Mapping-by-sequencing using NGS-based 3′-MACE-Seq reveals a new mutant allele of the essential nodulation gene Sym33 (IPD3) in pea (Pisum sativum L.)

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 F2-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 F2-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

Symbiotic Regulatory Genes Controlling Nodule Development in Pisum sativum L.

Analyses of natural variation and the use of mutagenesis and molecular-biological approaches have revealed 50 symbiotic regulatory genes in pea (Pisum sativum L.). Studies of genomic synteny using model legumes, such as Medicago truncatula Gaertn. and Lotus japonicus (Regel) K. Larsen, have identified the sequences of 15 symbiotic regulatory genes in pea. These genes encode receptor kinases, an ion channel, a calcium/calmodulin-dependent protein kinase, transcription factors, a metal transporter, and an enzyme. This review summarizes and describes mutant alleles, their phenotypic manifestations, and the functions of all identified symbiotic regulatory genes in pea. Some examples of gene interactions are also given. In the review, all mutant alleles in genes with identified sequences are designated and still-unidentified symbiotic regulatory genes of great interest are considered. The identification of these genes will help elucidate additional components involved in infection thread growth, nodule primordium development, bacteroid differentiation and maintenance, and the autoregulation of nodulation. The significance of symbiotic mutants of pea as extremely fruitful genetic models for studying nodule development and for comparative cell biology studies of legume nodules is clearly demonstrated. Finally, it is noted that many more sequences of symbiotic regulatory genes remain to be identified. Transcriptomics approaches and genome-wide sequencing could help address this challenge

Rhizobial Symbiosis in Crop Legumes: Molecular and Cellular Aspects

The production of high-value, environmentally friendly and healthy food has been the major global focus of sustainable agriculture in recent years [...

Comparison of the Formation of Plant–Microbial Interface in <i>Pisum sativum</i> L. and <i>Medicago truncatula</i> Gaertn. Nitrogen-Fixing Nodules

Different components of the symbiotic interface play an important role in providing positional information during rhizobial infection and nodule development: successive changes in cell morphology correspond to subsequent changes in the molecular architecture of the apoplast and the associated surface structures. The localisation and distribution of pectins, xyloglucans, and cell wall proteins in symbiotic nodules of Pisum sativum and Medicago truncatula were studied using immunofluorescence and immunogold analysis in wild-type and ineffective mutant nodules. As a result, the ontogenetic changes in the symbiotic interface in the nodules of both species were described. Some differences in the patterns of distribution of cell wall polysaccharides and proteins between wild-type and mutant nodules can be explained by the activation of defence reaction or premature senescence in mutants. The absence of fucosylated xyloglucan in the cell walls in the P. sativum nodules, as well as its predominant accumulation in the cell walls of uninfected cells in the M. truncatula nodules, and the presence of the rhamnogalacturonan I (unbranched) backbone in meristematic cells in P. sativum can be attributed to the most striking species-specific features of the symbiotic interface