Generation, Characterization And Molecular Analysis Of Low Phytate Mutations Germplasm In Soybean (Glycine Max L. Merr.)

Phytic acid (PA, myo-inositol 1,2,3,4,5,6-hexakisphosphate) is a ubiquitous component and the most abundant form of P in seeds, accounting for more than 60% of the total seed P. PA is a important anti-nutritional compound, which can chelate mineral nutrients, i.e. Fe and Zn, into salt compounds, which is deposited in protein bodies in seeds. In soybean (Glycine max), most of the PA is accumulated in cotyledons. Since phytates are largely indigestible by humans and other non-ruminant animals, therefore, the bioavailability of mineral nutrients is greatly reduced. More and more attentions have been drawn to the increase of feed cost and water pollution resulting from the decreased bioavailability of P deposited in phytic acid. Recently, breeding of low phytic acid (Ipa) crops has opened a new approach to settle PA-related nutritional and environmental issues.In this study, using seed gamma irradiation, two Ipa mutant lines were developed from two commercial soybean varieties using colorimetric assay of inorganic P (Pi) content. The main results were as follows:1. One mutant plant of about 5000 M₂ plants of Taiwan 75 produced M_2:3 seeds segregating for the high Pi (HIP) phenotype. This mutant became homozygous at M5 and named as Gm-lpa-TW-1. Simultaneously, one plant of about 25,000 M₂ plants of Zhechun # 3 was identified, of which its M_2:3 seeds all showed in the HIP phenotype. This mutant line was late named Gm-lpa-ZC-2s.2. The analysis of content of various seed P indicated that the mutants appeared to be consistently and significantly higher in inorganic P (Pi) and lower in phytic acid P (PA-P) than their corresponding parents. The two Ipa mutants also differed significantly in Pi content; Gm-lpa-TW-1 was about 7-8 times higher in Pi than its wild-type parent Taiwan 75, while Gm-lpa-ZC-2 was only 2-4 times higher than Zhechun # 3, which indicated the quantitative color appearance of Pi was well consistent with the qualitative colorimetric assay. The reduction of PA-P in Gm-lpa-ZC-2 were larger than the increase of Pi, which was complemented by the accompanied increase of lower inositol phosphates.3. There were significant environmental effects on the content and distribution of various P compounds in seeds. The highest total P content was observed in seeds produced in Hainan, while total P levels were similar among seeds of the same line grown in spring and autumn in Hangzhou. The environmental effect on Pi level was much larger than on total P; The Pi level, both of the mutant lines and parental varieties, differed significantly across 3 locations/seasons. Interestingly, the content of PA-P and inositol phosphates of both wild type and parent were rather stable, but in sharp contrast, those of the two mutant lines differed significantly among the three locations/seasons.4. The F₂ populations of crosses between mutants and wild type varieties, i.e. Gm-lpa-ZC-2×Wuxing # 4, Gm-lpa-TW-1×Zhechun # 3, Gw-lpa-TW-1×Zhongdou27, comprised plants, based on the Pi level of F_2:3 seeds, three types, i.e. homozygous for wild type, heterozygous, and homozygous for mutant type. The segregation of these three type plants well fit into a 1:2:1 ratio, which indicated the Ipa mutations were controlled by single recessive genes. The F₁ seeds of the cross between Ipa Gm-lpa-ZC-2 and lpa Gm-lpa-TW-1 showed a low Pi level as wild type varieties, suggesting that the two mutations were not allelic to each other.5. The F_2:3 population of Gm-lpa-ZC-2 x Wuxing # 4 was developed for molecular mapping of the Ipa mutation. The polymorphism between the mutant and Wuxing # 4 were first identified by screening about 700 microsatellite markers. The polymorphic markers were further subjected to bulk-segregant analysis; those potentially linked markers were analyzed using about 160 segregating F₂ plants. Finally, two micro-satellite markers, Satt168 and Satt416 of the B2 linkage group, were found to be linked to the Gm-lpa-ZC-2 Ipa mutation, at a distance of about 9 cM and 4.5 cM, respectively.6. The MIPS1 gene was identified as the candidate gene of the Ipa mutation, based on the similarity of PA-P reduction, little change of lower inositol phosphates, and concomitant reduction of raffmosaccharide with PA-P between Gm-lpa-TW-1 and another previously reported Ipa mutant LR 33, which was caused by a nucleotide substitution at position 1,188 from the start codon of its cDNA. Sequencing and linkage analysis results showed there was a 2 base pair deletion in the third extron of the Gm-lpa-TW-1 MIPS1 gene. This two base pair deletion could lead to a frame shift of the mRNA and to a loss of function at the molecular or biochemical level of the gene. The complete genomic sequences of Taiwan 75 and Gm-lpa-TW-1 have been deposited in GenBank under the accession numbers (Currently under "Bankit" numbers 862509 and 862513).7. In this experiment, no significant differences of plant density were observed among three types of the F_2:3 population derived from Gm-lpa-ZC-2×Wuxing # 4. This was consistent with the above observation on field emergence rate of the mutant and parent. No significant differences were detected between the two types of lines for other agronomic traits except plant height. Yield per individual of mutant type lines in one population (Gm-lpa-ZC-2 X Wuxing # 4) was significantly higher than wild type lines, but in the other population (Gm-lpa-ZC-2 X Wuxing # 1), the results were on the contrary. The seed size of different lines differed significantly in the latter population, but not in the former population.8. No significant changes in protein, amino acid and oil content were observed in seeds of mutants and their corresponding progenitors. However, the mean oleic acid content was increased while that of linoleic acid was decreased in Gm-lpa-TW-1, , resulting in an unchanged total unsaturated fatty acid content compared with Taiwan 75. The total isoflavones was increased by 29.5% in Gm-lpa-ZC-2, resulting from the increase of all individual compounds except Genistein over Zhechun # 3. However, the 42% reduction was observed in Gm-lpa-TW-1, accounted by decrease of all compounds compared with Taiwan 75. These results demonstrated that the two mutants are desirable for developing Ipa soybean varieties with an enhanced nutritional quality.