Three Soybean Yellow Foliage Loci: y11, y11-2, and CD-5 Encode Paralogous Magnesium Chelatase Subunits.

The soybean (Glycine max (L.) Merr.) chlorophyll deficient line MinnGold is a spontaneous mutant characterized by yellow foliage.  Two populations were developed to fine map the causative mutation to a 165.3-kb interval on chromosome 13.  Sequencing of a Mg-chelatase subunit candidate gene (ChlI1a) identified a non-synonymous single nucleotide polymorphism (SNP) in the third exon.  Mapping of the chlorophyll deficient mutation T219H (Y11y11) to chromosome 13 suggested that the mutants could be allelic, though the phenotypes of the MinnGold and T219H (Y11y11) are clearly distinct.  Sequencing of ChlI1a in y11 identified a different non-synonymous mutation in the third exon.  Whole plant transformation of MinnGold with a genomic clone of the wild-type allele fully rescued the wild-type phenotype.  The genetic position of CD-5, another chlorophyll deficient mutant, was previously mapped to chromosome 15, where a ChlI1a paralog resides.  Sequencing of this paralog, ChlI1b, identified a non-synonymous SNP in the third exon.  Analysis of progeny segregating for the MinnGold, T219H, and CD-5 phenotypes confirmed perfect co-segregation of the chlorophyll deficiency phenotypes and their respective candidate SNPs.  Protein sequence alignments of the two Mg-chelatase subunits indicated that the sites of amino acid modification in MinnGold, T219H, and CD-5 are highly conserved among photosynthetic species.  Collectively, these findings indicate that the MinnGold, T219H, and CD-5 phenotypes are caused by spontaneous non-synonymous mutations in the coding regions of the Mg-chelatase subunit genes ChlI1a and ChlI1b.  The negative interactions between the mutant and wild-type ChlI1a and ChlI1b proteins provide evidence for constrained sequence evolution in soybean paralogs.  To our knowledge, this is the first study to report the cloning and validation of chlorophyll deficiency loci in soybean.