Creation Of Barley Mutant With High Nitrogen Use Efficiency Based On Microspores Culture And Its Mechanism Of High Nitrogen Use Efficiency

In order to meet the food demands of the growing population,large amounts of nitrogen fertilizer have been put into the agricultural environment.However more than half of farmland nitrogen inputs are lost in air and water,negatively impacting the human health and environment.Improving the nitrogen use efficiency（NUE）of crops and reducing nitrogen fertilizer application in the field can greatly reduce environmental pollution caused by nitrogen leaching,which is a cost-effective measure in the sustainable development of agriculture.Barley（Hordeum vulgare L.）was one of the first domesticated crops.Due to the development of modern intensive breeding and cultivation,the genetic diversity of barley varieties is becoming narrower and narrower,which has become a bottleneck for genetic improvement.With the completion of barley genome sequencing,barley has become an important diploid model plant among the cereal crops.The sufficient genetic diversity materials are needed for high NUE breeding of barley.Therefore,the creation of germplasm resources with high NUE mutants is an effective method for high NUE breeding of barley.With the improvement of barley microspore culture technology,the method of microspore mutagenesis combined with low nitrogen stress culture to obtain excellent germplasm provides a shortcut for the creation of high NUE mutants.In this study,a batch of DH lines created by microspore chemical mutagenesis and low nitrogen stress culture.The mutant lines with significant higher NUE than wild type Hua30 obtained by performing field screening for many years.The phenotypic differences between the mutant line and wild type were identified by comparing the seedling dry weight,nitrogen accumulation,and root morphology.Furthermore,the expression of related genes involved in nitrogen uptake and assimilation was further analyzed and the reason for the enhanced nitrogen uptake in mutant line was elucidated at the molecular level.Meanwhile,the underlying differential mechanism of low nitrogen tolerance between wild type and mutant line was investigated by transcriptome sequencing,and some differentially expressed genes in response to low nitrogen stress were obtained from mutant line.The main research results are as follows:1 Field screening of high NUE mutants derived from microspore mutagenesis combined with low nitrogen stress culture.A total of 356 DH lines were produced from the barley cultivar Hua30 via microspore mutagenic treatment with ethyl methane sulfonate and pingyangmycin combined with low nitrogen stress culture.229 of these lines were subjected to field screening under high nitrogen（HN）and low nitrogen（LN）conditions,and the number of productive tillers was used as the main screening index.Five excellent mutant lines（A 1-28,A1-84,A1-226,A9-29 and A16-11）with high numbers of productive tillers were obtained over four consecutive years of screening,among which A1-28,A1-84,Al-226 and A9-29 were obtained by LN stress culture after microspore EMS mutagenesis and A16-11 was obtained by LN stress culture after microspores pingyangmycin mutagenesis.Subsequently,components related to NUE were characterized for these lines based on N uptake efficiency（NUpE）,N utilization efficiency（NUtE）,and N translocation efficiency（NTE）.Four mutant lines（A1-84,A1-226,A9-29 and A16-11）had higher NUpE under HN condition,while only two lines（A1-84 and A9-29）had higher NUpE under LN condition.As a result,the NUE of these lines were also significantly improved.Beside of these,A1-28 had a significantly higher NTE under LN condition.These results imply that high NUE mutants can be produced by microspore mutagenesis combined with LN stress culture and field screening,and the improvement of NUE of the mutant lines mainly depends on the enhancement of N uptake capacity.2 Analysis of morphological traits and genes involved in nitrogen metabolism in mutant line A9-29 at seedling stage.The high NUE mutant line A9-29 and the wild type barley cultivar Hua30 were subjected to hydroponic culture with HN（2.5mM NH4NO3）and LN（0.1mM NH4NO3）supply,the dry weight,nitrogen accumulation,and root morphology were determined at seedling stage,and the expression levels of the potential genes involved in nitrogen uptake and assimilation were analyzed at LN condition.The results showed that there was almost no significant difference in dry matter and N accumulation between A9-29 and Hua30 at HN condition,while A9-29 had a higher dry weight,N accumulation,N influx rate and larger root uptake area than Hua30 under LN condition.Moreover,A9-29 had higher expression of HvNRT2/3 genes than Hua30,especially HvNRT2.1,HvNRT2.5 and HvNRT3.3,under LN stress.Meanwhile,the expressions of the nitrogen assimilation genes,including HvNIA1,HvNIR1,HvGS1₁,HvGS13,and HvGLU2,were significantly increased in A9-29.In summary,the increase of root area,the upregulation of nitrate transporter and N assimilation genes of A9-29 may contribute to the improvement of nitrogen uptake capacity in the process of coping with LN stress,thereby promoting the growth and nitrogen accumulation of A9-29.3 Molecular mechanisms of root response to low nitrogen stress in mutant line of A9-29.In order to elucidate the response of roots to LN stress and its underlying molecular mechanisms,Transcriptome comparative analysis of A9-29 and Hua30 roots was performed under LN condition.The results showed that the root development of A9-29 was significantly affected by LN stress compared with that of Hua30,and the root morphological indexes including root length,root surface area and root volume were significantly increased in A9-29 with the extension of treatment time.Transcriptomic analysis showed that 1779 differentially expressed genes（DEGs）were up-regulated and 1487 DEGs were down-regulated in A9-29 compared with Hua30 under LN stress.Specific DEGs in A9-29 are mainly enriched in energy metabolism,lipid metabolism and the metabolism of other amino acid.In addition,transcription factor genes ERFs and IAA-related genes were specifically expressed in A9-29.These genes may be involved in regulating root growth and morphology to resist LN stress.In summary,A9-29 had more energy production and stronger stress resistance to promote the root growth,thereby enhancing the absorption of more nitrogen nutrients under LN conditions.This study provides a new idea for the molecular mechanism of barley root response to LN stress,and lays a theoretical foundation for breeding new barley varieties with LN tolerance.