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Microbial symbiosis in legumes is achieved through nitrogen-fixing root nodules, and these are important for sustainable agriculture. The molecular mechanisms underlying development of root nodules in polyploid legume crops are largely understudied. Through map-based cloning and QTL-seq approaches, we identified a pair of homoeologous GRAS transcription factor genes, Nodulation Signaling Pathway 2 (AhNSP2-B07 or Nb) and AhNSP2-A08 (Na), controlling nodulation in cultivated peanut (Arachis hypogaea L.), an allotetraploid legume crop, which exhibited non-Mendelian and Mendelian inheritance, respectively. The segregation of nodulation in the progeny of Nananbnb genotypes followed a 3:1 Mendelian ratio, in contrast to the 5:3~1:1 non-Mendelian ratio for nanaNbnb genotypes. Additionally, a much higher frequency of the nb allele (13%) than the na allele (4%) exists in the peanut germplasm collection, suggesting that Nb is less essential than Na in nodule organogenesis. Our findings reveal the genetic basis of naturally occurred non-nodulating peanut plants, which can be potentially used for nitrogen fixation improvement in peanut. Furthermore, the results have implications for and provide insights into the evolution of homoeologous genes in allopolyploid species.

Peanut root invasion by Bradyrhizobia is through a crack entry, which is different from many other legumes applying an infection thread entry in root hair. Understanding the role of root hair in the crack entry of Bradyrhizobia invasion of peanut root and subsequent peanut nodulation would facilitate improvement of biological nitrogen fixation in cultivated peanut. The objective of this study was to investigate the involvement of root hair in Bradyrhizobial invasion of peanut. Seedling roots of a nodulating peanut cultivar were observed for root hair emergence, its life span, and nodule formation at the base of the lateral roots with and without rhizobia inoculation for 14 days after germination (DAG). Scanning electron microscopy (SEM) was utilized to observe rhizobia accumulation at lateral roots at 24 hours after inoculation (HAI) before the emergence of root hair. Root hair emerged at 7 DAG with or without rhizobia inoculation. Two variations of rosette hair (RoH) were observed, the transient-thin RoH had life span of 3 days after root hair emergence and the thick and densely distributed RoH type stayed till the time of nodule emergence (9 days after inoculation). The lateral root devoid of root hair at the top 2 cm region was found to produce nodules. The SEM observation of seedling roots at 24 HAI showed that Bradyrhizobia invaded the roots at epidermis, protoplasm of cortical cell, and cortical cells of the main root near the newly emerged lateral root in the absence of RoH. The observations validated that root hair is not required in the Bradyrhizobia invasion of peanut root in the crack entry mode. Results from this study provided important morphological information for the hypothesis of close relationship between RoH and peanut nodulation for further genetic study of crack entry mechanism and signaling pathway of symbiosis between Bradyrhizobia and peanut.

The molecular mechanisms of symbiosis in cultivated peanut with a ‘crack entry’ infection process are largely understudied. In this study, we investigated the root transcriptional profiles of two pairs of non-nodulating (nod−) and nodulating (nod+) sister inbred peanut lines, E4/E5 and E7/E6, and their nod+ parents, F487A and PI262090 during rhizobial infection and nodule initiation by using RNA-seq technology. A total of 143, 101, 123, 215, 182, and 289 differentially expressed genes (DEGs) were identified in nod− E4, E7 and nod+ E5, E6, F487A, and PI262090 after inoculation with Bradyrhizobium sp. Different deficiencies at upstream of symbiotic signaling pathway were revealed in the two nod− genotypes. DEGs specific in nod+ genotypes included orthologs to some known symbiotic signaling pathway genes, such as NFR5, NSP2, NIN, ERN1, and many other novel and/or functionally unknown genes. Gene ontology (GO) enrichment analysis of nod+ specific DEGs revealed 54 significantly enriched GO terms, including oxidation-reduction process, metabolic process, and catalytic activity. Genes related with plant defense systems, hormone biosynthesis and response were particularly enriched. To our knowledge, this is the first report revealing symbiosis-related genes in a genome-wide manner in peanut representative of the ‘crack entry’ species.