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

More documents

Recommendations

Info

Table 2. Ethyl methane sulfonate–induced mutations identified in the Volano targeting induced local lesions in genomes (TILL- ING) population. Gene Plant identity † Nucleotide change Position from ATG ‡ Position in CDS ‡ Amino acid change SIFT score § Amino acid properties SD1 860 G/A 144 exon1 W > STOP 0.00 – 1427 C/T 473 exon1 S158F 0.09 N and P ® N and NP 921 T/C 802 exon2 Y234H 0.00 N and P ® B and P Hd1 782 G/A 242 exon1 C81Y 0.05 N and SP ® N and P SNAC1 1213 C/T 262 exon1 R88C 0.00 B and P ® N and SP 951 C/T 316 exon1 P106S 0.03 N and NP ® N and P 1523 C/T 467 exon1 S156F 0.00 N and P ® N and NP 269 G/A 756 exon2 E > E – – BADH2 1133 C/T 2690 exon6 L206F 0.00 N and NP ® N and NP 108 C/T 2829 intron6 – – – † M 2 line carrying the mutation. ‡ Positions refer to the genomic sequences (Michigan State University v.6.1 rice genome annotation [Ouyang et al., 2007]). CDS, coding DNA sequence. § Prediction based on SIFT (sorting tolerant from intolerant) algorithm (Kumar et al., 2009). An amino acid substitution predicted to impair the protein function has a score ≤0.05; if tolerated the score is >0.05. N, neutral; P, polar; SP, slightly polar; NP, nonpolar; B, basic. Table 3. Features of the observed mutations identified by the targeting induced local lesions in genomes (TILLING) screening of the Volano ethyl methane sulfonate–mutagenized population. Gene Amplicon size (bp) CDS † (bp) Total Silent Missense Nonsense HET ‡ HOM § SD1 1081 879 3 0 2 1 3 0 Hd1 972 828 1 0 1 0 1 0 SNAC1 1142 951 4 1 3 0 3 1 BADH2 840 350 2 1 1 0 2 0 Total 4035 3008 10 2 7 1 9 1 % 20% 70% 10% 90% 10% † CDS, coding DNA sequence. ‡ HET, heterozygous. § HOM, homozygous. Volano TILLING population observed in the pilot screen was 1/373 kb. TILLING for Crop Improvement of European Temperate Rice Four rice genes were selected to validate the Volano TILLING population based on the agronomic impact of the phenotypic effect expected from the alteration and/or inactivation of the encoded polypeptides. Reduction in plant height represents one of the most important goals of the current breeding programs to improve the field performance of the Volano variety. To identify Volano lines with shorter stature, we screened for EMS-induced mutations in the SD1 gene, which is the most important gene of the rice Green Revolution (Sasaki et al., 2002). SD1 encodes for a gibberellin 20-oxidase, a key enzyme in the gibberellin (GA) biosynthetic pathway, which plays a central role in determining plant height by affecting cellular and internode elongation. Alterations in the SD1 genomic sequence and encoded protein result in decreased levels of GAs due to the defective gibberellin 20-oxidase (GA20ox) enzyme, which lead to plants with shorter and thicker culms, improved lodging resistance, and a greater harvest index (Monna et al., 2002; Sasaki et al., 2002; Spielmeyer et al., 2002). The SD1 gene consists of three exons encoding a protein product of 389 amino acids with two predicted conserved functional domains, a non-heme dioxygenase N-terminal (DIOX_N) domain (IPR026992) spanning residues 64 to 168 and a oxoglutarate- and iron-dependent dioxygenase domain (IPR005123), typical of plant dioxygenases involved in hormone and pigment synthesis, spanning residues 224 to 324. The TILLING screening of SD1 in the Volano mutagenized population identified three independent point mutations, of which two were predicted to generate missense products and one a truncated protein (Table 2). The C/T transition in line M2_1427 led to a nonsynonymous change from a polar serine to a nonpolar phenylalanine at amino acid position 158 of the DIOX_N domain, which according to the SIFT prediction presumably does not affect protein function (Kumar et al., 2009). Based on sequence alignment, four residues other than serine were found at a corresponding position 158 in orthologous crop science, vol. 53, november–december 2013 www.crops.org 2555
dioxigenases from other plant species, among which are phenylalanine, threonine (less polar than serine), and to a lesser extent charged residues such as aspartic acid and histidine (data not shown). This sequence diversity indicates that this amino acid position may not be essential for enzyme function. In contrast, the T/C transition in line M2_921 caused a tyrosine®histidine substitution at position 234 in the oxoglutarate- and iron-dependent dioxygenase domain. The hydrophobic Tyr234 is highly conserved among plant dioxygenases and its replacement by any other residue is expected to be highly deleterious for the SD1 protein function (SIFT score: 0.00). The G/A transition in the M2_860 line created a premature stop codon at position 48 of the protein sequence, generating a predicted nonfunctional product lacking the majority of the polypeptide (341 amino acid residues), including the two functional domains. All the M 2 identified mutations in the SD1 gene were heterozygous. To confirm the inheritance of the induced mutations in the SD1 gene and to explore their phenotypic effect, 30 M 3 seeds derived from each M 2 mutant line were sown in the field and grown to maturity. The DNA was isolated from each M 3 plant and the single nucleotide polymorphism (SNP) alterations confirmed by sequencing. For the M 2 lines 921 and 860, M 3 progeny plants carrying the corresponding mutation in the homozygous state showed a statistically significant decrease in plant height when compared to homozygous wild-type plants (Fig. 2) (Wilcoxon signed-rank test: p < 0.01). An average height reduction of 19.1 ± 2.2 cm was observed in case of M 3 homozygous mutant progenies derived from M2_860 and 23.8 ± 4.7 cm in case of M2_921 (Fig. 3). Furthermore, the M 3 plants heterozygous for the mutations showed an intermediate stature between the homozygous mutant and the wild-type (Fig. 3) (Wilcoxon signed-rank test: p > 0.05), supporting the hypothesis that the observed phenotypes arose specifically from the EMS-induced alterations in the SD1 gene. In agreement with the predicted effect of the mutation on the protein function, the M 3 mutant progeny plants derived from the line M2_1427 did not differ significantly in plant height from the mutagenized lines not carrying the mutation (Fig. 3) (Wilcoxon signed-rank test: p > 0.05). Hd1, a rice ortholog of the A. thaliana flowering time gene CONSTANS (Putterill et al., 1995), encodes a transcriptional activator that promotes heading under shortday conditions and inhibits it under long-day conditions (Yano et al., 2000). By acting in a complex network with other flowering genes, such as Hd3a, Ehd1, and Gdh7, it plays a crucial role in determining flowering time variation in rice (Tsuji et al., 2011), which is a relevant trait in case of growth at northern latitudes such as Europe. The Hd1 protein contains an N-terminal zinc finger domain involved in DNA-binding and a C-terminal CCT domain Figure 2. Example of a “semidwarf” Volano mutant. A M 3 plant from line M2_921 carrying the homozygous mutation is shown (on the left) in comparison with a mutagenized plant not carrying the mutation (on the right) at maturity stage. Figure 3. Segregation of plant height among M 3 progenies of the three identified sd1 mutants (lines M2_860, M2_921, and M2_1427). For each M 2 line, the blue bars represent the average height of the homozygous wild-type plants, red bars represent plants carrying the mutation in the heterozygote state, and green bars represent the homozygous mutant plants. Error bars are indicated. Double asterisks (**) indicate significant differences between homozygous mutants and wild-type (wt) (p < 0.01; Wilcoxon signed-rank test). het, heterozygous. that functions as nuclear localization signal (Yano et al., 2000). Inactivation of the Hd1 gene results in earlier heading under long days with a reduction of the growing cycle (Yano et al., 2000). One missense mutation was identified in the Volano TILLING population in this gene (Table 2). The mutation was located in the zinc finger domain and caused the substitution of a conserved cysteine residue 2556 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: Figure 1. Examples of morphological
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

Create successful ePaper yourself

Delete template?

Save as template?