How Target-Sequence Enrichment and Sequencing (TEnSeq) Pipelines Have Catalyzed Resistance Gene Cloning in the Wheat-Rust Pathosystem

Abstract

The wheat-rust pathosystem has been well-studied among host-pathogen interactions since last century due to its economic importance. Intensified efforts toward cloning of wheat rust resistance genes commenced in the late 1990s with the first successful isolation published in 2003. Currently, a total of 24 genes have been cloned from wheat that provides resistance to stem rust, leaf rust, and stripe rust. Among them, more than half (15) were cloned over the last 4 years. This rapid cloning of resistance genes from wheat can be largely credited to the development of approaches for reducing the genome complexity as 10 out of the 15 genes cloned recently were achieved by approaches that are summarized as TEnSeq (Target-sequence Enrichment and Sequencing) pipelines in this review. The growing repertoire of cloned rust resistance genes provides new tools to support deployment strategies aimed at achieving durable resistance. This will be supported by the identification of genetic variation in corresponding Avr genes from rust pathogens, which has recently begun. Although developed with wheat resistance genes as the primary targets, TEnSeq approaches are also applicable to other classes of genes as well as for other crops with complex genomes.

Creation and judicious application of a wheat resistance gene atlas

“…The NLR gene family shares distinct protein motifs that can be used to guide the design of specific probes for R-gene enrichment sequencing (RenSeq; Jupe et al, 2013). Mutational genomics combined with RenSeq was used to isolate the wheat rust R genes Sr22, Sr26, Sr45, Sr61, Yr5a, Yr5b, and Yr7 and the powdery mildew R genes Pm21 and Pm1a (Supplemental Table 2; Steuernagel et al, 2016;Marchal et al, 2018;Xing et al, 2018;Zhang et al, 2020;Hewitt et al, 2021); however, capture-based methods are biased toward annotated genes for which probes have been designed. To reduce genome complexity in an unbiased and lossless manner, specific chromosomes can be flow sorted and sequenced from the wild type and several independent mutants, followed by a sequence comparison for the identification of a candidate gene (Sá nchez-Martín et al, 2016).…”

Application of Genomics Tools in Wheat Breeding to Attain Durable Rust Resistance

“…A recently developed molecular tool, MutChromSeq (Mutgenesis Chromosome flow sorting and short-read Sequencing), works based on mutagenesis followed by flow sorting of chromosomes and their sequencing to identify the possible induced mutations. This approach does not require a similar gene in bait library and hence enables reference-free forward genetics to open up the pan-genome to functional genomics ( Sanchez-Martin et al., 2016 ; Zhang et al., 2020 ). This rapid cloning approach which would need 18–24 months for mutagenesis and screening, approximately one month for chromosome flow sorting, sequencing and bioinformatics were successfully described for cloning of barley Eceriferum - q gene and wheat Pm2 gene ( Sanchez-Martin et al., 2016 ).…”

“…a ) on chromosome 2H from cultivated barley ( Hordeum vulgare ) cv Sudan was also rapidly cloned by combining this approach with genetic mapping. The successful application of this approach would need prior information on, chromosome location of the target gene ( Dracatos et al., 2019 ; Zhang et al., 2020 ), large mutant populations to identify the target genes among five to six mutants ( Mago et al., 2017 ; Dinh et al., 2020 ) and isolation of individual chromosomes ( Periyannan, 2018 ).…”

A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes

“…Conventional map-based cloning of Sr26 and Sr61 in wheat derivatives was not feasible due to the absence of recombination between common wheat and alien chromosome segments. Target-sequence Enrichment and Sequencing (TEnSeq) pipelines developed in recent years have overcome such constraints to map-based cloning 23 . Here we used mutational genomics and targeted exome capture of NLR immune receptor genes, a method termed MutRenSeq 8 , for isolation of Sr26 and Sr61 .…”