Abstract
Key message
This manuscript describes the identification, isolation and sequencing of a single chromosome containing high value resistance genes from a complex polyploid where sequencing the whole genome is too costly.
Abstract
The large complex genomes of many crops constrain the use of new technologies for genome-assisted selection and genetic improvement. One method to simplify a genome is to break it into individual chromosomes by flow cytometry; however, in many crop species most chromosomes cannot be isolated individually. Flow sorting of a single copy of a chromosome has been developed in wheat, and here we demonstrate its use to identify markers of interest in an Erianthus/Sacchurum hybrid. Erianthus/Saccharum hybrids are of interest because Erianthus is known to be highly resistant to soil borne diseases which cause extensive sugarcane yield losses in Australia. Sugarcane (Saccharum) cultivars are autopolyploids with a highly complex genome and over 100 chromosomes. Flow cytometry for sugarcane, as in most crops, does not resolve individual chromosomes to a karyotype peak for sorting. To isolate a single chromosome, we used genomic in situ hybridization (GISH) to identify the flow karyotype region containing the Erianthus chromosomes, flow sorted single chromosomes from this region, PCR screened for the Erianthus chromosomes and sequenced them. One Erianthus chromosome amplified and sequenced well, and from this data we could identify 57 resistant type genes and SNPs in nearly half of these genes. We developed KASP SNP assays and demonstrated that the identified SNP markers segregated as expected in a small introgression population. The pipeline we developed here to flow sort and sequence single chromosomes could be used in any crop with a large complex genome to rapidly discover and develop markers to important loci.
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All data are available on request to corresponding author.
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Abbreviations
- WGA:
-
Whole Genome Amplification
- FISHIS:
-
Fluorescence in situ Hybridization in Suspension
- GISH:
-
Genomic in situ Hybridization
- MDA:
-
Multiple Displacement Amplification
- SNP:
-
Single Nucleotide Polymorphism
- STP:
-
Single Tiling Path
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Acknowledgements
We would like to acknowledge the support of Sugar Research Australia who provided the genotype for flow sorting. We would also like to thank Dalia Khalil from the TRI Flow Cytometry Facility at the Translational Research Institute who was instrumental in getting the flow sorting parameters correct for sugarcane. This work was conducted in collaboration by the Joint Genome Institute and is supported by the Office of Science in the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
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Work was funded by CSIRO, Sugar Research Australia.
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Conceptualization, KSA and NP; Funding acquisition, KSA and NP; Investigation, CJM and JL; Formal analysis, CJM, AH, BZ and JS; Original draft preparation, CJM; Review and editing, KSA, CJM and NP; Supervision, KSA.
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Communicated by Maria Laura Federico.
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Metcalfe, C.J., Li, J., Zheng, B. et al. Isolation and sequencing of a single copy of an introgressed chromosome from a complex genome for gene and SNP identification. Theor Appl Genet 135, 1279–1292 (2022). https://doi.org/10.1007/s00122-022-04030-1
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DOI: https://doi.org/10.1007/s00122-022-04030-1