Screening of Diverse Ethiopian Durum Wheat Accessions for Aluminum Tolerance

Abstract

Acid soils and associated Al³⁺ toxicity are prevalent in Ethiopia where normally Al³⁺-sensitive durum wheat (Triticum turgidum ssp durum Desf.) is an important crop. To identify a source of Al³⁺ tolerance, we screened diverse Ethiopian durum germplasm. As a center of diversity for durum wheat coupled with the strong selection pressure imposed by extensive acid soils, it was conceivable that Al³⁺ tolerance had evolved in Ethiopian germplasm. We used a rapid method on seedlings to rate Al³⁺ tolerance according to the length of seminal roots. From 595 accessions screened using the rapid method, we identified 21 tolerant, 180 intermediate, and 394 sensitive accessions. When assessed in the field the accessions had tolerance rankings consistent with the rapid screen. However, a molecular marker specific for the D-genome showed that all accessions rated as Al³⁺-tolerant or of intermediate tolerance were hexaploid wheat (Triticum aestivum L.) that had contaminated the durum grain stocks. The absence of Al³⁺ tolerance in durum has implications for how Al³⁺ tolerance evolved in bread wheat. There remains a need for a source of Al³⁺-tolerance genes for durum wheat and previous work that introgressed genes from bread wheat into durum wheat is discussed as a potential source for enhancing the Al³⁺ tolerance of durum germplasm.

1. Introduction

Durum (Triticum turgidum ssp durum Desf.) wheat and bread wheat (Triticum aestivum L.) provide the Ethiopian population with a large proportion of its caloric intake [1,2]. Despite a general increase in production and productivity of wheat during the last two decades (1998–2016), Ethiopia imports a substantial amount of both durum and bread wheat. Durum production as a proportion of the total wheat produced in Ethiopia has declined over the past few decades from about 80% in the 1980s to an estimated current proportion of only about 20%. Despite a reduction in the proportion of grain produced in Ethiopia, durum constitutes 50% to 80% of the wheat grain imported in any given year [1], indicating that demand for durum remains strong. One of the abiotic stresses that may be contributing to the decline in durum production is the prevalence of acid soils in Ethiopia. Durum wheat is very sensitive of the toxic Al³⁺ found in acid soil and is the most sensitive of the small-grained crops to Al³⁺ toxicity [3]. By contrast, bread wheat has Al³⁺-tolerant alleles of TaALMT1 [4] and TaMATE1B [5] along with other yet to be cloned Al³⁺-tolerance genes [6].

Worldwide, acidic soils are one of the most important limitations to agricultural production [7]. In Ethiopia, acid soils account for about 34% of agricultural land area that range from slight to strongly acidic soils [8]. Acid soils generally occupy the western part of the country extending from southwest to northwest, although strongly acidic soils occur mainly in the western part of the country including the lowlands. Acid soils are particularly prevalent in the highlands of Ethiopia [9] and the application of lime commonly results in improved yields of various crops [10,11]. The productivity of wheat over acidic areas of Ethiopia is low as compared to parts of the country where soil acidity is absent. For instance, in areas with strongly acidic soils, which occur widely in the western and southwestern parts of the country, the productivity of wheat is as low as 0.8–2.0 t/ha. These areas include West Wallaga, Illu-Ababora, Jimma, Gamo Gofa, Asosa, and Metekel zones. On the other hand, a relatively higher grain yield, ranging from 2.5 to 3.1 t/ha, is obtained in regions with near neutral soil pH including West Arsi, Arsi, Siltie, East Shewa, and Bale zones (Supplementary Materials file, Table S1).

Aluminum is solubilized in acid soils into the toxic Al³⁺ which in sensitive plants typically affects the viability of the root apex. Al³⁺ inhibits root growth resulting in reduced uptake of water and nutrients ultimately hindering plant growth and development [12]. In addition to its toxic effects on plant tissues, Al³⁺ affects nutrient availability within the soil. All of these effects significantly reduce crop yield. While management, primarily by application of lime, is important for neutralizing acid soils, this can be costly and it can take years to correct acidity at depth. A complementary strategy for improving crop production on acid soils is the use of Al³⁺-tolerant germplasm developed through breeding or genetic modification along with liming practices. In several crop species, variation in Al³⁺ tolerance has been identified and selective breeding programs can be implemented to increase production on acid soils [13,14,15]. However as noted above, durum wheat is one of the most Al³⁺ sensitive of the small-grained crops and shows little variation in Al³⁺ tolerance [16]. Bread wheat is hexaploid and possesses the A-, B-, and D-genomes whereas durum wheat is tetraploid possessing only the A- and B-genomes. Although, as noted below, a major gene for Al³⁺ tolerance is found on the D-genome of bread wheat, there are other genes for Al³⁺ tolerance in bread wheat found on the A- and B-genomes [6]. Early reports that identified Al³⁺-tolerant durum genotypes with a level of tolerance similar to that of bread wheat [17] can now be attributed to misidentification or contamination of durum grain stocks with bread wheat [18], highlighting the importance of verifying the genetic identity of germplasm. Bread wheat, in contrast to durum, shows a large variation in Al³⁺ tolerance and much of this is conditioned by alleles of the TaALMT1 gene located on chromosome 4D [19] and TaMATE1B located on chromosome 4B. TaALMT1 encodes an Al³⁺-activated membrane channel permeable to malate with the malate exuded by root apices binding toxic Al³⁺ to protect the developing roots. Another Al³⁺ tolerance gene in bread wheat is TaMATE1B that encodes a citrate transporter in the plasma membrane and in some bread wheat genotypes confers a lower level of tolerance than TaALMT1 [20] whereas in other genotypes TaMATE1B appears to be the predominant Al³⁺ tolerance gene [21]. Both TaALMT1 and TaMATE1B have been introgressed from bread wheat into durum wheat. This required use of the pairing homeologous (ph1c) mutant to enable TaALMT1 to be transferred from chromosome 4D in bread wheat to chromosome 4B in durum wheat [20]. In contrast to bread wheat, for durum seedlings grown on acid soil, TaMATE1B appears to provide a greater level of Al³⁺ tolerance than TaALMT1 for reasons that are not understood.

Ethiopia has been considered as a center of diversity for durum germplasm and landraces grown by farmers are a potential source of agronomically important genes [22,23]. A 90 K single nucleotide polymorphism (SNP) chip analysis supports the notion that durum wheat in Ethiopia is particularly diverse [24]. Furthermore, others undertaking similar phylogenetic analyses based on SNP markers have suggested that Ethiopia is a second center of origin for durum wheat [25]. We speculated that because of the diversity of the durum germplasm coupled with a strong selection pressure imposed by extensive areas of acid soils in Ethiopia useful levels of Al³⁺ tolerance could have evolved in Ethiopian durum landraces. Cultivated durum genotypes sourced from various countries were found to be all Al³⁺ sensitive [16] whereas others [26] identified relatively tolerant lines in a similar population. The Ethiopian landraces are likely to be more diverse in their genetic makeup than the populations previously screened for tolerance and provide an opportunity to identify unique genes that confer tolerance of acid soils and Al³⁺ toxicity in particular. As an initial strategy it would be preferable to identify Al³⁺ tolerance in germplasm already adapted to Ethiopian conditions instead of introgressing genes from other sources. In this study we screened a diverse set of Ethiopian durum germplasm for Al³⁺ tolerance using a rapid hydroponic screen and then assessed selected lines more thoroughly in the hydroponic screen and finally in field trials on acid soil. Despite Ethiopia having conditions that would favor the evolution of Al³⁺ tolerance, the diverse durum germplasm was found to be Al³⁺ sensitive and highlighted the importance of verifying the identity of grain stocks. The absence of Al³⁺ tolerance genes in diverse durum germplasm suggests that genes encoding Al³⁺ tolerance found on the A- and B-genomes of bread wheat arose subsequent to the hybridization with the D-genome that produced hexaploid wheat.

2. Materials and Methods

2.1. Germplasm

A total of 595 durum wheat accessions obtained from the Ethiopian Biodiversity Institute (EBI: www.ibc.gov.et; Supplementary Materials file, Table S2), were screened in a series of non-replicated trials for their Al³⁺ tolerance using a rapid hydroponic screen. Based on their performance at this preliminary stage, accessions were classified as Al³⁺-tolerant, -intermediate, or -sensitive (see below). Subsequently all the tolerant as well as selected accessions of the intermediate and sensitive classes, totaling 150 accessions, were evaluated in a replicated hydroponic experiment and a field trial. For the experiments that screened previously identified Al³⁺-tolerant lines of durum the selected lines are described by Raman et al. [26]. A durum line (Langdon 4D (4B)) that has the 4B chromosome substituted by the 4D chromosome of bread wheat [27] was included as a positive control of a confirmed Al³⁺-tolerant durum line [18].

2.2. Hydroponic Culture

A rapid hydroponic screening was undertaken using an apparatus comprised of a plastic basin (for holding nutrient solution), a plate that held seed of different wheat accessions separately submerged in nutrient solution and an aeration system (Supplementary Materials file, Figure S1). The method is based on one described previously [16]. Seedlings grown submerged in the aerated nutrient solution remained viable and could be transplanted to soil when required to bulk up grain. Grain harvested from single plants of selected seedlings from this “preliminary” screen was then used in a replicated hydroponic experiment using the same growth conditions. For the replicated experiment, the average performance of 10 seedlings for each accession was determined and the experiment repeated three times.

The nutrient solution comprised of 500 μM KNO₃, 500 μM CaCl₂, 500 μM NH₄NO₃, 150 μM MgSO₄, 10 μM KH₂PO₄, 2 μM FeCl₃, and 5 μM of Al₂SO₄.18H₂O as described previously [28]. The pH of the solution was adjusted to about 4.3 with 1 M HCl. Dry grain was immersed in the solution and seedlings allowed to grow submerged for 5 days in the solution culture with the nutrient solution changed every day to maintain pH and Al³⁺ concentration relatively constant. After 5 days seedlings had typically developed three seminal roots and the total root length was recorded to the nearest 0.1 cm.

We used conventional hydroponics with the same nutrient solution composition as described above to compare the Al³⁺ tolerance of a set of durum and bread wheat genotypes in a range of AlCl₃ concentrations. Relative root length for the various genotypes was calculated after 3 days growth as root length in solution that contained Al³⁺ relative to root length in control solution that lacked Al³⁺. Errors associated with relative root length were calculated as described previously [29].

2.3. Determining the Identity of the Al³⁺-Tolerant Accessions

The morphological similarity between durum and bread wheat made it difficult to establish whether the identified Al³⁺-tolerant lines were durum or bread wheat by using phenotypic traits alone. Therefore, a polymerase chain reaction (PCR) assay that targeted the Dgas44 sequence was used to determine whether the lines were durum or bread wheat [30]. Dgas44 is a D-genome specific repetitive sequence that can be used to distinguish hexaploid wheat from tetraploid wheats that lack the D-genome [31]. The sequence of forward and reverse primers of Dgas44 marker, respectively, were 5′-CTTCTGACGGGTCAGGGGCAC-3′ and 5′-CTGAATGCCCCTGCGGCTTAAG-3′.

Ten grain of bulked up samples used in the field trial along with one verified bread wheat cultivar (Enkoy) and one durum wheat accession (8317), distinguished by its reddish/pinkish seed color, were planted in pots. Enkoy variety was included as a positive control (i.e., possesses the D-genome since it is a bread wheat cultivar), while accession 8317 was used as a negative control since it is a known durum accession that lacks the D-genome. Young green leaves were collected separately from three individual plants for DNA extraction. Leaf samples were freeze-dried in liquid nitrogen and pulverized with a Geno/Grinder 2000 and genomic DNA was extracted with a ZymoResearch kit (Plant/Seed MiniPrep) following the manufacturer’s protocol. The extracted genomic DNA was quantified with a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA) and PCR of samples undertaken as previously described [18].

2.4. Field Experiment

The field experiment was conducted at Bedi (38°36′3″ E, 9°5′59″ N), which is located in Watabicha Minjaro Kebele, Welmera District, West Shewa Zone, Oromia Regional State, Ethiopia (Supplementary Materials file, Figure S2). It is situated at approximately 35 km west of Addis Ababa and about 25 km away from Holeta town in the north-east. Crops are generally planted from around mid-June, though it varies with crop type and the time of onset of rains.

Samples of the reddish-brown soil to a depth of 20 cm were collected from the experimental plot with an auger in a regular pattern following a line transect. The soil samples were submitted the following day to JIJE LABOGLASS PLC (Addis Ababa, Ethiopia) for analysis of the major parameters using standard procedures as shown in

Table 1. Chemical and physical characteristics of the soils of the study area.

Soil Parameter		Value
pH (water extract; 1:1.25)		4.92
Buffer pH (water extract 1:2)		5.57
Electrical conductivity (dS/m)		0.07
Organic matter (%)		4.01
Total N (%)		0.78
Available P (mg/kg)		8.87
Exchangeable acidity (meq/100 g)		2.23
CEC and exchangeable bases (cmol (+)/kg)	CEC	27.19
	Ca	5.27
	Mg	0.66
	K	0.91
Texture	Clay (%)	59
	Silt (%)	28
	Sand (%)	13
	Soil class	Clay

. Characterized as a clay type, the pH of a water extract of the soils was found to be 4.92 indicating acidity and the likely presence of Al³⁺ toxicity. Note that a water extract will generally have a higher pH value than if a CaCl₂ solution is used for the extraction. For the limed treatments 451 kg of CaCO₃ was manually applied to the soil the year prior to the trial covering an area of 200 m². The lime was obtained from Guder Lime Factory (Guder, West Shewa Zone, Ethiopia) through collaboration with Oromia Agriculture and Rural Development Bureau. The plot was then ploughed immediately and re-ploughed after a week to thoroughly mix the lime with the soil to a depth of about 20 cm so that Al³⁺ would be detoxified in the soil solution.

A total of 150 accessions were selected based on their response to Al³⁺ toxicity in the preliminary hydroponic screen described above. These accessions were planted during the major rainy season on limed and un-limed plots in the field using a randomized complete block design. The two treatments of the experiment (limed and un-limed blocks) were each replicated twice, and the replicated blocks were spaced 1 m apart such that the long edge of the blocks were arranged adjacent to one another. All 150 accessions were planted side by side in each block resulting in a total area of 30 × 2 m for each block. Individual accessions were planted within the blocks randomly in single rows 2 m long with 20 cm spacing between rows. Contamination of un-limed plots with lime through erosion and splashing was avoided by considering slope and spacing factors in the experimental layout. Specifically, plots at a higher slope were assigned as un-limed plots and were separated from the limed plots by 2 m. N and P fertilizers were applied after 3–4 weeks of planting as urea and di-ammonium phosphate at blanket recommendation rate (100 kg/ha) since there was no pre-determined site-specific fertilizer application rate. Hand-weeding was done at early seedling stage (about one month) and before the booting stage. Mature plants were harvested to measure total biomass and grain yield.

2.5. Statistical Analysis

Normality of distributions of the replicated hydroponic and field data were tested with SPSS version 24 (IBM SPSS statistics for Windows 2016, Version 24.0. Armonk, NY, USA: IBM Corp.) and RStudio (version 1.0.143: Integrated Development for R. RStudio, Inc., Boston, MA, USA. Retrieved from www.rstudio.com). The non-parametric (Kruskal–Wallis) and parametric ANOVA were computed either with RStudio or SPSS in order to determine whether there was significant difference between accessions for their performance in Al³⁺ treatments. The general linear model in which both accessions and replications were considered as fixed factors was used for analysis of variance. The median absolute deviation, a non-parametric statistic, was computed with RStudio to assess the level of variability attributed to the trait. Furthermore, post-hoc analysis was conducted using Tukey’s honestly significant difference for parametric statistics, while a nonparametric post-hoc was done with Dunn’s test, a Kruskal–Wallis test based post-hoc with “agricolae” package of RStudio. The coefficient of determination (r²) was calculated to examine the relationship between various variables.

3. Results

3.1. Hydroponic Screen

Durum wheat accessions (595) were initially screened for Al³⁺ tolerance using a rapid hydroponic method of submerged seedlings. These accessions sometimes comprised of two or more genotypes since there was a large variation in performance between individual plants of the accession (Figure 1). Furthermore, there were visually observable differences within an accession such as variation in grain color. To take this heterogeneity into account an accession was scored based on its best-performing seedling. The use of average performance of plants in representing an accession would have resulted in rejection of many accessions because of a poor average performance such that a single plant within the accession with an acceptable level of Al³⁺ tolerance would be lost.

Accessions were classified into three phenotypic classes based on their total root length as tolerant (≥3.1 cm), intermediate (≤3.0 cm but ≥2.1 cm) and susceptible (≤2.0 cm). Moreover, accession 6956 was included in the tolerant class because of its exceptionally long primary seminal root. Using this criterion 21 accessions were classed as tolerant, 180 as intermediate, and 394 as susceptible to Al³⁺ toxicity (Supplementary Materials file, Table S3).

A total of 150 accessions that included all 21 of the Al³⁺-tolerant class, 79 of the best performers of the intermediate class, and a random selection of 50 of the sensitive class were evaluated in three replications to more precisely characterize their Al³⁺ tolerance. In this experiment accessions were represented in each experiment by the average performance of ≥10 seedlings using grain harvested from individually selected seedlings. The performance of accessions in the replicated experiment using the same hydroponic method as the preliminary screen was well correlated with the preliminary screen (r² = 0.72; Figure 2).

3.2. Field Experiment

The set of 150 selections used in the replicated hydroponic screen was assessed in the field on an acid soil. As there was limited grain harvested from single plants of the selected seedlings, the accessions were assessed as single rows 2 m long on both an un-amended plot and an equivalent plot that had been amended with lime to neutralize the acidity. The grain yield and biomass means for the limed plots of all accessions combined was greater than the means of the acid plot indicating that soil acidity was clearly present at this site. Grain yield on the acid site was reduced on average to only 18% of the limed site whereas biomass was reduced to 28% of the limed site (Figure 3).

The performance of the Al³⁺-tolerant lines on the un-limed acid plot was remarkable and clearly distinct from that of the sensitive accessions. Figure 4 shows the relative (un-limed/limed) biomass and grain yields of the accessions selected for the field trial against the root length as determined in the preliminary screen.

Expressing data in relative terms takes into account inherent differences in plant vigor when Al³⁺ is absent but a similar relationship was found when using unmodified data of only the acid plot (Supplementary Materials file, Figure S3). Biomass and final grain yield of genotypes grown in the acid trial were strongly correlated (Figure 5A). When biomass and grain yield data were combined within each grouping of lines selected from the preliminary screen (sensitive, intermediate, and tolerant), they showed rankings consistent with their grouping based on the screen (Figure 5B).

Since previous studies have found that stocks of durum wheat lines can be contaminated with bread wheat, we sought to verify that both Al³⁺-tolerant and -intermediate selections were indeed durum wheat. It can sometimes be difficult to distinguish the species based solely on the phenotypes so we used the Dgas molecular marker that is unique to the D-genome to distinguish the species. Using Dgas we found that all the Al³⁺-tolerant and -intermediate accessions were actually bread wheat (Figure 6), with only the most sensitive genotypes being durum wheat.

3.3. Other Potential Sources of Al³⁺-Tolerant Durum Wheat

As discussed in the Introduction, the Al³⁺ tolerance of durum has been enhanced by introgression of genes from bread wheat although not into Ethiopian germplasm [20]. As an alternate source of genes, a previous report identified three out of 420 tetraploid genotypes screened that were comparatively Al³⁺ tolerant although they did not approach the tolerance shown by a tolerant bread wheat used as a check line [26]. The lines had been confirmed as tetraploid with a molecular marker specific for the D-genome, so these genotypes were a potential source of genes that could be used in direct crosses to Ethiopian lines. To establish if these three lines had a useful level of Al³⁺ tolerance, we assessed their performance in hydroponic culture against bread wheat lines that varied in tolerance as well as a 4D (4B) substitution line of durum. However, all three lines were rated as sensitive with the best performer having a similar level of tolerance as Al³⁺-sensitive bread wheat (Figure 7). By contrast, a durum line where the 4B chromosome was substituted by the 4D chromosome of hexaploid wheat had a level of Al³⁺ tolerance comparable to the tolerant hexaploids.

4. Discussion

4.1. A Rapid Screen Proves Robust and Correlates with Grain Yield in Field Trials when Ranking Wheat Germplasm for Al³⁺ Tolerance

In this work we screened Ethiopian durum accessions for Al³⁺ tolerance first using a high throughput method with hydroponics, then with a replicated hydroponic screen and finally in the field on acid soil. Ethiopian durum accessions that include landraces are reported to show great diversity for many traits and molecular analyses have verified the unique nature of this germplasm [24]. However, despite this diversity our work shows that there does not appear to be useful levels of Al³⁺ tolerance in Ethiopian durum wheat and that seedlings identified to be Al³⁺ tolerant were in fact contaminating bread wheat. It should be noted that many of the accessions classed as tolerant only had a few Al³⁺-tolerant seedlings indicating that the durum grain stocks had been contaminated with bread wheat grain. This was verified by the use of a molecular marker that confirmed Al³⁺-tolerant seedlings to be bread wheat. We conclude that the rapid screen was sufficiently robust as a preliminary screen to identify Al³⁺-tolerant bread wheat contaminants. The accessions ranked as having intermediate tolerance in the preliminary screen could still have conferred a useful level of Al³⁺ tolerance for durum if they ranked similarly in the field. However, in the field the best performing accessions ranked as having intermediate Al³⁺ tolerance from the preliminary screen were all identified as bread wheat (Figure 6 and Supplementary Materials Figure S3B). Nevertheless, there exist Al³⁺-sensitive genotypes of bread wheat so the rapid screen does not replace the use of a molecular marker in identifying bread wheat lines. Verifying the identity of durum wheat grain stocks is of particular importance for germplasm banks but should also be confirmed by researchers. Bread wheat genotypes in the past have been mistakenly identified as durum wheat and the most relevant to this study was the incorrect identification of Al³⁺-tolerant durum lines that were subsequently found to be bread wheat [17,18]. In some cases, bread wheat has morphological characteristics in the field that are similar to durum wheat emphasizing the need for molecular analysis to establish the species identity [32].

The absence of Al³⁺ tolerance in unmodified Triticum turgidum ssp durum Desf. is consistent with previous studies that screened durum cultivars with presumably a lower level of diversity than the Ethiopian germplasm and also showed no or only a comparatively low level of Al³⁺ tolerance [16]. The simple and rapid screen undertaken with minimal equipment over only 5 days growth with seedlings submerged in nutrient solution showed a remarkable consistency in classifying the relative tolerance of germplasm grown in the field and measured for mature biomass and even grain yield (Figure 5B). Although there was a good general agreement of the hydroponic screens with performance of accessions in the field, there were exceptions. For instance, several accessions rated as having intermediate tolerance with the preliminary screen were the best or amongst the best for grain yield in the field (Figure 4B). This finding emphasizes the importance of verifying selections on acid soil whether in the field or in pots and that while hydroponic screens allow for rapid assessment of germplasm, the relative tolerance of genotypes can differ when grown in soil. For instance, a durum line carrying the TaMATE1B gene introgressed from hexaploid wheat (see below) shows marginal Al³⁺-tolerance in hydroponics that is considerably less than a line carrying TaALMT1, whereas the situation is reversed when the lines are grown on acid soil, with TaMATE1B lines outperforming TaALMT1 lines [20].

4.2. The Evolution of Al³⁺ Tolerance in Bread Wheat Occurred Subsequent to the Hybridization of the D-Genome

Here we show that durum lines previously reported to be relatively Al³⁺-tolerant [26] had a level of tolerance similar to sensitive wheat (Figure 7), confirming the absence of useful Al³⁺ tolerance in durum wheat germplasm (excluding lines where genes from hexaploid wheat have been introgressed as discussed below). This observation is puzzling given reports of the presence of Al³⁺ tolerance genes on the A- and B-genomes of bread wheat. Although TaALMT1, the major gene for Al³⁺ tolerance in bread wheat, is located on chromosome 4D, there are several loci on the A- and B-genomes reported to confer Al³⁺ tolerance [6]. For example, TaMATE1B the only Al³⁺ tolerance gene other than TaALMT1 that has been cloned from bread wheat, is located on chromosome 4B [33]. We surmise that the absence of Al³⁺ tolerance genes in diverse durum germplasm suggests that the multiple Al³⁺ tolerance genes found in bread wheat arose subsequent to the hybridization of the D-genome with the A- and B-genomes some 10,000 years ago. Transposable elements have been shown to enhance the level of expression of genes encoding transport proteins such as those of the MATE family [34] that confer Al³⁺ tolerance. The event of polyploidization commonly results in genome instability including the activation of transposable elements [35]. It is conceivable that activation of transposons when the hexaploid was formed has been key in enabling bread wheat to evolve Al³⁺ tolerance.

4.3. Strategies to Enhance the Al³⁺ Tolerance of Ethiopian Durum Wheat

To date the only durum germplasm verified to be Al³⁺ tolerant are those that were developed by introgression of Al³⁺ tolerance genes from bread wheat into durum wheat [20]. In those cases, the ph1c mutant was used to introgress the TaALMT1 gene located on chromosome 4D of bread wheat and steps taken to avoid the hybrid necrosis that can occur when crossing bread to durum wheat to introgress the TaMATE1B gene on chromosome 4B. This germplasm is not considered to be genetically modified since it was developed by so-called natural means and can therefore be used in the field without restrictions. The germplasm is a source of Al³⁺ tolerance genes in a durum background that could be used to introgress one or both genes derived from bread wheat into selected Ethiopian durum germplasm. Many of the farms that grow durum wheat in Ethiopia are small holdings and it has been found that landraces otherwise known as “farmer’s varieties” perform better than “improved” germplasm under many situations [36]. With this in mind it may be useful to cross the Al³⁺-tolerant germplasm described above into selected landraces adapted to local regions. Using local landraces as recurrent parents in multiple backcrosses while tracking the presence of Al³⁺ tolerance genes using molecular markers should maintain the germplasm with various valuable landrace traits that could otherwise be lost [37]. The TaMATE1B gene seems a preferred candidate at this stage since it appears to be more effective than TaALMT1 in a durum background and there exists a co-dominant marker that can be used to track the tolerant allele [20]. A recent publication has shown that the TaMATE1B gene introgressed into a durum genetic background confers a marked ability of roots from mature plants to withstand Al³⁺ toxicity when grown in an acid soil [38]. A simple backcrossing program where a single gene is introgressed into landraces could be useful in establishing whether or not TaMATE1B can improve durum grain production of landraces on acid soils of Ethiopia. We speculate that introgressing both TaALMT1 with TaMATE1B may provide the greatest level of Al³⁺ tolerance to durum wheat as has been found for some bread wheat genotypes.

5. Conclusions

Here we show that despite the diversity of Ethiopian durum germplasm and large regions of acid soils, conditions conducive for the evolution of Al³⁺ tolerance, a useful level of Al³⁺ tolerance was not detected. A high throughput screen identified Al³⁺-tolerant seedlings within accessions but all of them were shown to be contaminating bread wheat. This finding highlights the importance of correct identification of germplasm and confirmation of species identity with molecular markers particularly for closely-related species. The finding that Ethiopian durum germplasm is Al³⁺ sensitive is consistent with Al³⁺ tolerance having evolved in bread wheat subsequent to the hybridization of the D-genome with the A- and B-genomes. A field trial on acid soil showed biomass and grain yields that correlated with classifications based on a high throughput screen, confirming the utility of the rapid screen for preliminary assessment of germplasm.