In several crop species within the Triticeae tribe of the grass family Poaceae, single major aluminum (Al) tolerance genes have been identified that effectively mitigate Al toxicity, a major abiotic constraint to crop production on acidic soils. However, the trait is quantitatively inherited in species within other tribes, and the possible ancestral relationships between major Al tolerance genes and QTL in the grasses remain unresolved. To help establish these relationships, we conducted a molecular genetic analysis of Al tolerance in sorghum and integrated our findings with those from previous studies performed in crop species belonging to different grass tribes. A single locus, Alt_SB, was found to control Al tolerance in two highly Al tolerant sorghum cultivars. Significant macrosynteny between sorghum and the Triticeae was observed for molecular markers closely linked to putatively orthologous Al tolerance loci present in the group 4 chromosomes of wheat, barley, and rye. However, Alt_SB was not located within the homeologous region of sorghum but rather mapped near the end of sorghum chromosome 3. Thus, Alt_SB not only is the first major Al tolerance gene mapped in a grass species that does not belong to the Triticeae, but also appears to be different from the major Al tolerance locus in the Triticeae. Intertribe map comparisons suggest that a major Al tolerance QTL on rice chromosome 1 is likely to be orthologous to Alt_SB, whereas another rice QTL on chromosome 3 is likely to correspond to the Triticeae group 4 Al tolerance locus. Therefore, this study demonstrates a clear evolutionary link between genes and QTL encoding the same trait in distantly related species within a single plant family.

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GENETIC variation for tolerance to aluminum (Al) toxicity, a major limiting factor for plant growth on acidic soils, is well documented (DUNCAN 1988; PANDEY et al. 1994; CARVER and OWNBY 1995). However, the extent to which Al tolerance in different plant species derives from the action of orthologous or paralogous genes vs. that of distinctly different genes or gene ensembles has yet to be resolved.

For members of the grass tribe Triticeae including wheat, barley, and rye, comparative map data suggest that parallel mutations at a single orthologous locus on the group 4 chromosomes underlie Al tolerance (GARVIN and CARVER 2003). As such, Al tolerance in these crops can be readily evaluated by simple Mendelian analysis. In contrast, natural genetic variation for Al tolerance in other domesticated members of the Poaceae in different tribes, such as rice (WU et al. 2000; NGUYEN et al. 2001, 2002, 2003) and maize (MAGNAVACA et al. 1987; NINAMANGO-CáRDENAS et al. 2003) appears to be quantitative in nature. While intratribe conservation of Al tolerance genes seems likely, the absence of known major Al tolerance genes outside of the Triticeae suggests that Al tolerance in these other tribes may derive from genes wholly different from those found within the Triticeae. Nevertheless, there is compelling evidence that a number of agriculturally important traits may be controlled by orthologous loci in different grass species (LIN et al. 1995; PATERSON et al. 1995; PEREIRA and LEE 1995; HU et al. 2003). It is therefore desirable to develop a comprehensive model for Al tolerance gene evolutionary relationships in the Poaceae, to answer basic biological questions regarding the evolution of this trait, and to understand what opportunities may exist to use biotechnology to improve Al tolerance by pyramiding unique Al tolerance genes from different species.

In this study, we investigated the inheritance of Al tolerance in sorghum and determined the chromosome location of the major Al tolerance gene that was detected. The hypothesis that different Al tolerance genes exist outside the Triticeae was tested through synteny-based analysis of the genome locations of Al tolerance genes and quantitative trait loci (QTL) in sorghum, rice, and the Triticeae. Our results demonstrate that two apparently distinct major Al tolerance genes in the Triticeae and Andropogoneae are likely to be orthologous to two major QTL in rice, a member of the Oryzeae. Thus, this study establishes a framework for understanding the genetic complexity of Al tolerance across highly diverse domesticated members of the Poaceae.

Plant materials:

The Al tolerant sorghum inbred lines SC283 and SC566-14 (Sorghum bicolor ssp. bicolor) used in this research were collected from distinct regions within Africa and also differ distinctly in their classification. SC283 belongs to the guineae race (HARLAN and DE WET 1972), was classified by MURTY et al. (1967) as a conspicuum working group, and was collected in Tanzania, whereas SC566 (caudatum race) was classified as a caudatum working group and was collected in Nigeria. SC283 and SC566 were each crossed with BR007, an Al-sensitive line from the Embrapa Maize and Sorghum breeding program. F₁ plants derived from each cross were self-pollinated and two independent F₂ populations were generated for Al tolerance studies (n = 49 for F₂:BR007 x SC283; n = 135 for F₂:BR007 x SC566). F₂ individuals from the BR007 x SC283 population were also transplanted to pots with soil in a greenhouse and self-pollinated to obtain F_2:3 families.

Hydroponic analysis of Al tolerance:

Al-induced inhibition of seminal root growth in nutrient solution was used to quantify Al tolerance, using the basal nutrient solution described in MAGNAVACA et al. (1987). Seeds were surface sterilized in 0.1% NaOCl for 8 min, rinsed eight times with 50 ml of 18 M H₂O, and allowed to germinate in petri dishes covered with two layers of moist filter paper for 3 days at 26° in the dark. The seminal roots from the seedlings were then inserted through the mesh bottoms of polyethylene cups, covered with black beads, and placed into holes in the lids of polyethylene containers filled with 8 liters of nutrient solution under continuous aeration (48 seedlings/container). The experiments were carried out in a growth chamber with 26° day and 23° night temperatures, a light intensity of 550 µmol photons m^–2 sec^–1 and a 12-hr photoperiod.

A dose response analysis at 0, 60, 110, 148, and 222 µM Al was performed with SC283, SC566, and BR007 to define the level of Al to be used in genetic studies. These concentrations correspond to free Al⁺³ activities of {0}, {11}, {20}, {27}, and {39} µM Al⁺³ (braces indicate Al⁺³ activity), respectively, as estimated with the speciation software program GEOCHEM-PC (PARKER et al. 1995). Treatment with {27} µM Al⁺³ for 5 days elicited the largest growth differences between the Al-tolerant and Al-sensitive parents and was thus used for phenotypic evaluations of Al tolerance in the progeny (data not shown).

For genetic studies of Al tolerance, four seedlings of the relevant tolerant parent and four of the sensitive parent were planted together with 40 F₂ progeny in each container containing nutrient solution lacking Al, and the plants were given a 24-hr acclimation period. Subsequently, the initial length of each seedling's root growing in control solution (ilc) was measured and final lengths in control solution (flc) for the same roots were recorded 24 hr later. The solution was then replaced by a nutrient solution containing {27} µM Al⁺³, and final root lengths under Al treatment (flAl) were obtained after 5 days of exposure to Al. Intrinsic root growth rates were assessed for each individual using the root growth data obtained during the 24-hr growth period in control solution. Accordingly, a control root growth rate was obtained as [(crgr_1d) = flc – ilc]. The root growth rate under Al exposure over the 5-day period (Alrgr_5d) was then calculated as Alrgr_5d = flAl – flc and Al inhibition of root growth was calculated relative to the control root growth: RRG (% relative root growth) = [Alrgr_5d/(crgr_1d x 5)] x 100.

Seedlings of parents and F₂ progeny were also qualitatively scored for visual symptoms of root damage caused by Al and for root apical Al accumulation using hematoxylin staining (POLLE et al. 1978) as described by TANG et al. (2000). The combination of differences in mean percentage relative root growth inhibition (RRG), visual root damage, and hematoxylin staining pattern between Al-tolerant and Al-sensitive parents was used to classify F₂ progeny as Al tolerant or sensitive.

Progeny testing of F₂:BR007 x SC283 was completed on F_2:3 families, using visual symptoms of root damage and the RRG family means. Twelve F₃ plants from each F₂ individual were used for progeny testing, which for a dominant single gene model assures a probability of >95% for correctly classifying a heterozygous parent. Additionally, when only one plant in a family exhibited sensitivity to Al, testing was repeated with 20 progeny to ensure proper genotypic classification.

DNA isolation and restriction fragment length polymorphism analysis:

Genomic DNA was isolated from 4 g of leaf tissue using the protocol described by RIEDE and ANDERSON (1996). Parental survey membranes for DNA blot analysis were prepared according to TANG et al. (2000), but with restriction digestions consisting of 10 µg of DNA and 10 units of restriction enzyme (18 different restriction endonucleases in total). For restriction fragment length polymorphism (RFLP) analysis, cloned inserts were isolated by restriction digestion and labeled with [³²P]dCTP by the random hexamer method (FEINBERG and VOGELSTEIN 1984), denatured at 100° for 10 min, and hybridized to parental membranes at 65° overnight as described in BERNATZKY and TANKSLEY (1986). Membranes were sequentially washed at 65° for 30 min with 2x SSC, 1x SSC, and 0.5x SSC or 30 min with 2x SSC and 20 min with 1x SSC [for hybridization with genomic clones or cloned amplified fragment length polymorphism (AFLP) fragments]. All wash solutions also contained 0.1% (w/v) SDS.

For comparative mapping of sorghum vs. Triticeae for Al tolerance genes, progeny membranes containing subsets of the F₂ progeny derived from BR007 x SC283 (n = 23) and of the F₂ progeny from BR007 x SC566 (n = 25) were hybridized with a set of genomic and cDNA clones located in the Triticeae group 4 chromosomes and linked to Alt_BH in wheat (RIEDE and ANDERSON 1996), Alp in barley (TANG et al. 2000), and Alt3 in rye (MIFTAHUDIN et al. 2002), as well as clones located elsewhere on barley chromosome 4H (LANGRIDGE et al. 1995) and sorghum linkage group C (BOIVIN et al. 1999).

Bulked-segregant analysis with AFLP markers:

For bulked-segregant analysis (MICHELMORE et al. 1991) in the F₂ generation of BR007 x SC283, equal amounts of DNA from 10 tolerant and 10 sensitive progeny were combined to produce a tolerant bulk (TB) and a sensitive bulk (SB). The bulks were then screened for polymorphisms by AFLP analysis (VOS et al. 1995) using the GIBCO BRL AFLP Analysis System I kit (Life Technologies, Gaithersburg, MD) according to the manufacturer's recommendations. A total of 128 pairwise combinations between 8 EcoRI primers and 16 MseI primers (primers described in the GIBCO BRL protocol and M-CCA, M-CCT, M-CGA, M-CGT, M-CCC, M-CGC, M-CCG, and M-CGG) were assayed. After progeny testing for Al tolerance, another TB was assembled that eliminated heterozygous F₂ individuals, and the resulting homozygous TB and SB were screened with HindIII/MseI primers using adapter and primer sequences described by KASUGA et al. (1997). A total of 256 pairwise combinations between HindIII (H-ACT, H-AGC, H-AAC, H-AAG, H-ACA, H-ACC, H-ACG, H-AGG, H-AAA, H-AAT, H-AGA, H-AGT, H-ATA, H-ATC, H-ATG, and H-ATT) and MseI primers (same as those used for EcoRI/MseI amplifications) were assayed. Primer combinations revealing polymorphisms were subsequently tested on individual F₂ progeny to test for linkage to Al tolerance genes.

Cloning and conversion of AFLP markers:

AFLP fragments linked to Al tolerance genes were excised from gels, rehydrated in 100 µl of TE buffer overnight at 4°, reamplified, cloned into the pCR 2.1-TOPO vector (Invitrogen, Carlsbad, CA), and sequenced. The identity of the cloned fragments was verified by electroblotting to Hybond N+ (Amersham, Arlington Heights, IL) membranes followed by hybridization as described by PIERRE et al. (2000). Sorghum parental survey membranes were hybridized with the cloned AFLP markers to identify RFLPs differentiating the parents, and complete genotypic classification for the AFLP markers was obtained with progeny membranes.

Linkage analysis:

Genetic linkage maps were constructed using the Mapmaker Macintosh program V2.0 (LANDER et al. 1987), and genetic distances were estimated from recombination frequencies using the Kosambi function (KOSAMBI 1944). The two-point analysis with the "group" command (LOD = 3 and maximum recombination frequency, = 0.4) was used to infer linkage groups. Three-point analysis was used to calculate the likelihoods of possible orders of each linked triplet, and multipoint analysis with "First Order" and "Compare" commands was used to verify the results of the three-point analysis. Finally, "Ripple" was used to confirm the correct order of all triplets in the context of the final order.

Inheritance of Al tolerance in sorghum—BR007 x SC283:

Analysis of F₂ progeny:

Individuals exhibiting between 5 and 15% RRG suffered severe root damage due to Al exposure and were heavily stained by hematoxylin (Figure 1, A and B), which was a phenotype similar to that displayed by the sensitive parent BR007 (Figure 1C). In contrast, the progeny with 40–70% RRG exhibited minimal hematoxylin staining and negligible visual symptoms of root damage (Figure 1, A and B) that were similar to the tolerant parent SC283 (Figure 1C). Thus, progeny in the RRG range between 5 and 15% were considered Al sensitive, while those in the 40–70% RRG range were classified as Al tolerant.

View larger version (176K): In this window In a new window Download PPT slide   FIGURE 1.— Phenotypic analysis of Al tolerance. Comparisons of root damage and hematoxylin staining in Al-tolerant vs. Al-sensitive sorghum F2 seedlings grouped in the 5–15% RRG vs. 40–70% RRG classes after growth in nutrient solution containing {27} µM Al+3 for 5 days. (A) Visual symptoms of Al toxicity vs. Al tolerance. (B) Hematoxylin staining patterns showing differential Al accumulation in roots. (C) Visual symptoms of root damage of the Al-tolerant (SC283) and Al-sensitive (BR007) parents subjected to the same Al activity and exposure period.

 

Analysis of F_2:3 families:

The frequency distribution of mean RRG values in F_2:3 families (Figure 2) was bimodal, with a discontinuity present at the 20–25% RRG class. The F_2:3 families derived from Al-sensitive F₂ individuals uniformly exhibited strong visual symptoms of root damage after treatment with Al and low RRG means with a small variance, indicating that the F₂ parents were true breeding for sensitivity. Individuals within F_2:3 families derived from Al-tolerant F₂ plants were either uniformly undamaged by Al treatment or segregated for root damage symptoms. The data obtained from the F_2:3 families conformed to a 1:2:1 monogenic segregation model (0.10 < P < 0.25), thus indicating that SC283 harbors a single major Al tolerance gene henceforth designated Alt_SB.

View larger version (21K): In this window In a new window Download PPT slide   FIGURE 2.— Segregation for Al tolerance in BR007 x SC283. Mean percentage RRG frequency distribution for 49 F2:3 families (12 individuals per family) grown in nutrient solution containing {27} µM Al+3 for 5 days. RRG mean values and standard deviations were 8.7 ± 0.96 for BR007 (n = 12) and 52.6 ± 19.71 for SC283 (n = 19).

 

Gene action of Alt_SB:

Table 1 shows that in both the F₂ and the F₃ generations, the RRG mean of the heterozygous (Tt) class fell between the homozygote midclass mean [(TT + tt)/2] and the mean of the homozygous tolerant (TT) class. A similar observation was made from comparisons of the mean of the Tt class to the midparent [(SC283 + BR007/2)] RRG mean. These results suggest that Alt_SB is partially dominant under the experimental conditions for which the phenotype was assessed here. The addition of a tolerance allele from SC283 [additive effect (a)] increased RRG by 20%, and the degree of dominance (d/a, where d is the dominance effect) was estimated as 0.5.

View this table: In this window In a new window   TABLE 1 Estimates of gene action for AltSB

 

Molecular mapping of Alt_SB:

Comparative mapping of Alt_SB vs. major Al tolerance genes in the Triticeae:

To determine if Alt_SB is orthologous to the major Al tolerance locus located on group 4 chromosomes in members of the Triticeae, we searched for evidence that molecular markers previously found to be tightly linked to the Triticeae Al tolerance genes Alt_BH and Alp were also linked to Alt_SB (Figure 3). This analysis also included the rye Al tolerance gene Alt3 on chromosome 4R (MIFTAHUDIN et al. 2002), which is not depicted in Figure 3, but is most likely orthologous to Alt_BH in wheat 4DL and to Alp in barley 4H as all three Al tolerance loci are linked to the marker Xbcd1230. The marker loci Xbcd1230, Xbcd1117, and Xwg464, which are closely linked to the putative Triticeae orthologs Alt_BH, Alp, and Alt3, mapped to a single conserved region on sorghum LC (see F₂:BR007 x SC283 and F₂:BR007 x SC566 in Figure 3) that also contains Xcdo1395 (BOIVIN et al. 1999). This finding indicated that sorghum LC is the counterpart to the Triticeae group 4 chromosomes as suggested by GALE and DEVOS (1998). However, a ² analysis of goodness-of-fit to an independent segregation model did not support linkage to Alt_SB for any of these markers, including Xbcd1230 in the F₂:BR007 x SC283 map (Figure 3). Additionally, the positions of Xwg464, Xbcd1117, and Xcdo1395 in the conserved region of LC (Figure 3) indicate that those markers are also not linked to Alt_SB in F₂:BR007 x SC283 (data not shown). This finding suggests not only that Alt_SB resides elsewhere in the sorghum genome, but also that it may not be orthologous to the Triticeae Al tolerance genes.

View larger version (23K): In this window In a new window Download PPT slide   FIGURE 3.— Syntenic relationships for sorghum LC (BOIVIN et al. 1999 and BR007 crosses), barley 4H (TANG et al. 2000), and wheat 4DL (RIEDE and ANDERSON 1996). Sizes of the two selected progeny subsets were 23 for F2:BR007 x SC283 and 25 for F2:BR007 x SC566; markers were ordered with LOD > 3. The double arrow denoting Xbcd1230 and Xwg114 (*) in F2:BR007 x SC283 indicates that the relative order of these two markers could not be determined.

 

AFLP markers for Alt_SB and anchoring on the sorghum map:

Amplification of bulked DNA pools with E-ACG/M-CTA generated a 295-base-pair (bp) fragment (Figure 4) differentiating the tolerant and sensitive bulks and linked in repulsion to Alt_SB. This marker, designated AFS37-1, was found to be a single-copy sequence when the cloned fragment was used as a probe in Southern analysis (data not shown).

View larger version (79K): In this window In a new window Download PPT slide   FIGURE 4.— DNA sequence comparison of AFS37-1 and its allele in IS3620C. The AFLP patterns obtained from the amplification of the tolerant bulk (TB), sensitive bulk (SB), the tolerant parent SC283 (283), the sensitive parent BR007 (007), BTx623 (BTx), and IS3620C (IS) are shown on top. Sequence alignment between the 295-bp fragment amplified in the sensitive bulk and BR007 (=AFS37-1) and the 299-bp fragment amplified in IS3620C is shown below, with polymorphic repeats and nucleotide substitutions differentiating the alleles highlighted.

 
When DNA template from parents of a reference sorghum mapping population, BTx623 and IS3620C (PENG et al. 1999; MENZ et al. 2002), was amplified with E-ACG/M-CTA, a putative allele of AFS37-1 was identified in IS3620C but not in BTx623, and this was verified by sequence alignment to AFS37-1 from BR007 (Figure 4). We then utilized the BTx623 x IS3620C recombinant inbred line (RIL) population and the corresponding mapping data set of MENZ et al. (2002) to map AFS37-1 by AFLP analysis to the terminal region of sorghum linkage group C (LG-C; MENZ et al. 2002) depicted in Figure 5A. LG-C in the MENZ et al. (2002) map corresponds to linkage group G (LG) in BOIVIN et al. (1999) rather than to LC. This confirmed that Alt_SB is located on a sorghum chromosome that is not homeologous to that harboring the major Al tolerance locus in several Triticeae species. Sorghum LG-C (MENZ et al. 2002) is now being referred to as sorghum chromosome 3 (KLEIN et al. 2003), a designation that will be adopted here. The final Alt_SB linkage map was constructed in the BR007 x SC283 population solely with codominant segregation data (Figure 5B). To do that, AFS37-1 and additional linked AFLPs were cloned and used as probes for RFLP analysis (as RFLPs, loci designated as RFS37-x). These markers spanned a genetic distance of 13.7 cM in the vicinity of Alt_SB.

View larger version (31K): In this window In a new window Download PPT slide   FIGURE 5.— Chromosomal location of AltSB. (A) Position of marker AFS37-1 on sorghum chromosome 3 (LG-C in MENZ et al. 2002). (B) AltSB linkage map in F2: BR007 x SC283 (RFS37-1–9 are RFLP markers originated from the conversion of AFLP markers). Asterisk denotes that RFS37-1 refers to the cloned AFLP marker AFS37-1 that was anchored onto sorghum chromosome 3 (MENZ et al. 2002). (C) RFLP profile of IS3620C (IS) and BTx623 (BTx) DNA restricted with EcoRI and hybridized with isu52. (D) RFLP profile of BTx623 (BTx), IS3620C (IS), BR007 (007), and SC283 (283) DNA restricted with DraI and hybridized with isu52. Revised positions of the markers shown in A in the context of the complete sorghum chromosome 3 data set are found by selecting linkage group C at http://sorghumgenome.tamu.edu. Genetic distances are shown in centimorgans.

 

Validation of the chromosomal location for Alt_SB:

The segregation of isu52, a marker located at position 198.5 cM on sorghum chromosome 3 (MENZ et al. 2002; Figure 5A), was scored in F₂:BR007 x SC283 using the restriction enzyme DraI. No recombinants were detected between isu52 and Alt_SB (Figure 5B), and for this reason minimum linkage distances were estimated as 3 cM (p-0.05) and 4.6 cM (p-0.01) by the method of HANSON (1959).

Subsequently we sought to resolve an apparent discrepancy observed for the position of markers at the end of chromosome 3 where Alt_SB is located. The RFLP loci isu52 and isu68, both originally scored as single copy markers, were found to be tightly linked by PEREIRA and LEE (1993), whereas these same markers were genetically unlinked according to PENG et al. (1999). To clarify this discrepancy, both markers were rescored in the BTx623 x IS3620C RILs, which is the same population used in the study by PENG et al. (1999) and MENZ et al. (2002). The RFLP profile obtained with EcoRI in this population revealed that isu52 is duplicated on sorghum chromosome 3. One copy is located at position 198.5 cM (isu52.1; Figure 5A) and corresponds to the locus shown in Figure 5C that was also scored by Peng and co-workers (G. E. HART, personal communication). Segregation for the second copy, isu52.2, could be scored on the XbaI (not shown), EcoRI (Figure 5C), and DraI membranes (Figure 5D) and mapped at 175 cM (Figure 5A). The marker isu68 was found to be a single-copy marker and was tightly linked to isu52.2 at position 174.5 cM (Figure 5A). Thus, isu52.2 corresponds to isu52 on the map of PEREIRA and LEE (1993). Because DraI produced identical RFLP profiles with isu52 in both the BTx623 x IS3620C and the BR007 x SC283 parents (Figure 5D), it is isu52.2 (Figure 5A) that is tightly linked to Alt_SB (Figure 5B). A linear regression of F_2:3 RRG mean values as a function of the three genotypic classes for isu52.2 showed that this marker alone explained 79% of the phenotypic variance for Al tolerance in the progeny (r² = 0.79, P < 0.001).

Molecular genetics of Al tolerance in SC566:

The RRG frequency distribution of F₂ progeny derived from the cross of BR007 with a second Al tolerant parent, SC566, was clearly bimodal (Figure 6A). RRG values, symptoms of root damage caused by Al, and hematoxylin staining of F₂ progeny roots were compared to SC566 and BR007, and Al-sensitive individuals were identified in the 5–15% RRG classes while Al-tolerant progeny exhibited RRG values between 35 and 105%. Individuals exhibiting RRG values within intermediate classes (15–35% RRG) were also identified, but could not be unambiguously classified as tolerant or sensitive. To test whether Al tolerance in SC566 is due to the presence of Alt_SB, isu52.2 segregation was scored in this population (Figure 6B). F₂ individuals that were sensitive to Al (5–15% RRG) were all homozygous for the BR007 allele of isu52.2 (Figure 6B). In contrast, all but three of the Al-tolerant F₂ progeny (35–105% RRG) were either homozygous for the SC566 allele of isu52.2 or heterozygous. If a major Al tolerance gene and a marker locus are unlinked, the expected frequency of double homozygous individuals is 0.0625. Thus, in theory just 8 such individuals should be present in the BR007 x SC566 population, rather than the 22 that were observed (Figure 6B). This strong linkage disequilibrium specifically with isu52.2 indicates that SC566 harbors Alt_SB or an allele of this gene.

View larger version (50K): In this window In a new window Download PPT slide   FIGURE 6.— Molecular genetics of Al tolerance in SC566. (A) Frequency distribution for percentage RRG of 135 F2:BR007 x SC566 individuals exposed to a nutrient solution containing {27} µM Al+3 for 5 days. The RRG means for BR007 (5–10% RRG class) and SC566 (60–65% class) represent the mean of 12 and 38 individuals per parent, respectively. (B) Association between the SC566 Al tolerance gene and isu52.2. Shown are RFLP profiles of Al-tolerant and -sensitive parents SC566 and BR007, Al-sensitive F2 progeny (5–15% RRG), and Al-tolerant progeny in the 50–60% RRG range digested with DraI and hybridized to isu52. The RFLP profiles for the Al-tolerant progeny shown are representative of all Al-tolerant F2 progeny (35–105% RRG), with only three recombinants detected (asterisk denotes recombinant individual).

 

The grass family Poaceae is highly diverse and contains 10,000 species (KELLOGG 2001), many of which are our most important staple crops. The extremely broad adaptation of the grasses to diverse environments (KELLOGG 1998), including adaptation to the widespread Al-toxic acid soils, raises the question whether adaptation to Al toxicity in different grass species is associated with mutations in a limited number of genes or whether a far more diverse range of genes contributes to Al tolerance in the grasses.

Our genetic analysis of Al tolerance in sorghum, a member of the tribe Andropogoneae, revealed that this trait was encoded by a single major locus, Alt_SB, which behaved in a semidominant fashion under our experimental conditions. Thus, to date, Alt_SB is the only major Al tolerance gene that has been mapped to the genome of a grass species not in the tribe Triticeae. In addition, because Alt_SB was identified in SC283, which is considered to be a standard for Al tolerance (DUNCAN et al. 1983; FURLANI et al. 1987; DUNCAN 1988), it conditions perhaps the highest Al tolerance level within sorghum.

A molecular marker-based evaluation of intraspecific Al tolerance diversity in sorghum indicated that the single major Al tolerance loci in SC283 and SC566, another extremely tolerant sorghum cultivar, are the same. The fact that SC283 and SC566 exhibited very distinct morphological characteristics and that they were collected at different sites in Africa suggested that these cultivars may have different genetic origins. Indeed, the caudatum race to which SC566 belongs was proposed to have arisen from a domestication episode more recent than that from which the guinea race, which includes SC283, arose (HARLAN 1975; DOGGETT 1988). Thus, the presence of a common Al tolerance locus in these two highly diverse sorghum cultivars indicates that the genetic basis for Al tolerance in sorghum may be quite narrow. This is similar to results of a comprehensive study of Al tolerance gene diversity in barley (MINELLA and SORRELLS 1992), where different Al tolerance levels displayed by a large set of cultivars were found to be due to allelic variation at a common locus (Alp). These findings in both sorghum and barley suggest that in crop species that display single gene inheritance for Al tolerance, mutations in just one or a few genes may confer agriculturally significant levels of Al tolerance, although different alleles at a single locus may be present (MINELLA and SORRELLS 1992). In such species, intraspecific gene pyramiding may not be a feasible strategy for enhancing Al tolerance. Alternatively, combining distinct Al tolerance genes from different species may hold greater potential for Al tolerance improvement, provided that such interspecific diversity exists, that the genes can be isolated, and that they function in other genetic backgrounds.

Potential orthology between major Al tolerance genes in the Andropogonae and Triticeae was assessed by comparative mapping. Our results showed that while molecular markers linked to the Al tolerance loci on the Triticeae group 4 chromosomes mapped to the expected syntenic region in sorghum, Alt_SB mapped to sorghum chromosome 3, which is not homeologous to the Triticeae group 4 chromosomes. The absence of significant disruptions of macrocolinearity between the homeologous sorghum LC and Triticeae group 4 chromosomes in the region near the major Triticeae Al tolerance locus suggests that Alt_SB is a gene distinctly different from that identified in the Triticeae.

Interestingly, a wheat-rye chromosome 3R addition line showed a dramatic increase in tolerance (ANIOL and GUSTAFSON 1984). Considering that the Triticeae group 3 chromosomes are likely to be homeologous to sorghum chromosome 3 (NELSON et al. 1995; GALE and DEVOS 1998), it is possible that an Alt_SB ortholog is present and functioning in rye, but has not yet been mapped in this or other Triticeae species because of a lack of polymorphism among genotypes. Alternatively, because perturbations of gene colinearity caused by small-scale events such as gene duplications and deletions (BENNETZEN and RAMAKRISHNA 2002) occur in the grasses, and segmental translocations to nonhomeologous chromosomes have been found to disrupt colinearity between the sorghum genome and those of wheat and barley (LI and GILL 2002), we cannot rule out the possibility that Alt_SB is orthologous to the group 4 Triticeae Al tolerance genes and has been translocated to a nonhomeologous sorghum chromosome.

Alt_SB is located on sorghum chromosome 3, which is homeologous to rice chromosome 1 (VENTELON et al. 2001; KLEIN et al. 2003), and Al tolerance QTL have been repeatedly detected at the end of rice chromosome 1 (WU et al. 2000; NGUYEN et al. 2001, 2002, 2003). In particular, the major rice QTL detected by NGUYEN et al. (2001) explained 25% of the phenotypic variance and was linked to Xwg110, which is located 28 cM from isu52 on rice chromosome 1 [see http://www.gramene.org: Rice-Cornell RFLP 2001-1 and WILSON et al. (1999) for marker positions in rice]. Interestingly, isu68, which we found to be tightly linked to isu52.2 in the Alt_SB region of sorghum chromosome 3 (see PEREIRA and LEE 1993 and Figure 5A), is not tightly linked to isu52 in rice and falls within the confidence interval for the rice Xwg110 Al tolerance QTL (between Xwg110 and rg109 according to the Gramene database). A BLAST analysis (data not shown) indicated that isu52 is present as a single-copy gene on rice chromosome 1, whereas in sorghum we found isu52.2 tightly linked to Alt_SB and isu52.1 24 cM from the sorghum Al tolerance gene. This implies that the rice isu52 locus corresponds to isu52.1 that is loosely linked to Alt_SB in sorghum and that the major rice Al tolerance QTL on chromosome 1 is likely to correspond to Alt_SB due to their common proximity to isu68.

Another major rice Al tolerance QTL has been identified on rice chromosome 3 (WU et al. 2000; NGUYEN et al. 2003), which is homeologous to the Triticeae group 4 chromosomes (AHN et al. 1993; VAN DEYNZE et al. 1995). Because this rice QTL and the Al tolerance genes on the Triticeae group 4 chromosomes are both linked to Xcdo1395, these loci may be orthologous (NGUYEN et al. 2003). Thus, it appears that the more complex quantitatively inherited Al tolerance in rice (Oryzeae), one of the most Al-tolerant grasses (MA et al. 2002; NGUYEN et al. 2002), is in part due to the action of two major QTL, which in the Andropogoneae and the Triticeae act as two distinct major Al tolerance genes.

Our sorghum Al tolerance map data, when jointly analyzed with data obtained for both major Al tolerance genes or Al tolerance QTL in other domesticated grass crops from different tribes, suggest that the capacity to adapt to Al toxicity is associated with mutations in a small and common suite of genes, which is congruent with the results of PATERSON et al. (1995) for traits involved in crop domestication. However, a likely pattern of gene conservation between distinct major Al tolerance genes in sorghum and the Triticeae and two major rice QTL was revealed only by a broad intertribe comparative analysis of Al tolerance genes.

The use of comparative mapping to integrate information from genomes of a range of plant species to a reference genome such as that of rice or Arabidopsis has become pivotal to modern plant genomics. However, as noted previously (KILIAN et al. 1997), chromosomal rearrangements that disrupt colinearity (TIKHONOV et al. 1999) may reduce the likelihood of finding an ortholog of a gene of interest in the expected syntenic position of a single given reference genome. Our results suggest that this issue may be mitigated through broader evolutionary comparisons among different members of a plant family. Because of its small genome size, relatively distant evolutionary relationship with rice, and growing genome resources, sorghum serves as a useful complement to the rice genome to foster comparative genomics in the grasses.

The authors thank Randy T. Clark for assistance with the phenotypic analysis of Al tolerance, Holly S. Manslank for general technical support, and Owen A. Hoekenga for prospecting collaborations on sorghum genomics. The study was supported by U.S. Department of Agriculture-National Research Institute competitive grants 97-35100-4501 (to L.V.K., D.F.G., and M.E.S.) and 2001-35301-10647 (to L.V.K.), National Science Foundation Plant Genome grant DBI-0077713 (to P.E.K.), and The McKnight Foundation Collaborative Crop Research Program Project "New approach for improving phosphorus acquisition and aluminum tolerance of plants in marginal soils" (to R.E.S. and L.V.K.). J.V.M. gratefully acknowledges the Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES-Brasil) for providing financial support for his Ph.D. degree program.

¹ Present address: Embrapa Maize and Sorghum, Rod. MG 424, Km 65, 35701-970, Sete Lagoas-MG, Brazil.

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