Daqun Liu

Fine mapping of leaf rust resistance gene LrZH84 using expressed sequence tag and sequence-tagged site markers, and allelism with other genes on wheat chromosome 1B.

Zhou 8425B, possessing the leaf rust resistance gene LrZH84, is an elite wheat (Triticum aestivum) parental line in the Yellow-Huai Valley region of China. In the present study, 2,086 F(2) plants derived from Zhou 8425B/Chinese Spring were used for fine mapping of LrZH84 with expressed sequence tag (EST) and sequence-tagged site (STS) markers. Seventy inter-simple sequence repeat EST and STS markers on 1BL were used to screen the two parents and resistant and susceptible bulks; those polymorphic were used to analyze the entire F(2) population. Three EST markers (BF474863, BE497107, and CD373538) were closely linked to LrZH84, with genetic distances of 0.7, 0.7, and 1.7 cM, respectively. STS marker Hbsf-1 was developed from the sequences of polymerase chain reaction fragments amplified from EST marker BF474863. LrZH84 was 8.19 cM proximal to Lr44, but may be allelic to LrXi and LrG98 although they showed different reactions with some Puccinia triticina pathotypes.

Characterization of a Hypersensitive Response-Induced Gene TaHIR3 from Wheat Leaves Infected with Leaf Rust

Hypersensitive-induced reaction (HIR) proteins and more specific members of the proliferation, ion and death superfamily participate in response to pathogen attacks and development of spontaneous hypersensitive lesions in maize and barley leaves. In the present study, a full-length TaHIR3 (1,246-bp) was cloned and characterized from wheat near-isogenic line Thatcher-Lr15 infected by Puccinia triticina isolate 05-19-43② through a homology cloning strategy. A 4,203-bp sequence of TaHIR3 including five exons was also obtained from wheat Thatcher-Lr15 genomic DNA. TaHIR3 shared higher similarity with HIR3 isolated from wheat, barley, and maize. Gene expression of TaHIR3 was spatially measured in young wheat leaves, young roots, young stems, mature seeds, and temporally in wheat leaves inoculated with virulent and avirulent P. triticina. TaHIR3 was expressed in all samples except mature seeds and was upregulated in both combinations. More transcripts accumulated in the incompatible than compatible combination, implying a role in wheat growth and resistance to pathogens attack. A polyclonal antibody was prepared with recombinant protein purified from a prokaryotic expression system with the open reading frame of TaHIR3, and it detected the target protein in wheat leaves by Western blotting. Detection results at the protein level also showed that TaHIR3 was upregulated expression in wheat leaves infected with the leaf rust pathogen. Characterization of TaHIR3 and its expression profiles at the DNA and protein levels suggest that TaHIR3 and its deduced protein play a role in wheat HR causing by the leaf rust pathogen.

Characteristic expression of wheat PR5 gene in response to infection by the leaf rust pathogen, Puccinia triticina

TaLr35PR5 gene was obtained from the gDNA and cDNA of TcLr35 wheat. It was induced by Puccinia triticina, ABA and SA, but TaLr35PR5 was induced earlier and its expression level was higher in the incompatible interaction than that in the compatible interaction. In addition, the accumulations of TaLr35PR5 increased stably and showed significant peak challenged by P. triticina at different growth and development periods of TcLr35 wheat while it maintained similar level and changed little in mock inoculated. Western blottings were conducted to confirm that TaLr35PR5 be induced by P. triticina infection at the protein expression level. Similar to the expression patterns of TaLr35PR5 at RNA levels, the accumulations of TaLr35PR5 protein were weak in the seedling stage, then increased to the peak and kept constant levels at the mature stage which is consistent with the expression feature of Lr35 gene as an adult plant resistance gene.

Identification of AFLP Markers Linked to Lr19 Resistance to Wheat Leaf Rust

AFLP analyses were carried out on Thatcher, 23 near-isogenic lines and F2 generation of TcLr19 × Thatcher, to develop molecular markers for gene Lr19 resistance to wheat leaf rust. Seven markers linked to Lr19 resistance trait were obtained, which were P-AGT/M-GAG(subscript 289bp) (3.3cM), P-ACA/M-GGT(subscript 102bp) (4.1cM), P-ACA/M-GGT(subscript 106bp) (4.1cM), P-AAC/M-CAG(subscript 123bp) (4.9cM), P-AAC/M-GGT(subscript 203bp) (5.0cM), P-ACA/M-GGT(subscript 290bp) (5.7cM), and P-ATC/M-GAG(subscript 293bp) (9.6cM). All of these specific fragments were isolated from the polyacrylamide gels, reamplified, cloned, and sequenced. The research may facilitate genetic mapping, physical mapping, and the eventual cloning of Lr19.

Isolation and Characterization of NBS-LRR Class Resistance Homologous Gene from Wheat

Abstract One resistance gene analog fragment named RGA-CIN14 was isolated from TcLr19 wheat, which contains kinase-2, kinase-3a, and the GLPL motif of the NBS-spanning region, using degenerated primers according to the nucleotide binding site (NBS) conserved domain. Based on the RGA-CIN14, a full-length cDNA, CIN14, which was 2 987 bp encoding 880 amino acids, was obtained by using the method of the rapid amplification cDNA ends (RACE). Bioinformatics analysis showed that the deduced amino acids of CIN14 protein consisted of a NB-ARC conserved domain and many leucine-rich repeats (LRR) domains. The phylogenetic tree analysis indicated a considerable identity of the protein encoded by CIN14 with that of wheat leaf rust resistance gene Lr1, but a lower similarity with Lr21. The expression profile of the CIN14 gene detected by semi-quantitative RT-PCR showed that the CIN14 gene was not induced by Puccinia triticina and it was a constitutive gene with low abundance in the wheat leaf tissue. The resistance homology sequence was successfully obtained, which provides the shortcut for cloning of the resistance gene in TcLr19 wheat.

A novel homeobox-like gene associated with reaction to stripe rust and powdery mildew in common wheat.

Stripe rust and powdery mildew, caused by Puccinia striiformis f. sp. tritici and Blumeria graminis f. sp. tritici, respectively, are severe diseases in wheat (Triticum aestivum) worldwide. In our study, differential amplification of a 201-bp cDNA fragment was obtained in a cDNA-amplified fragment length polymorphism (AFLP) analysis between near-isogenic lines Yr10NIL and Avocet S, inoculated with P. striiformis f. sp. tritici race CYR29. A full-length cDNA (1,357 bp) of a homeobox-like gene, TaHLRG (GenBank accession no. EU385606), was obtained in common wheat based on the sequence of GenBank accession AW448633 with high similarity to the above fragment. The genomic DNA sequence (2,396 bp) of TaHLRG contains three exons and two introns. TaHLRG appeared to be a novel homeobox-like gene, encoding a protein with a predicted 66-amino-acid homeobox domain. It was involved in race-specific responses to stripe rust in real-time quantitative polymerase chain reaction (PCR) analyses with Yr9NIL, Yr10NIL, and Avocet S. It was also associated with adult-plant resistance to stripe rust and powdery mildew based on the field trials of doubled haploid lines derived from the cross Bainong 64/Jingshuang 16 and two F(2:3) populations from the crosses Lumai 21/Jingshuang 16 and Strampelli/Huixianhong. A functional marker, THR1 was developed based on the sequence of TaHLRG and located on chromosome 6A using a set of Chinese Spring nulli-tetrasomic lines.

Molecular Markers for Leaf Rust Resistance Gene Lr45 in Wheat Based on AFLP Analysis

Amplified fragment length polymorphism (AFLP) analysis was carried out in Thatcher, near isogenic lines (NILs) carrying different genes conferring resistance against wheat leaf rust, and TcLr45×Thatcher F2 progenies were used to develop markers for Lr45 gene. Sixty AFLP primer combinations were screened and most of them provided clear amplification products, 31 primer combinations displayed polymorphism of TcLr45 in 23 NILs. Two AFLP markers closely linked to the gene Lr45 were acquired: P-AGG/M-GAG(subscript 261 bp), which was found closely linked to the Lr45 locus at a distance of 0.6 cM on one side, and P-ACA/M-GGT(subscript 105 bp), which was found at a distance of 1.3 cM on the other side. The specific bands were cloned and subsequently sequenced. The 261-bp fragment produced by P-AGG/M-GAG showed 86% similarity with the sequence of Vulgare Hort Ⅰ gene; the 105-bp fragment produced by P-ACA/M-GGT showed 96% similarity with the phosphatidylserine decarboxylase gene of the Triticum monococcum. Both included an open reading frame (ORF).

A SSR Marker for Leaf Rust Resistance Gene Lr19 in Wheat

Abstract Microsatellite was carried out in Thatcher, six near-isogenic lines and F 2 progeny of TcLr19xThatcher to develop molecular markers for leaf rust resistance gene Lr19 . Thirteen primer pairs were screened, of which one primer pair Xgwm44 displayed polymorphsim in the population of TcLr19, Thatcher, and their F 2 generations. One marker closed linked to Lr19 resistance trait was obtained, and was named Xgwm44 139 bp with the genetic distance 0.9 cM. The research shows that Lr19 has more potential in marker-assisted breeding programs in wheat and provides a step stone for mapping genetic map, physical map and the eventual cloning.

QTL mapping of adult-plant resistance to leaf rust in the wheat cross Zhou 8425B/Chinese spring using high-density SNP markers

Wheat leaf rust is an important disease worldwide. Growing resistant cultivars is an effective means to control the disease. In the present study, 244 recombinant inbred lines from Zhou 8425B/Chinese Spring cross were phenotyped for leaf rust severities during the 2011–2012, 2012–2013, 2013–2014, and 2014–2015 cropping seasons at Baoding, Hebei province, and 2012–2013 and 2013–2014 cropping seasons in Zhoukou, Henan province. The population was genotyped using the high-density Illumina iSelect 90K SNP assay and SSR markers. Inclusive composite interval mapping identified eight QTL, designated as QLr.hebau-2AL, QLr.hebau-2BS, QLr.hebau-3A, QLr.hebau-3BS, QLr.hebau-4AL, QLr.hebau-4B, QLr.hebau-5BL, and QLr.hebau-7DS, respectively. QLr.hebau-2BS, QLr.hebau-3A, QLr.hebau-3BS, and QLr.hebau-5BL were derived from Zhou 8425B, whereas the other four were from Chinese Spring. Three stable QTL on chromosomes 2BS, 4B and 7DS explained 7.5–10.6%, 5.5–24.4%, and 11.2–20.9% of the phenotypic variance, respectively. QLr.hebau-2BS in Zhou 8425B might be the same as LrZH22 in Zhoumai 22; QLr.hebau-4B might be the residual resistance of Lr12, and QLr.hebau-7DS is Lr34. QLr.hebau-2AL, QLr.hebau-3BS, QLr.hebau-4AL, and QLr.hebau-5BL are likely to be novel QTL for leaf rust. These QTL and their closely linked SNP and SSR markers can be used for fine mapping, candidate gene discovery, and marker-assisted selection in wheat breeding.

Mapping of QTL conferring leaf rust resistance in Chinese wheat lines W014204 and Fuyu 3 at adult plant stage

Abstract Wheat leaf rust is a destructive foliar disease of common wheat (Triticum aestivum L.) worldwide. The most effective, economical s to control the disease is growing resistant cultivars with adult plant resistance (APR). The Chinese wheat lines W014204 and Fuyu 3 showed high leaf rust resistance in the field. To identify leaf rust APR genes in the two lines, two mapping populations with 215 and 163 F2:3 lines from the crosses W014204/Zhengzhou 5389 and Fuyu 3/Zhengzhou 5389, respectively, were phenotyped for leaf rust severities during the 2010–2011, 2011–2012 and 2012–2013 cropping seasons in the field at Baoding, Hebei Province, China. A total of 1 215 SSR markers were used to identify the quantitative trait loci (QTLs) for leaf rust APR in the two populations. In the W014204/Zhengzhou 5389 population, three QTLs were detected and designated as QLr.hbu-1BL.1, QLr.hbu-2BS.1 and QLr.hbu-7DS, and explained 2.9–8.4, 11.5–38.3 and 8.5–44.5% of the phenotypic variance, respectively; all the resistance alleles at these loci were derived from W014204. In the Fuyu 3/Zhengzhou 5389 population, three QTLs, QLr.hbu-1BL.2, QLr.hbu-2BS.2 and QLr.hbu-7BL, explained 12.0–19.2, 22.3–38.9 and 4.1–4.3% of the phenotypic variance, respectively, and all resistance alleles were contributed by Fuyu 3. Based on chromosome positions of closely linked markers, both QLr.hbu-1BL.1 and QLr.hbu-1BL.2 are Lr46, and QLr.hbu-7DS is Lr34. QLr.hbu-7BL was mapped on chromosome 7BL near to Lr68 and they are likely the same gene. Based on chromosome positions, pedigree and field reactions, the two 2BS QTLs are different from all the known APR genes and are likely to be new APR QTL for leaf rust. These QTLs and their closely linked markers are potentially useful for improving leaf rust resistance in wheat breeding.