Shavannor M. Smith

Recombination Events Generating a Novel Rp1 Race Specificity

Genes at the maize Rp1 rust resistance complex often mispair in meiosis, which allows genes to recombine unequally, creating recombinant haplotypes. Four recombinant haplotypes were identified from progeny of an Rp1-D/Rp1-I heterozygote that conferred a nonparental resistance specificity designated Rp1-I*. Sequence comparisons of paralogs in the recombinant and parental haplotypes demonstrated that all four recombinants were derived from intergenic (between gene) recombination events. The sequence of paralogs in the HRp1-I parental haplotype indicated this haplotype includes 41 or more rp1 genes, at least 31 of which are transcribed. The results indicate that most of the novel resistance specificities that have arisen spontaneously at Rp1 are the result of reassort ment of existing Rp1 genes.

High-Throughput Discovery of Mutations in Tef Semi-Dwarfing Genes by Next-Generation Sequencing Analysis

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ∼15–45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ∼6× fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ∼21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high-throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.

Recombinant Rp1 genes confer necrotic or nonspecific resistance phenotypes.

Genes at the Rp1 rust resistance locus of maize confer race-specific resistance to the common rust fungus Puccinia sorghi. Three variant genes with nonspecific effects (HRp1 -Kr1N, -D*21 and -MD*19) were found to be generated by intragenic crossing over within the LRR region. The LRR region of most NBS-LRR encoding genes is quite variable and codes for one of the regions in resistance gene proteins that controls specificity. Sequence comparisons demonstrated that the Rp1-Kr1N recombinant gene was identical to the N-terminus of the rp1-kp2 gene and C-terminus of another gene from its HRp1-K grandparent. The Rp1-D*21 recombinant gene consists of the N-terminus of the rp1-dp2 gene and C-terminus of the Rp1-D gene from the parental haplotype. Similarly, a recombinant gene from the Rp1-MD*19 haplotype has the N-terminus of an rp1 gene from the HRp1-M parent and C-terminus of the rp1-D19 gene from the HRp1-D parent. The recombinant Rp1 -Kr1N, -D*21 and -MD*19 genes activated defense responses in the absence of their AVR proteins triggering HR (hypersensitive response) in the absence of the pathogen. The results indicate that the frequent intragenic recombination events that occur in the Rp1 gene cluster not only recombine the genes into novel haplotypes, but also create genes with nonspecific effects. Some of these may contribute to nonspecific quantitative resistance but others have severe consequences for the fitness of the plant.

Haplotype Analysis and Linkage Disequilibrium at Five Loci in Eragrostis tef

Eragrostis tef (Zucc.), a member of the Chloridoideae subfamily of grasses, is one of the most important food crops in Ethiopia. Lodging is the most important production problem in tef. The rht1 and sd1 dwarfing genes have been useful for improving lodging resistance in wheat and rice, respectively, in what has been known as the “Green Revolution.” All homologs of rht1 and sd1 were cloned and sequenced from 31 tef accessions collected from across Ethiopia. The allotetraploid tef genome was found to carry two rht1 homologs. From sequence variation between these two putative homologs, an approximate ancestral divergence date of 6.4 million years ago was calculated for the two genomes within tef. Three sd1 homologs were identified in tef, with unknown orthologous/paralogous relationships. The genetic diversity in the 31 studied accessions was organized into a relatively small number of haplotypes (2−4) for four of these genes, whereas one rht1 homeologue exhibited 10 haplotypes. A low level of nucleotide diversity was observed at all loci. Linkage disequilibrium analysis demonstrated strong linkage disequilibrium, extending the length of the five genes investigated (2−4 kb), with no significant decline. There was no significant correlation between haplotypes of any of these genes and their recorded site of origin.

Isolation and Diversity Analysis of Resistance Gene Homologues from Switchgrass

Resistance gene homologs (RGHs) were isolated from the switchgrass variety Alamo by a combination of polymerase chain reaction and expressed sequence tag (EST) database mining. Fifty-eight RGHs were isolated by polymerase chain reaction and 295 RGHs were identified in 424,545 switchgrass ESTs. Four nucleotide binding site−leucine-rich repeat RGHs were selected to investigate RGH haplotypic diversity in seven switchgrass varieties chosen for their representation of a broad range of the switchgrass germplasm. Lowland and upland ecotypes were found to be less similar, even from nearby populations, than were more distant populations with similar growth environments. Most (83.5%) of the variability in these four RGHs was found to be attributable to the within-population component. The difference in nucleotide diversity between and within populations was observed to be small, whereas this diversity is maintained to similar degrees at both population and ecotype levels. The results also revealed that the analyzed RGHs were under positive selection in the studied switchgrass accessions. Intragenic recombination was detected in switchgrass RGHs, thereby demonstrating an active genetic process that has the potential to generate new resistance genes with new specificities that might act against newly-arising pathogen races.

Diversity and evolution of Rp1 rust resistance genes in four maize lines

Key messageThis manuscript provides genome-level analysis of disease resistance genes in four maize lines, including studies of haplotype and resistance gene number as well as selection and recombination.AbstractThe Rp1 locus of maize is a complex resistance gene (R-gene) cluster that confers race-specific resistance to Puccinia sorghi, the causal agent of common leaf rust. Rp1 NB-LRR disease resistance genes were isolated from two Rp1 haplotypes (HRp1-B and HRp1-M) and two maize inbred lines (B73 and H95). Sixty-one Rp1 genes were isolated from Rp1-B, Rp1-M, B73 and H95 with a PCR-based approach. The four maize lines carried from 12 to 19 Rp1 genes. From 4 to 9 of the identified Rp1 genes were transcribed in the four maize lines. The Rp1 gene nucleotide diversity was higher in HRp1-B and HRp1-M than in B73 and H95. Phylogenic analysis of 69 Rp1 genes revealed that the Rp1 genes maintained in HRp1-B, HRp1-M and H95 are evolving independently of each other, while Rp1 genes in B73 and HRp1-D appear more like each other than they do genes in the other lines. The results also revealed that the analysed Rp1 R-genes were under positive selection in HRp1-M and B73. Intragenic recombination was detected in Rp1 genes maintained in the four maize lines. This demonstrates that a genetic process that has the potential to generate new resistance genes with new specificities is active at the Rp1 locus in the four analysed maize lines and that the new resistance genes may act against newly arising pathogen races that become prevalent in the pathogen population.

A rapid and efficient method for assessing pathogenicity of ustilago maydis on maize and teosinte lines.

Maize is a major cereal crop worldwide. However, susceptibility to biotrophic pathogens is the primary constraint to increasing productivity. U. maydis is a biotrophic fungal pathogen and the causal agent of corn smut on maize. This disease is responsible for significant yield losses of approximately

First Report of Leaf Spot Caused by Bipolaris victoriae on Switchgrass in Georgia

1.0 billion annually in the U.S.(1) Several methods including crop rotation, fungicide application and seed treatments are currently used to control corn smut(2). However, host resistance is the only practical method for managing corn smut. Identification of crop plants including maize, wheat, and rice that are resistant to various biotrophic pathogens has significantly decreased yield losses annually(3-5). Therefore, the use of a pathogen inoculation method that efficiently and reproducibly delivers the pathogen in between the plant leaves, would facilitate the rapid identification of maize lines that are resistant to U. maydis. As, a first step toward indentifying maize lines that are resistant to U. maydis, a needle injection inoculation method and a resistance reaction screening method was utilized to inoculate maize, teosinte, and maize x teosinte introgression lines with a U. maydis strain and to select resistant plants. Maize, teosinte and maize x teosinte introgression lines, consisting of about 700 plants, were planted, inoculated with a strain of U. maydis, and screened for resistance. The inoculation and screening methods successfully identified three teosinte lines resistant to U. maydis. Here a detailed needle injection inoculation and resistance reaction screening protocol for maize, teosinte, and maize x teosinte introgression lines is presented. This study demonstrates that needle injection inoculation is an invaluable tool in agriculture that can efficiently deliver U. maydis in between the plant leaves and has provided plant lines that are resistant to U. maydis that can now be combined and tested in breeding programs for improved disease resistance.

RESISTANCE GENE COMPLEXES: Evolution and Utilization

Switchgrass (Panicum virgatum) is a native North American perennial C4 grass and was selected by the Department of Energy as a model bioenergy crop. However, for the purposes of bioenergy production, switchgrass will be grown on a large number of hectares for multiple years. As a result of extended production periods, foliar diseases may impact biomass yield and quality. In the summer of 2010, light-to-dark brown, irregularly-shaped leaf spots with white necrotic centers and chlorosis were observed on the Southern Switchgrass Diversity Panel (SSDP) maintained at the University of Georgia in Watkinsville. The SSDP consists of 480 switchgrass genotypes, and has been characterized at phenotypic (plant height, plant color) and genotypic levels (Evans et al. 2014). Disease symptoms increased in the field each year and were observed on 30% of the SSDP entries during the summer of 2016. Symptomatic leaf tissue was collected from the field in 2016, surface sterilized, air dried and plated on 2% water agar. After ...