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.)

Abstract

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.

Innovative transcriptome‐based genotyping highlights environmentally responsive genes for phenology, growth and yield in a non‐model grain legume

“…Namely, an average coverage per library in published studies ranged from 5.0 to 10.4 mln reads (1.3 to 11.6 mln reads per particular libraries) (Bojahr et al, 2016;Parreira et al, 2018;Zajac et al, 2015;Zawada et al, 2014;Zhernakov et al, 2019). MACE read coverage reported here was similar, as it ranged from 3.4 to 21.2 mln reads per sample (9.6 mln on average).…”

“…Likewise, MACE protocol was applied in pea (Pisum sativum) for detection of single nucleotide variants which were further converted into CAPS markers (Zhernakov et al, 2017). Further application of MACE in pea resulted in identification of new mutant allele of the key nodulation gene Sym33, evidenced both by linkage mapping with CAPS or dCAPS markers and by differential gene expression profiling (Zhernakov et al, 2019).…”

Candidate Domestication-Related Genes Revealed by Expression Quantitative Trait Loci Mapping of Narrow-Leafed Lupin (Lupinus angustifolius L.)

“…The MACE protocol was also applied in pea ( Pisum sativum ) providing single nucleotide variants subsequently converted into CAPS markers [38]. Furthermore, MACE-based studies in pea resulted in the identification of a new mutant allele of the key nodulation gene Sym33 [107]. The MACE was also used for transcriptomic profiling of Phaseolus vulgaris seeds and Solanum lycopersicum pollen [39,40].…”

Symbiotic Regulatory Genes Controlling Nodule Development in Pisum sativum L.

“…Many symbiotic genes in pea have been localized using morphological and molecular markers ( Table 1 ). The largest number of genes are located in the linkage group I, which includes PsSym2 [ 25 ], PsSym5 , PsSym10 , PsSym19/PsSym41 [ 29 ], PsSym33/PsSym11 [ 59 , 60 ], PsSym35 [ 15 ], PsSym37 , PsK1 [ 89 ], and PsNod3 [ 76 ]. It is interesting to note that all genes encoding the components of Nod factor reception (see below), such as PsSym2 , PsSym10 , PsSym19/PsSym41 , PsSym37 , and PsK1 , are mapped to this linkage group.…”

“…In PsSym33 , four independent ineffective mutants were obtained: RisFixU ( Pssym33-1 ) [ 31 ]; SGEFix − -5 ( Pssym33-2 ) [ 49 ]; SGEFix − -2 ( Pssym33-3 ) [ 58 ]; and N24 ( Pssym11 = Pssym33-4 ) [ 32 , 60 ] ( Table 1 ). The PsSym33 gene is orthologous to the M .…”