Spinach, one of the most popular leafy vegetables in the world, belongs to the Chenopodiaceae family, and in spite of having high economic value and many nutritional benefits, is classified as one of the high oxalate accumulating agricultural crops. Oxalate is a non-nutritive chemical compound that has harmful effects related to kidney stone diseases. Most of these adverse effects are the result of consuming the soluble form of oxalate. On the other hand, this chemical composition has an undeniable biological role on the biological functions of spinach. Therefore, considering these issues and the approach of improving agricultural products to increase nutritional value, it is inevitable to produce new varieties of spinach that have less oxalate accumulation or a non-harmful accumulation of this compound. The presence of both domesticated populations and wide species of spinach in Iran, the most significant source of this plant, make it possible to modify traits related to oxalate accumulation. Therefore, finding the mechanism of action of the genes involved in the accumulation of oxalate in spinach plants was considered the first step in the implementation of future breeding projects for this trait.
Material and Methods
In order to achieve the objectives of this research, a complete seven by seven diallel cross experiment was conducted using selected cultivars and wild related species (Spinacia tukestanica and S. tetrandra) that possessed different patterns of oxalate accumulation. These patterns were divided into three groups: low oxalate, high insoluble oxalate, and high soluble oxalate. High-performance liquid chromatography was used to measure total and soluble oxalate, while ascorbic acid was measured through a colorimetric method by a spectrophotometer device. The genetic variance components of four chemical compounds including total, soluble and insoluble oxalate, as well as ascorbic acid as the most important components in creating oxalate accumulation were estimated in 49 progenies, obtained from the crossing of spinach plants. The estimations were based on the variances of general and specific combinability using the first method of the Griffing's model one. The narrow and broad sense heritability and also Baker's coefficient, were calculated. Baker's index was known as an indicator to estimate the action mechanism of the genes. The values of this index are between zero and one. The numerical valued that are close to one indicate additive action, while values that are close to zero indicate non-additive action or dominance and epistasis actions.
As a general rule, the possibility of interspecies crosses in spinach was confirmed due to the same chromosomal level (2n=12) in all three species. The results of analysis of variance of the diallel cross showed the existence of significant differences for all four evaluated chemical compounds. This meant that the diallel crossings confirmed the possibility of genetic analysis based on this method. Also, the effects of GCA, SCA, the reciprocal crosses and type of maternal on mean values of all evaluated chemical compounds were significant. The highest variance of general and specific combinability and Baker's coefficient value (0.43) were achieved for the total oxalate trait. The greatest narrow and broad sense heritability was obtained with values of 0.46 and 0.57, respectively, for this trait.
Due to the greater contribution of genetic variance in the creation of total oxalate, it can be comprehended that it is possible to modify the total oxalate trait in the early breeding generations. Also considering the mentioned Baker's coefficient, both the additive and dominance/epistasis genes actions play a role in creating this trait, although the trend is more towards the effect of relative dominance. Therefore, modification of new spinach varieties based on hybridization methods and then selection in the first generations after hybridization is recommended. The second trait with the highest probability of occurrence in the generations after breeding was the insoluble oxalate followed by soluble oxalate. However, due to the very low narrow sense heritability (0.33), soluble oxalate is not recommended as a proper breeding trait for improving oxalate accumulation in spinach. Finally, the lowest narrow-sense heritability (0.32) was estimated for the ascorbic acid trait as one of the precursors of oxalate production, especially its insoluble form. So, it can be said that the role of ascorbic acid in improving oxalate accumulation in spinach plants is practically ruled out.
In this research, the gene action mechanisms involved in the formation of four essential compounds during the oxalate accumulation process in spinach were determined. Therefore, the modification of new varieties of this plant in terms of not having oxalic acid anti-nutrient problems by hybridization methods and then selection in the first generations is recommended.