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Type: Journal article
Title: Detection of somaclonal variation during cocoa somatic embryogenesis characterised using cleaved amplified polymorphic sequence and the new freeware Artbio
Author: rodriguez lopez, C.
Bravo, H.S.
Wetten, A.C.
Wilkinson, M.J.
Citation: Molecular Breeding, 2010; 25(3):501-516
Publisher: Springer
Issue Date: 2010
ISSN: 1380-3743
Statement of
Carlos M. Rodríguez López, Hector Sicillia Bravo, Andrew C. Wetten and Michael J. Wilkinson
Abstract: 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 soft- ware 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 x 10- 3 substitutions/screened base. This elevated rate is of a similar order of magnitude to previous reports of denovo microsatellite length mutations arising in the crop and suggests caution should be exercised when applying somatic embryogenesis for the conservation of plant germplasm.
Rights: © Springer Science+Business Media B.V. 2009
RMID: 0030007043
DOI: 10.1007/s11032-009-9348-x
Appears in Collections:Agriculture, Food and Wine publications

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