Screening Of High Nitrogen Efficient Germplasms And Its Mechanism In New-type Brassica Napus

Currently, large amounts of nitrogenous fertilizer are required in oilseed rape to achieve high seed yield, but substantial nitrogen (N) is leached and thus lead to the low N fertilizer utilization efficiency. Screening and breeding N-efficient rapeseed is the effective way to improve N fertilizer use efficiency (NUE). New-type Brassic napus has been developed by selfing or interspecific crosses between traditional B. napus (AnAnCnCn), B. carinata (BcBcCcCc) and B. rapa (ArAr). The introgressed excellent alleles from Ar genome of B. rapa (ArAr) and Cc genome of B. carinata (BcBcCcCc) make it has stronger heterosis and was more resistant and tolerant to biotic and abiotic stresses. In this study, the screening of N-efficient new-type B. napus germplasm of homozygous double haploid (DH) lines was conducted at different stage using hydroponics and pot experiments. The plant growth, N absorption and utilization, root N uptake kinetics, N assimilation involved in enzyme activity and gene expression, N use efficiency, photosynthetic efficiency and the exogenous gene composition introgressed were determined under low-N stress to explore the effective mechanism in the N efficient new-type B. napus. The main results were as follows:1. Screening N-efficient and N-inefficient genotypes of new-type Brassic napus1.1 The screening of N-efficient genotype was employed in 46 DH lines of new-type B. napus and 2 traditional B. napus under low-N and high-N conditions at the seedling stage using hydroponic experiment.19 parameters involved in N efficiency were determined and the data was analyzed by principal component analysis and indexes variation analysis. Shoot dry weight under low-N condition was the key determinant factor and the shoot N accumulation and N utilization efficiency (low-N/high-N) were the reference indexes. The new-type B. napus D4-9 and D4-15 were the N-efficient genotypes whereas the D1-1 and D2-1 were the N-inefficient genotypes based on three times of hydroponic screening.1.2 The performance of the screened new-type B. napus N-efficient and N-inefficient genotypes at the maturity was consistent with that of seedling stage, new-type B. napus D4-9 and D4-15 being the N-efficient genotypes and the D1-1 and D2-1 being the N-inefficient genotypes. The D4-15 and D-1 were used to reveal the possible mechanism involved in the N efficient.2. Nitrogen efficient mechanism of new-type Brassic nap us2.1 Under low-N stress, the root morphology, shoot dry weight, shoot N accumulation, shoot N use efficiency of D4-15 were significantly higher than those of D1-1. Compared with N-inefficient genotype D1-1, N-efficient genotype D4-15 had the more developed root system, stronger ability of N absorption, higher accumulation and utilization efficiency under the low-N conditions, suggesting D4-15 being more adaptable to the low-N stress.2.2 The uptake kinetics was employed to dissect the absorption difference between the N-efficient genotype D4-15 and N-inefficient genotype D1-1. The results showed that the uptake of 15NH4+ and 15NO3-in D4-15 were significantly higher than in D1-1. Under the low-N condition, compared with N-inefficient genotype D1-1, N-efficient genotype D4-15 root had lower Km and Cmin values and higher Imax, suggesting that genotypes D4-15 had the stronger ability of N absorption.2.3 Under low-N stress, nitrate reductase activity (NRA) and glutamine synthetase activity (GSA) in the N-efficient genotype D4-15 leaves were significantly higher than that of N-inefficient genotype D1-1, and NRA and GSA in leaves were significantly higher than that in roots. The results suggested that N-efficient genotype D4-15 had stronger ability of NO3 reduction and NH4+ assimilation in leaves and roots.2.4 Compared with the values in high-N condition, net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (Tr) and stomatal limitation (Ls) of rapeseed were significantly lower under low-N stress, while the intercellular CO2 concentration (Ci) was higher significantly. Under low-N condition, Pn, gs, the lower leaf SPAD value and Leaf photosynthetic nitrogen use efficiency (PNUE) in D4-15 leaves were significantly higher than those of D1-1, suggesting that N-efficient genotype D4-15 can absorb more CO2 to create more carbohydrates, and its leaf had the ability to maintain alonger photosynthesis to product more photosynthetic materials, eventually yielded higher seed setting.2.5 Under low-N condition, compared with N-inefficient genotype D1-1, the expression conditions of nitrate transporter (BnNRT1;1, BnNRT2;5, BnNRT2;6, BnNRT2;7) and ammonium transporter (BnAMT1;1) in roots, and BnNRT2;5 in leaves were significantly higher in new-type B. napus N-efficient genotype D4-15. Higher expression conditions of nitrogen metabolism genes (BnNR2 and BnGln2) were observed in the leaves and roots of D4-15.2.6 The amount of Ar genome and Cc genome introgression in new-type B. napus N-efficient genotype D4-15 were 32.7% and 32.2% respectively, the sum was 64.9%, while N-efficient genotype D1-1 were 17.7%, 14.6% and total was 32.3%. Obviously, N-efficient genotype D4-15 had the higher amount of exotic genome components, which might be responsible for N efficient physiological and molecular mechanism of new-type B. napus N-efficient genotype D4-15.To sum up, 2 N-efficient genotypes and 2 N-inefficient genotypes of new-type B. napus DH lines were obtained based on the germplasm screening for N efficiency. Under the low-N condition, compared with N-inefficient genotype D1-1, N-efficient germplasm D4-15 had the larger root system, stronger ability of N absorption and utilization, thus forming a high biomass and seed yield, which had a higher N efficiency, stronger adaptability to low-N stress. The morphological and physiological characteristics might lead to higher activity and expression capacity of N absorption metabolism key protein (enzyme)/gene under low-N stress in N-efficient genotype D4-15. It was coincided with that N-efficient genotype D4-15 has more amount of exogenous genes introgression. However, more direct and sufficient evidence remains to be the revealed for N-efficient germplasm and gene cloning and characterization.