Large collections of pea symbiotic mutants were accumulated in the 1990s, but the causal genes for a large portion of the mutations are still not identified due to the complexity of the task. We applied a Mapping-by-Sequencing approach including Bulk Segregant Analysis and Massive Analysis of cDNA Ends (MACE-Seq) sequencing technology for genetic mapping the Sym11 gene of pea which controls the formation of symbioses with both nodule bacteria and arbuscular-mycorrhizal fungi. For mapping we developed an F 2-population from the cross between pea line N24 carrying the mutant allele of sym11 and the wild type NGB1238 (=JI0073) line. Sequencing libraries were prepared from bulks of 20 plants with mutant and 12 with wild-type phenotype. MACE-Seq differential gene expression analysis between mutant-phenotype and wild-type-phenotype bulks revealed 2,235 genes, of which 514 (23%) were up-regulated and 1,721 (77%) were down-regulated in plant roots inoculated with rhizobia as a consequence of sym11 mutation. MACE-Seq also detected single nucleotide variants between bulks in 217 pea genes. Using a novel mathematical model we calculated the recombination frequency (RF) between the Sym11 gene and these 217 polymorphic genes. Six genes with the lowest RF were converted into CAPS or dCAPS markers and genetically mapped on the complete mapping population of 108 F 2-plants which confirmed their tight linkage to Sym11 and to each other. The Medicago truncatula Gaertn. (Mt) homologs of these genes are located in a distinct region of Mt chromosome 5, which corresponds to linkage group I of pea. Among 94 candidate genes from this region only one was down-regulated-the pea Sym33 homolog of the Mt IPD3 gene which is essential for nodulation. Sequencing of the Sym33 allele of the N24 (sym11) mutant revealed a single nucleotide deletion (c.C319del) in its third exon resulting in a codon shift in the open reading frame and premature translation termination. Thus, we identified a novel mutant allele sym33-4 most probably responsible for the mutant phenotype of the N24 (sym11) line, thereby demonstrating that mapping by MACE-Seq can be successfully used for genetic mapping of mutations and identification of candidate genes in pea.
Keywords: Mapping-by-sequencing; Massive analysis of cDNA Ends; Next generation sequencing; Pea; RNA-Seq; Symbiotic genes.