Resistance Gene Enrichment Sequencing for plants (RenSeq)

Overview

The Resistance gene enrichment sequencing (RenSeq) workflow allows for the comprehensive study of highly complex disease resistance gene (R gene) families within crop plant genomes (Jupe et al 2013). R genes are often known as “NB-LRR” or “NLR” structural class proteins because they usually have two types of domains, nucleotide-binding (“NB”) and leucine-rich repeat (“LRR”) (Jupe et al 2013). These genes are important as they function to protect against a wide variety of pathogens such as fungi, oomycetes, bacteria, insects, viruses, and more (Schulze-Lefert & Panstruga, 2011). Therefore, disease resistance genes are critical to understand from an agricultural perspective so they can be harnessed for maximum utility in breeding programs and related research endeavors.

Watch our Part Two March 10 webinar on:

Advances in Resistance Gene Sequencing (RenSeq) for Crops Using Targeted NGS

Watch our Part One February 10 webinar on:

Advances in Resistance Gene Sequencing (RenSeq) for Crops Using Targeted NGS

Evolution of RenSeq

While RenSeq provides enrichment of R genes, understanding the role of individual genes defense mechanisms against specific pathogens is the ultimate goal of this discovery process. AgRenSeq (Associated genetics R gene enrichment sequencing) is the next evolution in identifying the role R genes by providing correlation between disease resistant traits and the highly specific immune receptor domains within R genes. The video below from the John Innes Centre provides a clear explanation how screening of wild and ancient varietals uncovers DNA domains delivering resistance to various pathogens.

RenSeq applications continue to broaden into new and unique techniques to review R genes, their functions, as well as identifying pathogens themselves which turn on expression of these genes. A brief timeline in R gene identification and analysis:

• MutRenSeq – Mutagenesis of R genes prior to cloning and identification with target capture sequencing.
• PenSeq – Pathogen virulent gene capture and sequencing for identification of genes encoding pathogenicity.
• AgRenSeq – Association genetics RenSeq to identify correlation between traits and genes.
• dRenSeq – Diagnostic RenSeq for high-confidence identification and complete sequence validation of known R genes in crops.

Why Hybridization Capture?

Crop plant genomes typically have hundreds or more distinct NLR regions, each of which are several Kb in length. These regions can be independent, or found in clusters close to other NLR regions (Jupe et al 2013). As a large, complex gene family with relatively high sequence similarity between members, this creates challenges for resolving these regions with certain next-generation sequencing (NGS) methods, such as low-pass whole genome sequencing or amplicon-based studies. The technique of hybridization-based capture, on the other hand, provides an ideal balance of robust target recovery in terms of both breadth and depth.

In hybridization capture, NGS libraries are denatured and allowed to hybridize to customized biotinylated probes which are complimentary to regions of interest as shown in Figure 1. Affordable oligo synthesis technologies from vendors such as Daicel Arbor Biosciences, can generate custom probe sets containing tens of thousands of unique probes, allowing a single RenSeq experiment to comprise the full sequence diversity of all known R genes in a given genome. This approach delivers recovery of both known NLR regions as well as novel ones, due to the nature of hybridization-based capture to enrich molecules that have sequence divergence up to 20% from a given probe. Such R genes may have been missing during annotation of a given genome sequence, or missing altogether from the reference genome. Thus hybridization capture can function both as a tool for discovery of novel R gene genomic content, as well as a tool to help improve the annotation of NLR regions in a genome (Jupe et al 2013).

The ability to fully sequence one or more complete NLR genes, typically 3.2 kb in length, improves the fine resolution of genomic regions relevant to R gene studies. An advantage of hybridization capture with myBaits^® Custom probe libraries is they are fully compatible with any sequencing platform, meaning that the same probe set can be used to enrich DNA samples prepared for  Illumina^® short-read sequencing or long-read sequencing platforms such as Oxford Nanopore^® or PacBio^®. Daicel Arbor Biosciences is happy to discuss your RenSeq project and help design a custom panel or create a full service project performed by scientists on our myReads^® NGS services team.

Publications

The following selection of papers utilized myBaits^® Custom kits from Daicel Arbor Biosciences among their toolkits for the study of disease resistance genes. Over the last several years, many variations on the original RenSeq approach have been invented, several of which are included below.

• Bettgenhaeuser, Hernández-Pinzón, et al. 2021. The barley immune receptor Mla recognizes multiple pathogens and contributes to host range dynamics. Nature Communications, 12(1), pp.1-14.
• Upadhyaya, Mago, et al. 2021. Genomics accelerated isolation of a new stem rust avirulence gene–wheat resistance gene pair. Nature Plants, 7(9), pp.1220-1228.
• Seong, Seo, et al. 2020. Evolution of NLR resistance genes with noncanonical N‐terminal domains in wild tomato species. New Phytologist, 227(5), pp.1530-1543.
• Narang et al. 2020. Discovery and characterisation of a new leaf rust resistance gene introgressed in wheat from wild wheat Aegilops peregrina. Scientific Reports, 10(1), pp.1-9.
• Lin et al. 2020. RLP/K enrichment sequencing; a novel method to identify receptor‐like protein (RLP) and receptor‐like kinase (RLK) genes. New Phytologist, 227(4), pp.1264-1276.
• Armstrong et al. 2019. Tracking disease resistance deployment in potato breeding by enrichment sequencing. Plant Biotechnology Journal
• Arora et al. 2019. Resistance gene cloning from a wild crop relative by sequence capture and association genetics. Nature Biotechnology
• Cevik et al. 2019. Transgressive segregation reveals mechanisms of Arabidopsis immunity to Brassica-infecting races of white rust (Albugo candida). PNAS
• Grech-Baran et al. 2019. Extreme resistance to Potato Virus Y in potato caaryying the Ry(sto) gene is mediated by a TIR-NLR immune receptor. Plant Biotechnology Journal
• Jouanin et al. 2019. Development of GlutEnSeq capture system for sequencing gluten gene families in hexaploid bread wheat with deletions or mutation induced by gamma-irradiation or CRISPR/Cas9. Journal of Cereal Science
• Narang et al. 2019. Fine mapping of Aegilops peregrina co-segregating leaf and stripe rust resistance genes to distal-most end of 5DS. Theoretical and Applied Genetics
• Van de Weyer et al 2019. A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana. Cell
• Brabham et al. 2018. An ancient integration in a plant NLR is maintained as a trans-species polymorphism. bioRxiv
• Giolai et al. 2018. Targeted capture and sequencing of gene-sized DNA molecules. Biotechniques
• Marchal et al. 2018. BED-domain-containing immune receptors confer diverse resistance spectra to yellow rust. Nature Plants
• Thilliez et al. 2018. Pathogen enrichment sequencing (PenSeq) enables population genomic studies in oomycetes. New Phytologist
• Giolai et al. 2017. Comparative analysis of targeted long read sequencing approaches for characterization of a plant’s immune receptor repertoire. BMC Genomics
• Witik et al. 2016. Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing. Nature Biotechnology
• Steuernagel et al. 2016. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture. Nature Biotechnology

References

1. Jupe et al. 2013. Resistance gene enrichment sequencing (RenSeq) enables reannotation of the NB‐LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations. The Plant Journal
2. Schulze-Lefert and Panstruga. 2011. A molecular evolutionary concept connecting nonhost resistance, pathogen host range, and pathogen speciation. Trends in Plant Science