Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio
- 237 Downloads
The scarcity and stochastic nature of genetic mutations presents a significant challenge for scientists seeking to characterise de novo mutation frequency at specific loci. Such mutations can be particularly numerous during regeneration of plants from in vitro culture and can undermine the value of germplasm conservation efforts. We used cleaved amplified polymorphic sequence (CAPS) analysis to characterise new mutations amongst a clonal population of cocoa plants regenerated via a somatic embryogenesis protocol used previously for cocoa cryopreservation. Efficacy of the CAPS system for mutation detection was greatly improved after an ‘a priori’ in silico screen of reference target sequences for actual and potential restriction enzyme recognition sites using a new freely available software called Artbio. Artbio surveys known sequences for existing restriction enzyme recognition sites but also identifies all single nucleotide polymorphism (SNP) deviations from such motifs. Using this software, we performed an in silico screen of seven loci for restriction sites and their potential mutant SNP variants that were possible from 21 restriction enzymes. The four most informative locus-enzyme combinations were then used to survey the regenerant populations for de novo mutants. We characterised the pattern of point mutations and, using the outputs of Artbio, calculated the ratio of base substitution in 114 somatic embryo-derived cocoa regenerants originating from two explant genotypes. We found 49 polymorphisms, comprising 26.3% of the samples screened, with an inferred rate of 2.8 × 10−3 substitutions/screened base. This elevated rate is of a similar order of magnitude to previous reports of de novo microsatellite length mutations arising in the crop and suggests caution should be exercised when applying somatic embryogenesis for the conservation of plant germplasm.
KeywordsArtbio Somaclonal variation SNPs CAPS PCR–RFLP Theobroma cacao
We thank Cocoa Research UK for funding this study.
- Blondon F, Marie D, Brown S, Kondorosi A (1994) Genome size and base composition in Medicago sativa and M. Truncatula species. Plant Genet Breed 37(2):264–270Google Scholar
- Charters YM (2000) The potential of anchored microsatellite analysis for cocoa germplasm characterization. PhD thesis, School of Plant Sciences, Reading UniversityGoogle Scholar
- Cotton RGH (1997) Mutation detection. Oxford University Press, OxfordGoogle Scholar
- Graur D, Li WH (2000) Fundamentals of molecular evolution. Sinauer, SunderlandGoogle Scholar
- Jin N, Chow CY, Liu L, Zolov SN, Bronson R, Davisson M, Petersen JL, Zhang Y, Park S, Duex JE, Goldowitz D, Meisler MH, Weisman LS (2008) VAC14 nucleates a protein complex essential for the acute interconversion of PI3P and PI(3, 5)P2 in yeast and mouse. EMBO J 27(24):3221–3234CrossRefPubMedGoogle Scholar
- Kaeppler SM, Phillips RL, Olhoft P (1998) Molecular basis of heritable tissue culture-induced variation in plants. Current plant science and biotechnology in agriculture. Kluwer, DordrechtGoogle Scholar
- Lopez-Baez O, Bollon H, Eskes A (1993) Embryogenèse somatique de cacaoyer Theobroma cacao L. à partir de pièces florales. C R Acad Sci Paris 316:579–584Google Scholar
- Sahasrabudhe SR, Luo X, Humayun MZ (1991) Specificity of base substitutions induced by the acridine mutagen ICR-191: mispairing by guanine N7 adducts as a mutagenic mechanism. Genetics 119:981–989Google Scholar
- Thomas RK, Nickerson E, Simons JF, Jänne PA, Tengs T, Yuza Y, Garraway LA, LaFramboise T, Lee JC, Shah K, O’Neill K, Sasaki H, Lindeman N, Wong K-K, Borras AM, Gutmann EJ, Dragnev KH, DeBiasi R, Chen T-H, Glatt KA, Greulich H, Desany B, Lubeski CK, Brockman W, Alvarez P, Hutchison SK, Leamon JH, Ronan MT, Turenchalk GS, Egholm M, Sellers WR, Rothberg JM, Meyerson M (2006) Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nature Med 12:852–855CrossRefPubMedGoogle Scholar
- Thomas RK, Baker AC, Debiasi RM, Winckler W, Laframboise T, Lin WM, Wang M, Feng W, Zander T, Macconnaill LE, Lee JC, Nicoletti R, Hatton C, Goyette M, Girard L, Majmudar K, Ziaugra L, Wong KK, Gabriel S, Beroukhim R, Peyton M, Barretina J, Dutt A, Emery C (2007) High-throughput oncogene mutation profiling in human cancer. Nature Genet 39:347–351CrossRefPubMedGoogle Scholar