Multi-trait association mapping for phosphorous efficiency reveals flexible root architectures in sorghum.

BACKGROUND: On tropical regions, phosphorus (P) fixation onto aluminum and iron oxides in soil clays restricts P diffusion from the soil to the root surface, limiting crop yields. While increased root surface area favors P uptake under low-P availability, the relationship between the three-dimensional arrangement of the root system and P efficiency remains elusive. Here, we simultaneously assessed allelic effects of loci associated with a variety of root and P efficiency traits, in addition to grain yield under low-P availability, using multi-trait genome-wide association. We also set out to establish the relationship between root architectural traits assessed in hydroponics and in a low-P soil. Our goal was to better understand the influence of root morphology and architecture in sorghum performance under low-P availability. RESULT: In general, the same alleles of associated SNPs increased root and P efficiency traits including grain yield in a low-P soil. We found that sorghum P efficiency relies on pleiotropic loci affecting root traits, which enhance grain yield under low-P availability. Root systems with enhanced surface area stemming from lateral root proliferation mostly up to 40 cm soil depth are important for sorghum adaptation to low-P soils, indicating that differences in root morphology leading to enhanced P uptake occur exactly in the soil layer where P is found at the highest concentration. CONCLUSION: Integrated QTLs detected in different mapping populations now provide a comprehensive molecular genetic framework for P efficiency studies in sorghum. This indicated extensive conservation of P efficiency QTL across populations and emphasized the terminal portion of chromosome 3 as an important region for P efficiency in sorghum. Increases in root surface area via enhancement of lateral root development is a relevant trait for sorghum low-P soil adaptation, impacting the overall architecture of the sorghum root system. In turn, particularly concerning the critical trait for water and nutrient uptake, root surface area, root system development in deeper soil layers does not occur at the expense of shallow rooting, which may be a key reason leading to the distinctive sorghum adaptation to tropical soils with multiple abiotic stresses including low P availability and drought.

Association mapping and genomic selection for sorghum adaptation to tropical soils of Brazil in a sorghum multiparental random mating population.

KEY MESSAGE: A multiparental random mating population used in sorghum breeding is amenable for the detection of QTLs related to tropical soil adaptation, fine mapping of underlying genes and genomic selection approaches. Tropical soils where low phosphorus (P) and aluminum (Al) toxicity limit sorghum [Sorghum bicolor (L.) Moench] production are widespread in the developing world. We report on BRP13R, a multiparental random mating population (MP-RMP), which is commonly used in sorghum recurrent selection targeting tropical soil adaptation. Recombination dissipated much of BRP13R's likely original population structure and average linkage disequilibrium (LD) persisted up to 2.5 Mb, establishing BRP13R as a middle ground between biparental populations and sorghum association panels. Genome-wide association mapping (GWAS) identified conserved QTL from previous studies, such as for root morphology and grain yield under low-P, and indicated the importance of dominance in the genetic architecture of grain yield. By overlapping consensus QTL regions, we mapped two candidate P efficiency genes to a ~ 5 Mb region on chromosomes 6 (ALMT) and 9 (PHO2). Remarkably, we find that only 200 progeny genotyped with ~ 45,000 markers in BRP13R can lead to GWAS-based positional cloning of naturally rare, subpopulation-specific alleles, such as for SbMATE-conditioned Al tolerance. Genomic selection was found to be useful in such MP-RMP, particularly if markers in LD with major genes are fitted as fixed effects into GBLUP models accommodating dominance. Shifts in allele frequencies in progeny contrasting for grain yield indicated that intermediate to minor-effect genes on P efficiency, such as SbPSTOL1 genes, can be employed in pre-breeding via allele mining in the base population. Therefore, MP-RMPs such as BRP13R emerge as multipurpose resources for efficient gene discovery and deployment for breeding sorghum cultivars adapted to tropical soils.

The genetic architecture of phosphorus efficiency in sorghum involves pleiotropic QTL for root morphology and grain yield under low phosphorus availability in the soil.

BACKGROUND: Phosphorus (P) fixation on aluminum (Al) and iron (Fe) oxides in soil clays restricts P availability for crops cultivated on highly weathered tropical soils, which are common in developing countries. Hence, P deficiency becomes a major obstacle for global food security. We used multi-trait quantitative trait loci (QTL) mapping to study the genetic architecture of P efficiency and to explore the importance of root traits on sorghum grain yield on a tropical low-P soil. RESULTS: P acquisition efficiency was the most important component of P efficiency, and both traits were highly correlated with grain yield under low P availability. Root surface area was positively associated with grain yield. The guinea parent, SC283, contributed 58% of all favorable alleles detected by single-trait mapping. Multi-trait mapping detected 14 grain yield and/or root morphology QTLs. Tightly linked or pleiotropic QTL underlying the surface area of fine roots (1-2 mm in diameter) and grain yield were detected at positions 1-7 megabase pairs (Mb) and 71 Mb on chromosome 3, respectively, and a root diameter/grain yield QTL was detected at 7 Mb on chromosome 7. All these QTLs were near sorghum homologs of the rice serine/threonine kinase, OsPSTOL1. The SbPSTOL1 genes on chromosome 3, Sb03g006765 at 7 Mb and Sb03g031690 at 60 Mb were more highly expressed in SC283, which donated the favorable alleles at all QTLs found nearby SbPSTOL1 genes. The Al tolerance gene, SbMATE, may also influence a grain yield QTL on chromosome 3. Another PSTOL1-like gene, Sb07g02840, appears to enhance grain yield via small increases in root diameter. Co-localization analyses suggested a role for other genes, such as a sorghum homolog of the Arabidopsis ubiquitin-conjugating E2 enzyme, phosphate 2 (PHO2), on grain yield advantage conferred by the elite parent, BR007 allele. CONCLUSIONS: Genetic determinants conferring higher root surface area and slight increases in fine root diameter may favor P uptake, thereby enhancing grain yield under low-P availability in the soil. Molecular markers for SbPSTOL1 genes and for QTL increasing grain yield by non-root morphology-based mechanisms hold promise in breeding strategies aimed at developing sorghum cultivars adapted to low-P soils.

Repeat variants for the SbMATE transporter protect sorghum roots from aluminum toxicity by transcriptional interplay in cis and trans.

Acidic soils, where aluminum (Al) toxicity is a major agricultural constraint, are globally widespread and are prevalent in developing countries. In sorghum, the root citrate transporter SbMATE confers Al tolerance by protecting root apices from toxic Al3+, but can exhibit reduced expression when introgressed into different lines. We show that allele-specific SbMATE transactivation occurs and is caused by factors located away from SbMATE Using expression-QTL mapping and expression genome-wide association mapping, we establish that SbMATE transcription is controlled in a bipartite fashion, primarily in cis but also in trans Multiallelic promoter transactivation and ChIP analyses demonstrated that intermolecular effects on SbMATE expression arise from a WRKY and a zinc finger-DHHC transcription factor (TF) that bind to and trans-activate the SbMATE promoter. A haplotype analysis in sorghum RILs indicates that the TFs influence SbMATE expression and Al tolerance. Variation in SbMATE expression likely results from changes in tandemly repeated cis sequences flanking a transposable element (a miniature inverted repeat transposable element) insertion in the SbMATE promoter, which are recognized by the Al3+-responsive TFs. According to our model, repeat expansion in Al-tolerant genotypes increases TF recruitment and, hence, SbMATE expression, which is, in turn, lower in Al-sensitive genetic backgrounds as a result of lower TF expression and fewer binding sites. We thus show that even dominant cis regulation of an agronomically important gene can be subjected to precise intermolecular fine-tuning. These concerted cis/trans interactions, which allow the plant to sense and respond to environmental cues, such as Al3+ toxicity, can now be used to increase yields and food security on acidic soils.

Exploiting sorghum genetic diversity for enhanced aluminum tolerance: Allele mining based on the AltSB locus.

Root damage due to aluminum (Al) toxicity restricts crop production on acidic soils, which are extensive in the tropics. The sorghum root Al-activated citrate transporter, SbMATE, underlies the Al tolerance locus, AltSB, and increases grain yield under Al toxicity. Here, AltSB loci associated with Al tolerance were converted into Amplification Refractory Mutation System (ARMS) markers, which are cost effective and easy to use. A DNA pooling strategy allowed us to identify accessions harboring rare favorable AltSB alleles in three germplasm sets while greatly reducing genotyping needs. Population structure analysis revealed that favorable AltSB alleles are predominantly found in subpopulations enriched with guinea sorghums, supporting a possible Western African origin of AltSB. The efficiency of allele mining in recovering Al tolerance accessions was the highest in the largest and highly diverse germplasm set, with a 10-fold reduction in the number of accessions that would need to be phenotyped in the absence of marker information. Finally, Al tolerant accessions were found to rely on SbMATE to exclude Al3+ from sensitive sites in the root apex. This study emphasizes gene-specific markers as important tools for efficiently mining useful rare alleles in diverse germplasm, bridging genetic resource conservation efforts and pre-breeding for Al tolerance.

Duplicate and conquer: multiple homologs of PHOSPHORUS-STARVATION TOLERANCE1 enhance phosphorus acquisition and sorghum performance on low-phosphorus soils.

Low soil phosphorus (P) availability is a major constraint for crop production in tropical regions. The rice (Oryza sativa) protein kinase, PHOSPHORUS-STARVATION TOLERANCE1 (OsPSTOL1), was previously shown to enhance P acquisition and grain yield in rice under P deficiency. We investigated the role of homologs of OsPSTOL1 in sorghum (Sorghum bicolor) performance under low P. Association mapping was undertaken in two sorghum association panels phenotyped for P uptake, root system morphology and architecture in hydroponics and grain yield and biomass accumulation under low-P conditions, in Brazil and/or in Mali. Root length and root surface area were positively correlated with grain yield under low P in the soil, emphasizing the importance of P acquisition efficiency in sorghum adaptation to low-P availability. SbPSTOL1 alleles reducing root diameter were associated with enhanced P uptake under low P in hydroponics, whereas Sb03g006765 and Sb03g0031680 alleles increasing root surface area also increased grain yield in a low-P soil. SbPSTOL1 genes colocalized with quantitative trait loci for traits underlying root morphology and dry weight accumulation under low P via linkage mapping. Consistent allelic effects for enhanced sorghum performance under low P between association panels, including enhanced grain yield under low P in the soil in Brazil, point toward a relatively stable role for Sb03g006765 across genetic backgrounds and environmental conditions. This study indicates that multiple SbPSTOL1 genes have a more general role in the root system, not only enhancing root morphology traits but also changing root system architecture, which leads to grain yield gain under low-P availability in the soil.

Association mapping provides insights into the origin and the fine structure of the sorghum aluminum tolerance locus, AltSB.

Root damage caused by aluminum (Al) toxicity is a major cause of grain yield reduction on acid soils, which are prevalent in tropical and subtropical regions of the world where food security is most tenuous. In sorghum, Al tolerance is conferred by SbMATE, an Al-activated root citrate efflux transporter that underlies the major Al tolerance locus, AltSB, on sorghum chromosome 3. We used association mapping to gain insights into the origin and evolution of Al tolerance in sorghum and to detect functional variants amenable to allele mining applications. Linkage disequilibrium across the AltSB locus decreased much faster than in previous reports in sorghum, and reached basal levels at approximately 1000 bp. Accordingly, intra-locus recombination events were found to be extensive. SNPs and indels highly associated with Al tolerance showed a narrow frequency range, between 0.06 and 0.1, suggesting a rather recent origin of Al tolerance mutations within AltSB. A haplotype network analysis suggested a single geographic and racial origin of causative mutations in primordial guinea domesticates in West Africa. Al tolerance assessment in accessions harboring recombinant haplotypes suggests that causative polymorphisms are localized to a ∼6 kb region including intronic polymorphisms and a transposon (MITE) insertion, whose size variation has been shown to be positively correlated with Al tolerance. The SNP with the strongest association signal, located in the second SbMATE intron, recovers 9 of the 14 highly Al tolerant accessions and 80% of all the Al tolerant and intermediately tolerant accessions in the association panel. Our results also demonstrate the pivotal importance of knowledge on the origin and evolution of Al tolerance mutations in molecular breeding applications. Allele mining strategies based on associated loci are expected to lead to the efficient identification, in diverse sorghum germplasm, of Al tolerant accessions able maintain grain yields under Al toxicity.

Incomplete transfer of accessory loci influencing SbMATE expression underlies genetic background effects for aluminum tolerance in sorghum.

Impaired root development caused by aluminum (Al) toxicity is a major cause of grain yield reduction in crops cultivated on acid soils, which are widespread worldwide. In sorghum, the major Al-tolerance locus, AltSB , is due to the function of SbMATE, which is an Al-activated root citrate transporter. Here we performed a molecular and physiological characterization of various AltSB donors and near-isogenic lines harboring various AltSB alleles. We observed a partial transfer of Al tolerance from the parents to the near-isogenic lines that was consistent across donor alleles, emphasizing the occurrence of strong genetic background effects related to AltSB . This reduction in tolerance was variable, with a 20% reduction being observed when highly Al-tolerant lines were the AltSB donors, and a reduction as great as 70% when other AltSB alleles were introgressed. This reduction in Al tolerance was closely correlated with a reduction in SbMATE expression in near-isogenic lines, suggesting incomplete transfer of loci acting in trans on SbMATE. Nevertheless, AltSB alleles from the highly Al-tolerant sources SC283 and SC566 were found to retain high SbMATE expression, presumably via elements present within or near the AltSB locus, resulting in significant transfer of the Al-tolerance phenotype to the derived near-isogenic lines. Allelic effects could not be explained by coding region polymorphisms, although occasional mutations may affect Al tolerance. Finally, we report on the extensive occurrence of alternative splicing for SbMATE, which may be an important component regulating SbMATE expression in sorghum by means of the nonsense-mediated RNA decay pathway.

The relationship between population structure and aluminum tolerance in cultivated sorghum.

BACKGROUND: Acid soils comprise up to 50% of the world's arable lands and in these areas aluminum (Al) toxicity impairs root growth, strongly limiting crop yield. Food security is thereby compromised in many developing countries located in tropical and subtropical regions worldwide. In sorghum, SbMATE, an Al-activated citrate transporter, underlies the Alt(SB) locus on chromosome 3 and confers Al tolerance via Al-activated root citrate release. METHODOLOGY: Population structure was studied in 254 sorghum accessions representative of the diversity present in cultivated sorghums. Al tolerance was assessed as the degree of root growth inhibition in nutrient solution containing Al. A genetic analysis based on markers flanking Alt(SB) and SbMATE expression was undertaken to assess a possible role for Alt(SB) in Al tolerant accessions. In addition, the mode of gene action was estimated concerning the Al tolerance trait. Comparisons between models that include population structure were applied to assess the importance of each subpopulation to Al tolerance. CONCLUSION/SIGNIFICANCE: Six subpopulations were revealed featuring specific racial and geographic origins. Al tolerance was found to be rather rare and present primarily in guinea and to lesser extent in caudatum subpopulations. Alt(SB) was found to play a role in Al tolerance in most of the Al tolerant accessions. A striking variation was observed in the mode of gene action for the Al tolerance trait, which ranged from almost complete recessivity to near complete dominance, with a higher frequency of partially recessive sources of Al tolerance. A possible interpretation of our results concerning the origin and evolution of Al tolerance in cultivated sorghum is discussed. This study demonstrates the importance of deeply exploring the crop diversity reservoir both for a comprehensive view of the dynamics underlying the distribution and function of Al tolerance genes and to design efficient molecular breeding strategies aimed at enhancing Al tolerance.

A gene in the multidrug and toxic compound extrusion (MATE) family confers aluminum tolerance in sorghum.

Crop yields are significantly reduced by aluminum toxicity on highly acidic soils, which comprise up to 50% of the world's arable land. Candidate aluminum tolerance proteins include organic acid efflux transporters, with the organic acids forming non-toxic complexes with rhizosphere aluminum. In this study, we used positional cloning to identify the gene encoding a member of the multidrug and toxic compound extrusion (MATE) family, an aluminum-activated citrate transporter, as responsible for the major sorghum (Sorghum bicolor) aluminum tolerance locus, Alt(SB). Polymorphisms in regulatory regions of Alt(SB) are likely to contribute to large allelic effects, acting to increase Alt(SB) expression in the root apex of tolerant genotypes. Furthermore, aluminum-inducible Alt(SB) expression is associated with induction of aluminum tolerance via enhanced root citrate exudation. These findings will allow us to identify superior Alt(SB) haplotypes that can be incorporated via molecular breeding and biotechnology into acid soil breeding programs, thus helping to increase crop yields in developing countries where acidic soils predominate.

Comparative mapping of a major aluminum tolerance gene in sorghum and other species in the poaceae.

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, AltSB, 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, AltSB was not located within the homeologous region of sorghum but rather mapped near the end of sorghum chromosome 3. Thus, AltSB 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 AltSB, 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.

Growth and Nutrition of Mollicute-Infected Maize.

Maize bushy stunt phytoplasma (MBSP) and corn stunt spiroplasma (CSS) diseases are widespread in Brazil. The leafhopper Dalbulus maidis is the insect vector for these pathogenic mollicutes. The effects of these diseases on the development of maize plants and the possible interaction of soil water availability on these effects were evaluated in two experiments carried out on potted plants. Experiment 1 was carried out in a 2 × 4 factorial, where factor 1 corresponded to healthy and mollicute-infected plants and factor 2 to the maintenance of 40, 60, 80, and 100% of the total soil water availability. Leafhoppers collected from a field with high incidence of these diseases were used to inoculate plants with mollicutes. There were three treatments in experiment 2: healthy plants, plants infected with phytoplasma, and plants infected with spiroplasma. MBSP was predominant in experiment 1. The infected plants grew less and lowered nutrient uptake, in distinct proportions, indicating a differential effect of mollicutes on nutrient uptake independent of available soil water. Soil water availability did not significantly affect plant growth and nutrient uptake or mollicute infection. The results indicated that plants infected by mollicutes contained less protein than healthy plants. Experiment 2 showed a reduction in growth of plants infected with mollicutes and less nutrient uptake by spiroplasma-infected plants. The results showed a detrimental effect of the spiroplasma on Mg uptake. Both experiments showed more water retention by infected plants than by healthy ones. These experiments clearly demonstrated that reduced plant growth and nutrient uptake are major effects on plants infected with MBSP and CSS.