Casella et al. - 2013 - TILLING in European Rice Hunting Mutations for Cr

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ice involves a complex network of genes (reviewed in Tsuji et al., 2011) and variation at other important regulators is expected also to contribute at determining adaptation of rice cultivars to high-latitude climate regions (Xue et al., 2008; Takahashi et al., 2009). The isolation of additional natural or induced Hd1 mutants from European rice accessions will clarify the role of Hd1 in regulating the growth cycle in temperate areas. A rapid and effective stomatal closure in a crop is considered a positive trait for the improvement of water use efficiency in drought conditions (Sinclair and Muchow, 2001). A previous study in A. thaliana reported the cloning of a quantitative trait locus regulating transpiration efficiency while maintaining biomass production, hence uncoupling the usually negative association between transpiration efficiency and both stomatal conductance and biomass production (Masle et al., 2005). Based on this finding the SNAC1 gene, which has been shown to be involved in stomatal closure mechanisms in the early stages of drought stress (Hu et al., 2006), was selected as a target for TILLING screening of the Volano mutagenized population. Three independent M 2 lines carrying missense mutations in the NAC domain of the SNAC1 gene were identified and the M 3 progeny plants will be evaluated in water-limited conditions in the next growing season. A “positive” phenotypic effect would be predicted only in case a gain-of-function mutation occurred, causing an altered expression of the gene that constitutively activates it. However, those kinds of mutations are expected to occur more frequently in regulatory rather than coding regions, where loss of function mutations are more probable to take place. In this sense, the next experiment to be undertaken would be the screening for mutations in the regulatory sequence of SNAC1. A quality trait of rice that is highly appreciated both in Asian and European countries is the presence of aromatic compounds leading to scented rice grains. The major gene for grain fragrance ( fgr) encodes the enzyme BADH2, inactivation of which leads to the accumulation of the precursor 2-AP that releases the typical “basmatilike” perfume (Bradbury et al., 2005). One single base substitution in the BADH2 gene was identified in the Volano TILLING population causing a missense mutation. The point mutation identified differs from all the polymorphisms described so far that have been shown to impair the enzymatic function, leading to the fragrant phenotype (Kovach et al., 2009), and does not affect the functional NAD-binding domain. In this work we have shown that TILLING, based on classical mutagenesis to create new genetic variation without the use of transgenic technologies, represents a powerful technique for crop improvement that can be applied to temperate japonica rice grown in European areas. The initial screening of the EMS-mutagenized Volano collection identified a number of mutations in agronomically and quality relevant genes that can provide genetic material of direct use in marker-assisted breeding programs. The validated Volano TILLING EMS-mutagenized population represents a valuable tool to assess the function of candidate genes in controlling target traits of high relevance for cultivation of rice in temperate areas, such as disease resistance, abiotic stress tolerance, and nutritional properties of the rice seeds. The use of next generation sequencing (NGS) approaches will facilitate the discovery of mutants in a large set of candidate genes (Rigola et al., 2009; Tsai et al., 2011). In the future, NGS technologies will also facilitate the resequencing of mutagenized lines showing phenotypes of relevance in agronomic and quality traits (forward genetics) to identify novel but yet unknown genetic components. Supplemental Information Available Supplemental material is available at http://www.crops. org/publications/cs. Acknowledgments We would like to thank Prof. Chun-Ming Liu for sharing the EMS mutagenesis protocol and Dr. Laura Rossini and Dr. John Williams for critical reading of the manuscript. This work was financially supported by the Italian Ministry of Agriculture, Food and Forestry Policies within the framework of the project VALORYZA (D.M. 301/7303/2006), by the CARIPLO Foundation within the DRYRICE project (2008-3179) and by the AGER Foundation within the RISINNOVA project (2010-2369). 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Page 1 and 2: Published August 26, 2013 RESEARCH
Page 3 and 4: Table 1. Target genes and primers u
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Casella et al. - 2013 - TILLING in European Rice Hunting Mutations for Cr

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