Detecting durable resistance to rice bacterial blight

2016 Spring.Includes bibliographical references.The productivity of rice, a staple crop worldwide, is limited by pathogens such as Xanthomonas oryzae pv oryzae (Xoo). Controlling yield loss to the resulting disease, bacterial blight, is most effective through growing genetically pathogen resistant rice varieties. However, widespread deployment of varieties containing single gene resistance to bacterial blight places an immense selection pressure on Xoo to evolve virulence. The major virulence factors employed by Xoo to drive infection are transcription activator like (TAL) effectors. TAL effectors are secreted into the host cells where they target the transcription of particular host susceptibility genes to favor infection. Previous TAL effector research indicates that not all TALs are created equal and some are crucial to the virulence of Xoo. By breeding for resistance genes targeting necessary TAL effectors we may find more durable resistance as selection pressure on the pathogen will result in loss of the TAL effector function and therefore a decrease in virulence and pathogen fitness. In the present study, we characterized a novel and widespread TAL effector through quantitative trait loci (QTL) mapping. We used the indica rice Multi-Parent Advanced Generation Inter-Cross (MAGIC) population to screen for resistance to the cloned TAL effector, TAL7b, and the Philippine race 6 Xoo strain PXO99A. Our results confirm that TAL7b is a virulence enhancing factor and that the MAGIC population contains six loci targeting resistance to TAL7b.We also identified another seven resistance QTL to the highly virulent Xoo strain, PXO99A

Representing true plant genomes: haplotype-resolved hybrid pepper genome with trio-binning

As sequencing costs decrease and availability of high fidelity long-read sequencing increases, generating experiment specific de novo genome assemblies becomes feasible. In many crop species, obtaining the genome of a hybrid or heterozygous individual is necessary for systems that do not tolerate inbreeding or for investigating important biological questions, such as hybrid vigor. However, most genome assembly methods that have been used in plants result in a merged single sequence representation that is not a true biologically accurate representation of either haplotype within a diploid individual. The resulting genome assembly is often fragmented and exhibits a mosaic of the two haplotypes, referred to as haplotype-switching. Important haplotype level information, such as causal mutations and structural variation is therefore lost causing difficulties in interpreting downstream analyses. To overcome this challenge, we have applied a method developed for animal genome assembly called trio-binning to an intra-specific hybrid of chili pepper (Capsicum annuum L. cv. HDA149 x Capsicum annuum L. cv. HDA330). We tested all currently available softwares for performing trio-binning, combined with multiple scaffolding technologies including Bionano to determine the optimal method of producing the best haplotype-resolved assembly. Ultimately, we produced highly contiguous biologically true haplotype-resolved genome assemblies for each parent, with scaffold N50s of 266.0 Mb and 281.3 Mb, with 99.6% and 99.8% positioned into chromosomes respectively. The assemblies captured 3.10 Gb and 3.12 Gb of the estimated 3.5 Gb chili pepper genome size. These assemblies represent the complete genome structure of the intraspecific hybrid, as well as the two parental genomes, and show measurable improvements over the currently available reference genomes. Our manuscript provides a valuable guide on how to apply trio-binning to other plant genomes

Genetic and genomic tools for improving end-use quality in wheat

Doctor of PhilosophyGenetics Interdepartmental ProgramJesse PolandWheat accounts for 20% of daily caloric intake of the world population and has one of the widest cultivation distributions of any crop. With increasing demand for both quantity and quality, wheat yields must increase while also maintaining acceptable end-use quality. However, measuring end-use quality is complex, requires large volumes grain and significant effort. The overarching goal of this dissertation research was to develop genetic and genomic tools to facilitate breeding for end-use quality in wheat. Building on initial work with genomic prediction of wheat quality, we continued application of genomic prediction models to the International Maize and Wheat Improvement Center (CIMMYT) wheat breeding program. For practical application in the breeding program to advance selection, we focused on forward prediction in each cycle of the bread wheat program. Models were built on 12 years of past data including over 18,000 entries with quality data. Predictions for 10,000 yield trial lines were generated each year for selection, with forward prediction accuracies of 0.40 to 0.73, and approached heritability. This is one of the largest scale applications of genomic selection. We also studied the interaction of climate change and the important quality genes, high-molecular weight glutenins (HMW-GS) and low-molecular weight glutenins (HMW-GS). A diverse panel of 54 CIMMYT wheat varieties were grown in 2 levels of drought stress, heat stress and optimal growth conditions. Quality traits, HMW-GS and LMW-GS alleles were measured. We fit a mixed linear model for each quality trait with HMW-GS, LMW-GS, environment, and the interactions of those as predictors. Overall, the superior glutenin alleles either maintained or increased quality in stressful environments. This work confirmed that superior alleles should always be selected for, regardless of target environment. To increase the genetic diversity for wheat quality, we analyzed Glu-D1 gene diversity on the wheat D genome donor, Aegilops tauschii. We constructed Glu-D1 molecular haplotypes from sequence data of 234 Ae. tauschii accessions and found 15 subclades and over 45 haplotypes, representing immense gene diversity. We found evidence that the 5+10 allele originated from a newly described Lineage 3 of Ae. tauschii, further supporting that this unique lineage contributed to modern bread wheat. We also observed rare recombinant haplotypes between the x and y subunits of any HMW-GS locus. This work will facilitate incorporation of Ae. tauschii Glu-D1 alleles into modern wheat. Given that certain HMW-GS alleles are highly desirable, we set out to develop a high-throughput, high resolution genotyping method for HMW-GS alleles that would fit within genotyping already done for genomic prediction models. This ‘sequence based genotyping’ approach uses diagnostic k-mers developed to predict alleles in skim-sequenced breeding material. Prediction accuracies for Glu-D1 and Glu-A1 were very good, but lower for the Glu-B1 alleles where many alleles are highly related. Overall, SBG offers a high throughput method to call alleles from existing data. These genetic and genomic tools developed and implemented for end-use quality selection in wheat offer promising resources for continued improvement of both yield and quality in wheat breeding

High molecular weight glutenin gene diversity in Aegilops tauschii demonstrates unique origin of superior wheat quality.

Central to the diversity of wheat products was the origin of hexaploid bread wheat, which added the D-genome of Aegilops tauschii to tetraploid wheat giving rise to superior dough properties in leavened breads. The polyploidization, however, imposed a genetic bottleneck, with only limited diversity introduced in the wheat D-subgenome. To understand genetic variants for quality, we sequenced 273 accessions spanning the known diversity of Ae. tauschii. We discovered 45 haplotypes in Glu-D1, a major determinant of quality, relative to the two predominant haplotypes in wheat. The wheat allele 2 + 12 was found in Ae. tauschii Lineage 2, the donor of the wheat D-subgenome. Conversely, the superior quality wheat allele 5 + 10 allele originated in Lineage 3, a recently characterized lineage of Ae. tauschii, showing a unique origin of this important allele. These two wheat alleles were also quite similar relative to the total observed molecular diversity in Ae. tauschii at Glu-D1. Ae. tauschii is thus a reservoir for unique Glu-D1 alleles and provides the genomic resource to begin utilizing new alleles for end-use quality improvement in wheat breeding programs

Broad-spectrum resistance and susceptibility to bacterial blight and bacterial leaf streak of rice

Quantitative trait loci (QTL) that confer broad-spectrum resistance (BSR) have been elusive targets of crop breeding programs. Bacterial leaf streak (BLS) and bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzicola (Xoc) and Xanthomonas oryzae pv. oryzae (Xoo), respectively, are responsible for major losses in rice production in Asia and Africa. Controlling these two diseases is particularly important in Sub-Saharan Africa, where no sources of BSR are available in currently deployed varieties. Our goal is to identify novel, broad-spectrum resistance sources to control BLS and BB in rice, using a Multi-parent Advanced Generation Inter-Cross (MAGIC) population, derived from eight elite indica cultivars. MAGIC populations have an increased level of recombination and provide higher precision and resolution to detect QTL. The MAGIC parents and lines were genotyped and phenotyped in both greenhouse and field conditions by screening with diverse strains of Xoc and Xoo. Using genome-wide association and interval mapping analysis, we identified 37 strain-specific QTL, and 14 QTL effective against multiple X. oryzae strains. From these, three QTL are pathovar-specific and 11 confer resistance to both pathovars. By detecting phenotypic effects of causal alleles, we have identified resources that will facilitate a better understanding of how the involved genes contribute to resistance or susceptibility. Because the MAGIC founders are elite varieties, the BSR QTL identified can be rapidly incorporated into breeding programs to achieve more durable resistance to BLS and BB

Resistance and susceptibility QTL identified in a rice MAGIC population by screening with a minor-effect virulence factor from Xanthomonas oryzae pv. oryzae

Summary Effective and durable disease resistance for bacterial blight (BB) of rice is a continuous challenge due to the evolution and adaptation of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), on cultivated rice varieties. Fundamental to this pathogens’ virulence is transcription activator-like (TAL) effectors that activate transcription of host genes and contribute differently to pathogen virulence, fitness or both. Host plant resistance is predicted to be more durable if directed at strategic virulence factors that impact both pathogen virulence and fitness. We characterized Tal7b, a minor-effect virulence factor that contributes incrementally to pathogen virulence in rice, is a fitness factor to the pathogen and is widely present in geographically diverse strains of Xoo. To identify sources of resistance to this conserved effector, we used a highly virulent strain carrying a plasmid borne copy of Tal7b to screen an indica multi-parent advanced generation inter-cross (MAGIC) population. Of 18 QTL revealed by genome-wide association studies and interval mapping analysis, six were specific to Tal7b (qBB-tal7b). Overall, 150 predicted Tal7b gene targets overlapped with qBB-tal7b QTL. Of these, 21 showed polymorphisms in the predicted effector binding element (EBE) site and 23 lost the EBE sequence altogether. Inoculation and bioinformatics studies suggest that the Tal7b target in one of the Tal7b-specific QTL, qBB-tal7b-8, is a disease susceptibility gene and that the resistance mechanism for this locus may be through loss of susceptibility. Our work demonstrates that minor-effect virulence factors significantly contribute to disease and provide a potential new approach to identify effective disease resistance

Quantitative resistance to bacterial pathogens of rice

Disease resistance is the foundation for managing many plant diseases, because resistant varieties have the strongest impact with minimal environmental effects or cost. Consequently, sources of broad-spectrum resistance (BSR), or resistance that is effective against multiple and/or diverse pathogens is of particular interest. However, achieving BSR depends on having effective resistance sources to introgress into elite germplasm. Multi-parent Advanced Generation Inter-Cross (MAGIC) populations are powerful tools for identifying resistance because they have high levels of recombination and enhanced resolution relative to biparental populations. We screened an indica rice MAGIC population developed from eight elite founders for BSR to diverse strains of the rice bacterial blight and leaf streak pathogens Xanthomonas oryzae pv. oryzae (Xoo) and X. o. pv. oryzicola (Xoc), respectively. In addition, building on our hypothesis that durable disease resistance is attainable by targeting key microbial virulence factors, we screened for resistance to Xoo strains isogenic for the known and common virulence factor TAL7b. A combination of genome-wide association studies and interval mapping analyses revealed a number of loci that conferred BSR to both Xoo and Xoc, as well as resistance targeted at TAL7b. These BSR QTL are excellent sources for durable, broadly effective resistance in the field

De Novo Genome Assembly of the Japanese Wheat Cultivar Norin 61 Highlights Functional Variation in Flowering Time and Fusarium Resistance Genes in East Asian Genotypes

Bread wheat is a major crop that has long been the focus of basic and breeding research. Assembly of its genome has been difficult because of its large size and allohexaploid nature (AABBDD genome). Following the first reported assembly of the genome of the experimental strain Chinese Spring (CS), the 10+ Wheat Genomes Project was launched to produce multiple assemblies of worldwide modern cultivars. The only Asian cultivar in the project is Norin 61, a representative Japanese cultivar adapted to grow across a broad latitudinal range, mostly characterized by a wet climate and a short growing season. Here, we characterize key aspects of its chromosome-scale genome assembly spanning 15 Gb with a raw scaffold N50 of 23 Mb. Analysis of the repetitive elements identified chromosomal regions unique to Norin 61 that encompass a tandem array of the pathogenesis-related-13 family. We report novel copy-number variations in the B homeolog of the florigen gene FT1/VRN3, pseudogenization of its D homeolog, and the association of its A homeologous alleles with the spring/winter growth habit. Further, the Norin 61 genome carries typical East Asian functional variants from CS ranging from a single nucleotide to multi-Mb scale. Examples of such variation are the Fhb1 locus, which confers Fusarium head-blight resistance, Ppd-D1a, which confers early flowering, Glu-D1f for Asian noodle quality, and Rht-D1b, which introduced semi-dwarfism during the green revolution. The adoption of Norin 61 as a reference assembly for functional and evolutionary studies will enable comprehensive characterization of the underexploited Asian bread wheat diversity.ISSN:0032-0781ISSN:1471-905