Rapid identification of angulata leaf mutations using next-generation sequencing
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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).
KeywordsArabidopsis Chloroplast mutants Cloning-by-sequencing NGS SHOREmap Whole-genome sequencing
Days after stratification
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.
- Dong H, Fei GL, Wu CY, Wu FQ, Sun YY, Chen MJ, Ren YL, Zhou KN, Cheng ZJ, Wang JL, Jiang L, Zhang X, Guo XP, Lei CL, Su N, Wang H, Wan JM (2013) A rice virescent-yellow leaf mutant reveals new insights into the role and assembly of plastid caseinolytic protease in higher plants. Plant Physiol 162:1867–1880PubMedCrossRefPubMedCentralGoogle Scholar
- Esteve-Bruna D, Pérez-Pérez JM, Ponce MR, Micol JL (2013) incurvata13, a novel allele of AUXIN RESISTANT6, reveals a specific role for auxin and the SCF complex in Arabidopsis embryogenesis, vascular specification, and leaf flatness. Plant Physiol 161:1303–1320PubMedCrossRefPubMedCentralGoogle Scholar
- Geisler M, Kolukisaoglu HU, Bouchard R, Billion K, Berger J, Saal B, Frangne N, Koncz-Kalman Z, Koncz C, Dudler R, Blakeslee JJ, Murphy AS, Martinoia E, Schulz B (2003) TWISTED DWARF1, a unique plasma membrane-anchored immunophilin-like protein, interacts with Arabidopsis multidrug resistance-like transporters AtPGP1 and AtPGP19. Mol Biol Cell 14:4238–4249PubMedCrossRefPubMedCentralGoogle Scholar
- Mollá-Morales A, Sarmiento-Manús R, Robles P, Quesada V, Pérez-Pérez JM, González-Bayón R, Hannah MA, Willmitzer L, Ponce MR, Micol JL (2011) Analysis of ven3 and ven6 reticulate mutants reveals the importance of arginine biosynthesis in Arabidopsis leaf development. Plant J 65:335–345PubMedCrossRefGoogle Scholar
- Nelissen H, Fleury D, Bruno L, Robles P, De Veylder L, Traas J, Micol JL, Van Montagu M, Inze D, Van Lijsebettens M (2005) The elongata mutants identify a functional elongator complex in plants with a role in cell proliferation during organ growth. Proc Natl Acad Sci USA 102:7754–7759PubMedCrossRefPubMedCentralGoogle Scholar
- Ochando I, Jover-Gil S, Ripoll JJ, Candela H, Vera A, Ponce MR, Martínez-Laborda A, Micol JL (2006) Mutations in the microRNA complementarity site of the INCURVATA4 gene perturb meristem function and adaxialize lateral organs in Arabidopsis. Plant Physiol 141:607–619PubMedCrossRefPubMedCentralGoogle Scholar
- Quesada V, Sarmiento-Manús R, González-Bayón R, Hricová A, Pérez-Marcos R, Gracía-Martínez E, Medina-Ruíz L, Leyva-Díaz E, Ponce MR, Micol JL (2011) Arabidopsis RUGOSA2 encodes an mTERF family member required for mitochondrion, chloroplast and leaf development. Plant J 68:738–753PubMedCrossRefGoogle Scholar
- Rédei GP, Hirono Y (1964) Linkage studies. Arabidopsis Inf Serv 1:9–10Google Scholar
- Shimoni-Shor E, Hassidim M, Yuval-Naeh N, Keren N (2010) Disruption of Nap14, a plastid-localized non-intrinsic ABC protein in Arabidopsis thaliana results in the over-accumulation of transition metals and in aberrant chloroplast structures. Plant Cell Environ 33:1029–1038PubMedCrossRefGoogle Scholar
- Tabata R, Kamiya T, Shigenobu S, Yamaguchi K, Yamada M, Hasebe M, Fujiwara T, Sawa S (2012) Identification of an EMS-induced causal mutation in a gene required for boron-mediated root development by low-coverage genome re-sequencing in Arabidopsis. Plant Signal Behav 8:18–24Google Scholar