RNA-seq bulked segregant analysis combined with KASP genotyping rapidly identified PmCH7087 as responsible for powdery mildew resistance in wheat.

Powdery mildew causes considerable yield losses in common wheat (Triticum aestivum L.) production. Mapping and cloning powdery mildew-resistant quantitative trait loci can benefit stable yield production by facilitating the breeding of resistant varieties. In this study, we used the powdery mildew resistance introgression line 'CH7087' (harboring the resistance gene PmCH7087) and developed a large F2 population and a corresponding F2:3 segregation population containing 2,000 family lines for molecular mapping of PmCH7087. Genetic analysis demonstrated that the resistance phenotype was controlled by a single dominant gene. According to the performance exhibited by the F2:3 lines, 50 resistant lines and 50 susceptible lines without phenotype segregation were chosen for pooling and bulked segregant RNA sequencing (BSR-Seq) analysis. A region spanning 42.77 Mb was identified, and genotyping of an additional 183 F2:3 lines with extreme phenotypes using 20 kompetitive allele-specific polymerase chain reaction (KASP) markers in the BSR-Seq mapping regions confirmed this region and narrowed it to 9.68 Mb, in which 45 genes were identified and annotated. Five of these transcripts harbored nonsynonymous single nucleotide polymorphisms between the two parents, with the transcripts of TraesCS2B01G302800 being involved in signal transduction. Furthermore, TraesCS2B01G302800.2 was annotated as the closest homologue of serine/threonine-protein kinase PBS1, a typical participant in the plant disease immune response, indicating that TraesCS2B01G302800 was the candidate gene of PmCH7087. Our results may facilitate future research attempting to improve powdery mildew resistance in wheat and to identify candidate genes for further verification and gene cloning.

Molecular identification and mapping of a novel stripe rust resistance gene in wheat resistance line CH5389 / Identificação e mapeamento de um novo gene de resistência a ferrugem linear na linhagem de trigo CH5389

ABSTRACT: Stripe rust, caused by Puccinia striiformis is one of the most destructive diseases of wheat worldwide. CH5389 is a wheat-Thinopyrum intermedium derived line conferring stripe rust resistance. Genetic analyses of seedlings of F2 populations and F2:3 families developed by crossing CH5389 and susceptible common wheat revealed that stripe rust resistance in CH5389 was controlled by a single dominant gene that was designated YrCH5389. Eight SSR and EST-PCR polymorphic markers on chromosome 3AL were identified in F2 population of CH5389/Taichung29. The YrCH5389 was flanked by EST marker BE405348 and SSR marker Xwmc388 on chromosome 3AL with genetic distances of 2.2 and 4.6 cM, respectively. Comparative genomic analysis demonstrated that the orthologous genomic region of YrCH5389 covered 990 kb in rice, 640 kb in Brachypodium, and 890 kb in sorghum. Based on the locations of the markers, the resistance gene was located to chromosome deletion bin 3AL-0.85-1.00. Because there are no officially named stripe rust resistance genes on the 3AL chromosome, the YrCH5389 should be designated as a new resistance gene. These linkage markers could be useful for marker-assisted selection in wheat resistance breeding.

RESUMO: A ferrugem linear causada por Puccinia striiformis é uma das doenças mais destrutivas do trigo no mundo. A linhagem CH5389 é derivada do cruzamento de trigo com Thinopyrum intermedium e confere resistência a ferrugem linear. Análises genéticas de indivíduos da população F2 e família F2:3 obtida a partir do cruzamento entre CH5389 e trigo comum suscetível revelaram que a resistência à ferrugem linear na linhagem CH5389 foi controlada por um único gene dominante, designado YrCH5389. Oito marcadores polimórficos SSR e EST-PCR no cromossomo 3AL foram identificados na população F2 de CH5389/Taichung29. O gene YrCH5389 foi delimitado pelos marcadores EST BE405348 e SSR Xwmc388 no cromossomo 3AL com distâncias genéticas de 2,2 e 4,6 cM, respectivamente. Análises genômicas comparativas demonstraram que regiões genômicas ortólogas do gene YrCH5389 compreendem 990 kb em arroz, 640 kb em braquipódio e 890 kb em sorgo. Com base nas localizações dos marcadores, o gene de resistência foi localizado no cromossomo 3AL-0.85-1.00. Como não há genes oficialmente nomeados de resistência à ferrugem linear no cromossomo 3AL, o YrCH5389 deve ser designado como um gene novo de resistência. Esses marcadores de ligação podem ser úteis para a seleção assistida de genótipos de trigo resistentes a ferrugem linear.

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 ß5) 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 > ω 0), 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.

Molecular Mapping of the Stripe Rust Resistance Gene Yr69 on Wheat Chromosome 2AS.

Wheat is one of the major food crops in the world. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an economically important disease that affects wheat worldwide. The discovery of novel resistance genes and the deployment of effectively resistant cultivars are important for the ongoing control of wheat stripe rust and the maintenance of the agricultural productivity of wheat. CH7086, a new stripe rust-resistant wheat introgression line, was selected by crossing susceptible cultivars with the resistant Thinopyrum ponticum-derived partial amphiploid Xiaoyan 7430. The resistance of CH7086 is effective against all current Chinese P. striiformis f. sp. tritici races. CH7086 was crossed with the stripe rust-susceptible cultivars to develop F1, F2, F3, and BC1 populations for genetic analysis. Segregation in the F2 and BC1 populations and F2:3 lines were tested for resistance against the P. striiformis f. sp. tritici race CYR32. This test showed that CH7086 carries a single dominant gene for stripe rust resistance, which was temporarily designated YrCH86. The closest of the eight simple sequence repeat (SSR) and expressed sequence tag-SSR markers flanking the locus were X2AS33, which is 1.9 cM distal, and Xmag3807, which is 3.1 cM proximal. The resistance gene and its polymorphic markers were placed in deletion bin 2AS-0.78-1.00 using the 'Chinese Spring' nullisomic-tetrasomic, ditelosomic, and deletion lines. The tests of both allelism and resistance specificity suggested that the resistance gene found in CH7086 was not Yr17, which was the only current formally named Yr gene on chromosome 2AS. Thus, YrCH86 appeared to be a new locus and was permanently designated Yr69.

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.

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.

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.

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.

Putative Thinopyrum intermedium-derived stripe rust resistance gene Yr50 maps on wheat chromosome arm 4BL.

Stripe rust-resistant wheat introgression line CH223 was developed by crossing the resistant partial amphiploid TAI7047 derived from Thinopyrum intermedium with susceptible cultivars. The resistance is effective against all the existing Chinese stripe rust races, including the most widely virulent and predominant pathotypes CYR32 and CYR33. Cytological analyses using GISH detected no chromosomal segments from Th. intermedium. It was presumed that the segment was too small to be detected. Normal bivalent pairing at meiosis in CH223 and its hybrids confirmed its stability. Genetic analysis of the F(1), F(2), F(3) and BC(1) populations from crosses of CH223 with susceptible lines indicated that resistance was controlled by a single dominant gene. The resistance gene was mapped using an F(2:3) population from Taichung 29/CH223. The gene was linked to five co-dominant genomic SSR markers, Xgwm540, Xbarc1096, Xwmc47, Xwmc310 and Xgpw7272, and flanked by Xbarc1096 and Xwmc47 at 8.0 and 7.2 cM, respectively. Using the Chinese Spring nulli-tetrasomic and ditelosomic lines, the polymorphic markers and the resistance gene were assigned to chromosome arm 4BL. As no permanently named stripe rust resistance genes had been assigned to chromosome 4BL, this new resistance gene is designated Yr50. The gene, together with the identified closely linked markers, could be used in marker-assisted selection to combine two or more resistance genes in a single genotype.

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.

Inheritance and mapping of powdery mildew resistance gene Pm43 introgressed from Thinopyrum intermedium into wheat.

Powdery mildew resistance from Thinopyrum intermedium was introgressed into common wheat (Triticum aestivum L.). Genetic analysis of the F(1), F(2), F(3) and BC(1) populations from powdery mildew resistant line CH5025 revealed that resistance was controlled by a single dominant allele. The gene responsible for powdery mildew resistance was mapped by the linkage analysis of a segregating F(2) population. The resistance gene was linked to five co-dominant genomic SSR markers (Xcfd233, Xwmc41, Xbarc11, Xgwm539 and Xwmc175) and their most likely order was Xcfd233-Xwmc41-Pm43-Xbarc11-Xgwm539-Xwmc175 at 2.6, 2.3, 4.2, 3.5 and 7.0 cM, respectively. Using the Chinese Spring nullisomic-tetrasomic and ditelosomic lines, the polymorphic markers and the resistance gene were assigned to chromosome 2DL. As no powdery mildew resistance gene was previously assigned to chromosome 2DL, this new resistance gene was designated Pm43. Pm43, together with the identified closely linked markers, could be useful in marker-assisted selection for pyramiding powdery mildew resistance genes.