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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,  Alt   SB  , and increases grain yield under Al toxicity. Here,  Alt   SB   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  Alt   SB   alleles in three germplasm sets while greatly reducing genotyping needs. Population structure analysis revealed that favorable  Alt   SB   alleles are predominantly found in subpopulations enriched with guinea sorghums, supporting a possible Western African origin of  Alt   SB  . 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 Al 3+  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.

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