**3.1 Nodule markers**

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 capacity and hence plant performance.

Identification and Application of Phenotypic

and Molecular Markers for Abiotic Stress Tolerance in Soybean 189

Fig. 6. A comparison of root morphology traits in three soybean cultivars after plants had

been exposed to drought for 1 month under field conditions.

Fig. 5. A comparison of the root phenotype in three soybean cultivars grown under conditions of drought. A representative shoot phenotype is shown for each of the three soybean cultivars studied: Jackson, A-5409RG and Prima 2000. Photos were taken after plants had been exposed to drought for 1 month under field conditions.

Fig. 5. A comparison of the root phenotype in three soybean cultivars grown under conditions of drought. A representative shoot phenotype is shown for each of the three soybean cultivars studied: Jackson, A-5409RG and Prima 2000. Photos were taken after

plants had been exposed to drought for 1 month under field conditions.

Fig. 6. A comparison of root morphology traits in three soybean cultivars after plants had been exposed to drought for 1 month under field conditions.

Identification and Application of Phenotypic

synthesis or leghemoglobin (Naya et al., 2007).

**4. Molecular markers for drought** 

markers for stress tolerance.

and Molecular Markers for Abiotic Stress Tolerance in Soybean 191

inhibition of nitrogenise activity. (Durand et al., 1987, Gonzalez et al., 1998, Arrese-Igor et al., 1999, King & Purcell, 2005). There is a close relationship between leaf and nodule water potentials (Durand et al., 1987). Water stress directly inhibits nitrogenase activities because of increased resistance to oxygen diffusion in the nodule. The increase in oxygen diffusion resistance is often linked to a decrease in nodule respiration as well as nitrogenase activity. In these conditions respiratory substrate accumulate as do oxidized lipids, and there is enhanced expression of antioxidant genes suggesting that impaired respiratory activity in the bacteroids under drought leads to enhanced oxidation prior to effects on sucrose

In an earlier study on the stress tolerance of different soybean varieties, we investigated the responses of symbiotic nitrogen fixation to dark chilling (van Heerden et al., 2008). Nodule structure, respiration and carbon-nitrogen interactions were compared in the two soybean genotypes that differed in chilling sensitivities: PAN809 (PAN) is chilling sensitive and Highveld Top (HT) is chilling resistant. We found that nodule numbers and the abundance of nitrogenase and leghemoglobin proteins were unaffected by dark chilling. However, the chilling stress caused a large decrease in nodule respiration rates and nitrogenase activities. The large chilling-dependent decrease in ureide contents observed in the PAN nodules was linked to decreases in respiration and an inability to maintain the oxygen diffusion barrier (van Heerden et al., 2008). These data suggest that nodule respiration and mitochondrial markers could be useful tolls in assisting conventional breeding efforts aimed at the

development of higher yielding soybean genotypes with better chilling tolerance.

Accurate phenotypic markers must ultimately be associated with molecular markers to aid and accelerate current plant breeding efforts to select improved soybean varieties with better stress tolerance. The effectiveness of morphological and physiological markers can vary greatly according to the growth stage of the plant and the many variables in the environment, particularly under field conditions. The selection of molecular markers is based either on variations in genomic DNA or on variations in gene expression (transcriptome) patterns. Comparisons of the transcriptome and also proteome signatures of organs or tissues under stress conditions can provide a direct assessment of processes and/or components than can be developed into a useful marker for stress tolerance in breeding programs. In general, a molecular marker might identify variations in plant responses to stress at the gene level, or in certain regions of DNA, the composition of DNA or in the degree of DNA methylation. While the DNA content of a cell is independent of environmental conditions, organ specificity or growth stage, DNA methylation can be regulated by these endogenous and environmental conditions and so alter also the patterns of gene expression. Variable regions of the genome (transposons), which can include single copy and repetitive genome regions, can also have considerable impact on plant stress tolerance. Such genomic regions can be functional or 'silent' without any obvious function. More recently, next generation sequencing with the identification of single nucleotide polymorphisms, is increasingly being considered as a tool to identify useful molecular

Further, Quantitative trait loci (QTLs) are chromosomal regions (genomic loci) that might regulate the expression levels of proteins. QTLs can be associated with DNA segments that are considered to make a significant contribution to the expression of complex phenotypic traits

Root length (A), root surface area (B) and root volume (C) were measured in soybean cultivars, Jackson, A-5409RG and Prima 2000. Roots were harvested in the field to a depth of 60 cm under drought conditions. The data were obtained from the roots as illustrated in Figure 7 and are the mean SE of 3 individual root systems per cultivar. Scanned root images were analyzed using the winRHIZO 2008a software which is an image analysis system specially designed for root measurement in different forms (Regent Instruments Canada Inc.). Using this software, root morphological data of root length, average diameter, total area and volume were determined. Furthermore, nodules were counted from the root images. Significance was determined using mean separation student's t-test (α = 0.05) Means with the same letter above the bars are not significantly different.

Fig. 7. A comparison of the root architecture traits in three soybean cultivars: Jackson, A-5409RG and Prima 2000 (Prima). Measurements of primary (tap) root branching (open bars) and lateral root branching density (closed bars) were performed after plants had been exposed to drought for 1 month under field conditions. Data represent the mean SE for values obtained from 3 individual plants. The student's t-test (α = 0.05) was applied. The letters above the bars indicate significant differences.

When a large collection (> 3,000) of soybean lines was screened for variations in leaf ureide contents under drought only eight were found to maintain low ureide contents under conditions of water deprivation (Sinclair et al., 2000). Thus, leaf and nodule ureide contents could be used as markers for alterations in nitrogen fixation under drought.

Of the factors that are important in preventing the inhibition of nitrogen fixation under drought perhaps the most important is the maintenance of the oxygen diffusion barrier. Other important factors include a continued supply of assimilated carbon from the leaves, together with high nodule sucrose synthase activities. In addition the export of ureides from the nodules is also important in preventing the accumulation of these metabolites and also amino acids in the nodules. Increases in nodule ureide or amino acid levels would lead to an

Root length (A), root surface area (B) and root volume (C) were measured in soybean cultivars, Jackson, A-5409RG and Prima 2000. Roots were harvested in the field to a depth of 60 cm under drought conditions. The data were obtained from the roots as illustrated in Figure 7 and are the mean SE of 3 individual root systems per cultivar. Scanned root images were analyzed using the winRHIZO 2008a software which is an image analysis system specially designed for root measurement in different forms (Regent Instruments Canada Inc.). Using this software, root morphological data of root length, average diameter, total area and volume were determined. Furthermore, nodules were counted from the root images. Significance was determined using mean separation student's t-test (α = 0.05) Means

Fig. 7. A comparison of the root architecture traits in three soybean cultivars: Jackson, A-5409RG and Prima 2000 (Prima). Measurements of primary (tap) root branching (open bars) and lateral root branching density (closed bars) were performed after plants had been exposed to drought for 1 month under field conditions. Data represent the mean SE for values obtained from 3 individual plants. The student's t-test (α = 0.05) was applied. The

When a large collection (> 3,000) of soybean lines was screened for variations in leaf ureide contents under drought only eight were found to maintain low ureide contents under conditions of water deprivation (Sinclair et al., 2000). Thus, leaf and nodule ureide contents

Of the factors that are important in preventing the inhibition of nitrogen fixation under drought perhaps the most important is the maintenance of the oxygen diffusion barrier. Other important factors include a continued supply of assimilated carbon from the leaves, together with high nodule sucrose synthase activities. In addition the export of ureides from the nodules is also important in preventing the accumulation of these metabolites and also amino acids in the nodules. Increases in nodule ureide or amino acid levels would lead to an

could be used as markers for alterations in nitrogen fixation under drought.

letters above the bars indicate significant differences.

with the same letter above the bars are not significantly different.

inhibition of nitrogenise activity. (Durand et al., 1987, Gonzalez et al., 1998, Arrese-Igor et al., 1999, King & Purcell, 2005). There is a close relationship between leaf and nodule water potentials (Durand et al., 1987). Water stress directly inhibits nitrogenase activities because of increased resistance to oxygen diffusion in the nodule. The increase in oxygen diffusion resistance is often linked to a decrease in nodule respiration as well as nitrogenase activity. In these conditions respiratory substrate accumulate as do oxidized lipids, and there is enhanced expression of antioxidant genes suggesting that impaired respiratory activity in the bacteroids under drought leads to enhanced oxidation prior to effects on sucrose synthesis or leghemoglobin (Naya et al., 2007).

In an earlier study on the stress tolerance of different soybean varieties, we investigated the responses of symbiotic nitrogen fixation to dark chilling (van Heerden et al., 2008). Nodule structure, respiration and carbon-nitrogen interactions were compared in the two soybean genotypes that differed in chilling sensitivities: PAN809 (PAN) is chilling sensitive and Highveld Top (HT) is chilling resistant. We found that nodule numbers and the abundance of nitrogenase and leghemoglobin proteins were unaffected by dark chilling. However, the chilling stress caused a large decrease in nodule respiration rates and nitrogenase activities. The large chilling-dependent decrease in ureide contents observed in the PAN nodules was linked to decreases in respiration and an inability to maintain the oxygen diffusion barrier (van Heerden et al., 2008). These data suggest that nodule respiration and mitochondrial markers could be useful tolls in assisting conventional breeding efforts aimed at the development of higher yielding soybean genotypes with better chilling tolerance.
