Mutational Enhancement, Molecular Mapping And Genetic Improvement Of Low Phytic Acid Mutant Germplasm In Rice (Oryza Sativa L.)

Phytic acid (PA) is the primary storage form of phosphorus (P) in cereal seeds; it chelates important minerals such as Zn²⁺,Fe³⁺,K⁺ and Ca²⁺ and virtually renders them indigestible by human and non-ruminant animals. Therefore, PA is commonly regarded as the major anti-nutritional component in cereal grains and can result in P pollution. In the present study, several low phytic acid (lpa) mutant lines were developed through physical and chemical mutagenesis; the screening procedure of mutant lpa seeds was simplified; the genetic control of lpa mutations was investigated; and two lpa mutations were molecularly mapped; and the effect of two lpa mutations on agronomic traits including yield and seed viability was examined. The results are summarized below:1. A simplified protocol for screening of high inorganic P (HIP) mutant seeds was established through studies on the influencing factors, i.e., sample pre-treatment, extraction time and condition, color development time, on the measurement of inorganic P (Pi) in seeds of Xieqingzao and its lpa mutant line Os-lpa-XQZ-1.2. Throughγ-rays irradiation or additionally with NaN₃ treatment of indica rice varieties Xieqingzao (XQZ), Minghui 86 (MH86), R6547 and japonica variety Xiushui 110 (XS110), thirteen lpa mutant lines were independently developed. Eleven of them are non-lethal, i.e., Os-lpa-XQZ-1-1～2, Os-lpa-MH86-1-1～7, Os-lpa-XS110-1 and Os-lpa-XS110-2, the others are homozygous lethal, i.e., Os-lpa-XQZ-3, Os-lpa-R6547-3 and Os-lpa-XS110-3. No homozygous lpa plants could be obtained through conventional seed germination and growth under field conditions; but some plants were successfully produced through in vitro rescue of mature embryo for Os-lpa-XS110-3 and Os-lpa-R6547-3. In these homozygous non-lethal mutants, seed phytic acid P content is reduced approximately from 34% to 64%, and inorganic P is increased about 3⁶-fold but total P is unaltered.3. Genetic analysis showed that the lpa trait is inherited in a single recessive gene controlled mode. Allelism tests further showed the mutations in Os-lpa-XQZ-1,Os-lpa-XS110-1,Os-lpa-XS110-2 and Os-lpa-MH86-1 are mutually non-allelic. The lpa mutation in Os-lpa-XQZ-1 is allelic to the previously reported lpa1-1 locus in KBNT lpa1-1; two lpa mutations in the two mutants from XQZ, i.e., Os-lpa-XQZ-1-1～2, are allelic, and all lpa mutations in the 7 mutant lines of MH86, i.e., Os-lpa-MH86-1-1～7, are also mutually allelic. In the progenies of crosses between lines with non-allelic mutations, the segregation modes except the one of Os-lpa-XQZ-1 and Os-lpa-MH86-1 which fit into a two independent gene mode, were very complex, which indicated the genetic linkage and functional relationship are complicated among them. 4. The two lpa mutations in Os-lpa-XS110-land Os-lpa-XQZ-1 were mapped using SSR markers. The two bulk DNA pools were constructed for F₂ seedlings, of both lpa mutant type and wild type, of the sub-species cross between G0133 and Os-lpa-XS110-1. Each F₂ seed was cut into endosperm part and embryo part, the former was used for determination of Pi phenotype (HIP or wild type), and the latter was used for production of seedlings through in vitro culture. A total of 358 SSR loci were analyzed using the two DNA pools and the loci RM143, -571, -468 and -3199 were found linked with the lpa mutation. Further analysis using individual plants mapped the mutation on the chromosome 3S with the genetic distance of～1.907cM to RM3199. Since RM3199 is very close to LOC-Os03g52760, a locus encoding the putative myo-inositol kinase (MIK, EC 2.7.1.64). Since the lpa3 in maize resulted from the MIK, the lpa mutation in Os-lpa-XS110-1 is very likely occurred in the LOC-Os03g52760 locus. Similar analysis of the Os-lpa-XQZ-1/Huahui No.7 F₂ populations found the mutation is linked to RM208 and RM3542 on chromosome 2L, where the KBNT lpa1-1 mutation was previously mapped. The result was consistent with allelism test of the mutations.5. In addition to comparisons between the two lpa mutants and their corresponding parent varieties, the agronomic, yield and seed viability was investigated for mutant and wild type the F2 plants and the F2:3 lines in three populations. The results indicated that the lpa traits severely affected the yield, 1000-grain weight (TGW) and the vigor of seeds. However, some mutant lines were found having superior yield and TGW to lpa parent, and even to wild type parent, which implied these agronomic traits could be improved through crossing breeding.