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Background  Peanut is an important legume crop growing worldwide. With the published allotetraploid genomes, further functional studies of the genes in peanut are very critical for crop improvement. CRISPR/Cas9 system is emerging as a robust tool for gene functional study and crop improvement, which haven’t been extensively utilized in peanut yet. Peanut plant forms root nodules to fix nitrogen through a symbiotic relationship with rhizobia. In model legumes, the response of plants to rhizobia is initiated by Nod factor receptors (NFRs). However, information about the function of  NFRs  in peanut is still limited. In this study, we applied the CRISPR/Cas9 tool in peanut hairy root transformation system to explore the function of  NFR  genes.    Results  We firstly identified four  AhNFR1  genes and two  AhNFR5  genes in cultivated peanut ( Tifrunner ). The gene expression analysis showed that the two  AhNFR1  and two  AhNFR5  genes had high expression levels in nodulating (Nod+) line E5 compared with non-nodulating (Nod-) line E4 during the process of nodule formation, suggesting their roles in peanut nodulation. To further explore their functions in peanut nodulation, we applied CRISPR technology to create knock-out mutants of  AhNFR1  and  AhNFR5  genes using hairy root transformation system. The sequencing of these genes in transgenic hairy roots showed that the selected  AhNFR1  and  AhNFR5  genes were successfully edited by the CRISPR system, demonstrating its efficacy for targeted mutation in allotetraploid peanut. The mutants with editing in the two  AhNFR5  genes showed Nod- phenotype, whereas mutants with editing in the two selected  AhNFR1  genes could still form nodules after rhizobia inoculation.    Conclusions  This study showed that CRISPR-Cas9 could be used in peanut hairy root transformation system for peanut functional genomic studies, specifically on the gene function in roots. By using CRISPR-Cas9 targeting peanut  AhNFR  genes in hairy root transformation system, we validated the function of  AhNFR5  genes in nodule formation in peanut.

The application of CRISPR/Cas9 in hairy roots to explore the functions of AhNFR1 and AhNFR5 genes during peanut nodulation