Chongmei Dong

Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor

BackgroundTILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes.ResultsWe demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation.ConclusionPolyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.

Oligonucleotide-directed gene repair in wheat using a transient plasmid gene repair assay system

Oligonucleotide-directed gene repair is a potential technique for agricultural trait modification in economically important crops. However, large variation in the repair frequencies among the scientific reports indicates that there are many factors influencing the repair process. We report here a transient assay system using GFP as a reporter for testing the efficiency of plasmid DNA repair in cultured wheat cells. This assay showed that osmotic medium supplemented with 2,4-D increased the oligo-targeting frequency, and that the repair of a point mutation was more efficient than repair of a single base deletion mutation in cultured scutellum cells of immature wheat embryos. This study provides the first evidence that oligonucleotide-directed mutagenesis is applicable to regenerable cultured wheat scutellum cells.

Wheat stripe rust resistance genes Yr5 and Yr7 are allelic

Stripe rust is one of the most destructive diseases of wheat. Breeding for resistance is the most economical and environmentally acceptable means to control stripe rust. Genetic studies on resistance sources are very important. Previous inheritance studies on Triticum aestivum subsp. spelta cv. album and wheat cultivar Lee showed that each possessed a single dominant gene for stripe rust resistance, i.e., Yr5 and Yr7, respectively. Both were located on the long arm of chromosome 2B, but due to the complexities caused by genetic background effects there was no clear evidence on the allelism or linkage status of these genes. Our study, involving an intercross of Avocet S near-isogenic lines possessing the genes, provided clear evidence for allelism or extremely close linkage of Yr5 and Yr7 based on phenotypic and molecular studies.

Mutation Surveyor: An In Silico Tool for Sequencing Analysis

DNA sequencing is widely used for DNA diagnostics and functional studies of genes of interest. With significantly increased sequencing outputs, manual reading of sequence results can impede an efficient and accurate analysis. Mutation Surveyor is a useful in silico tool developed by SoftGenetics that assists the detection of sequence variations within Sanger sequencing traces. This tool can process up to 400 lanes of data at a time with high accuracy and sensitivity. It can effectively detect SNPs and indels in their homozygous or heterozygous states as well as mosaicism. In this chapter, we describe the general application of Mutation Surveyor for DNA sequencing analysis and its unique features.

Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat

Fungal effectors of wheat stem rust The fungal pathogen Ug99 (named for its identification in Uganda in 1999) threatens wheat crops worldwide. Ug99 can kill entire fields of wheat and is undeterred by many of the disease-resistance genes that otherwise protect wheat crops. Two papers describe two peptides secreted by the fungus as it attacks the wheat (see the Perspective by Moscou and van Esse). Chen et al. show that fungal AvrSr50 binds to the plants immune receptor Sr50, and Salcedo et al. show that fungal AvrSr35 binds to Sr35. Successful binding activates the plants immune defenses. Removing or inactivating these Avr effectors leaves the plant defenseless and susceptible to disease. Science, this issue p. 1607, p. 1604; see also p. 1541 Fungal genes encode factors that interact with receptors in the wheat host to trigger resistance to disease. Race-specific resistance genes protect the global wheat crop from stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) but are often overcome owing to evolution of new virulent races of the pathogen. To understand virulence evolution in Pgt, we identified the protein ligand (AvrSr50) recognized by the Sr50 resistance protein. A spontaneous mutant of Pgt virulent to Sr50 contained a 2.5 mega–base pair loss-of-heterozygosity event. A haustorial secreted protein from this region triggers Sr50-dependent defense responses in planta and interacts directly with the Sr50 protein. Virulence alleles of AvrSr50 have arisen through DNA insertion and sequence divergence, and our data provide molecular evidence that in addition to sexual recombination, somatic exchange can play a role in the emergence of new virulence traits in Pgt.

WM5: Isolation and characterisation of a gene expressed during early meiosis and shoot meristem development in wheat

Wheat Meiosis 5 (WM5), isolated from an early meiosis anther cDNA library of wheat by cDNA subtraction encodes a novel glycine-serine-proline-alanine-rich protein. The corresponding homologous genes are located on the short arms of chromosomes 3A, 3B and 3D of allohexaploid wheat (Triticum aestivum L.). The copy on 3DS is located within the region deleted in the wheat mutant ph2a that displays increased homoeologous chromosome pairing in crosses with alien species. While WM5 is expressed primarily in young flower buds during early meiosis it is also expressed in shoot meristems, thus indicating functional roles in both meiosis and meristem development. Overall, the WM5 amino acid sequence shares no significant similarity with other known proteins in the NCBI database. However, the carboxyl-terminal region does have similarity with the Arabidopsis PDF1 (Protodermal Factor 1) protein. Comparing WM5 and PDF1 reveals that the two proteins share 33% identity and have similar hydropathy plots and predicted secondary structures. In situ immuno-staining locates the protein to the nuclei of pollen mother cells undergoing meiosis and the epidermal layer of the shoot and flower meristem, including the cell wall and cuticle. We propose that the WM5 protein has a role in shoot and flower development within this economically important cereal crop.

Two new Magnaporthe species pathogenic to warm-season turfgrassses in Australia

Two new species of ectotrophic root-infecting fungi pathogenic to warm-season turfgrasses are described. Magnaporthe garrettii P. T. W. Wong & M. L. Dickinson sp. nov. causes a serious patch disease on couch (Cynodon dactylon) bowling greens in South Australia, and Magnaporthe griffinii P. T. W. Wong & A.M. Stirling sp. nov. is associated with a disease complex (“summer decline”) of hybrid couch (C. dactylon × C. transvaalensis) golf greens in New South Wales and Queensland. Both are homothallic, producing perithecia readily on potato dextrose agar. They differ from other Magnaporthe spp. in having uniseriate rather than biseriate or multiseriate ascospores, and the absence of a conidial anamorph. Analysis of nuclear rRNA ITS sequences has shown that M. griffinii is a new taxon with low homology to M. grisea, M. poae, M. rhizophila, M. salvinii and Gaeumannomyces graminis. This could not be carried out with M. garrettii because there were no living cultures available and the genomic DNA extracted from dead mycelia and perithecia was totally degraded. However, the two new species can be readily distinguished by morphological differences in their perithecia and ascospores. Examination of earlier herbarium specimens has shown that M. garrettii was associated with a patch disease of buffalo grass (Stenotaphrum secundatum) in New South Wales and M. griffinii was associated with diseases of South African couch grass (C. tranvaalensis) in South Australia and of kikuyu grass (Pennisetum clandestinum) in New South Wales.

Molecular identification of the wheat male fertility gene Ms1 and its prospects for hybrid breeding

The current rate of yield gain in crops is insufficient to meet the predicted demands. Capturing the yield boost from heterosis is one of the few technologies that offers rapid gain. Hybrids are widely used for cereals, maize and rice, but it has been a challenge to develop a viable hybrid system for bread wheat due to the wheat genome complexity, which is both large and hexaploid. Wheat is our most widely grown crop providing 20% of the calories for humans. Here, we describe the identification of Ms1, a gene proposed for use in large-scale, low-cost production of male-sterile (ms) female lines necessary for hybrid wheat seed production. We show that Ms1 completely restores fertility to ms1d, and encodes a glycosylphosphatidylinositol-anchored lipid transfer protein, necessary for pollen exine development. This represents a key step towards developing a robust hybridization platform in wheat.Heterosis can rapidly boost yield in crop species but development of hybrid-breeding systems for bread wheat remains a challenge. Here, Tucker et al. describe the molecular identification of the wheat Ms1 gene and discuss its potential for large-scale hybrid seed production in wheat.

Comparative Genomics Integrated with Association Analysis Identifies Candidate Effector Genes Corresponding to Lr20 in Phenotype-Paired Puccinia triticina Isolates from Australia

Leaf rust is one of the most common and damaging diseases of wheat, and is caused by an obligate biotrophic basidiomycete, Puccinia triticina (Pt). In the present study, 20 Pt isolates from Australia, comprising 10 phenotype-matched pairs with contrasting pathogenicity for Lr20, were analyzed using whole genome sequencing. Compared to the reference genome of the American Pt isolate 1-1 BBBD Race 1, an average of 404,690 single nucleotide polymorphisms (SNPs) per isolate was found and the proportion of heterozygous SNPs was above 87% in the majority of the isolates, demonstrating a high level of polymorphism and a high rate of heterozygosity. From the genome-wide SNPs, a phylogenetic tree was inferred, which consisted of a large clade of 15 isolates representing diverse presumed clonal lineages including 14 closely related isolates and the more diverged isolate 670028, and a small clade of five isolates characterized by lower heterozygosity level. Principle component analysis detected three distinct clusters, corresponding exactly to the two major subsets of the small clade and the large clade comprising all 15 isolates without further separation of isolate 670028. While genome-wide association analysis identified 302 genes harboring at least one SNP associated with Lr20 virulence (p < 0.05), a Wilcoxon rank sum test revealed that 36 and 68 genes had significant (p < 0.05) and marginally significant (p < 0.1) differences in the counts of non-synonymous mutations between Lr20 avirulent and virulent groups, respectively. Twenty of these genes were predicted to have a signal peptide without a transmembrane segment, and hence identified as candidate effector genes corresponding to Lr20. SNP analysis also implicated the potential involvement of epigenetics and small RNA in Pt pathogenicity. Future studies are thus warranted to investigate the biological functions of the candidate effectors as well as the gene regulation mechanisms at epigenetic and post-transcription levels. Our study is the first to integrate phenotype-genotype association with effector prediction in Pt genomes, an approach that may circumvent some of the technical difficulties in working with obligate rust fungi and accelerate avirulence gene identification.

TILLING for Plant Breeding

TILLING is widely used in plant functional genomics. Mutagenesis and SNP detection is combined to allow for the isolation of mutations in genes of interest. It can also be used as a plant breeding tool, whereby variation in known or candidate genes of interest to breeding programs is generated. Here we describe a simple low-cost TILLING procedure.