Genetic Diversity Analysis And Molecular Mechanism Of Low Nitrogen Tolerance Of Shanghai Barley Landraces

Nitrogen is an essential element for plant growth and is also the primary influencing factor for crop yield.Therefore,people often apply more nitrogen fertilizer to increase crop yield.With the increasing application of nitrogen fertilizer,increasing nitrogen application cannot continue to improve crop yield.In addition,excessive application of nitrogen fertilizer not only increases the cost of agriculture,but also causes a series of environmental problems such as soil acidification,water eutrophication and global warming.Therefore,new solutions are needed that can increase crop yields even while maintaining or reducing N fertilizer application.Improving crop yield through improving nitrogen use efficiency is recognized as an effective method.There are differences in nitrogen use efficiency between different crops or different varieties of the same crop.Therefore,obtaining nitrogen efficient germplasm resources is crucial for crop nitrogen efficient breeding.Barley landraces are early domesticated populations that have been adapted to the local environment for a long time and have more genetic diversity and stronger adaptability compared with modern cultivars.It may contain many nitrogen tolerance genes due to long-term growth at low nitrogen fertilizer levels.Therefore,it will be important to clarify the genetic background of local barley landraces,screen and identify low-nitrogen tolerant germplasm resources and conduct related molecular mechanism studies.This will provide germplasm resources and scientific basis for subsequent breeding.The main research results are as follows.1.Genetic diversity and population structure of Shanghai barley landraces.The 112barley landraces in Shanghai were genotyped by Genotyping-by-Sequencing,and their genetic diversity and population structure were analyzed.Cluster analysis showed that the 112 barley landraces could be roughly divided into three groups,I,II,and III,of which I was mainly six-row-naked barley and II was mainly six-row-hulled barley and III was mainly two-row-hulled barley.Population structure analysis indicated that 112 barley landraces could be divided into three subgroups,SP1,SP2,and SP3,of which SP1 mainly included six-row-naked barley,SP2 mainly included six-row-hulled barley and SP3mainly included two-row-hulled barley,and this subgroup seemed to be consistent with the classification by row-type and naked or hulled type;The Fst values show that there is a large genetic differentiation between these three subgroups,with SP1 and SP2 being more closely related to each other than they are to SP3,where both SP1 and SP2 are dominated by six-row barley,while SP3 is dominated by two-row barley.2.The screening of barley varieties with different nitrogen use efficiency based on chlorate treatment.Based on the genetic relationship of 112 barley landraces,we randomly selected 18 barley landraces with relatively distant genetic relationships,and three cultivars bred in our laboratory.Two treatments,chlorate stress(2 mmol·L^-1 KCl O₃)and normal nitrogen supply(2 mmol·L^-1KNO₃),were set up at the one-leaf stage,and seedlings were collected after 4 days of treatment to determine the seedling height,root length,shoot dry mass,and root dry mass.The results showed that chlorate had a significant inhibitory effect on barley seedling growth,especially on seedling height and dry mass.The coefficients of variation of the different indicators showed that the coefficients of variation of root length and dry mass were relatively large,while the coefficients of variation of seedling height were relatively small under either chlorate treatment or normal culture conditions.The correlation analysis also showed that these morphological indicators were positively correlated under both conditions.Chlorate sensitivity based on seedling height and shoot dry mass could separate the different varieties,indicating that it could be used as an indicator for efficient screening and identification of barley for nitrogen.Combining these two indicators,B104,B002,B068,B092,and B053 were among the six most chlorate-sensitive varieties,indicating that their nitrogen use efficiency might also be high,while B016,B006,and B008 were among the six least chlorate-sensitive varieties,indicating that their nitrogen use efficiency might also be low.3.The analysis of response mechanism of barley landraces to low nitrogen by transcriptome and translatome.Based on the previous screening results,we randomly selected one nitrogen-efficient barley variety（B068）and one nitrogen-inefficient barley variety（B016）and subjected them to different nitrogen supply treatments at the three-leaf stage:normal nitrogen supply(1.43 mmol·L^-1 NH₄NO₃)and low nitrogen stress(0.24mmol·L^-1 NH₄NO₃),respectively.14 days later,the shoot was taken and analyzed by combined transcriptome and translational sequencing.Compared with normal nitrogen supply,569 up-regulated genes and 204 down-regulated genes were detected in B068 at the transcriptional level;147 up-regulated genes and 108 down-regulated genes were detected at the translational level.634 up-regulated genes and 124 down-regulated genes were detected in B016 at the transcriptional level;213 up-regulated genes and 111 down-regulated genes were detected at the translational level.In addition to the enriched common pathways,the significantly different genes in B068 were mainly enriched in nitrogen compound metabolism,nucleic acid metabolism,gene expression and transcriptional regulation and modification;the significantly different genes in B016 were mainly enriched in signal transduction,phosphorus metabolism and other processes.The differences in low nitrogen tolerance between B068 and B016 were mainly in（1）uptake,transport,and assimilation of nitrogen,such as nitrate transporter,nitrate reductase,nitrite reductase and glutamate synthase;（2）differences in transcription factor gene expression,such as NLP1,NAC067,MYB-like,and WRKY-like transcription factors.（3）differential expression of antioxidant enzyme genes,such as superoxide dismutase（SOD）.Quantitative PCR results showed general agreement with RNA-seq results and revealed that genes such as HORVU.MOREX.r3.7HG0729020（NRT2.4）,HORVU.MOREX.r3.6HG0570640（HKT6）and HORVU.MOREX.r3.1HG0029360（WRKY50）play an important role in response to low nitrogen stress.