Genetic dissection of domestication traits in interspecific chickpea populations.

Chickpea (Cicer arietinum) is a pulse crop that provides an integral source of nutrition for human consumption. The close wild relatives Cicer reticulatum and Cicer echinospermum harbor untapped genetic diversity that can be exploited by chickpea breeders to improve domestic varieties. Knowledge of genomic loci that control important chickpea domestication traits will expedite the development of improved chickpea varieties derived from interspecific crosses. Therefore, we set out to identify genomic loci underlying key chickpea domestication traits by both association and quantitative trait locus (QTL) mapping using interspecific F2 populations. Diverse phenotypes were recorded for various agronomic traits. A total of 11 high-confidence markers were detected on chromosomes 1, 3, and 7 by both association and QTL mapping; these were associated with growth habit, flowering time, and seed traits. Furthermore, we identified candidate genes linked to these markers, which advanced our understanding of the genetic basis of domestication traits and validated known genes such as the FLOWERING LOCUS gene cluster that regulates flowering time. Collectively, this study has elucidated the genetic basis of chickpea domestication traits, which can facilitate the development of superior chickpea varieties.

Genome-wide identification of Sclerotinia sclerotiorum small RNAs and their endogenous targets.

BACKGROUND: Several phytopathogens produce small non-coding RNAs of approximately 18-30 nucleotides (nt) which post-transcriptionally regulate gene expression. Commonly called small RNAs (sRNAs), these small molecules were also reported to be present in the necrotrophic pathogen Sclerotinia sclerotiorum. S. sclerotiorum causes diseases in more than 400 plant species, including the important oilseed crop Brassica napus. sRNAs can further be classified as microRNAs (miRNAs) and short interfering RNAs (siRNAs). Certain miRNAs can activate loci that produce further sRNAs; these secondary sRNA-producing loci are called 'phased siRNA' (PHAS) loci and have only been described in plants. To date, very few studies have characterized sRNAs and their endogenous targets in S. sclerotiorum. RESULTS: We used Illumina sequencing to characterize sRNAs from fungal mycelial mats of S. sclerotiorum spread over B. napus leaves. In total, eight sRNA libraries were prepared from in vitro, 12 h post-inoculation (HPI), and 24 HPI mycelial mat samples. Cluster analysis identified 354 abundant sRNA clusters with reads of more than 100 Reads Per Million (RPM). Differential expression analysis revealed upregulation of 34 and 57 loci at 12 and 24 HPI, respectively, in comparison to in vitro samples. Among these, 25 loci were commonly upregulated. Altogether, 343 endogenous targets were identified from the major RNAs of 25 loci. Almost 88% of these targets were annotated as repeat element genes, while the remaining targets were non-repeat element genes. Fungal degradome reads confirmed cleavage of two transposable elements by one upregulated sRNA. Altogether, 24 milRNA loci were predicted with both mature and milRNA* (star) sequences; these are both criteria associated previously with experimentally verified miRNAs. Degradome sequencing data confirmed the cleavage of 14 targets. These targets were related to repeat element genes, phosphate acetyltransferases, RNA-binding factor, and exchange factor. A PHAS gene prediction tool identified 26 possible phased interfering loci with 147 phasiRNAs from the S. sclerotiorum genome, suggesting this pathogen might produce sRNAs that function similarly to miRNAs in higher eukaryotes. CONCLUSIONS: Our results provide new insights into sRNA populations and add a new resource for the study of sRNAs in S. sclerotiorum.

The broad host range pathogen Sclerotinia sclerotiorum produces multiple effector proteins that induce host cell death intracellularly.

Sclerotinia sclerotiorum is a broad host range necrotrophic fungal pathogen, which causes disease on many economically important crop species. S. sclerotiorum has been shown to secrete small effector proteins to kill host cells and acquire nutrients. We set out to discover novel necrosis-inducing effectors and characterize their activity using transient expression in Nicotiana benthamiana leaves. Five intracellular necrosis-inducing effectors were identified with differing host subcellular localization patterns, which were named intracellular necrosis-inducing effector 1-5 (SsINE1-5). We show for the first time a broad host range pathogen effector, SsINE1, that uses an RxLR-like motif to enter host cells. Furthermore, we provide preliminary evidence that SsINE5 induces necrosis via an NLR protein. All five of the identified effectors are highly conserved in globally sourced S. sclerotiorum isolates. Taken together, these results advance our understanding of the virulence mechanisms employed by S. sclerotiorum and reveal potential avenues for enhancing genetic resistance to this damaging fungal pathogen.

Association Mapping Combined with Whole Genome Sequencing Data Reveals Candidate Causal Variants for Sclerotinia Stem Rot Resistance in Brassica napus.

Canola (Brassica napus) yield can be significantly reduced by the disease sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum, a necrotrophic fungal pathogen with an unusually large host range. Breeding cultivars that are physiologically resistant to SSR is desirable to enhance crop productivity. However, the development of resistant varieties has proved challenging due to the highly polygenic nature of S. sclerotiorum resistance. Here, we identified regions of the B. napus genome associated with SSR resistance using data from a previous study by association mapping. We then validated their contribution to resistance in a follow-up screen. This follow-up screen also confirmed high levels of SSR resistance in several genotypes from the previous study. Using publicly available whole genome sequencing data for a panel of 83 B. napus genotypes, we identified nonsynonymous polymorphisms linked to the SSR resistance loci. A qPCR analysis showed that two of the genes containing these polymorphisms were transcriptionally responsive to S. sclerotiorum infection. In addition, we provide evidence that homologues of three of the candidate genes contribute to resistance in the model Brassicaceae species Arabidopsis thaliana. The identification of resistant germplasm and candidate genomic loci associated with resistance are important findings that can be exploited by breeders to improve the genetic resistance of canola varieties.

Field Pea (Pisum sativum) Germplasm Screening for Seedling Ascochyta Blight Resistance and Genome-Wide Association Studies Reveal Loci Associated with Resistance to Peyronellaea pinodes and Ascochyta koolunga.

Ascochyta blight is a damaging disease that affects the stems, leaves, and pods of field pea (Pisum sativum) and impacts yield and grain quality. In Australia, field pea Ascochyta blight is primarily caused by the necrotrophic fungal species Peyronellaea pinodes and Ascochyta koolunga. In this study, we screened 1,276 Pisum spp. germplasm accessions in seedling disease assays with a mix of three isolates of P. pinodes and 641 accessions with three mixed isolates of A. koolunga (513 accessions were screened with both species). A selection of three P. sativum accessions with low disease scores for either pathogen, or in some cases both, were crossed with Australian field pea varieties PBA Gunyah and PBA Oura, and recombinant inbred line populations were made. Populations at the F3:4 and F4:5 generation were phenotyped for their disease response to P. pinodes and A. koolunga, and genotypes were determined using the diversity arrays technology genotyping method. Marker-trait associations were identified using a genome-wide association study approach. Trait-associated loci were mapped to the published P. sativum genome assembly, and candidate resistance gene analogues were identified in the corresponding genomic regions. One locus on chromosome 2 (LG1) was associated with resistance to P. pinodes, and the 8 Mb genomic region contains 156 genes, two of which are serine/threonine protein kinases, putatively contributing to the resistance trait. A second locus on chromosome 5 (LG3) was associated with resistance to A. koolunga, and the 35 Mb region contains 488 genes, of which five are potential candidate resistance genes, including protein kinases, a mitogen-activated protein kinase, and an ethylene-responsive protein kinase homolog.

The evolutionary and molecular features of the broad-host-range plant pathogen Sclerotinia sclerotiorum.

Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of plant species, including many of the world's most important crops. Key features of S. sclerotiorum include its extraordinary host range, preference for dicotyledonous plants, relatively slow evolution, and production of protein effectors that are active in multiple host species. Plant resistance to this pathogen is highly complex, typically involving numerous polymorphisms with infinitesimally small effects, which makes resistance breeding a major challenge. Due to its economic significance, S. sclerotiorum has been subjected to a large amount of molecular and evolutionary research. In this updated pathogen profile, we review the evolutionary and molecular features of S. sclerotiorum and discuss avenues for future research into this important species.

Identification of Sclerotinia stem rot resistance quantitative trait loci in a chickpea (Cicer arietinum) recombinant inbred line population.

Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum , is one of the most economically devastating diseases in chickpea (Cicer arietinum L.). No complete resistance is available in chickpea to this disease, and the inheritance of partial resistance is not understood. Two hundred F7 recombinant inbred lines (RILs) derived from a cross between a partially resistant variety PBA HatTrick, and a highly susceptible variety Kyabra were characterised for their responses to SSR inoculation. Quantitative trait locus (QTL) analysis was conducted for the area under the disease progress curve (AUDPC) after RIL infection with S. sclerotiorum . Four QTLs on chromosomes, Ca4 (qSSR4-1, qSSR4-2), Ca6 (qSSR6-1) and Ca7 (qSSR7-1), individually accounted for between 4.2 and 15.8% of the total estimated phenotypic variation for the response to SSR inoculation. Candidate genes located in these QTL regions are predicted to be involved in a wide range of processes, including phenylpropanoid biosynthesis, plant-pathogen interaction, and plant hormone signal transduction. This is the first study investigating the inheritance of resistance to S. sclerotiorum in chickpea. Markers associated with the identified QTLs could be employed for marker-assisted selection in chickpea breeding.

Identification of Sources of Sclerotinia sclerotiorum Resistance in a Collection of Wild Cicer Germplasm.

Sclerotinia sclerotiorum is an important fungal pathogen of chickpea (Cicer arietinum L.), and it can cause yield losses up to 100%. The wild progenitors are much more diverse than domesticated chickpea, and this study describes how this relates to S. sclerotiorum resistance. Initially, the pathogenicity of nine Australian S. sclerotiorum isolates was examined on three Cicer lines to develop a robust phenotyping assay, and significant differences in isolate aggressiveness were identified with six isolates being classed as highly aggressive and three as moderately aggressive. We identified two S. sclerotiorum isolates, CU8.20 and CU10.12, to be highly aggressive and moderately aggressive, respectively. A subsequent phenotyping assay was conducted using the two isolates to evaluate 86 wild Cicer accessions (Cicer reticulatum and Cicer echinospermum) and two C. arietinum varieties for resistance to S. sclerotiorum. A subset of 12 genotypes was further evaluated, and subsequently, two wild Cicer accessions with consistently high levels of resistance to S. sclerotiorum were examined using the initially characterized nine isolates. Wild Cicer accessions Karab_084 and Deste_063 demonstrated consistent partial resistance to S. sclerotiorum. There were significant differences in responses to S. sclerotiorum across wild Cicer collection sites. The Cermik, Karabahce, and Destek sites' responses to the aggressive isolate CU8.20 ranged from resistant to susceptible, highlighting an interaction between isolate genotype and chickpea collection site for sclerotinia stem rot resistance. This is the first evidence of partial stem resistance identified in wild Cicer germplasm, which can be adopted in chickpea breeding programs to enhance S. sclerotiorum resistance in future chickpea varieties.

Modeling first order additive × additive epistasis improves accuracy of genomic prediction for sclerotinia stem rot resistance in canola.

The fungus Sclerotinia sclerotiorum infects hundreds of plant species including many crops. Resistance to this pathogen in canola (Brassica napus L. subsp. napus) is controlled by numerous quantitative trait loci (QTL). For such polygenic traits, genomic prediction may be useful for breeding as it can capture many QTL at once while also considering nonadditive genetic effects. Here, we test application of common regression models to genomic prediction of S. sclerotiorum resistance in canola in a diverse panel of 218 plants genotyped at 24,634 loci. Disease resistance was scored by infection with an aggressive isolate and monitoring over 3 wk. We found that including first-order additive × additive epistasis in linear mixed models (LMMs) improved accuracy of breeding value estimation between 3 and 40%, depending on method of assessment, and correlation between phenotypes and predicted total genetic values by 14%. Bayesian models performed similarly to or worse than genomic relationship matrix-based models for estimating breeding values or overall phenotypes from genetic values. Bayesian ridge regression, which is most similar to the genomic relationship matrix-based approach in the amount of shrinkage it applies to marker effects, was the most accurate of this family of models. This confirms several studies indicating the highly polygenic nature of sclerotinia stem rot resistance. Overall, our results highlight the use of simple epistasis terms for prediction of breeding values and total genetic values for a complex disease resistance phenotype in canola.