The Study Of Screening And Identification Indices And Molecular Mechanisms Of Low Nitrogen Tolerance In Barley Landraces Of Shanghai Region

Nitrogen is one of most necessary macro elements for crop growth and development.Thus,the first Green Revolution base on chemical fertilizers?especially the nitrogen fertilizers?and dwarfing breeding has made a great success in increasing crop production.However,the extensive or excess use of nitrogen fertilizers has also caused many problems at the same time,such as lower nitrogen use efficiency,soil hardening,environmental pollution,and so on.Therefore,agriculture urgently needs to reduce its dependence on nitrogen fertilizer on the one hand,and it also needs to ensure crop yield on the other hand.This contradiction has become increasingly prominent.While,above problems can be effectively solved by cultivating and planting low nitrogen tolerant crop varieties.However,the phenotypic evaluation and molecular mechanisms of low nitrogen tolerance for plants are very complex,and they are still unclear.Barley with tolerance to avariety of biotic or abiotic stresses?especially tolerance to poor soil?is the fourth largest cereal crop in the world,and it is also one of the model plants.Thus,use of barley to carry out low nitrogen tolerance research will help to better promote crop breeding for low nitrogen tolerance.In previously,our group?Barley Cell Engineering Research Lab of Biotechnology Research Institute in Shanghai Academy of Agricultural Sciences?had collected a batch of barley landraces from Shanghai region,and the genetic diversity analysis showed that there were great genetic variations in these germplasms.Considering their adaptation in Shanghai Region,these barley germplasms would play important roles in barely breeding for low nitrogen tolerance in this area.The main results are as follows:1.19 barley landraces from Shanghai region were selected for low nitrogen tolerance screening and identification.A hydroponic experiment was used for low nitrogen evaluation by investigating root length,plant height,tiller number,root dry weight and shoot dry weight under normal nitrogen supply and low nitrogen stress in barley at seedling stage.The ANOVA analysis showed that there were significant differences between the different nitrogen supply levels and among different barley landraces and there were also significant interactions between nitrogen supply and barley landrace.The coefficient of variation indicated that the indices of tiller number and dry weights with high coefficients of variation were more suitable for screening barley landraces with low nitrogen tolerance,while the plant height with low coefficient of variation was not a suitable screening index.The correlation analysis suggested that there were significantly positive correlations between dry weights and other indices,although there were some reductions of correlation under low nitrogen stress.Based on the importance of biomass for low nitrogen tolerance evaluation,we compared shoot dry weight between normal nitrogen supply and low nitrogen stress,and the t-test revealed that there were five barley landraces of B929,B942,B945,B968 and B974 with low nitrogen tolerance,meanwhile this criteria was more precise for evaluation of low nitrogen tolerance than that by the relative value.Furthermore,we investigated the correlations between shoot dry weight and nitrogen uptake or utilization related traits,and found that these traits should be considered together for evaluation of low nitrogen tolerance in barley.2.Three of the five with low nitrogen tolerance under 0.50 mM NH₄NO₃ low nitrogen stress were randomly selected for further identification of low nitrogen tolerance under two lower nitrogen stresses?they were 0.24 mM and 0.10 mM NH₄NO₃,respectively?.The results showed that barley landrace of B929 was still tolerant to both of the low nitrogen stresses,and B968 was only tolerant to low nitrogen stress of 0.24mM NH₄NO₃,while B974 was not tolerant to the two lower nitrogen stresses.Coefficient of variation analysis suggested that dry weights related traits were more suitable as screening index of low nitrogen tolerance,while the trait of plant height was not.In addition,this study further explored the possibility of low nitrogen tolerance identification by physiology traits related nitrogen uptake or utilization,and discussed the importance of nitrogen uptake or utilization on low nitrogen tolerance.3.B968,a low nitrogen tolerant barley landrace,was used for transcriptome analysis by using RNA-seq technology after 1h of different nitrogen treatments,they were normal nitrogen supply?NN?,low nitrogen stress-moderate?LNM?and low nitrogen stress-lower?LNL?.The results showed that there were 12295 and 12497differentially expressed genes?DEGs?in LNM and LNL,respectively,and DEGs were very similar in the two low nitrogen treatments.GO analysis of DEGs suggested that the terms were mainly enriched in cell components in both the two low nitrogen treatments.KEGG analysis of DEGs also indicated that the metabolic pathways of the two different low nitrogen treatments were similar.The enrichment factors of carbon fixation pathway in photosynthetic organisms were higher,and there were also two genes related photosynthesis in top ten up-regulated genes under low nitrogen stresses.Therefore,we inferred that the genes related to photosynthesis pathway might be related to low nitrogen tolerance in barley,and these genes would be used as candidates for further research.In addition,we also found that there were differences between qPCR and RNA-seq results,and made a further discussion.