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

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at position 81 of the second B-box type domain with a tyrosine, predicted to impair protein function (Table 2). The assessment of M 3 progeny mutant plants for phenotypic variation in life cycle length did not show significant changes when compared to sister lines not carrying the point mutation (data not shown). SNAC1 is a NAM, ATAF, and CUC (NAC) transcription factor expressed in guard cells of rice upon dehydration and involved in regulating stomatal closure (Hu et al., 2006). Its overexpression in transgenic rice plants was shown to greatly enhance tolerance to drought and salt stress while inactivation resulted in increased stomatal aperture and sensitivity to dehydration (Hu et al., 2006; You et al., 2013), suggesting that only gain-of-function mutations in this gene would result in a desirable phenotype. All the three missense mutations identified in the SNAC1 gene in the Volano TILLING population were located within the conserved NAC DNA-binding domain (Hu et al., 2006; Chen et al., 2011) and they were all generating changes in amino acid polarity (Table 2). The C/T transition at position 88 led to the substitution of a very conserved arginine (very hydrophilic) with cysteine (hydrophobic) in a putative nuclear localization signal within the NAC domain (Hu et al., 2006). The other two missense mutations resulted in a proline®serine and a serine®phenylalanine replacements at amino acid positions 106 and 156, respectively. According to the SIFT scores, all the three amino acid substitutions are predicted to affect protein function (Table 2). The progeny of the three M 2 lines carrying the missense mutations are currently under investigation to assess the response under water-limited conditions. The BADH2 gene encodes the enzyme betaine aldehyde dehydrogenase (BADH2) that catalyses the oxidation of a precursor of 2-acetyl-1-pyrroline (2-AP). Inactivation of this enzyme leads to accumulation of 2-AP, the compound responsible of the typical aroma of fragrant rice (Bradbury et al., 2005). The BADH2 enzyme belongs to the nicotinamide adenine dinucleotide (NAD)-dependent aldehyde dehydrogenase family and is predicted to contain three domains based on its three-dimensional structure: a NAD binding, a substrate binding, and an oligomerization domain (Chen et al., 2008). While Volano is a nonfragrant variety carrying the functional BADH2 gene, the missense mutation identified in the TILLING population caused a leucine to phenylalanine substitution at amino acid position 206 in the NAD binding domain, likely to impair protein function according to SIFT prediction (Table 2). Assessment of the M 3 progeny from the missense M 2 mutant line did not reveal the appearance of the fragrance phenotype (data not shown). DISCUSSION Volano is one of the most widely cultivated and important European rice varieties. In Italy, it is the most cultivated variety of the Long A grain group and the second most cultivated variety at national level (Ente Nazionale Risi, 2012a). Volano is representative of those temperate japonica genotypes that are adapted to south European pedoclimatic conditions above the 45th parallel, requiring early maturation and low photoperiod sensitivity (Okumoto et al., 1996; Ichitani et al., 1997). Europe is one of the most northern areas where rice is grown extensively as a crop, together with northern China and Japan (Lu and Chang, 1980). Belonging to the Arborio class, which is the most popular rice for risotto, Volano is considered representative of the traditional Italian high quality rice and is exported worldwide. Due to its strategic relevance for Italian and European breeding programs, this traditional variety was chosen for the development of the first rice TILLING platform of a European temperate japonica accession. The Volano variety has various agronomic traits that may benefit to be improved, including plant height, the duration of the growth cycle, and the resistance to biotic (blast disease) and abiotic (growth in water-limited conditions) stresses. To create the TILLING population EMS was chosen as a mutagenic agent, which has been used to create mutant collections of rice and other cereal species. Different mutagens can produce a different spectrum of mutations and therefore different series of alleles. Gamma and fast neutron irradiation have been shown to induce small (few base pairs) and large (hundreds to thousands of base pairs) deletions, respectively, and mainly result in gene knockout (Li et al., 2001; Sato et al., 2006; Morita et al., 2009). On the other hand, chemical mutagens such as EMS and MNU typically induce SNPs randomly throughout the genome (Henikoff and Comai, 2003; Cooper et al., 2008; Suzuki et al., 2008). These point mutations lead to the generation of a series of polymorphic alleles, including loss of function, that provides a range of different phenotypes with a potential use in crop improvement (Gilchrist and Haughn, 2005). In rice, MNU-induced mutations have been shown to occur at a rate two times higher (1/135 kb) (Suzuki et al., 2008) than those obtained with EMS (1/300 kb) (Till et al., 2007). However, to obtain such a high mutation frequency, the treatment with MNU requires that the flowers are exposed to the mutagen rather than the seeds (as for EMS), making the experimental procedure less amenable to large-scale mutagenesis. The density of one mutation every 373 kb estimated in the Volano TILLING population described here, based on a pilot screening of four target genes in 1152 M 2 lines, was comparable to what has previously been obtained in other rice EMS-mutagenized populations. This observed rate is higher than that reported for the indica rice variety IR64 (1/1000 kb) (Wu et al., 2005) and slightly lower than that obtained in Nipponbare (1/300 kb) (Till et al., 2007). However, this difference is likely to be due to the higher dose of EMS (1.5%) used by Till et al. (2007) compared to that used in this study (0.75%). Overall, the results crop science, vol. 53, november–december 2013 www.crops.org 2557
obtained confirmed the efficiency of the mutagenic treatment and the suitability of the Volano TILLING platform as a source of new genetic variation in temperate japonica. Besides the validation of the TILLING platform, the present work provides genetic material that can be directly exploited for the agronomic improvement of Volano. Currently, one of the main objectives of the breeding programs is the reduction in plant stature. Reduction in total height has been shown to increase plant responses to N inputs, resulting in a higher yield without culm elongation and lodging problems (Ashikari et al., 2002). Moreover, a shorter stature can be beneficial for the plant in terms of tolerance to water-limited conditions, as reduced plant height leads to a significant reduction of the area involved in loss of water by transpiration, which represents one of the main strategies of drought escape (Levitt, 1980). In this study we identified three independent mutations in the SD1 (semidwarf 1) gene, which in rice plays a crucial role in determining plant height (Sasaki et al., 2002). SD1 encodes a GA20-oxidase (GA20ox), a key enzyme in the biosynthesis of gibberellins. In particular, it catalyzes the sequential oxidation and elimination of C-20 in the GA biosynthetic pathway, providing a substrate for the GA3b-hydroxylase (GA3ox) that catalyzes the last step of the synthesis of active GAs (Hedden and Phillips, 2000). Two GA20ox genes (GA20ox-1 and GA20ox-2) have been shown to be present in the rice genome (Monna et al., 2002; Sasaki et al., 2002). GA20ox-1 is predominantly expressed in unopened flowers and is necessary for flowers to develop and fertilize normally (hence ensuring yield) while GA20ox-2 (corresponding to SD1) is highly expressed in the leaf blade and stems. This functional redundancy explains why loss of SD1 function (and consequently GA deficiency) can result in reduction of plant height without seed yield being affected (Monna et al., 2002; Sasaki et al., 2002). Two of the three mutations in the SD1 gene identified in this study displayed a strong phenotypic effect, resulting in a significant reduction (about 21% on average) of plant height. The level of reduction in height is correlated with the allelic status of the mutation, with a stronger effect associated with the homozygous state when compared to the heterozygote. Both the mutations were predicted to affect protein activity: in the line M2_860, the premature insertion of a stop codon generates an inactive truncated GA20ox enzyme and therefore leaf- and stem-specific reduced GA biosynthesis while the line M2_921 carried a substitution of a highly conserved tyrosine, likely to generate a loss of protein function. Unexpectedly, the phenotypic effect on plant stature associated to the missense mutation appeared to be slightly stronger than that observed for the nonsense mutant. This observation would deserve a thorough investigation to unravel the underlying cellular and regulatory mechanisms. Several sd1 alleles that cause semidwarfism in rice have been described and used in breeding programs worldwide to improve the agronomic performance of local varieties (Asano et al., 2007). Dee-geo-woo-gen, the Chinese semidwarf rice cultivar in which sd1 was first identified and the derived high-yielding cultivar IR8 (IRRI, 1967), the first Green Revolution rice variety, carry the same sd1 allele harboring a 383-bp deletion from exon 1 to exon 2 that originates a stop codon (Monna et al., 2002, Sasaki et al., 2002). An independent deletion of 280 bp was found in the coding region of SD1 in the indica semidwarf variety Doongara (Spielmeyer et al., 2002). In addition, several point mutations occurring at different positions in the SD1 coding sequence were shown to cause single amino acid substitutions resulting in semidwarfism, as found in the japonica semidwarf varieties Jikkoku, Reimei, and Calrose 76 (Spielmeyer et al., 2002). The two sd1 mutants identified in the Volano TILLING population described here represent valuable genetic material for breeding programs to enhancing yield performance and adaptation to changing climatic conditions (water scarcity during the plant maturation stage) of European temperate japonica rice varieties. Rice is a short day plant exhibiting a high level of natural variation in flowering time that contributed to its adaptation to grow in a wide range of different environments while maintaining yield production. The Hd1 (Heading date-1) gene targeted for TILLING screening in this study plays a key role in rice by determining flowering time (Yano et al., 2000; Tsuji et al., 2011) and hence represents a good candidate to study the genetic components involved in controlling growth cycle duration in European temperate japonica rice. A short basic vegetative growth phase, together with a low sensitivity to photoperiod, are the most important traits determining the adaptability of rice varieties to cultivation at high latitudes, such as Italy and south Europe in general (Okumoto et al., 1996; Ichitani et al., 1997). Moreover, as with plant height reduction, reducing the growth cycle also represents an effective strategy for drought escaping (Levitt, 1980). In wheat and barley, shortening the crop life-cycle duration has been shown to be an effective breeding strategy to reduce water consumption (Cattivelli et al., 2008). Allelic diversity at the Hd1 locus was demonstrated to be one of the major determinants of flowering time variation in cultivated rice (Takahashi et al., 2009). The TILLING screening of the Volano population yielded one missense mutation in the Hd1 coding sequence that was predicted to affect protein function although no significant phenotypic variation in growth cycle duration was revealed in the progeny. These preliminary results may indicate that the missense mutation does not affect the protein function or that Hd1 is not the most relevant gene involved in controlling the life cycle duration in European temperate japonica germplasm. The photoperiodic control of flowering in 2558 www.crops.org crop science, vol. 53, november–december 2013
Page 1 and 2: Published August 26, 2013 RESEARCH
Page 3 and 4: Table 1. Target genes and primers u
Page 5 and 6: Figure 1. Examples of morphological
Page 7: dioxigenases from other plant speci
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Page 13: Till, B.J., J. Cooper, T.H. Tai, P.

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

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