Tracking disease resistance deployment in potato breeding by enrichment sequencing

Following the molecular characterisation of functional disease resistance genes in recent years, methods to track and verify the integrity of multiple genes in varieties are needed for crop improvement through resistance stacking. Diagnostic resistance gene enrichment sequencing (dRenSeq) enables the highconfidence identification and complete sequence validation of known functional resistance genes in crops. As demonstrated for tetraploid potato varieties, the methodology is more robust and cost-effective in monitoring resistances than whole-genome sequencing and can be used to appraise (trans)gene integrity efficiently. All currently known NB-LRRs effective against viruses, nematodes and the late blight pathogen Phytophthora infestans can be tracked with dRenSeq in potato and hitherto unknown polymorphisms have been identified. The methodology provides a means to improve the speed and efficiency of future disease resistance breeding in crops by directing parental and progeny selection towards effective combinations of resistance genes

Effector Genomics Accelerates Discovery and Functional Profiling of Potato Disease Resistance and Phytophthora Infestans Avirulence Genes

Potato is the world's fourth largest food crop yet it continues to endure late blight, a devastating disease caused by the Irish famine pathogen Phytophthora infestans. Breeding broad-spectrum disease resistance (R) genes into potato (Solanum tuberosum) is the best strategy for genetically managing late blight but current approaches are slow and inefficient. We used a repertoire of effector genes predicted computationally from the P. infestans genome to accelerate the identification, functional characterization, and cloning of potentially broad-spectrum R genes. An initial set of 54 effectors containing a signal peptide and a RXLR motif was profiled for activation of innate immunity (avirulence or Avr activity) on wild Solanum species and tentative Avr candidates were identified. The RXLR effector family IpiO induced hypersensitive responses (HR) in S. stoloniferum, S. papita and the more distantly related S. bulbocastanum, the source of the R gene Rpi-blb1. Genetic studies with S. stoloniferum showed cosegregation of resistance to P. infestans and response to IpiO. Transient co-expression of IpiO with Rpi-blb1 in a heterologous Nicotiana benthamiana system identified IpiO as Avr-blb1. A candidate gene approach led to the rapid cloning of S. stoloniferum Rpi-sto1 and S. papita Rpi-pta1, which are functionally equivalent to Rpi-blb1. Our findings indicate that effector genomics enables discovery and functional profiling of late blight R genes and Avr genes at an unprecedented rate and promises to accelerate the engineering of late blight resistant potato varieties

Discovery and characterization of two new stem rust resistance genes in Aegilops sharonensis

Stem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goat grass species Aegilops sharonesis (Sharon goatgrass) as a substantial reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F6 recombinant inbred line and an F2 population, two genes were identified that mapped to the short arm of chromosome 1Ssh, designated as Sr-1644-1Sh, and the long arm of chromosome 5Ssh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (one of the Ug99 lineage races), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors

Aegilops sharonensis genome-assisted identification of stem rust resistance gene Sr62

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance

Aegilops sharonensis genome-assisted identification of stem rust resistance gene Sr62

The wild relatives and progenitors of wheat have been widely used as sources of disease resistance (R) genes. Molecular identification and characterization of these R genes facilitates their manipulation and tracking in breeding programmes. Here, we develop a reference-quality genome assembly of the wild diploid wheat relative Aegilops sharonensis and use positional mapping, mutagenesis, RNA-Seq and transgenesis to identify the stem rust resistance gene Sr62, which has also been transferred to common wheat. This gene encodes a tandem kinase, homologues of which exist across multiple taxa in the plant kingdom. Stable Sr62 transgenic wheat lines show high levels of resistance against diverse isolates of the stem rust pathogen, highlighting the utility of Sr62 for deployment as part of a polygenic stack to maximize the durability of stem rust resistance

Effector-driven marker development and cloning of resistance genes against Phytophthora infestans in potato breeding clone SW93-1015

Key message: We show the usefulness of integrating effector screening in a breeding program and in resistance gene cloning, withPhytophthoraresistance in the Swedish potato breeding clone SW93-1015 as an example. Abstract: Phytophthora infestans is one of the most devastating plant pathogens worldwide. We have earlier found that the SW93-1015 potato breeding clone has an efficient resistance against P. infestans under field conditions in Sweden, which has an unusually high local diversity of the pathogen. This potato clone has characteristics that are different from classical R-gene-mediated resistance such as elevated levels of hydrogen peroxide (H2O2) under controlled conditions. Analysis of 76 F1 potato progenies from two individual crosses resulted in nearly 50 % resistant clones, from both crosses. This result suggests that the SW93-1015 clone has a simplex genotype for this trait. Screening with over 50 different P. infestans effectors, containing the conserved motif RXLR (for Arg, any amino acid, Leu, Arg), revealed a specific response to Avr2, which suggests that SW93-1015 might contain a functional homolog of the R2 resistance gene. We cloned eight R2 gene homologs from SW93-1015, whereof seven have not been described before and one gene encoded a protein identical to Rpi-ABPT. Expression of this gene in potato cultivar Désirée provided R2-specific resistance, whereas other homologues did not. Using RNAseq analyses we designed a new DNA marker for the R2 resistance in SW93-1015. In summary, we have demonstrated the use of effector screening in practical breeding material and revealed the key resistance mechanism for SW93-1015

Characterisation and Analysis of the <i>Aegilops</i><i> sharonensis</i> Transcriptome, a Wild Relative of Wheat in the <i>Sitopsis</i> Section

<div><p><i>Aegilops</i><i>sharonensis</i> Eig (Sharon goatgrass) is a wild diploid relative of wheat within the <i>Sitopsis</i> section of <i>Aegilops</i>. This species represents an untapped reservoir of genetic diversity for traits of agronomic importance, especially as a source of novel disease resistance. To gain a foothold in this genetic resource, we sequenced the cDNA from leaf tissue of two geographically distinct <i>Ae</i><i>. sharonensis</i> accessions (1644 and 2232) using the 454 Life Sciences platform. We compared the results of two different assembly programs using different parameter sets to generate 13 distinct assemblies in an attempt to maximize representation of the gene space in <i>de novo</i> transcriptome assembly. The most sensitive assembly (71,029 contigs; N50 674 nts) retrieved 18,684 unique best reciprocal BLAST hits (BRBH) against six previously characterised grass proteomes while the most specific assembly (30,609 contigs; N50 815 nts) retrieved 15,687 BRBH. We combined these two assemblies into a set of 62,243 non-redundant sequences and identified 139 belonging to plant disease resistance genes of the nucleotide binding leucine-rich repeat class. Based on the non-redundant sequences, we predicted 37,743 single nucleotide polymorphisms (SNP), equivalent to one per 1,142 bp. We estimated the level of heterozygosity as 1.6% in accession 1644 and 30.1% in 2232. The <i>Ae</i><i>. sharonensis</i> leaf transcriptome provides a rich source of sequence and SNPs for this wild wheat relative. These sequences can be used with existing monocot genome sequences and EST sequence collections (e.g. barley, <i>Brachypodium</i>, wheat, rice, maize and <i>Sorghum</i>) to assist with genetic and physical mapping and candidate gene identification in <i>Ae</i><i>. sharonensis</i>. These resources provide an initial framework to further build on and characterise the genetic and genomic structure of <i>Ae</i><i>. sharonensis</i>.</p></div

Best reciprocal BLAST hits of <i>Ae</i><i>. sharonensis</i> transcripts against barley unigenes, wheat full-length cDNAs and the exomes of sequenced grasses.

<p>Best reciprocal BLAST hits of <i>Ae</i><i>. sharonensis</i> transcripts against barley unigenes, wheat full-length cDNAs and the exomes of sequenced grasses.</p

Predicted NB-LRR proteins from <i>Ae</i><i>. sharonensis</i> assemblies and BRBH against grass NB-LRRs.

<p>Predicted NB-LRR proteins from <i>Ae</i><i>. sharonensis</i> assemblies and BRBH against grass NB-LRRs.</p