Study On Genetic Diversity And Mechanism Of Differential Response To Nitrogen In Populus Deltoides Marsh.

Poplar（Populus spp.）is the main afforestation tree species in the mid-latitudes of the world.The characteristics of rapid growth,easy reproduction and wide range of suitable growth make it have important ecological and economic value.The germplasm resources of Populus deltoides have become an important gene donor of the main poplar varieties in the world.Nitrogen is an essential element for plant growth.In the process of large-scale popularization and planting of poplar,it is often faced with the problem of low soil nitrogen content in soil,and it can not meet the growth needs.The selection of nitrogen-efficient clones is the key to solve the problem of poplar production.Based on the germplasm resources of P.deltoides collected from the main natural distribution area by research group,the genetic diversity of germplasm resources was comprehensively evaluated,and the mechanism of nitrogen efficient utilization of fine germplasm was analyzed.The molecular genetic diversity of P.deltoides was analyzed and evaluated by SSR markers.The phenotypic genetic diversity of P.deltoides was evaluated by analyzing the phenotypic and physiological characteristics of different provenances and clones.The phenotypic and physiological characters of P.deltoides were measured under different nitrogen treatments,and the genetic diversity of nitrogen use characteristics of P.deltoides was analyzed and evaluated.Different nitrogen use types of clones were used as materials to conduct different nitrogen culture treatments to reveal the mechanism of nitrogen efficient utilization of excellent germplasm,and to explore the key genes of plant response to low nitrogen stress and nitrogen utilization characteristics.It provides scientific basis for the rational protection,development and utilization of P.deltoides germplasm resources.The main research conclusions are as follows:（1）A total of 119 alleles were detected in 338 clones of P.deltoides with 15 pairs of polymorphic and effective SSR primers.The average polymorphism information content（PIC）was 0.574,and the molecular genetic diversity was rich.There was a moderate level of genetic differentiation among provenance populations（Fst=0.135）,and 85.33%of genetic variation was from individual differences.The results of genetic structure analysis showed that 338 clones of P.deltoides from six provenances could be divided into three groups:northern type P.deltoides germplasm resources（‘Que’and‘Was’）,central P.deltoides germplasm resources（‘Iow’）and southern type P.deltoides germplasm resources（‘Lou’,‘Ten’and‘Mis’）,which were related to the geographical distribution of provenances.The strategy of gradual clustering,maximizing the number of alleles and priority sampling of rare alleles were used to construct a genetic optimization core collection including 34 clones.The percentage of alleles retained was100.00%,which could preserve more complete genetic information of P.deltoides germplasm resources.（2）338 clones from 6 provenances of P.deltoides were used to evaluate the phenotypic genetic diversity.Finally,based on the effective data analysis of 258 clones,the Shannon Wiener index of 22 phenotypic and physiological traits ranged from 1.844 to 2.080,which had rich diversity.Compared with the physiological characters of leaves,biomass characters and morphological characters of leaves showed greater variation among provenances.In addition to the chlorophyll fluorescence parameters（Fv/Fm）,plant height,ground diameter,photosynthetic characteristics,leaf carbon and nitrogen content,leaf morphology,leaf biomass,root biomass and stem biomass were significantly different between provenances and among individuals Within provenance（p<0.01）.Based on the results of comprehensive evaluation and analysis,the clones were divided into four categories:excellent,good,medium and poor.Population structure analysis showed that the phenotypic and physiological characteristics of clones from‘Mis’,‘Lou’and‘Ten’provenances were similar,those of‘Iow’and‘Que’provenances were similar,while those of‘Was’provenances were quite different from those of other provenances.Based on the idea of maximizing the proportion of phenotypic retention,a phenotypic optimization core collection including 32 clones was constructed by using the trait frequency sampling strategy.The variation rate of coefficient of variation was 142.19%,which could preserve more complete phenotypic information of P.deltoides germplasm resources.（3）With NH₄NO₃as nitrogen source,the growth of P.deltoides was inhibited,and the positive correlation among the growth traits of stem,leaf and root increased,while the negative correlation between leaf physiological indexes and stem growth traits,and between leaf physiological indexes and leaf growth traits weakened,and the allocation of nutrients in plants changed adaptively under the condition of low nitrogen supply.By constructing a comprehensive evaluation model of clone growth under different nitrogen growth conditions,it was found that leaf growth correlation could be used as one of the evaluation indexes of nitrogen use characteristics.The responses of clones from different provenances to low nitrogen stress were different.Based on the evaluation results under different treatments,338 clones were divided into 9 different nitrogen use characteristic groups,among which 26 clones belonged to nitrogen efficient（N-efficient）group;24 clones belonged to nitrogen inefficiency（N-inefficient）group.The nitrogen use characteristics of P.deltoides were rich in genetic diversity.（4）Under different nitrogen（NH₄NO₃）treatments,the leaf chlorophyll content,leaf morphology,biomass related traits of different parts of plant（root,stem and leaf）and enzyme activities related to nitrogen metabolism of N-efficient clones were higher than those of N-inefficient clones.The root growth of N-inefficient clones was inhibited under low nitrogen conditions and promoted under high nitrogen conditions,while that of N-efficient clones was the opposite,indicating that there were differences in the response of the two clones to different nitrogen treatments.During the treatment of low nitrogen stress,2 988 and 2 044 DEGs were detected in the leaves of N-efficient and N-inefficient clones respectively.KEGG enrichment analysis showed that more DEGs were associated with glutathione metabolism,phenylpropanoid biosynthesis and plant hormone signal transduction in the leaves of N-efficient clones.Twenty two key genes,including 12 transcription factors were found to be significantly negatively correlated with GS enzyme activity,GOGAT enzyme activity and amino acid contents by weighted correlation network analysis（WGCNA）.They were related to plant stress response and the negative regulation of life process.Among them,Podel.03G153500 has the strongest connectivity,and the protein encoded by Podel.03G153500 is involved in cell development and programmed cell death.At the same time,Podel.18G019200 is second only to the core gene in connectivity and belongs to WRKY transcription factor,which mediates multiple hormone signaling pathways.Under different nitrogen treatments,5 270 and 204 specific DEGs were detected in the leaves of N-efficient and N-inefficient clones,respectively.KEGG analysis showed that the specific DEGs in the leaves of N-efficient clones were specifically enriched in glutathione metabolism,fatty acid metabolism,RNA polymerase and plant hormone signal transduction pathways.Through association analysis,14 key genes,including 4 transcription factors,which may play a positive role in plant nitrogen efficient utilization characteristics,were discovered.At the same time,12 key genes,including 3 transcription factors,which may play a negative role in plant nitrogen efficient utilization were found by association analysis and gene co-expression network.Among them,Podel.04G159900 is not only the core gene that plays a negative regulatory role,but also an ARR-B transcription factor involved in cytokinin signal transduction.These key genes may play a key role in maintaining high nitrogen use efficiency and strong nitrogen adaptation of plants,which provides scientific basis for analyzing the mechanism of high nitrogen use efficiency of plants.In this study,the genetic diversity of germplasm resources in terms of phenotype,molecular and nitrogen use characteristics were systematically evaluated.The mechanism of nitrogen efficient utilization of elite germplasm were analyzed in terms of phenotype,physiology and gene expression aspects.Through the establishment of gene co-expression network,the key genes involved in plant response to low nitrogen stress and nitrogen efficient utilization were explored.It provides an important reference for the breeding and improvement of nitrogen efficient clones,which is of great significance to the efficient and sustainable development of poplar breeding.