Cloning And Functional Characterization Of Gene Conferring Tolerance To Nitrogen Deficiency In Rice

Nitrogen, the most amount macronutrient element required by crops, is crucial in agricultural production systems. N deficiency severely affects rice growth and reduces rice production. However, excessive use of N fertilizer has caused severe pollution to agricultural and ecological environments. The necessity of breeding of crops that require lower input of N fertilizer has been recognized.The Yuanjiang common wild rice introgression lines used in this study were derived from a across between an elite indica cultivar Teqing and an accession of common wild rice (O. rufipogon Griff.). This set of introgression lines were used to analyze QTLs for low nitrogen tolerance and screened for low nitrogen tolerant or sensitive lines. Here we identified a major quantitative trait locus on chromosome12, Tolerance Of Nitrogen Deficiency1(TOND1), that confers tolerance to N deficiency in the indica cultivar Teqing. The main results were as follows:1. A total of28QTLs for six low nitrogen tolerance-related traits which explained7%-22%of the phenotypic variance were detected in hydroponic codndition and a total of22QTLs for seven low nitrogen tolerance-related traits were detected in the field. The alleles from O. rufipogon at seventeen (77.3%) loci were in the direction of improving low nitrogen tolerance-related traits in Teqing background.2. The putative tolerance of N deficiency QTL was primarily mapped between SSR markers RM1300and RM17on the long arm of chromosome12using311F2individuals derived from the cross between Teqing and YIL105. According to the genotypes and phenotypes of the recombinants, we further delimited the mapping region to an about269kb physical region in cultivated rice genome. Comprehensively considering genome sequences and expression analysis datas of genes in the mapping region, we finally regarded a putative thaumatin protein with fingerprint motif as Tolerane Of Nitrogen Deficiency1(TOND1). Down-regulation of TOND1in Teqing leads to a decreased tolerance to N deficiency:the RNAi transgenic plants (pTOND1-RNAi) exhibited an obvious reduction in plant dry weight (DW), N concentration (NC), total N amount per plant (TN), SPAD, plant height and root length in comparison with that of the control plants, under N-deficient hydroponic conditions. Furthermore, panicle number per plant (PN), grain number per plant (GN) and grain yield per plant (GY) of the RNAi transgenic plants were lower than that of control plants under a variety of N-deficiency levels. Over-expression of the TOND1gene significantly enhanced tolerance of N-deficiency in hydroponic condition and grain yield in the field condition.3. Through GUS staining and qRT-PCR in various organs, TOND1was preferentially expressed in the anther and spikelet hull, leaf sheath and younger leaf. The TOND1-GFP fusion protein localized to the plasma membrane.4. Sequence verification of75indica and75japonica cultivars from18countries and regions demonstrated that only27.3%of cultivars (41indica cultivars) contain TOND1whereas72.7%of cultivars, including the remaining34indica cultivars and all75japonica cultivars, do not harbor the TOND1allele. Therefore, it has broad prospect for TOND1in rice breeding. A further test of association showed that three SNPs in the promotor and two SNPs in the coding region lead to the increase in N-deficiency tolerance in cultivars harboring with TOND1.