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 species1,2,3,4. 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 long-range linkage5 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.
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We are grateful to the staff at Dovetail Genomics for constructing the CH Campala Lr22a scaffolds. We thank M. Karafiátová for supervising chromosome 2D flow sorting and estimation of purity in flow sorted fractions, and Z. Dubská, R. Šperková and J. Weiserová for technical assistance. We also thank B. Senger and L. Luthi for assistance with field experiments and B. Keller for continuous support. This work was financed by an Ambizione fellowship of the Swiss National Science Foundation. J.V., H.Š., and J.D. were supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant award LO1204 from the National Program of Sustainability I).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Phenotypic response conferred by the Lr22a leaf rust resistance gene against ten Swiss P. triticina isolates.
The third leaf of ‘Thatcher’ (left) and RL6044 (right) is shown ten days after inoculation. The infection type was scored according to a 0-4 scale (bottom)1. The isolate number is indicated in the top right corner.1. Roelfs, A.P. Race specificity and methods of study. in The Cereal Rusts Vol. I; Origins, specificity, structure, and physiology (eds. Roelfs, A.P. & Bushnell, W.R.) (Academic Press, Orlando, 1984).
Supplementary Figure 2 Comparison of transposable element (TE) fraction in the ‘CH Campala Lr22a’ assembly with that of a quantitative survey performed with Roche/454 sequencing.
For those TE families where data was available, we compared the contributions of annotated TE families. Note that the overall contribution of the high-copy Copia element RLC_Angela is much lower in the ‘CH Campala Lr22a’ assembly, indicating that repetitive sequences derived from high-copy TEs are collapsed in the ‘CH Campala Lr22a’ assembly. This may explain why the total length of the ‘CH Campala Lr22a’ assembly was ~160 Mb shorter than the estimated size of chromosome 2D. For this comparison, we annotated 150 Mb (positions 100-250 Mb) of the ‘CH Campala Lr22a’ pseudomolecule (‘CH Campala Lr22a’ scaffolds anchored to the genetic map of Ae. tauschii). The Roche/454 was done on Ae. tauschii whole-genome DNA2.2. Middleton, C.P., Stein, N., Keller, B., Kilian, B. & Wicker, T. Comparative analysis of genome composition in Triticeae reveals strong variation in transposable element dynamics and nucleotide diversity. Plant J 73, 347-356 (2013).
Supplementary Figure 3 Comparison of the ‘CH Campala Lr22a’ sequence assembly (blue) to the Ae. tauschii genetic map (red).
(a) Comparison over the entire 2D chromosome and (b) the region containing the mapped Lr22a markers. The Lr22a target interval between markers gwm455 and wmc503 is indicated in red on the ‘CH Campala Lr22a’ assembly.
The amino acid sequence of Lr22a from RL6044 (NLR1-ThLr22a) is compared to the predicted NLR1 protein version found in the susceptible wheat cultivar ‘Thatcher’ (NLR1-Th). The Lr22a gene sequences in RL6044 and ‘CH Campala Lr22a’ were identical. CC = coiled-coil, NB-ARC = nucleotide-binding, LRR = leucine-rich repeat. The predicted LRR motifs are indicated in yellow and blue, respectively.
The LRR domains of the respective proteins were used to construct the tree. Numbers indicate how many times the sequences to the right of the fork occurred in the same group out of 100 trees. AtRPM1 was identified as the closest homolog of Lr22a in Arabidopsis by using a BLASTP search and the two proteins show 33% amino acid identity. The Arabidopsis NLR protein At5g45510 was used to root the tree.
Shown are the regions that contain unique amino acid (AA) residues in Lr22a in the N-terminal region (AA 123 and 140) and in the LRR region (AA 637-664 and AA 732-759). ‘Ostro’ and ‘Oberkulmer’ are spelt wheat cultivars (Triticum aestivum ssp. spelta).
Supplementary Figure 7 Simulation of probabilities for a target gene being flanked by two recombination events on a single sequence scaffold.
(a) Recombination frequencies along chromosome 2D. The x-axis is the position on the 2D pseudomolecule (‘CH Campala Lr22a’ scaffolds anchored to Ae. tauschii genetic map – see Supplementary Fig. 3 and Supplementary Table 3) in Mb while the y-axis shows the recombination frequency. Recombination frequencies were calculated based on the Ae. tauschii genetic map3 (see online methods). The obtained values are similar to previously reported recombination frequencies in wheat4,5. For subsequent simulations, chromosome 2D was divided into two telomeric bins (0-100 Mb and 430-521 Mb) where recombination rates were highest, two pericentromeric bins (100-150 Mb and 250-430 Mb) and one centromeric bin (150-250 Mb) with almost no recombination. For each bin, median and average recombination frequencies are indicated. For chromosome 2D, the telomeric 100 Mb had median recombination rates of 1.2 Mb/cM for 2DS and 2.75 Mb/cM for 2DL, respectively. Data from chromosome 3B indicate that these two regions may contain well over 60% of the genes4. (b) Simulations to calculate population sizes required for a target gene being flanked by two recombination events on a single sequence scaffold. Simulations are based on the sizes of sequence scaffold used in the 2D pseudomolecule. The dashed lines indicate population sizes necessary to reach 90% or 95% chances of finding a target gene and its closest flanking markers on a single sequence scaffold. Blue = telomeric bin 2DS, red = telomeric bin 2DL, orange = pericentromeric bins (compiled data from both pericentromeric bins). For the simulation, a random and equal distribution of recombination events along the respective bin was assumed.3. Luo, M.C. et al. A 4-gigabase physical map unlocks the structure and evolution of the complex genome of Aegilops tauschii, the wheat D-genome progenitor. Proc Natl Acad Sci U S A 110, 7940-7945 (2013).4. Choulet, F. et al. Structural and functional partitioning of bread wheat chromosome 3B. Science 345, 1249721 (2014).5. Gardner, K.A., Wittern, L.M. & Mackay, I.J. A highly recombined, high-density, eight-founder wheat MAGIC map reveals extensive segregation distortion and genomic locations of introgression segments. Plant Biotechnol J 14, 1406-1417 (2016).
Supplementary Figure 8 Flow cytometric analysis and sorting chromosome 2D from ‘CH Campala’ (left) and ‘CH Campala Lr22a’ (right).
Bivariate flow karyotypes DAPI vs GAA-FITC were generated and sort windows delimiting the populations of chromosome 2D were set. Insets: Representative images of flow sorted chromosomes 2D that were identified after fluorescence in situ hybridization (FISH) with probes for GAA microsatellites (yellow-green) and Afa family repeat (red). Chromosomal DNA was stained by DAPI (blue).
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Thind, A., Wicker, T., Šimková, H. et al. Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly. Nat Biotechnol 35, 793–796 (2017). https://doi.org/10.1038/nbt.3877
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