Defining the mutation sites in chickpea nodulation mutants PM233 and PM405

Area of Science:

• Plant genetics
• Legume symbiosis
• Molecular biology

Background:

• Chickpeas, like other legumes, form root nodules for symbiotic nitrogen fixation with rhizobium bacteria.
• Understanding the host plant genetics of this symbiosis is crucial for both basic science and agricultural applications.
• Mutant chickpea lines PM233 (rn1) and PM405 (rn4) exhibit nodulation-deficient phenotypes, indicating defects in key nodulation genes.

Purpose of the Study:

• To identify and characterize the specific mutation sites responsible for the nodulation-deficient phenotypes in chickpea mutants PM233 and PM405.
• To elucidate the genetic basis of root nodulation in chickpeas by defining the rn1 and rn4 alleles.

Main Methods:

• Utilized Illumina and Nanopore sequencing to analyze genomic DNA from mutant and wild-type chickpea lines.
• Performed de novo assembly of Nanopore reads and alignment of Illumina reads to the chickpea reference genome.
• Conducted variant calling and genotyping of segregating progeny populations to pinpoint candidate mutations.

Main Results:

• Identified a ~35 kb deletion encompassing the CaNSP2 gene in mutant PM233 (rn1).
• Discovered a single base deletion causing a frameshift mutation in the NIN nodulation gene in mutant PM405 (rn4).
• Nanopore sequencing was critical for accurately defining the deletion boundaries in the CaNSP2 region.

Conclusions:

• The rn1 mutation in chickpea PM233 is caused by the complete deletion of the CaNSP2 gene.
• The rn4 mutation in chickpea PM405 results from a frameshift mutation in the NIN gene.
• Genomic misassemblies in reference genomes previously hindered the characterization of these nodulation genes.