Haixian Zhan

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.

New St-chromosome-specific molecular markers for identifying wheat–Thinopyrum intermedium derivative lines

Many grass species in Triticeae serve as important gene pools for forage and cereal crops breeding. Extensive natural and artificial interspecific hybridization have given rise to different ploidy of Triticeae species (Wang et al. 1994). The St genome was defined in diploid Pseudoroegneria species, and is a donor genome of at least seven genera including Douglasdeweya, Roegneria, Elytrigia, Thinopyrum, Elymus, Kengyilia and Pascopyrum according to different taxonomic systems (Wang et al. 1994; Yen et al. 2005). In the past decades, numerous studies on species containing St-chromosome were focussed on genomic relationship and molecular phylogeny based on molecular markers including chloroplast DNA, high-copy nuclear genes, singlecopy nuclear genes, as well as genomic in situ hybridization (GISH) (Kellogg and Appels 1995; Mason-Gamer et al. 2002; Mason-Gamer 2005). Among these species, Thinopyrum intermedium with genome constitution of E1E2St (Wang et al. 1994) or JJsSt (Chen et al. 1998) was widely used in wheat breeding programme (Chen 2005; Li and Wang 2009). Molecular markers, particularly genome-specific markers, are useful in identifying the genome constitution of the unknown species, and also provide efficient tools to check the target alien genes transferred to wheat (Schwarzacher et al. 1992; Wang et al. 2010). Only a few St-chromosomespecific RFLP (Zhang et al. 2001), SCAR (Liu et al. 2007) and ISSR (Zeng et al. 2008) markers were developed to identify genome composition and the individual St-chromosomes. However, the most available markers are lacking in information on the corresponding homologous linkage group relative to wheat. Based on the orthologous gene conservation between rice and wheat, Ishikawa et al. (2007) reported an array of PCR-

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.

Characterization of a partial wheat–Thinopyrum intermedium amphiploid and its reaction to fungal diseases of wheat

Partial amphiploids between wheat (Triticum aestivum L.) and Thinopyrum species play an important role in the transfer and use of traits from alien species. A wheat-Thinopyrum intermedium partial amphiploid, TAI8335, and its alien parent were characterized by a combination of genomic in situ hybridization (GISH) and cytological observations. Evidence from GISH indicated that the donor parent Th. intermedium possessed seven pairs of S, seven J(s) and 21 J chromosomes. Mitotic observation showed that the majority of TAI8335 plants had 56 chromosomes, but a few had 54 to 55, in some cases with two to three additional telochromosomes. The chromosomes in most pollen mother cells of plants with 2n = 56 formed 28 bivalents, averaging 27.12 in 223 cells, suggesting a basic cytological stability. Sequential GISH patterns using genomic Pseudoroegneria spicata and genomic Th. intermedium DNA as probes revealed that TAI8335 had fourteen chromosomes derived from Th. intermedium and its alien genome consisted of one pair of S-, three pairs of J(s) - and one pair of J-genome chromosomes as well as two translocated chromosome pairs, one being a Robertsonian translocation and another an intercalary translocation, both of which involved J and S genome. Two of the telochromosomes in the aneuploid plants originated from the J genome and one from wheat. Disease screening demonstrated this line was highly resistant to leaf rust, stem rust, stripe rust and powdery mildew. This study showed that the partial amphiploid TAI8335 appears to serve as a novel source for the transfer of resistance genes for multiple fungal pathogens to wheat.

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.

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.