Wenxiang Yang

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.

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.

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.

TaRAR1 is Required for Lr24-Mediated Wheat Leaf Rust Resistance

Abstract Virus-induced gene silencing (VIGS) offers a rapid and high throughout technology platform for the analysis of gene function in plants. The barley stripe mosaic virus (BSMV) VIGS system was optimized in studies silencing phytoene desaturase expression in wheat, and demonstrated that infection with BSMV construct carrying a 412 bp fragment of TaRAR1 caused conversion of incompatible to compatible interactions to Lr24-mediated resistance in wheat TcLr24 and cultivar 5R615 harboring Lr24 whereas infection with a control construct had no effect on resistance or susceptibility. RT-PCR analysis showed that BSMV-induced gene silencing could be detected at mRNA levels. These studies indicated that TaRAR1 was a required component for Lr24-mediated race-specific resistance and the BSMV-VIGS was a powerful tool for dissecting the genetic pathways of disease resistance in hexaploid wheat.

Identification and Molecular Tagging of Leaf Rust Resistance Gene (Lr24) in Wheat

Abstract This research was aimed to develop AFLP markers co-segregated with gene Lr24 and validate the using for marker assisted selection (MAS). An F2 population developed from the cross between the resistant line TcLr24 and the susceptible line Thatcher was tested for resistance to the Puccinia triticina races BGQQ and SHRT using for genetic analysis and molecular marker. A total of 224 AFLP primer combinations were used to test the resistant and susceptible parents, as well as the resistant bulk and the susceptible bulk. Four AFLP markers, P-AGA/M-CTT289 bp, P-AGC/M-CAC188 bp, P-AGC/M-CAC162 bp and P-ACG/M-CGC239 bp were co-segregated with Lr24. The AFLP fragment from the primer combination P-ACG/M-CGC was cloned, sequenced and converted into a STS marker named as ASTS212. Thatcher backgrounded NILs and 115 varieties were examined by using this STS marker and the marker SCS1302607 developed by Gupta. 5R615, 5R616, 1R13, and 1R17 were identified and validated to contain gene Lr24. The marker is dominant and may be useful in identification the resistance gene Lr24 in wheat and wheat breeding programs.

Effects of the leaf rust pathogen on expression of TaHIR4 at the gene and protein levels in wheat

Abstract The hypersensitive response (HR) is one of the most-efficient forms of plant defense against biotrophic pathogens. However, the molecular mechanisms leading to HR remain largely unknown. A putative hypersensitive-induced response (HIR) protein gene TaHIR4 cDNA (919 bp) and a DNA sequence (2520 bp) including at least five exons were isolated and identified from wheat. Phylogenetic analysis showed that TaHIR4 was highly homologous to HvHIR4 from barley at both the cDNA and deduced amino-acid levels. Reverse transcription quantitative polymerase chain reaction (qRT-PCR) results showed that TaHIR4 transcripts were highly upregulated by infection of the leaf rust pathogen and expressed in all tested samples except the mature seeds. Western blotting results also showed that TaHIR4 protein was upregulated in wheat leaves infected with the leaf rust pathogen. Characterization and expression profiles at the gene and protein levels suggested that TaHIR4 plays a role in wheat HR, caused by the leaf rust pathogen.

Postulation of Leaf Rust Resistance Genes in Seven Chinese Spring Wheat Cultivars

To detect the leaf rust resistance genes in the 7 Chinese spring wheat clultivars Shenmian 99025, Shenmia 99042, Shenmian 85, Shenmian 91, Shenmian 96, Shenmian 1167 and Shenmian 962, Thatcher, Thatcher backgrounded near-isogenic lines and 15 pathotypes of P. triticina were used for gene postulate at the seedling stage, and 9 of the 15 pathotypes were used in the field tests. Molecular markers closely linked to, or co-segregated with resistance genes Lr1, Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, Lr26, Lr28, Lr29, Lr32, Lr34, Lr35, Lr37, Lr38, and Lr47 were screened to assist detection of the resistance genes. As results, 4 known resistance genes, including Lr1, Lr9, Lr26, and Lr34, and other unknown resistance genes were postulated singly or in combination in the tested cultivars. Shenmian 85, Shenmian 91, Shenmian 96, Shenmian 962, Shenmian 1167, and Shenmian 99042 are potentially useful for wheat production and breeding programs. The result suggested that combining gene postulation, molecular markers and pedigrees is effective and more accuracy method to know the resistance genes in cultivars.

Analysis of Wheat Leaf Rust Resistance Genes in 14 Wheat Cultivars or Lines: Analysis of Wheat Leaf Rust Resistance Genes in 14 Wheat Cultivars or Lines

选用16个小麦叶锈菌菌系对14个小麦品种（系）进行抗叶锈性鉴定和苗期抗叶锈基因推导,初步分析这些品种（系）的抗性和携带的抗病基因;进一步利用21个与Lr基因紧密连锁或共分离的分子标记,对这14个品种（系）中可能含的抗叶锈基因进行鉴定。结果表明,s98351-2-2-2-1可能含Lr3a、Lr28和Lr50;9629-03A-4-1-1可能含Lr37;97167-1-2-1-1-2-1、919-20-2c2、9589、免中438、9916-8-6和9916-8-18含Lr26;96104-1-5-1c2可能含Lr28;00-55-3-1-1含Lr1;1R13可能含Lr24、Lr37和Lr38;1R17可能含Lr24和Lr38;1R35含Lr10和Lr34,还可能含Lr3a和Lr50;9524-1-2-2-1含未知抗叶锈基因或本试验使用的已知抗病基因以外的抗叶锈基因。所有品种（系）均不含Lr9、Lr19、Lr20、Lr21、Lr29、Lr35、Lr42和Lr47基因。测试的14个品种（系）中有比较丰富的抗叶锈病基因,可为育种提供丰富的抗源。

Evaluation of Wheat Leaf Rust Resistance Genes in 10 Wheat Genotypes

Wheat leaf rust (Puccinia triticina) is one of the most important plant diseases in the world, and growing resistant cultivars is the most economical, effective and environmentally safe method for controlling this disease. With the aim of evaluating leaf rust resistance, 10 uncharacterized wheat genotypes, as well as 35 lines with known leaf rust resistance genes (Lr genes), were exposed to 12 Chinese P. triticina pathotypes at both seedling and adult stages. Two wheat genotypes, 09p205 and 09p200, showed high resistance to all the tested pathotypes at both seedling and adult stages. Three accessions, Xinmai 16, 00-225-2-1-2-2, and 21941-6, showed resistance at the adult stage. Four genotypes, 21898-3, 21924-4, 21915-3, and 21982-1, showed slow rusting resistance. In addition, 24 molecular markers were used to assess the presence of 20 known Lr genes. In the 10 genotypes analyzed Lr2c, Lr3ka, Lr9, Lr14a, Lr26, Lr34, and Lr37 were detected, either alone or in combination.