Abstract
Breeding new and sustainable crop cultivars of high yields and desirable traits has been a major challenge for ensuring food security for the growing global human population. For polyploid crops such as wheat, introducing genetic variation from wild relatives of its subgenomes is a key strategy to improve the quality of their breeding pools. Over the past decades, considerable progress has been made in speed breeding, genome sequencing, high-throughput phenotyping and genomics-assisted breeding, which now allows us to realize whole-genome introgression from wild relatives to modern crops. Here, we present a standardized protocol to rapidly introgress the entire genome of Aegilops tauschii, the progenitor of the D subgenome of bread wheat, into elite wheat backgrounds. This protocol integrates multiple modern high-throughput technologies and includes three major phases: development of synthetic octaploid wheat, generation of hexaploid A. tauschii–wheat introgression lines (A-WIs) and homozygosis of the generated A-WIs. Our approach readily generates stable introgression lines in 2 y, thus greatly accelerating the generation of A-WIs and the introduction of desirable genes from A. tauschii to wheat cultivars. These A-WIs are valuable for wheat-breeding programs and functional gene discovery. The current protocol can be easily modified and used for introgressing the genomes of wild relatives to other polyploid crops.
Key points
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This protocol describes a method to introgress the entire genome of the wild Aegilops tauschii into elite bread wheat with the aim of introducing genetic variation and desirable traits.
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This protocol allows more efficient introgression than existing methods by using a rapid, high-throughput introgression platform.
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Data availability
All raw data of exome sequencing of AW-I and parent lines have been deposited in the National Center for Biotechnology Information under BioProject number PRJNA961342. Original 55K SNP array genotype data in derived C1, BC1F1, BC2F1 and BC3F1 generations of A. tauschii T093 and bread wheat AK58 are available in the Supplementary Data.
Code availability
A code resource used in the project has been deposited in GitHub (https://github.com/haoli01/Introgress-analysis).
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Acknowledgements
We are grateful to Jan Dvorak (UC Davis, USA) and Le Wang (Jilin University, China) for help with the manuscript. This work was supported by grants from the National Natural Science Foundation of China (32230079), the National Key Research and Development Program of China (2022YFF1001600) and the Natural Science Foundation of Henan Province (222301420102).
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Contributions
C.-P.S., Y. Zhou and H. Li provided conceptualization. H. Li, L.Z., R.F., Z.L., Y. Liu, A.S., Can Li, F.N. and X.L. provided methodology. The investigation was performed by H. Li, L.Z., R.F., Z.L., Y. Liu, A.S., Can Li, F.N., X.L., Y.Y., T.G., Y. Zhu, M.B., Y. Li, W.L., Chenglin Li, H. Liang, S.B., F.M., G.G. and Z.Z. The original draft was prepared by H. Li, L.Z. and Y. Zhou. The draft was reviewed and edited by C.-P.S., H. Li, Y. Zhou and J.H. Funding was acquired by C.-P.S., Y. Zhou and H. Li. Resources and supervision were provided by C.-P.S., Y. Zhou and H. Li.
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Nature Protocols thanks Zhongfu Ni, Nils Stein, Awais Rasheed and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Zhou, Y. et al. Nat. Plants 7, 774–786 (2021): https://doi.org/10.1038/s41477-021-00934-w
Ma, F. et al. Crop J. 11, 1521–1532 (2023): https://doi.org/10.1016/j.cj.2023.05.001
Extended data
Extended Data Fig. 1 An SOW pool developed by crossing 85 A. tauschii accessions from all five sublineages with bread wheat cultivar.
a, Sublineage information of 85 A. tauschii accessions. The accession number of each sublineage is indicated in parentheses. b, Spike and grain phenotype of 18 SOWs and parent lines. AK58 and T093 represent wheat cultivar and A. tauschii, respectively. c, ND-FISH karyotypes of 12 SOWs. Most of the SOWs carried a complete chromosome set of A. tauschii, while several SOWs occasionally lose chromosomes. Green: oligo-pSc119.2; red: oligo-pTa535; yellow: oligo-(GAA)10. Adapted with permission from ref. 3, Springer Nature Limited.
Extended Data Fig. 2 Meiotic pairing of A. tauschii T093 × common wheat AK58 F1 hybrids.
a–c, The averaged univalent (I) and bivalent (II) number in each meiotic cell of A. tauschii T093 (n = 85 cells), common wheat AK58 (n = 50 cells) and their F1 hybrids (n = 72 cells). d, A meiotic cell of A. tauschii T093 × common wheat AK58 F1 hybrids showing seven bivalents and 14 univalents. Arrows represent the bivalents of 1D with 1Dt, and 2D with 2Dt, respectively. Green: oligo-(GAA)10; red: oligo- pTa535-1. Bar = 20 μm. ANPC, average number per cell.
Extended Data Fig. 3 Graphical representation of the whole-genome coverage in BC1F1, BC2F1 and BC3F1 in seven homologous groups based on a 55K SNP array.
a–c, Whole-genome coverage in BC1F1 (a), BC2F1 (b) and BC3F1 (c) by using original population size. d–f, Whole-genome coverage in BC1F1 (d), BC2F1 (e) and BC3F1 (f) by using calculated saturation population size. Each row represents an A-WI, and each column represents a SNP locus. Pink: heterozygous for A. tauschii T093 alleles; blue: homozygous for bread wheat AK58 alleles; white: missing data. 0: genotype consistent with bread wheat cultivar AK58; 1: missing data; 2: genotype consistent with A. tauschii T093.
Extended Data Fig. 4 Distribution of introgressed fragment size in different generations.
a and b, Histogram of a foreign fragment size of BC4F1 (a) and BC4F2 (b) in seven homologous groups with 10 Mb as the length interval. c and d, count of introgressed fragments with length <10 Mb. The width of each column represents a span of 1 Mb. chr, chromosome.
Supplementary information
Supplementary Information
Supplementary Figs. 1 and 2 and Table 2
Supplementary Video 1
A video file showing how to emasculate the spikes of A. tauschii
Supplementary Table 1
The inheritance frequencies of A. tauschii T093 alleles evaluated by 55K SNP array in derived BC1F1, BC2F1 and BC3F1 generations
Supplementary Table 3
Statistics of introgressed fragment sizes in derived BC4F1, BC4F2 and BC4F4 generations
Supplementary Data 1
Supplementary Data
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Li, H., Zhu, L., Fan, R. et al. A platform for whole-genome speed introgression from Aegilops tauschii to wheat for breeding future crops. Nat Protoc 19, 281–312 (2024). https://doi.org/10.1038/s41596-023-00922-8
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DOI: https://doi.org/10.1038/s41596-023-00922-8
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