Low-cost assembly of a cacao crop genome is able to resolve complex heterozygous bubbles

Abstract

Cacao (Theobroma cacao) is a tropical tree that produces the essential raw material for chocolate. Because yields have been stagnant, land use has expanded to provide for increasing chocolate demand. Assembled genomes of key parents could modernize breeding programs in the remote and under-resourced locations where cacao is grown. The MinION, a long read sequencer that runs off of a laptop computer, has the potential to facilitate the assembly of the complex genomes of high-yielding F₁ hybrids. Here, we validate the MinION’s application to heterozygous crops by creating a de novo genome assembly of a key parent in breeding programs, the clone Pound 7. Our MinION-only assembly was 20% larger than the latest released cacao genome, with 10-fold greater contiguity, and the resolution of complex heterozygosity and repetitive elements. Polishing with Illumina short reads brought the predicted completeness of our assembly to similar levels to the previously released cacao genome assemblies. In contrast to previous cacao genome projects, our assembly required only a small scientific team and limited reagents. Our sequencing and assembly methods could easily be adopted by under-resourced breeding programs, speeding crop improvement in the developing world.

References

[1] Michael, T. P. & Jackson, S. The first 50 plant genomes. Plant Genome 6, 1-7 (2013).

[2] Ong-Abdullah, M. et al. Loss of Karma transposon methylation underlies the mantled somaclonal variant of oil palm. Nature 525, 533-537 (2015).

[3] Argout, X. et al. The genome of Theobroma cacao . Nat. Genet. 43, 101-108 (2011).

[4] Argout, X. et al. The cacao Criollo genomev2.0: an improved version of the genome for genetic and functional genomic studies. BMC Genom. 18, 730 (2017).

[5] Motamayor, J. C. et al. The genome sequence of the most widely cultivated cacao type and its use to identify candidate genes regulating pod color. Genome Biol. 14, r53 (2013).

[6] Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17, 239 (2016).

[7] Quick, J. et al. Real-time, portable genome sequencing for Ebola surveillance. Nature 530, 228-232 (2016).

[8] Pound, F. J. Cacao and witches’ broom disease. Report on a recent visit to the Amazon territory of Peru, September 1942-February 1943. Arch. Cocoa Res. 1, 73-91 (1943).

[9] Martinez, S. F. CATIE Progress Report 1979. (Tropical Agriculture Research and Training Center, Turrialba, Costa Rica., 1979).

[10] Hunter, J. R. The status of cacao (Theobroma cacao, sterculiaceae) in the western hemisphere . Econ. Bot. 44, 425-439 (1990).

[11] Romero Navarro, J. A. et al. Application of genome wide association and genomic prediction for improvement of cacao productivity and resistance to black and frosty pod diseases. Front. Plant Sci. 8, 1905 (2017).

[12] Motamayor, J. C. et al. Geographic and genetic population differentiation of the Amazonian chocolate tree (Theobroma cacao L) . PLoS ONE 3, e3311 (2008).

[13] Motamayor, J. C. et al. Cacao domestication I: the origin of the cacao cultivated by the Mayas. Heredity 89, 380-386 (2002).

[14] Cheesman, E. E. Notes on the nomenclature, classification and possible relationship of cacao populations. Tropical Agriculture 21, 144-59 (1944).

[15] Michael, T. P. et al. High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell . Nat. Commun. 9, 541 (2018).

[16] Schmidt, M. H.-W. et al. De novo assembly of a new solanum pennellii accession using nanopore sequencing . Plant Cell 29, 2336-2348 (2017).

[17] Livingstone, D. et al. Making a chocolate chip: development and evaluation of a 6K SNP array for Theobroma cacao . DNA Res. 22, 279-291 (2015).

[18] Rastas, P., Calboli, F. C. F., Guo, B., Shikano, T. & Merilä, J. Construction of ultradense linkage maps with Lep-MAP2: stickleback F2 recombinant crosses as an example. Genome Biol. Evol. 8, 78-93 (2015).

[19] Tang, H. et al. ALLMAPS: robust scaffold ordering based on multiple maps. Genome Biol. 16, 3 (2015).

[20] Livingstone, D. et al. A larger chocolate chip-development of a 15K Theobroma cacao L. SNP array to create high-density linkage maps . Front. Plant Sci. 8, 2008 (2017).

[21] Simão, F. A., Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. Bioinformatics 31, 3210-3212 (2015).

[22] Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014).

[23] Stanke, M. & Morgenstern, B. AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Res. 33, W465-W467 (2005).

[24] Collard, B. C. Y. & Mackill, D. J. Marker-assisted selection: an approach for precision plant breeding in the twenty-first century. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 363, 557-572 (2008).

[25] dos Santos Fernandes, L. et al. Mapping of a major QTL for Ceratocystis wilt disease in an F1 population of Theobroma cacao . Front. Plant Sci. 9, 155 (2018).

[26] Araújo, I. S. et al. Mapping of quantitative trait loci for butter content and hardness in cocoa beans (Theobroma cacao L.) . Plant. Mol. Biol. Rep. 27, 177-183 (2009).

[27] Royaert, S. et al. Identification of candidate genes involved in Witches’ broom disease resistance in a segregating mapping population of Theobroma cacao L. in Brazil . BMC Genom. 17, 107 (2016).

[28] Lanaud, C. et al. A meta-QTL analysis of disease resistance traits of Theobroma cacao L. . Mol. Breed. 24, 361-374 (2009).

[29] Li, H. Minimap and miniasm: fast mapping and de novo assembly for noisy long sequences. Bioinformatics 32, 2103-2110 (2016).

[30] Vaser, R., Sović, I., Nagarajan, N. & Šikić, M. Fast and accurate de novo genome assembly from long uncorrected reads. Genome Res. 27, 737-746 (2017).

[31] Sović, I. et al. Fast and sensitive mapping of nanopore sequencing reads with GraphMap. Nat. Commun. 7, 11307 (2016).

[32] Koren, S. et al. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 27, 722-736 (2017).

[33] Ruan, J. Ultra-fast de novo assembler using long noisy reads. 2015. https://github.com/ruanjue/smartdenovo. (accessed 17 Feb. 2019).

[34] Kurtz, S. et al. Versatile and open software for comparing large genomes. Genome Biol. 5, R12 (2004).

[35] Simpson, J. Signal-level algorithms for MinION data. 2014. https://github.com/jts/nanopolish. (accessed on 17 Feb. 2019).

[36] Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv.[q-bio.GN]. 00, 1-3 (2013).

[37] Kent, W. J. BLAT-the BLAST-Like alignment tool. Genome Res. 12, 656-664 (2002).

[38] R Core Team . R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria (2018).

[39] Tarailo-Graovac, M. & Chen, N. Using RepeatMasker to identify repetitive elements in genomic sequences. Curr. Protoc. Bioinforma. Unit4, 10 (2009). Chapter 4.

[40] Krumsiek, J., Arnold, R. & Rattei, T. Gepard: a rapid and sensitive tool for creating dotplots on genome scale. Bioinformatics 23, 1026-1028 (2007).

[41] Cornejo, O. E. et al. Genomic insights into the domestication of the chocolate tree, Theobroma cacao L. (2017). https://doi.org/10.1101/223438

[42] Sveinsson, S., Gill, N., Kane, N.C., Cronk, Q. Transposon fingerprinting using low coverage whole genome shotgun sequencing in Cacao (Theobroma cacao L.) and related species . BMC Genomics 14, 1-12 (2013).