V. Molecular Mapping and QTL Analysis

30. Molecular markers linked to genes for tolerance to ferrous toxicity in rice seedlings

P. WU1, A. LOU1, J. ZHU1, L. ZHANG1, Y. WU1, N. HUANG2 and D. SENADHIRA2

1 ) Department of Biological Science, Zhejiang Agricultural University, Hangzhou, 310029 China
2) The International Rice Research Institute, P.O. Box 933, 1099 Manila, Philippines

Molecular markers linked to genes for tolerance to ferrous (Fe2+) toxicity in rice seedlings were identified by using 175 DNA markers mapped on all chromosomes of a double haploid population derived from a cross between an upland variety, Azucena and an indica variety, IR64 (Huang et al. 1997).

In preliminary screening using toxic and non-toxic solution cultures, no leaf bronzing was observed in Azucena under Fe2+ stress with 250 mg Fe2+/L at pH 4.5 for 4 weeks, but clear symptoms appeared in IR64. IR64 also had a large decrease in shoot biomass when compared with that of Azucena. Experiments were conducted from April to May, 1996 for testing the population (135 lines) under the same conditions as in screening experiment. Leaf bronzing index (LBI) defined as the ratio of bronzed leaf number to total leaf number and relative decrease in shoot dry weight (RDS) from stress to normal culture were used to score the phenotypic tolerance in the population. Segregation for bronzing was observed among the lines. Distribution of LBT was not normal with about 40 lines showing no bronzing (LBI score < 0.05). The values of LBI from 0.05 to 0.85 showed normal distribution. The distribution of RDS showed same skewness toward the tolerant genotype. The results indicated that the tolerance may be controlled by a major gene and some modifiers.

Fig. 1. The most likely positions of gene loci for tolerance to Fe2+ detected from a doubled haploid population from IR64/Azucena. Chromosomal segments associated with gene loci for leaf bronzing index (LBI), relative decrease in shoot dry weight (RDS) from normal culture to Fe2+ stress, ascorbate peroxidase activity (APA), glutathione reductase activity (GRA), dehydroascorbate concentation (DHA) and the ratio of DHA to ascorbate concentration (AS), respectively, under Fe2+stress are indicated as above.

Single locus analysis (SAS/GLM) and interval mapping analysis (mapmaker/QTL) were used to detect QTLs conditioning the tolerance. Two QTLs were detected on chromosome 1, with one linked to RG345 and the other flanked by RG810 and RG331 for both LBI and RDS. The one linked to RG345 explained about 31 % of the total variation in LBI and about 20% in RDS and for the other, about 15% and 14% respectively. The third QTL detected on chromosome 8 linked to RG978 explained about 10% of total variation in RDS (Fig. 1 ).

To investigate the tolerance mechanism, activities of enzymes and concentrations of substrates involved in the scavenging system for H2O2 that could be generated through the Fenton Reaction (Gutteridge and Halliwell 1992) were measured for IR64 and Azucena under both normal and Fe2+ stress. No significant differences were found in ascorbate peroxidase activity (APA), glutathione reductase activity (GRA) and in concentrations of ascorbale (AS) and dehydroascorbate (DHA) under normal culture between the parents. Under Fe2+ stress. Azucena was about twice higher in activities of the enzymes and lower in concentration of DHA than those of IR64. APA, GRA, DHA and the ratio of DHA to AS under Fe2+ stress were measured for the DH population in 1997 April. Wide segregation for the parameters was found. The loci located in the interval between RG345 and RZ19 were detected for APA, GRA, DHA and DHA/AS with about 23%, 10%, 10% and 25, respectively, of the total variations in the parameters. For GRA, DHA and DHA/AS, one locus linked to RG769 on chromosome 7 was detected with about 16%, 10% and 14%, respectively, of the total variations. The results suggested that tissue tolerance in rice for Fe2+ toxicity may be, at least partially, attributable to H2O2 scavenging system.

References

Huang, N., A. Parco, T. Mew, G. Magpantay, S. McCouch, E. Guilderdoni. J. Xu, P. Subudhi, R. Angeles and G.S. Khush. 1997. RFLP mapping of isozymes, RAPD and QTLs for grain shape, brown planthopper resistance in a doubled haploid rice population. Molecular Breeding 3: 105-1 13.

Gutteridge, J.M.C. and B. Halliwell. 1992. Iron and Oxygen: A dangerous mixture. In Iron Transport and Storage, P. Ponka. H.M. Schulman and R.C. Woodworth (eds.). CRC Press p57.