Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly

Abstract

Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago longrange linkage to assemble complex genomes. We applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.DOI: https://doi.org/10.1038/nbt.3877Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-144182 Journal Article Accepted Version Originally published at: Thind, Anupriya Kaur; Wicker, Thomas; Šimková, Hana; Fossati, Dario; Moullet, Odile; Brabant, Cécile; Vrána, Jan; Doležel, Jaroslav; Krattinger, Simon G (2017). Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly. Nature Biotechnology, 35(8) Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene using molecular marker information and EMS mutants and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis RPM1 protein.While the world population continues to grow, the arable land per capita is decreasing 6 . To ensure food security, agriculture will require high-yielding crops that can withstand diseases, pests and adverse climatic conditions. A better understanding of genes that control these important traits may enable breeding of crop cultivars capable of feeding the 9-10 billion people expected by 2050. triticina isolates [13][14][15][16] . The Lr22a-mediated resistance is not present in young seedlings (<20 days) but is only visible in wheat plants from ~25 days of age.First, in order to evaluate the effectiveness of Lr22a against Swiss P. triticina isolates we inoculated the Lr22a-containing backcross line RL6044 ('Thatcher Lr22a') and the spring wheat cultivar 'Thatcher' with ten P. triticina isolates that were collected in Switzerland. The first leaves of RL6044 developed leaf rust pustules of similar size as the susceptible control 'Thatcher', while we observed complete to moderate resistance on the third leaves of 30-dayold RL6044 plants in comparison to 'Thatcher' (Fig. 1, Supplementary Fig. 1).Lr22a was previously mapped to the short arm of wheat chromosome 2D using microsatellite analysis of the Lr22a-containing wheat line 98B34-T4B 13 . In order to...

A review of wheat diseases—a field perspective

“…The cloning of 10 race‐specific genes in wheat ( Sr22 , Sr33 , Sr35 , Sr45 , Sr50 , Yr10 , Lr1 , Lr21 , Lr10 , Lr22 ) has demonstrated that, as in other plants, these genes encode nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) proteins (Ellis et al ., ; Mago et al ., ; Steuernagel et al ., ; Thind et al ., ), and hence resistance responses must be governed by the direct or indirect recognition of cognate Avr factors. So far, no Avr factor has been characterized in the wheat rust fungi; however, genome sequencing and transcript predictions indicate the presence of rich effector repertoires (Bruce et al ., ; Cantu et al ., ; Duplessis et al ., ; Garnica et al ., ; Upadhyaya et al ., ).…”

Mechanisms of powdery mildew resistance of wheat – a review of molecular breeding

“…Wheat powdery mildew resistance gene ( Pm gene) Pm21 encodes a typical NLR protein, though it confers a broad‐spectrum Bgt resistance at both seedling and adult plant stages (He et al , 2018). Some genes are involved in quantitative resistance such as adult‐plant resistance (APR) gene LR22a (Thind et al , 2017) which, however, also encodes an NLR protein. Moreover, decoupling of race‐specific resistance and life‐long resistance was observed in wheat lines carrying Pm6 and Pm8 , in which resistance was present at the adult plant stage but not in young seedlings (Golzar et al , 2016).…”

“…The advances in DNA sequencing and bioinformatics technologies have also made approaches available for rapid isolation of resistance genes in large‐genome species such as wheat. When prior map information of the targeted gene and isolation of individual chromosomes are accessible, targeted chromosome‐based cloning via long‐range assembly (TACCA) can be used and the success of this approach has been demonstrated by the isolation of the wheat leaf rust R gene Lr22a (Thind et al , 2017). There are also some novel isolation techniques that use mutational genomics for resistance gene cloning and can bypass the construction of high‐density maps.…”

Applying the latest advances in genomics and phenomics for trait discovery in polyploid wheat

“…The target chromosomes of independent mutants are then flow‐sorted and sequenced before being compared to identify candidate genes. A second approach, TACCA (Thind et al ., ), relies on knowledge of a specific target interval (based on flanking markers) identified by traditional genetic mapping, and as such is amenable to both qualitative and quantitative traits (Box 1). The aim of this method is to build, in one step, long‐range contiguous assemblies that completely span the target interval, thereby eliminating the need for BAC clones.…”