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

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was stopped after the extension step of 10 min at 72°C. Before sequencing, the PCR products were purified from free primers and nucleotides using ExoSAP-IT (GE Healthcare), following the manufacturer’s instructions. Sequencing reactions were set up in a final volume of 10 μL with 10 to 40 ng of purified PCR product and a primer concentration of 0.32 μM using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Life Technologies Corp.). The following conditions were used: 96°C for 1 min and 25 cycles of 96°C for 10 sec, 55°C for 5 sec, and 60°C for 4 min. Samples were then ethanol and ethylenediaminetetraacetic acid precipitated and analyzed on the ABI3730 DNA sequencer (Applied Biosystems, Life Technologies Corp.). The output sequences were analyzed with the software Mutation Surveyor (SoftGenetics, 2010) to identify and validate the mutations. To predict whether a point mutation would have an effect at the protein level, the target amino acid sequences (semidwarf 1 [SD1], Heading date-1 [Hd1], Stress-responsive NAC 1 [SNAC1], and betaine aldehyde dehydrogenase [BADH2]) were analyzed with SIFT (Sorting Tolerant From Intolerant) (Kumar et al., 2009) to identify the regions that do not tolerate substitutions. Phenotypic Field Evaluations Rice plants were grown in the field at the Consiglio per la Ricerca e la Sperimentazione in Agricoltura-Rice Research Unit in Vercelli (Italy). Seeds were sown directly into dry soil and conventional submersion was established at the third to fourth leaf developmental stage until 1 mo before harvesting. Standard fertilization was performed with 150 kg N ha –1 , 40 kg P 2 O 5 ha –1 , and 150 kg K 2 0 ha –1 . The total rate of N and K was fractionated in two times (one-third before sowing and two-thirds at panicle initiation stage) to improve the efficiency of the two fertilizers. During the growing season the following agronomical traits were assessed according to the International Union for the Protection of New Varieties of Plants guidelines (UPOV): total plant height (cm), measured at maturity on the primary tiller, considering the distance from the soil to the tip of the panicle (excluding the awn); panicle length (cm), measured at maturity on the primary tiller, considering the distance from the base to the tip of the panicle (excluding the awn); panicle type and panicle attitude in relation to stem, both assessed after ripening; grain characteristics (length, width, and weight), assessed after ripening; and growth cycle, considering the days from seed sowing till seed ripening. At maturity, paddy rice samples were harvested by hand and stored in controlled conditions. RESULTS Development of the Volano TILLING Population The Volano TILLING platform was developed using the chemical mutagen EMS, shown to be effective for rice by Till et al. (2007) using the japonica cultivar Nipponbare, in which a density of induced mutations of 1/300 kb was reported. However, EMS toxicity and efficiency is genotype specific and can vary greatly within the same species and subspecies, as shown by the lower mutation rate observed in the EMS-mutagenized indica variety IR64 (1/1000 bp) (Wu et al., 2005). A pilot test using different doses of EMS was performed on small batches of Volano seeds to identify the optimal quantity of EMS to be used for large-scale mutagenesis (data not shown). Based on the results obtained, the mutagenesis was performed using 0.75% EMS on 20,000 seeds of Volano. The mutagenized seeds were sown directly in field conditions and the surviving M 1 plants were grown to maturity and self-fertilized. Approximately 2000 fertile M 1 lines were harvested and 20 seeds from each line were planted to generate the M 2 generation. During the growth of the M 2 population, several mutant phenotypes were observed at different developmental stages, ranging from seedling to maturity stage. The most common mutant phenotypes were related to dwarfism, plant sterility, alteration in plant architecture, growth cycle duration, and seed morphology. Examples of the phenotypes observed are shown in Fig. 1. A summary of the phenotypic data collected for plant height, panicle length, growth cycle, and grain size and shape is reported in Supplemental Table S2 and Supplemental Fig. S1. While the majority of the M 2 lines had an average plant height of 98.6 ± 9.7 cm typical of Volano, 2.1% of the mutagenized lines showed a semidwarf phenotype with a reduction in height >20 cm (Supplemental Fig. S1). Similarly, 1.5% of the M 2 mutagenized plants showed increased panicle length (>25 cm) with respect to that of Volano (22 cm) (Supplemental Fig. S1). Variability in growth cycle duration was also observed, with a range difference of 40 d between the earliest and the latest M 2 lines. Few “early” mutants showing a shortening of at least 20 d between the sowing and the ripening time were recorded (Supplemental Fig. S1). From the 20 M 2 seeds sown for each M 1 line, one M 2 fertile and healthy-looking individual plant was chosen for DNA isolation and for seed harvest. In total 1860 M 2 lines were selected to constitute the Volano mutagenized TILLING collection. Detection of Mutations in Candidate Genes To estimate the efficiency of the EMS treatment and to evaluate the mutation frequency in the Volano EMS-treated population, TILLING was initially performed on DNA isolated from 1152 M 2 lines organized using to a two-dimensional pooling strategy. A similar strategy was successfully used to screen another rice mutagenized population (Till et al., 2007), where eightfold pools were used in both dimensions. The advantage of using a 2D pooling strategy is that the M 2 line containing the mutation can be directly identified without sequencing all the samples in the pool. For this pilot screening, four genes of relevance for agronomic and quality traits were selected: SD1 (semidwarf 1), involved in plant height determination (Sasaki et al., 2002), Hd1 (Heading date-1), which plays a crucial role in determining the flowering time (Yano et al., 2000), SNAC1 (Stressresponsive NAC 1), which was shown to be a central player in stomata guard cell closure under water stress conditions (Hu crop science, vol. 53, november–december 2013 www.crops.org 2553
Figure 1. Examples of morphological mutant phenotypes observed in the M 2 generation of the Volano ethyl methane sulfonate–mutagenized population. (a) Abnormal spikelet pigmentation, (b) altered grain shape (c and f) dwarfism, (d) early flowering, and (e) late flowering. et al., 2006), and BADH2, which is responsible of the typical aroma of fragrant rice (Bradbury et al., 2005). The TILLING screening identified three mutations in the SD1 gene, one in Hd1, four in SNAC1, and two in BADH2 (Table 2). The mutations observed were predominantly G/C to A/T transitions (90%), except for one T/C transition detected in the SD1 gene. These results are in line with other EMS mutagenesis studies reported in literature. In Arabidopsis thaliana, maize (Zea mays L.), and wheat, G/C to A/T transitions were shown to make up more than 99% of all EMS-induced mutations (Greene et al., 2003; Henikoff and Comai, 2003) whereas in other species such as tomato, barley (Hordeum vulgare L.), and rice, these transitions represent only 70% of the observed mutations (Caldwell et al., 2004; Till et al., 2007; Minoia et al., 2010). The only T/C transition detected was heterozygous and therefore is unlikely to have been a naturally occurring mutation, considering the low estimated rate of spontaneous mutations (10 –7 to 10 –8 bp per generation) (Greene et al., 2003). All but one of the mutations identified occurred in exons (Table 2), which may be expected considering that 25% of the 4035 nucleotides used for the screening spanned introns (Table 3). Based on the predicted effect on the protein product, we found 70% missense, 20% silent, and 10% nonsense mutations. Besides the nonsense mutation, the majority of the missense mutations detected (six out of seven) were likely to generate nonfunctional protein products, according to SIFT predictions (Kumar et al., 2009). The SIFT algorithm predicts whether an amino acid substitution in a protein will have a phenotypic effect based on its degree of conservation in alignments derived from closely related sequences. The assumption is that amino acid residues at positions important for protein function should be conserved throughout evolution among members of a protein family (Kumar et al., 2009). All but one (the silent base change in SNAC1 mentioned above) of the mutations identified were found to be heterozygous (Table 3). To estimate the mutation rate, 200 bp were subtracted from the length of each screened amplicon according to Greene et al. (2003), who reported the difficulty to reliably detect mutations close to the TILLING primers. The resulting estimated average mutation density in the 2554 www.crops.org crop science, vol. 53, november–december 2013
Page 1 and 2: Published August 26, 2013 RESEARCH
Page 3: Table 1. Target genes and primers u
Page 7 and 8: dioxigenases from other plant speci
Page 9 and 10: obtained confirmed the efficiency o
Page 11 and 12: Sorghum bicolor L. Moench resulting
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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