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Dep. of Agronomy, Iowa State Univ., Ames, IA 50011-1010
* Corresponding author (wfehr{at}iastate.edu).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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4.3 g kg–1 phytate P and 0.7 g kg–1 inorganic P (Wilcox et al., 2000). Erdman (1979) suggested that because monogastric animals, including humans, swine, and poultry, have little or no phytase activity in their digestive system, it would be desirable to remove phytate from cereals and oilseeds. By reducing phytate and increasing inorganic P in soybean seed, the amount of supplemental P or phytase enzyme added to a ration containing soybean meal could be reduced, and there would be less undigested phytate P in the animal waste (Cromwell et al., 2000; Spencer et al., 2000; Cromwell, 2002). Lowering the P content of animal waste is important for avoiding excess accumulations of the mineral in soils that can result in reduced water quality (Parry, 1998).
A procedure for the development of mutants of soybean with low phytate was patented by Raboy (2000). It was used successfully by Wilcox et al. (2000) to develop mutant soybean lines that had 1.9 g kg–1 phytate P and 3.1 g kg–1 inorganic P. The lines originated from two M2 plants designated M153 and M766. Their analysis of segregation in the progeny from M153 and M766 suggested that they contained independent mutations for low phytate.
A breeding program for the development of low-phytate soybean cultivars was initiated at Iowa State University in 2001 with a low-phytate breeding line, CX1834-1-6, obtained from J. R. Wilcox. CX1834-1-6 was an F3:5 line selected from the cross of the cultivar ‘Athow’ with the mutant line M153-1-4-6-14. On the basis of the research by Wilcox et al. (2000), it was assumed that low phytate was controlled by a single major allele. Crosses were made at Iowa State University between CX1834-1-6 and three lines that had normal phytate. When the genotype of F2 plants were evaluated by testing their individual F3 seeds, the genotypic ratio of the F2 generation for the three populations did not fit the expected single-gene model. The objective of this study was to determine the inheritance of the low-phytate trait in the breeding line CX1834-1-6.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The line with normal phytate used for this study was A00-711013 developed by Iowa State University. The breeding line A00-711013 is an F4 plant selection from the cross of ‘AP1953’ to LN94-10470. AP1953 was developed by Agripro Seeds, Ames, IA. LN94-10470 is a line developed by the University of Illinois from the cross of ‘Jack’ x ‘Hartwig’ (Nickell et al., 1990; Anand, 1992).
The cross of CX1834-1-6 to A00-711013 was made at the Agricultural Engineering and Agronomy Research Center near Ames, IA, during July 2001 to develop low- and normal-phytate lines for evaluation of the influence of the low-phytate trait on agronomic and seed characteristics of soybean. The F1 and parent seeds were planted in the Iowa State University–University of Puerto Rico soybean nursery at Isabela, PR, in October 2001. The soil type is a Coto clay (very-fine, kaolinitic, isohyperthermic Typic Eutrustox). Simple-sequence repeat markers were used to confirm that the F1 plants were hybrids. A random sample of 350 F2 seed harvested from the F1 plants was planted at Isabela in February 2002, and each F2 plant was harvested individually to obtain F2:3 lines.
A random sample of 210 F2:3 lines and the parents were planted at two locations near Ames in May 2002 in two sets of 110 entries. Each set included 105 random lines, each of the parents, and three breeding lines and cultivars used as checks for agronomic and seed traits. The two sets were grown adjacent to each other as separate experiments in a randomized complete-block design with one replication at each location. The soil type at both locations is a Nicollet loam (fine-loamy, mixed, superactive, mesic Aquic Hapludoll). The entries were planted in single-row plots 0.76 m long with 1.02 m between rows and an alley of 1.07 m between the ends of plots. There were 20 seeds planted in each plot.
After planting, the 210 F2:3 lines were evaluated for phytate with 11 individual F3 seeds. With a single-gene model, there was a 95% probability that F2:3 lines segregating for alleles at one locus could be identified with 11 F3 seeds (Sedcole, 1977). On the basis of the progeny test, there were 127 lines homogeneous for normal phytate, 73 lines segregating, and 10 lines homogeneous for low phytate, which did not satisfactorily fit the expected 1:2:1 ratio. Analysis of additional F3 seeds from some of the heterogeneous lines suggested that low phytate may be controlled by recessive alleles at two independent loci that exhibit duplicate dominant epistasis.
There was insufficient remnant F3 seeds to evaluate a two-gene model. To genotype the F2–derived lines for a two-gene model, a random pod was harvested from each F3 plant in the two replications of each line planted at Ames. One F4 seed from each of 23 pods was evaluated for phytate content to identify segregating lines with 95% certainty (Sedcole, 1977).
To evaluate dominance and maternal effects for phytate, reciprocal crosses of CX1834-1-6 with A00-711013 were made at Ames in July 2002. The female and male plants used for crossing were identified. Each of 10 F1 seeds from the reciprocal crosses and a selfed seed from the male and female parent plants of each F1 seed were cut into two portions with a razor blade. The one-third of the seed without the embryonic axis was analyzed for phytate content and the other part was planted at Isabela in October 2002. Each F1 and parent plant was harvested individually. A random sample of 105 F2 seeds from each of the reciprocal crosses was evaluated for phytate content.
The method of analysis for phytate content of all seeds in the study was adapted from Chen et al. (1956), Raboy (2000), and Israel (2001, personal communication). Individual seeds or chips of individual seeds were placed in envelopes and crushed with a hammer. Each crushed seed was placed in a 12- by 75-mm disposable glass test tube. A 1-mL aliquot of 12.5% trichloroacetic acid and 25 mM magnesium chloride was added to each test tube. Seeds were soaked in the solution for 3 to 5 min. A 1-mL aliquot of colorimetric reagent that was 1 vol 3 M sulfuric acid, 1 vol 2.5% ammonium molybdate, 1 vol 10% ascorbic acid, and 2 vol redistilled water was added to each test tube. The colorimetric agent was prepared daily and the dilution of 10% ascorbic acid was stored at 4°C. Samples were allowed to sit for 15 to 20 min before scoring. Seeds with high inorganic P and low phytate produced a solution that was dark blue. Seeds with normal phytate produced a solution that was clear or light blue.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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There were 197 F2 seeds with normal phytate and 13 with low phytate. The segregation satisfactorily fit a phenotypic ratio of 15:1 normal to low phytate with a Chi-square value of 0.0013 (P > 0.97). The segregation indicated that low phytate was controlled by recessive alleles at two independent loci exhibiting duplicate dominant epistasis.
The phenotype of the 23 F4 seeds analyzed from each of the 210 F2–derived lines was used to divide the lines into three classes: homogeneous for normal phytate, segregating for phytate, and homogeneous for low phytate. There were 86 lines homogeneous for normal phytate, 114 segregating, and 10 homogeneous for low phytate, which satisfactorily fit a 7:8:1 ratio (Table 1). The results confirmed the two-gene model for inheritance of low phytate in CX1834-1-6. The recessive alleles for low phytate were designated pha1 and pha2.
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The duplicate dominant epistasis exhibited by the alleles at the two loci makes it necessary for both recessive alleles to be homozygous before an individual can express the low-phytate trait. This simplifies selection of individuals that are true breeding for low phytate in a cultivar development program. Individual seeds in a segregating population can be analyzed by the nondestructive technique used in this study. All low-phytate seeds will produce true-breeding individuals and lines.
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Received for publication March 11, 2003.
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