Comparison of Grain Zinc and Iron Concentration between Synthetic Hexaploid Wheats and Their Parents

Deficiencies of iron (Fe) and zinc (Zn) in human food afflict a large proportion of the world’s population. Wheat is a major food source of minerals. One way to enhance bread wheat’s ability to enrich these minerals would be to take advantage of diversity of wild species by creating synthetic hexaploid wheat (SW). In this study, two minerals (Fe and Zn) concentrated in the grain of Aegilops tauschii Coss. (2n = 2x = 14, DD), Triticum turgidum L. (2n = 4x = 28, AABB), and 33 lines of their corresponding SW (2n = 2x = 42, AABBDD) were evaluated. The results showed that Fe concentration was decreased in most of SW lines compared with their parental Aegilops tauschii accessions, while Zn concentration was greatly increased in most of SW lines compared with their parental Aegilops tauschii accessions. Aegilops tauschii had stronger Fe enrichment than Triticum turgidum while they expressed the same ability for Zn enrichment. The genotypic variance based on their physiological performance was analyzed. SW lines showed less genotypic variance of Fe and Zn concentration than Aegilops tauschii. SW lines showed less genotypic variance of Fe concentration than Triticum turgidum L. lines while they had more genotypic variance of Zn concentration than Triticum turgidum L. lines. Regardless of the fact that the traits expressed in wild relatives of wheat may not predict the traits that will be expressed in SW lines derived from them, production of SW could be a powerful method creating genotypes with enhanced trait expression.


Introduction
Micronutrient malnutrition is a serious health problem worldwide [1].Zinc (Zn) and iron (Fe) deficiencies are the most common micronutrient deficiencies in human populations affecting health of over three billion people worldwide [1].Cereals are an important source of micronutrient minerals for humans.Wheat is a major staple food crop and its nutritional quality have a significant impact on human health and well-being, especially in developing countries [2] [3].Breeding of wheat cultivars with increased micronutrient concentration is a low-cost and sustainable strategy for alleviating micronutrient malnutrition.However, common wheat cultivars usually have low grain Fe and Zn contents [2] [4] [5], with a narrow genotypic variations [6]- [10].
Common wheat is an allohexaploid species that originated from natural hybridization between tetraploid wheat (Triticum turgidum L.) and Aegilops tauschii Cosson [11] [12].Ae. tauschii, the D-genome donor of common wheat, has a very wide geographic distribution extending westwards to Turkey and eastwards to Afghanistan and China [13]- [15] and shows abundant genetic variations [16]- [18].Since only a few Ae.tauschii accessions was involved in the origin of common wheat, a lot of genetic diversities in this species are not represented in common wheat population.Ae. tauschii provides important genetic resources for the common wheat improvement of Fe and Zn concentrations [7]- [10] [19].
Artificially synthetic hexaploid wheat (SHW) between T. turgidum and Ae.tauschii has been used as a bridge for transfer the gene from Ae. tauschii into common wheat.In the present study, Fe and Zn concentration of Ae. tauschii, T. turgidum, and their corresponding synthesized lines were compared to investigate their expression in hexaploid level and to choose synthetic hexaploid for nutritional quality improvement of common wheat.

Plant Materials
Thirty-three synthetic hexaploid wheat and their parents, including 13 Ae.tauchii accessions and 23 T. turgidun lines, were produced by Dr. Dengcai Liu (Table 1).These Ae. tauchi I and T. turgidun have diverse geographic origins and belongs to different subspecies.Synthesized hexaploid wheat lines were produced through spontaneous chromosome doubling via union of unreduced gametes [20].All these materails were grown at 1 row 1 m length plot in irrigated field trials with 2 replications at Wen Jiang of Triticeae Research Institute of Sichuan Agricultural University in the 2009-2010 crop season.

Chemical Analysis
Grain samples were analyzed for Fe and Zn concentrations by atomic absorption spectrometry according to Orhan et al. [21].The grain samples were washed with distillated water, a 0.1 mol•L −1 HCl solution and deionized water.After being dried in a laboratory oven at 65˚C, the dry matter was quantified, and then submitted to grinding.Fe and Zn were quantified in an extract obtained by nitro-perchloric digestion and their content was determined by conventional atomic absorption spectrometry run with an air-acetylene flame.

Statistical Analysis
Data were analyzed using analysis of variance (ANOVA).The correlations among the physiological traits were estimated based on Pearson correlation coefficient values.

Distribution of Fe and Zn Contents in Aegilops tauschii, Triticum turgidum Accessions, and SHW Lines
For the Fe element, of 33 SW lines, 82% displayed the concentration of between their corresponding two parents, 12% displayed the concentration of higher than their corresponding two parents, while only 6% of them displayed concentrations lower than their corresponding parents.For the Zn element, of 33 SW lines, 82% displayed the concentration higher than their corresponding parents, 15% of them displayed concentration between their corresponding parents, 3% of them showed decreased Zn concentration after allopolyploidization compared with corresponding parents (Tables 2-4, Figure 1 and Figure 2).The mean grain Fe concentration in Aegilops tauschii was significantly higher than that in Triticum turgidum L. The mean grain Zn concentration was in the same level in Aegilops tauschii and Triticum turgidum (Table 3 and Table 4).The mean concentration of grain Fe in 33 SW lines decreased by 66% compared with the corresponding diploid parent Aegilops tauschii (Table 3; Figure 1).While the mean concentration of grain Zn in 33 SW lines increased by 113% compared with the corresponding diploid parent Aegilops tauschii (Table 3, Figure 2).

The SW Lines Showed Lower Variance of Fe and Zn Concentration
The genotypic variance of the three group lines was analyzed based on their physiological performance, using the coefficient of variation as a parameter.SW lines showed less genotypic variance than Aegilops tauschii in Fe, Zn, and Se concentration.SW lines showed less genotypic variance than Triticum turgidum L. lines in Fe and Se concentration while they had more genotypic variance than Triticum turgidum L. lines in Zn concentration (Table 3 and Table 4; Figure 3, Figure 4).

Correlation of Traits of Ae. tauschii and Triticum turgidum L. Accessions with Those of Their Corresponding SW Lines
No physiological traits (Fe and Zn concentration) of the SW lines were significantly correlated with the corresponding traits of their parental Ae. tauschii and Triticum turgidum L. lines (Table 3 and Table 4).

Discussion
Successful expression of useful characters of wild wheat relatives in synthetic hexaploid level is a key step for common wheat improvement.The narrow genetic variation in Fe and Zn concentrations of wheat has limited the breeding of wheat to enhance the Fe and Zn concentrations in grain.Wheat relatives show wide genetic variation in these characters [22] [23].In this study, comparative studies of two mineral traits among and between the synthetics and their parental Ae. tauschii and Triticum turgidum L. accessions were conducted.The results showed that Ae. tauschii had wider variations of Fe concentration than SW lines and SW lines had wider variations of Zn concentration than Ae.tauschii accessions.The variations observed at the diploid genome level were not necessarily reflected in the synthetics (Table 2, Figures 1-4).Significant variation in most of the morphological and physiological characteristics was measured in the Ae.tauschii accessions [18] [24].Meanwhile, Aegilops tauschii had stronger Fe enrichment ability than Triticum turgidum while they had the same ability for Zn enrichment.The average concentrations of grain Fe in SW lines were 50% lower than Ae.tauschii, respectively (Figure 1), indicating that allopolyploidization reduced the expression of Fe enrichment characters in Ae. tauschii.Interestingly, the variation of Zn concentration was wider in SW lines compared with their corresponding tetraploid and diploid parental lines.Zn concentration was significantly increased after allopolyploidization in most of the combinations.In 33 SW lines, 82% of them had higher Zn concentration than their corresponding tetraploid and diploid parental lines (Table 2), indicating that grain Zn accumulation was significantly stimulated by allopolyploidization. Previous research work indicated that alien chromosome addition wheat lines derived from 6 species of Aegilops tauschii showed increased grain Zn concentration of be-tween 50% and 248% compared with the recipient cultivar, Chinese Spring [25].This is consistent with our result of Zn.
Repression of D genome variation has been commonly observed for most morphological traits in synthetic hexaploid wheats [26] [27], indicating that the expression of D genome variation was masked.Allopolyploidization is often accompanied by genetic and epigenetic modification of the genome as previously observed in synthetic polyploids of wheat and Arabidopsis [25] [28]- [31].In the present study, the changes of high Fe concentration character and low Zn concentration of Aetauschhi by allopolyploidization should be the results of genome interaction between A, B, and D genome because our recent data from similar SW lines indicated that allohexaploidization of wheat did not increase the SSR mutation rate [32].
Regardless of the fact that the traits expressed in wild relatives of wheat may not predict the traits that will be expressed in SW lines derived from them, production of SW could be a powerful method creating genotypes with enhanced trait expression.

Figure 1 .
Figure 1.Mean concentration of grain Fe in 33 Triticum turgidum L., Aegilops tauschii accessions and their corresponding SW lines.

Figure 2 .
Figure 2. Mean concentration of grain Zn in 33 Triticum turgidum L., Aegilops tauschii accessions and their corresponding SW lines.

Figure 3 .
Figure 3. Comparisons of variability Fe concentration in parental Aegilops tauschii accessions and synthetic hexaploids.

Figure 4 .
Figure 4. Comparisons of variability Zn concentration in parental Aegilops tauschii accessions and synthetic hexaploids.

Table 2 .
Distribution of 33 SW lines according to Fe, Zn, and Se contents compared with their parents.

Table 3 .
Variation in morphological traits in synthetics and corresponding Aegilops tauschii parental accessions.

Table 4 .
Variation in morphological traits in synthetics and corresponding Triticum turgidum L. parental accessions.Ythetic Wheat Lines AccessionsTriticum turgidum L.1Coefficients of variation are based on SD/Mean.