| In this paper, gene resources of Populus nigra were used as materials, nitrogen use efficiency were evaluated and the high-effective-nitrogen-genotypes were selected by analyzing the growth of various clones under two different nitrogen treatments, which provides high-effective-nitrogen-genotypes of poplar for our country, and the basic mechanism of their efficiency to nitrogen and evolution of the ability to absorb nutrients during poplar breeding, also provides more accurate phenotype data for the functional SNP markers. The three main results are described as following:1. Study on evaluation of nitrogen efficiency within P.nigra gene resources and selection of excellent genotypes. The variation in nitrogen utilization efficiency of 104 P.nigra genotypes was studied by analyzing growth and physiological parameters under nitrogen fertilization or non-fertilization conditions. The results showed that the variation of nitrogen utilization efficiency among seedlings of varied black poplar genotypes were significant at different nitrogen supply. All seedlings with varied genotypes in this study were divided into four categories according to annual average individual volume increment in the two nitrogen treatments. These categories are high efficient in both nitrogen applications, efficient in high N application, efficient in low N application, and low efficient in both nitrogen applications. Eight high efficient genotypes were selected from each of the first two categories. These seedlings were highly efficient to nitrogen utilization with excellent growth and productivity; therefore they were highly valuable for poplar breeding. Comparison analysis of the related parameters of nitrogen utilization efficiency showed that the value of nitrogen response to parameters including net photosynthetic rate, nitrate reductase activity, GS activity, root dry weight, root volume and specific root area of seedlings within the first category were all higher than those within the second category, which revealed preliminarily the mechanisms underlying nitrogen utilization of different P.nigra genotypes.2. Discovery and analysis of SNPs from genes that are involved in nitrogen utilization. A total of 205 single nucleotide polymorphism (SNP) sites are found from nine genes, three of which related to ammonium nitrogen absorption (AMT1;2, AMT1;3 and AMT1;5), four of which related to nitrate nitrogen absorption (NRT1.2, NRT2.1 NRT2.3 and NRT2.4), two of which related to nitrogen assimilation (GW1 and GW2), The media SNP frequency was one site per 50 bp. The average nucleotide diversity for the sequenced regions was calculated to beθw=0.00525 andπT=0.00380, between P. tremula and P. trichocarpa. The Ka:Ks ratios for all nine gene lower than one , indicating the action of purifying selection. Tests of neutrality for each gene revealed that the results reject neutral evolution at NRT1.2 and AMT1;2. Nucleotide sequence of NRT1.2 and AMT1;2 were likely to be undergoing negative selection which caused hitchhiking effect. Recombination analyzing for the nine candidate genes revealed nine recombination events of SNP in P. nigra, which was similar to P. tomentosa. The linkage disequilibrium of SNPs in five candidate genes was detected and the result showed that LD declined rapidly within the gene regions. These rules were similar to those in other trees. Which suggested that wide LD mapping in the whole genome of Populus might be impossible and unnecessary, but LD mapping based on candidate genes could be particularly useful in breeding programs of poplar. The results showed that P. nigra were suitable for association analysis with candidate genes and development of SNP markers.3. Association analysis between SNPs and NUE traits. A total of 17 SNPs were genotyped using MALDI-TOF MS technology. These SNPs were from eight genes. Association analysis between marker and growth traits was carried out using One-way Analysis of Variance (ANOVA) and General Linear Model (GLM) techniques. The growth traits included volume, seedling height and ground diameter under the two nitrogen treatments. The results showed seven SNPs associations between markers and growth traits, both SNP2 and SNP7 were very significantly correlated with growth traits under the two nitrogen treatments, which concluded that AMT1;2 and AMT1;5 could be the potential major gene affecting NUE of P.nigra or correlate with the major gene, and polymorphic loci SNP2 and SNP7 might be used as molecular markers for improving growth of P. nigra. SNP3, SNP4, SNP5, SNP6 and SNP14 were significantly associated with growth traits under the two nitrogen conditions, these loci might be assist functional markers with NUE in poplar. SNP2 of AMT1;2 showed C-T transition, which resulted in changes to amino acid (Thr-Ile). The trees with genotype CT at SNP2 had higher growth (volume, seedling height and ground diameter) than CC genotype. SNP7 of AMT1;5 showed A-G transition, which was a synonymous mutation. The trees with genotype GA at SNP7 had higher growth (volume and ground diameter) than AA genotype. SNP7 of AMT1;5 shows A-G transition, which also was a synonymous mutation. SNP3, SNP4, SNP5 and SNP6 were all located in coding region of AMT1;3, SNP3, SNP4 and SNP6 which resulted in changes of amino acid. The trees with genotype GG at SNP3, genotype TT at SNP4, genotype GA at SNP5, genotype CC at SNP6 had higher growth (volume, seedling height and ground diameter) than those with other genotypes. SNP14 was located in non-coding region of NRT2.4, The tree with genotype GA at SNP 14 had higher ground diameter than AA genotype.
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