Biochemical and molecular characterization of two low-phytate pea lines
Phytate is the major storage form of phosphorus in crop seeds, but is not well digested by humans and non-ruminant animals. In addition, phytate chelates several essential micronutrients which are also excreted contributing to phosphorus pollution in the environment. This research was aimed at the biochemical and molecular characterization of two low phytate pea mutant lines, 1-150-81 and 1-2347-144 developed at the Crop Development Centre, University of Saskatchewan in collaboration with Dr. Victor Raboy, USDA, Idaho. Low phytic acid (lpa) crops are low in phytic acid and high in inorganic phosphorus (Pi). In Study I, two lpa pea genotypes, 1-150-81, 1-2347-144, and their progenitor CDC Bronco were evaluated in field trials for two years. The lpa genotypes did not significantly differ from CDC Bronco in all agronomic traits assessed except for lower seed weight and grain yield. The concentration of IP6 at 14 DAF was not significantly different among CDC Bronco, 1-150-81 and 1-2347-144. However, the concentrations of IP6 among CDC Bronco, 1-150-81 and 1-2347-144 started to differ significantly from 21 DAF onwards. The lpa genotypes 1-150-81 and 1-2347-144 showed 65% and 60% reduction in IP6, respectively, when compared to their progenitor CDC Bronco at 49 DAF. The Pi concentrations between the lpa genotypes were similar and significantly higher than CDC Bronco from 21 DAF to 49 DAF. At 49 DAF, 1-150-81 and 1-2347-144 were 72 and 84% higher in Pi, respectively, than CDC Bronco. The total P concentration was similar in lpa genotypes and CDC Bronco throughout the seed development. This study elucidated the rate and accumulation of phosphorus compounds in lpa genotypes. In Study II, aiming at understanding the genetic basis of the lpa mutation in pea lines 1-150-81 and 1-2347-144, a 1530 bp open reading frame of myo-inositol phosphate synthase gene (MIPS) was amplified from CDC Bronco and the lpa genotypes. Sequencing results showed no difference in coding sequence in MIPS between CDC Bronco and lpa genotypes. Transcript levels of both MIPS and myo-inositol tetrakisphosphate1-kinase (ITPK1) were relatively lower at 49 DAF than at 14 DAF for CDC Bronco and lpa lines. There was no difference in expression level of both MIPS and ITPK1 between CDC Bronco and the lpa genotypes at 49 DAF. The data demonstrated that mutation in MIPS was not responsible for lpa trait in pea. Study III was aimed at developing a single nucleotide polymorphism (SNP) based genetic linkage map and mapping genomic regions associated with phytic acid-phosphorus (PA-P) concentration using PR-15 recombinant inbred lines (RILs) derived from a cross between a low phytate (lpa) pea genotype, 1-2347-144 and normal phytate pea cultivar CDC Meadow. A total of 163 RILs were genotyped using 1536 SNP markers in an Illumina GoldenGate array. Three hundred and sixty seven polymorphic SNP markers, ordered into 7 linkage groups (LGs), generated a linkage map with a total length of 437.2 cM. The phytic acid locus was mapped on to LG5. A quantitative trait locus (QTL) for iron bioavailability was mapped on to the same location in LG5 as phytic acid concentration. Potential benefits arising out of this research include improved bioavailability of phosphorus, iron and zinc in foods and feeds, less phosphorus excretion and environmental pollution and a saving in feed costs.
DegreeDoctor of Philosophy (Ph.D.)
CommitteeTar'an, Bunyamin; Gray, Gordon R.; Beattie, Aaron; Classen, Henry; Sharpe, Andrew; Bai, Yuguang
Copyright DateAugust 2014
Low-phytate field pea, biochemistry, molecular genetics