, Volume 240, Issue 5, pp 1113–1122 | Cite as

Rapid identification of angulata leaf mutations using next-generation sequencing

  • Eduardo Mateo-Bonmatí
  • Rubén Casanova-Sáez
  • Héctor Candela
  • José Luis Micol
Original Article


Map-based (positional) cloning has traditionally been the preferred strategy for identifying the causal genes underlying the phenotypes of mutants isolated in forward genetic screens. Massively parallel sequencing technologies are enabling the rapid cloning of genes identified in such screens. We have used a combination of linkage mapping and whole-genome re-sequencing to identify the causal mutations in four loss-of-function angulata (anu) mutants. These mutants were isolated in a screen for mutants with defects in leaf shape and leaf pigmentation. Our results show that the anu1-1, anu4-1, anu9-1 and anu12-1 mutants carry new alleles of the previously characterized SECA2, TRANSLOCON AT THE OUTER MEMBRANE OF CHLOROPLASTS 33 (TOC33), NON-INTRINSIC ABC PROTEIN 14 (NAP14) and CLP PROTEASE PROTEOLYTIC SUBUNIT 1 (CLPR1) genes. Re-sequencing the genomes of fine mapped mutants is a feasible approach that has allowed us to identify a moderate number of candidate mutations, including the one that causes the mutant phenotype, in a nonstandard genetic background. Our results indicate that anu mutations specifically affect plastid-localized proteins involved in diverse processes, such as the movement of peptides through chloroplast membranes (ANU1 and ANU4), metal homeostasis (ANU9) and protein degradation (ANU12).


Arabidopsis Chloroplast mutants Cloning-by-sequencing NGS SHOREmap Whole-genome sequencing 





Days after stratification


Ethylmethane sulfonate


Next-generation sequencing



We thank J.M. Serrano, F.M. Lozano, T. Trujillo, R. Sarmiento-Mañús, D. Navarro, L. Serna, J.M. Sánchez-Larrosa and A. Torregrosa for their excellent technical assistance. Research in the laboratory of J.L.M. is supported by Grants from the Ministerio de Economía y Competitividad of Spain [BFU2011-22825 and CSD2007-00057 (TRANSPLANTA)], the Generalitat Valenciana (PROMETEO/2009/112) and the European Commission [LSHG-CT-2006-037704 (AGRON-OMICS)]. HC is a recipient of a Marie Curie International Reintegration Grant (PIRG03-GA-2008-231073). RCS holds a fellowship from the Ministerio de Economía y Competitividad of Spain (BES-2009-014106). EMB holds a predoctoral fellowship from the Universidad Miguel Hernández.

Supplementary material

425_2014_2137_MOESM1_ESM.pdf (226 kb)
Supplementary material 1 (PDF 226 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Eduardo Mateo-Bonmatí
    • 1
  • Rubén Casanova-Sáez
    • 1
    • 2
  • Héctor Candela
    • 1
  • José Luis Micol
    • 1
  1. 1.Instituto de BioingenieríaUniversidad Miguel HernándezElcheSpain
  2. 2.Department of Forest Genetics and Plant Physiology, Umeä Plant Science CentreSwedish University of Agricultural SciencesUmeaSweden

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