Characterization of the CCT family and analysis of gene expression in Aegilops tauschii

Flowering is crucial for reproductive success in flowering plant. The CCT domain-containing genes widely participate in the regulation of flowering process in various plant species. So far, the CCT family in common wheat is largely unknown. Here, we characterized the structure, organization, molecular evolution and expression of the CCT genes in Aegilops tauschii, which is the D genome donor of hexaploid wheat. Twenty-six CCT genes (AetCCT) were identified from the full genome of A. tauschii and these genes were distributed on all 7 chromosomes. Phylogenetic analysis classified these AetCCT genes into 10 subgroups. Thirteen AetCCT members in group A, C, H and G achieved rapid evolution based on evolutionary rate analysis. The AetCCT genes respond to different exogenous hormones and abiotic treatments, the expression of AetCCT4, 7, 8, 11, 12, 16, 17, 19, 21 and 22 showed a significant 24 h rhythm. This study may provide a reference for common wheats evolution, domestication and evolvement rules, and also help us to understand the ecological adaptability of A. tauschii.

Evolution of the Aux/IAA Gene Family in Hexaploid Wheat

The Aux/IAA (IAA) gene family, involved in the auxin signalling pathway, acts as an important regulator in plant growth and development. In this study, we explored the evolutionary trajectory of the IAA family in common wheat. The results showed ten pairs of paralogs among 34 TaIAA family members. Seven of the pairs might have undergone segmental duplication, and the other three pairs appear to have experienced tandem duplication. Except for TaIAA15-16, these duplication events occurred in the ancestral genomes before the divergence of Triticeae. After that point, two polyploidization events shaped the current TaIAA family consisting of three subgenomic copies. The structure or expression pattern of the TaIAA family begins to differentiate in the hexaploid genome, where TaIAAs in the D genome lost more genes (eight) and protein secondary structures (α1, α3 and β5) than did the other two genomes. Expression analysis showed that six members of the TaIAA family were not expressed, and members such as TaIAA8, 15, 16, 28 and 33 exhibited tissue-specific expression patterns. In addition, three of the ten pairs of paralogs (TaIAA5–12, TaIAA15–16 and TaIAA29–30) showed similar expression patterns, and another five paralog pairs displayed differential expression patterns. Phylogenetic analysis showed that paralog pairs with high rates of evolution (ω > ω0), particularly TaIAA15–16 and TaIAA29–30, experienced greater motif loss, with only zero to two interacting IAA proteins. In contrast, most paralogous genes with low ω, such as TaIAA5–12, had more complete motifs and higher degrees of interaction with other family members.

Development of NBS-related microsatellite (NRM) markers in hexaploid wheat

NBS (nucleotide binding site) genes, one type of the most important disease-resistance genes in the plant kingdom, are usually found clustered in genome. In this study, a total of 2288 full-length NBS protein-coding sequences were isolated from the wheat (Triticum aestivum L.) genome, and 903 TaNBSs of which were found expressed in wheat. Meanwhile, 2203 microsatellite loci were detected within 1061 scaffolds containing TaNBS. The distribution of these microsatellite loci across wheat homologous groups (HG) is 20% HG2, 16% HG7, 15% HG1, 15% HG6, 12% HG4, 12% HG5 and 10% HG3. We developed 1830 NBS-related microsatellite (NRM) markers for the microsatellite loci on TaNBS-scaffold sequences.Among them, 342 NRM markers were developed for HG2 with the largest number of microsatellite loci, and 69 out of these markers were anchored to the wheat genetic map using mapping population. Then, a total of 26 2AS-NRM markers, nine 2BL-NRM markers and nine 2DL-NRM markers were integrated into the genetic maps carrying Yr69, Pm51 and Pm43, respectively. Finally, candidate sequences, within the gene clusters where Yr5 and Sr21 located, were analyzed according to the genomic position information of TaNBS and NRM markers. These NRM markers have clear chromosome locations and are correlated with potential disease resistance sequences, which can be manipulated to mapping or adding linkage markers of disease-resistance genes or QTLs, especially for those in the NBS gene clusters.