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National Small Grains Germplasm Research Facility, Agricultural Research Service, United States Department of Agriculture, 1691 S. 2700 W., Aberdeen, ID 83210 USA
* Corresponding author (pbregit{at}uidaho.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Low phytate mutants have been isolated in several cereal crop species (barley; rice, Oryza sativa L.; maize, Zea mays L.; and wheat, Triticum aestivum L.) and soybean, Glycine max (L.) Merr. (Hitz et al., 2002; Guttieri et al., 2004; Raboy et al., 2000, 2001; Rasmussen and Hatzack, 1998; Wilcox et al., 2000). These mutants produce seed that contain normal levels of total P but a significant fraction of the phytic acid has been replaced by inorganic P or by myo-inositol phosphates containing five or fewer P esters. Feeding trials that include LP cereal grains have been conducted on swine, fish, and poultry. These studies have associated the LP trait with improved P and mineral availability, associated improvements in animal growth, and/or reductions in fecal P (Ertl et al., 1998; Spencer et al., 2000; Li et al., 2001a,2001b; Veum et al., 2002; Jang et al., 2003; Overturf et al., 2003; Thacker et al., 2003; Leytem et al., 2004).
Field evaluations of LP and wild-type (WT) maize containing moderate (65%) reductions of phytate showed that germination and stand establishment were not affected, but eight of 14 LP-WT hybrid pairs showed grain yield reductions associated with the LP trait; overall, LP lines averaged 94% of the WT grain yield (Ertl et al., 1998). Evaluations of LP soybean with similar reductions in phytate performed similarly, but the LP trait was associated with significant reductions in seedling emergence (Meis et al., 2003; Oltmans et al., 2005). Preliminary agronomic evaluations of several LP mutations in barley have suggested that agronomic performance is negatively correlated with the degree of phytate reduction (Raboy, unpublished data). This study was conducted to examine the agronomic performance of barleys homozygous for four independent LP mutations that result in phytate reductions ranging from 35 to 95% of wild type, when grown in irrigated or rain-fed production environments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The lines examined in this study were developed by backcrossing each mutant line to the wild-type (WT) parent, Harrington. The lpa1-1 lines were derived from BC4 populations, lpa2-1 and lpa3-1 from BC3 populations, and M955 from BC2 populations. Selections were based solely on the presence of the LP mutant allele in all backcross cycles except for the final one. For the final backcross cycle, F2.3 lines were grown as 1.3-m headrows under irrigation at Aberdeen, ID, in 2000. Heads were selected at random from rows that were visually similar to Harrington. Heads homozygous for either the LP or the WT allele for each of the four mutations were identified on the basis of inorganic P assays of five individual kernels as described (Dorsch et al., 2003), and the remainder of the seed from each head was grown in 2001 as F3.4 1.3-m headrows at Aberdeen, ID. Individual headrows were selected on the basis of visual similarity to Harrington, harvested in bulk, and their LP or WT status verified as described above. For this study, six WT and six LP sib lines were selected from each of the populations containing either lpa1-1, lpa2-1, lpa3-1, or M955. Lines evaluated in 2002 and 2003 were BCxF3.5 and BCxF3.6 populations, respectively.
Test Sites
The 24 WT and 24 LP lines, plus the Harrington WT parent, were grown at Aberdeen and Tetonia, ID (2002 and 2003) and Soda Springs and Filer, ID (2003 only). These locations offer diverse environments and differ significantly with respect to elevation above sea level (Filer, 1064 m; Aberdeen, 1338 m; Soda Springs, 1763 m; Tetonia, 1794 m), growing season, temperatures, and water availability. The growing season for Filer was late March to mid-July, and at Aberdeen early April to early August. For both locations, daily low to high temperatures during anthesis and grain filling generally ranged from 10 to 15°C to 27 to 35°C. The growing seasons for the Soda Spring and Tetonia locations were mid-May to late August, and temperatures during anthesis and grain filling were approximately 3 to 5°C cooler than for Aberdeen and Filer.
Prevailing agronomic practices for the growth of spring malting barley were used at all locations, including the use of sprinkler irrigation at Aberdeen and furrow irrigation at Filer. No irrigation was provided at Soda Springs or Tetonia. At the University of Idaho Research and Extension Center in Aberdeen, barley was planted on as part of a 3-yr rotation of potatoes-green manure (oat)-barley. Soil tests at Aberdeen showed similar levels of N, P, and K in both years (approximately 40 µg g–1 or 180 kg ha–1 N in the top 60 cm and 20 µg g–1 P and approximately 255 µg g–1 K in the top 30 cm); no additional fertilizer was added. At the University of Idaho Research and Extension Center in Tetonia, barley was planted as part of a 2-yr small grains-fallow rotation. Soil tests at Tetonia showed similar levels of N, P, and K in both years (approximately 2 µg g–1 or 9 kg ha–1 N in the top 60 cm and 40 µg g–1 P and approximately 255 µg g–1 K in the top 30 cm). No additional P or K was added. Nitrogen was added as a combination of ammonium nitrate and ammonium sulfate to bring available N to 100 kg ha–1. At Filer, barley was planted on a commercial farm following sweet corn. Soil tests at Filer showed 38 µg g–1 N (or 170 kg ha–1) N, 20 kg ha–1 P, and 230 µg g–1 K; 12.3 kg ha–1 N and 58.2 kg ha–1 P2O5 were added as monoammonium phosphate. At Soda Springs, barley was planted on a commercial farm as part of a continuous malt barley operation with no rotation; no soil tests were conducted. Fertility management was based on long-term averages of previous soil tests, and the precipitation, fertility management practices, and grain yield and quality at that site in the previous year. Fifty-six kilograms per hectare N and 22.4 kg ha–1 P2O5 were added as a combination of ammonium nitrate, ammonium sulfate, and monoammonium phosphate.
Experimental Design and Statistical Analysis
The experimental design at all locations was a randomized complete block with two replicates. Plots were planted with a small-plot drill (Hege) equipped with double disc openers in ranges of 10 side-by-side plots, 2.4 m in length, consisting of 7 rows on 17.8-cm centers, with approximately 36 cm between plots. Planting rates at Aberdeen and Filer were approximately 112 kg ha–1 and at Tetonia and Soda Springs approximately 90 kg ha–1. The outside plots of each range were bordered by other plots that were not part of the test. Each range was separated by a 1.2 m alley. Harvesting was done with a small plot combine (Wintersteiger). Agronomic data were collected for days to heading, plant height, percent lodging, grain yield, grain test weight, and the percentage plump kernels (defined as kernels retained on a 2.38- by 19.1-mm slotted screen).
All data were analyzed by SAS v. 8.0 Proc GLM (1999. SAS Institute Inc., Cary, NC, USA). Sibset (lpa1-1, lpa2-1, lpa3-1, or M955), genotype (WT or LP), and environment (irrigated or rain-fed), and their interactions were considered to be fixed effects. Subenvironments (locations and years) within environments and all other sources of variance were considered random. Comparisons of WT sibsets to Harrington were based Dunnett's comparisons of sibset x environment means, with sibset x subenvironment(environment) as the error term. Comparisons of LPA vs. WT were made using single-degree-of-freedom contrasts. All declarations of significance were based on p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The WT sibs for each mutation were not significantly different than Harrington for any of the measured traits, in both the irrigated and rain-fed environments. This suggest that the source of any observed differences between the backcross-derived WT and LP sibs was the presence or absence of the mutant alleles. Therefore, the effects of each LP mutation could be assessed on the basis of WT vs. LP sib comparisons.
Performance under Irrigated Conditions
No differences for seedling emergence, stand establishment, and general appearance throughout the growing season could be detected on the basis of visual observations of the Harrington recurrent parent, WT lines, and LP lines. Analyses of heading dates and plant heights did not detect significant differences among LP sibsets nor between LP or WT genotypes. Mean heading dates for Aberdeen and Filer were 169 and 158 d after 1 January, respectively. Mean plant heights for Aberdeen and Filer were 94 and 107 cm, respectively. The lpa2-1 LP sibset was not different from its WT sibset for yield, test weight, or percentage plump kernels (Table 1). There was a significant reduction in percentage lodging, which was surprising given the equal heights and yields of the WT and LP sibsets. Visual observations did not detect morphological differences that might explain this reduction. LP sibsets containing the lpa1-1 and lpa3-1 mutations showed relatively small changes when compared with their respective WT sibsets. No differences were detected for yield or percentage plump kernels between WT and LP sib lines, and for lpa1-1, no differences were detected for lodging. Both sibsets showed significant and similar reductions of test weight (Table 1).
Mutant M955, which conditions a 90% reduction in phytate, as compared with the 35 to 70% reductions conditioned by the other mutants, clearly had a negative impact on agronomic performance (Table 1). Substantial reductions in grain yield and percentage plump kernels were recorded, and the reduction in test weight was more severe than for lpa1-1 or lpa3-1. Lodging percentage was reduced also, most likely as a consequence of the significantly reduced yield.
Performance under Rain-Fed Conditions
No differences for seedling emergence or stand establishment were detected on the basis of visual observations of the Harrington recurrent parent, WT lines, and LP lines for any of the mutant classes. However, the LP sibsets, particularly for lpa2-1, lpa3-1, and M955, tended to appear less vigorous and exhibit more signs of stress than their respective WT sibsets or Harrington. These differences were slight and could not consistently be noted. No differences could be detected for heading dates and plant heights among LP sibsets nor were any detected between the LP or WT genotypes. The mean heading date for Tetonia was 194 d after 1 January; heading dates were not recorded at Soda Springs because the frequency of observations was insufficient; however, qualitative assessments conducted at several dates surrounding head emergence also suggested that all lines had similar heading dates. Mean plant heights for Tetonia and Soda Springs were 48 and 45 cm, respectively.
Additional data for agronomic performance under rain-fed conditions are shown in Table 2. The mutant lpa1-1 yielded well, but the LP sibset had lower test weight and percentage plump kernels relative to the WT sibset. In contrast, mutants lpa2-1, lpa3-1, and M955 were associated with significant, and substantial, reductions in grain yield. The LP sibsets for all three mutations showed significant reductions in test weights and percentages plump kernels.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One limitation of these data is that the effects of the mutant alleles were studied in a single genetic background. However, all of these mutations have been evaluated in several other genetic backgrounds as part of an extensive effort to produce commercially competitive LP cultivars (Bregitzer, unpublished data). Selections for good agronomic appearance within early generations of recombinant LP populations—including two- and six-rowed lines, hulled, and hulless lines—grown at Aberdeen have consistently favored lines derived from crosses to lpa1-1, lpa2-1, and lpa3-1. For instance, of 140 single plant selections from the cross M955/86Ab2626//HB317 on the basis of vigorous plant appearance and plump kernels, only six (4%) were homozogyous for the LP trait. Lines developed from all of these selections had very poor yields and small kernels. In contrast, selection among populations segregating for the other three mutations, the ratio of LP/LP:LP/WT:WT/WT plants closely approximates the expected segregation ration of 1:2:1. Yield trials of lines derived from lpa1-1 have shown their yields to be generally higher than those derived from lpa2-1 or lpa3-1. For instance, preliminary yield trials at Aberdeen in 2002 lines derived from backcrosses of lpa1-1 to ‘Colter’ showed their mean yield to be 92% of Colter, compared with 66% for lines derived from lpa2-1. Lines developed from backcrosses of lpa2-1 and lpa3-1 to ‘Stander’ yielded, respectively, 100% and 82% of Stander. More extensive testing of one line, 00ID1550 (Colter*3/lpa1-1), showed that relative to Colter the mean yields under irrigated conditions (6 location-years) and rain-fed locations (6 location-years) was, respectively, 101 and 97%. Mean test weight for both irrigated and rain-fed conditions was 98% of Colter, consistent with the test weight reductions noted for lpa1-1 in this study. Thus, it is reasonable to conclude that, at least on a qualitative basis, the results of this study may be predictive of those that would be obtained for these mutations when tested in other genetic backgrounds. Further studies will be necessary to verify this conclusion.
It was expected that the alterations of phosphorus profiles of the LP sibsets examined for this study would be similar to their mutant parent, under both the irrigated and rain-fed production environments. Previous characterizations of the original mutant lines, using seed produced in two different environments, Aberdeen, ID, and Saskatoon, SK, Canada, (Dorsch et al., 2003; Leytem et al., 2004; Overturf et al., 2003; Thacker et al., 2003), showed similar results with respect to the alterations in phosphorus profiles conditioned by these four mutations. Furthermore, semiquantitative analyses of seed from recombinant breeding populations for Pi content generally resulted in the ranking lpa2-1 equal to or slightly less than lpa1-1; populations containing lpa3-1 have slightly higher Pi content, and M955 populations have the highest Pi content. Qualitative evaluations of total P and Pi were conducted on grain produced in this study that confirmed this expectation and will be published elsewhere. In brief, the these studies showed Pi/total P concentrations (in mg g–1) for the LP sibsets for Harrington, lpa1-1, lpa2-1, lpa3-1, and M955 to be 0.20/3.31, 1.06/3.00, 0.89/3.61, 1.86/3.49, and 2.63/3.54, respectively, for irrigated environments and 0.28/3.94, 1.05/3.39, 1.27/4.43, 1.89/4.04, and 3.04/4.24, respectively, for rain-fed environments. Statistical analyses revealed the following relationships: for Pi, Harrington < lpa1-1 = lpa2-1 < lpa3-1 < M955; for total P, lpa1-1 < Harrington = lpa2-1 = lpa3-1 = M955.
The data produced in this study indicate that the development of agronomically competitive cultivars carrying the lpa1-1, lpa2-1, or the lpa3-1 mutations will be possible, but only the lpa1-1 mutation may be compatible with the production of LP barley grain under conditions of moisture stress that are typical of rain-fed production environments, even those located in relatively cool, high altitude locations that are otherwise suitable for barley production. Because of the reductions in test weight associated with either lpa1-1 or lpa3-1, the development of cultivars with acceptable test weight may be facilitated by introgressing these alleles into genotypes with relatively higher test weights, e.g., two-rowed types typically have significantly higher test weights than six-rowed types.
The reaction of lines to moisture stress suggest that the LP mutations are associated with significant perturbations of essential physiological functions in the whole plant, in addition to the desirable changes in seed P chemistry they condition. Phytate is the most abundant representative of a class of compound referred to as inositol phosphates (Shears, 2004). Both myo-inositol (the backbone of phytic acid) and inositol phosphates including phytate play important roles in hormonal responses in plants, and in signal transduction and stress response in many eukaryotes (Flores and Smart, 2000; Ishitani et al., 1996; Lemtiri-Chlieh et al., 2003; Perera et al., 2004). Not surprisingly, lines containing the mutant with the most extreme phenotype (M955; 90% reduction of phytate) fared relatively worse than lines carrying less extreme mutations.
Interestingly, lpa1-1, which had the best performance under moisture stress, has been shown to have a tissue-specific phenotype (Ockenden et al., 2004). Phytic acid has been shown to accumulate primarily in aleurone tissues, with lesser amounts accumulating in the germ (O'Dell et al., 1972). The lpa1-1 mutation only affects P and phytate P in the aleurone layer. Phytic acid P levels in lpa1-1 germ are equal to or greater than wild type, whereas each of the other three mutations affect both the germ and aleurone layer. Although a causal relationship has yet to be established, and the impact of lpa1-1 on whole plant processes has not yet been studied, it is not surprising that a mutation limited to a specific seed tissue might have more impact on grain quality than on grain yield. This restricted tissue specificity of barley lpa1-1 represents a potential strategy to engineer low phytate crops without significant yield reductions; i.e., to development of germplasm where the block in phytic acid synthesis is restricted to the aleurone.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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