**2.1 Morphological and physiological markers**

Of the wide range of possible morphological characteristics that can be used in the selection of soybean varieties for enhanced drought tolerance (Figures 1 and 2), shoot parameters are generally considered to be the easiest to assess under field conditions. Shoot markers remain major targets in breeding programs, particularly in developing countries, where variations in shoot morphology are often determined subjectively under field or glasshouse conditions. Often this involves visual monitoring of easily detectable plant characteristics such as the number of leaves per plant or the shoot height. These simple parameters can be measured easily in soybean at different intervals during the growing period, and they can be assessed together with a range of other less easily determined parameters such as dry matter yield per plant (Udensi et al., 2010) or photosynthetic capacity and water use efficiency (Gilbert et al., 2011). Rapid growth is often directly related to the supply of water during the growing season. Hence, early maturity, early vigour, stomatal regulation, leaf area maintenance and osmotic adjustment of roots and shoots are generally considered to be useful and effective markers of good plant performance under drought conditions.

Fig. 2. Measurable phenotypic markers for drought tolerance.

A wide range of morphological, architectural, and physiological parameters are associated with water conservation and these can be used as markers for drought tolerance in soybean. Water deprivation leads to changes in turgor, osmotic pressure, leaf water potential, stomatal conductance and transpiration (Basra et al., 1999; Earl, 2002; Ribas-Carbo et al., 2005). Stomatal closure as a result of leaf water loss results in higher leaf water potentials

Identification and Application of Phenotypic

and Molecular Markers for Abiotic Stress Tolerance in Soybean 185

Fig. 3. Instantaneous water use efficiency of soybean. Comparison of instantaneous water use efficiency (IWUE) values measured in three soybean cultivars at day 0 (A) and after 18 days growth under drought conditions (B). Data are the means ±SEM of four independent replicates. Means with the same letter above the bars are not significantly different.

and allows better maintenance of the leaf water (Nakayama et al., 2007). However, stomatal closure also results in reduced CO2 movement for carboxylation within the chloroplast and this can be a major cause of drought-induced decreases in CO2 assimilation capacity, particularly in C3 plants (Chaves & Oliveira, 2004; Flexas et al., 2006; Warren, 2008). General plant performance parameters, such as biomass production, shoot and root length or seed yield but also leaf movements and phenological plasticity as an adaptation to drought (Acosta-Gallegos & White, 1995; Pastenes et al., 2005) might be useful as indicators for drought tolerance. Such physiological traits are therefore potential candidates for development as breeding markers for the selection of superior drought tolerant varieties. However, breeders can often be reluctant to use such traits, particularly if the associated measurements are time-consuming and technically demanding.

### **2.2 Water use efficiency**

Traits that serve to conserve water (conservative traits), including low stomatal conductance, low leaf growth rate, high water use efficiency (WEU), or deep but sparse root systems, favour better water use efficiencies. Research efforts in this regard have often tended to focus on the use of instantaneous water use efficiency (IWUE) values as a physiological marker for drought tolerance. The IWUE of a plant is determined from the ratio between CO2 assimilation rate and stomatal conductance values (Soares-Cordeiro et al., 2009). It is considered that higher IWUE values provide an indication of improved drought tolerance because varieties with high IWUE values are better able to assimilate carbon at low stomatal conductance and hence attain a greater yield using less water than other varieties. Genetic variation has been observed in soybean with regard to the amount of dry matter produced per given amount of water (Hufstetler et al., 2007). Moreover, soybean genotypes with a quick recovery after drought stress were more productive than genotypes with slower recovery (Hufstetler et al., 2007). Rapid recovery after drought stress is considered as an important trait in varieties grown under drier conditions with periods of drought. Water use efficiency (WUE) is increased by reduced transpiration and water use and biomass production is tightly linked to transpiration, WUE, and nitrogen accumulation. Breeding plants for high WUE under drought might actually result in low-yielding genotypes (Blum, 2011). It has been argued biomass production under most drought conditions can only be enhanced by an effective use of water which will allow maximal soil moisture capture for transpiration and minimizing water loss by soil evaporation (Blum, 2011).

Breeding efforts to improve drought tolerance in soybeans must ensure that a high level of water use efficiency is maintained in new varieties. In a study carried out in our group we compared a range of physiological shoot and root traits under optimal and drought conditions in three soybean varieties: Prima 2000, a commercial variety registered in South Africa that is suitable for cultivation in areas with longer growing seasons but suffering water stress during the growth period; A5409RG, a commercial variety that harbours a glyphosate-resistance gene and that is better suited to areas that do not experience drought or where crops are grown under irrigation; and Jackson, a drought-escaping cultivar with a shorter life cycle (Chen et al., 2007 a,b; Sall & Sinclair, 1991). Of the three genotypes studied, Prima had the highest IWUE values and shoot biomass under both well-watered and drought conditions (Fenta et al., 2011, Figure 3). Prima maintained highest photosynthetic CO2 assimilation rates under drought. The IWUE values in Prima leaves experiencing long term drought stress were twice those measured under water-replete conditions. The drought-induced increase in IWUE values observed in Jackson was smaller under drought,

and allows better maintenance of the leaf water (Nakayama et al., 2007). However, stomatal closure also results in reduced CO2 movement for carboxylation within the chloroplast and this can be a major cause of drought-induced decreases in CO2 assimilation capacity, particularly in C3 plants (Chaves & Oliveira, 2004; Flexas et al., 2006; Warren, 2008). General plant performance parameters, such as biomass production, shoot and root length or seed yield but also leaf movements and phenological plasticity as an adaptation to drought (Acosta-Gallegos & White, 1995; Pastenes et al., 2005) might be useful as indicators for drought tolerance. Such physiological traits are therefore potential candidates for development as breeding markers for the selection of superior drought tolerant varieties. However, breeders can often be reluctant to use such traits, particularly if the associated

Traits that serve to conserve water (conservative traits), including low stomatal conductance, low leaf growth rate, high water use efficiency (WEU), or deep but sparse root systems, favour better water use efficiencies. Research efforts in this regard have often tended to focus on the use of instantaneous water use efficiency (IWUE) values as a physiological marker for drought tolerance. The IWUE of a plant is determined from the ratio between CO2 assimilation rate and stomatal conductance values (Soares-Cordeiro et al., 2009). It is considered that higher IWUE values provide an indication of improved drought tolerance because varieties with high IWUE values are better able to assimilate carbon at low stomatal conductance and hence attain a greater yield using less water than other varieties. Genetic variation has been observed in soybean with regard to the amount of dry matter produced per given amount of water (Hufstetler et al., 2007). Moreover, soybean genotypes with a quick recovery after drought stress were more productive than genotypes with slower recovery (Hufstetler et al., 2007). Rapid recovery after drought stress is considered as an important trait in varieties grown under drier conditions with periods of drought. Water use efficiency (WUE) is increased by reduced transpiration and water use and biomass production is tightly linked to transpiration, WUE, and nitrogen accumulation. Breeding plants for high WUE under drought might actually result in low-yielding genotypes (Blum, 2011). It has been argued biomass production under most drought conditions can only be enhanced by an effective use of water which will allow maximal soil moisture capture for transpiration and minimizing water loss by soil

Breeding efforts to improve drought tolerance in soybeans must ensure that a high level of water use efficiency is maintained in new varieties. In a study carried out in our group we compared a range of physiological shoot and root traits under optimal and drought conditions in three soybean varieties: Prima 2000, a commercial variety registered in South Africa that is suitable for cultivation in areas with longer growing seasons but suffering water stress during the growth period; A5409RG, a commercial variety that harbours a glyphosate-resistance gene and that is better suited to areas that do not experience drought or where crops are grown under irrigation; and Jackson, a drought-escaping cultivar with a shorter life cycle (Chen et al., 2007 a,b; Sall & Sinclair, 1991). Of the three genotypes studied, Prima had the highest IWUE values and shoot biomass under both well-watered and drought conditions (Fenta et al., 2011, Figure 3). Prima maintained highest photosynthetic CO2 assimilation rates under drought. The IWUE values in Prima leaves experiencing long term drought stress were twice those measured under water-replete conditions. The drought-induced increase in IWUE values observed in Jackson was smaller under drought,

measurements are time-consuming and technically demanding.

**2.2 Water use efficiency** 

evaporation (Blum, 2011).

Fig. 3. Instantaneous water use efficiency of soybean. Comparison of instantaneous water use efficiency (IWUE) values measured in three soybean cultivars at day 0 (A) and after 18 days growth under drought conditions (B). Data are the means ±SEM of four independent replicates. Means with the same letter above the bars are not significantly different.

Identification and Application of Phenotypic

in the field.

system architecture of soybean.

capacity and hence plant performance.

**3.1 Nodule markers** 

and Molecular Markers for Abiotic Stress Tolerance in Soybean 187

that Prima 2000 and Jackson have deeper roots with a greater branching density than A-5409RG under drought conditions (Figure 5). A-5409RG had a shallower and thicker root system under drought conditions, with lower overall numbers of roots and a lower branching density. This cultivar also had the highest shoot to root ratio under drought conditions. The production of a lower root biomass compared to the shoot would result in a lower water harvesting capacity. Prima 2000 and Jackson 2000 also had larger root systems under drought than A-5409RG with a greater root length, root surface area and a greater root volume (Figure 6). Both varieties were therefore able to extend the root system in response to drought and so withstand water stress. This study indicates the importance of root traits in the field performance of different soybean cultivars under drought conditions. The association of root traits with the higher productivity of Prima 2000 under drought conditions suggests that these parameters could be used as a marker for drought tolerance

Fig. 4. Soybean root system architecture. Schematic representation of the allorhizic root

For soybean, the ability to maintain not only high rates of photosynthesis but also symbiotic nitrogen fixation under drought can be important "in terms of yield". Symbiotic nitrogen fixation is rapidly inhibited by drought and thus soybean plants exposed to drought are also deprived of nitrogen (Sinclair et al., 2007). Like photosynthesis, nitrogenase activity can therefore be used as a sensitive marker for drought tolerance. Metabolic markers, such as nitrogenase activity, detect the early plant responses to drought and they are more sensitive than morphology markers. Soybean genotypes can be selected for increased yield on the basis of leaf or nodule numbers (Udensi et al., 2010). Other nodule parameters such as size, leghemoglobin content, ureide accumulation can be used as markers for nitrogen fixation

even though Jackson had lower stomatal conductance rates than Prima. In contrast, no significant differences (P > 0.05) in IWUE values were found in A-5409RG either under water-replete or drought conditions.
