【作者】 刘希华；

【导师】 苏晓华；

【作者基本信息】 南京林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 本研究是对欧洲黑杨(Populus nigra L.)基因资源氮素利用效率进行研究,通过对比分析施氮前后各无性系的生长表现和测定分析与氮素利用相关的生理指标,筛选出氮素利用效率高效型欧洲黑杨基因型,为我国资源高效型杨树新品种选育提供优良种质,并为杨树氮素利用机理研究和育种资源的养分利用能力评价提供依据,为功能性单核苷酸多态性(single nucleotide polymorphism,SNP)分子标记提供较为准确的表型数据。主要研究结果为以下三个方面:1、欧洲黑杨氮素利用效率评价及优良基因型筛选。本研究以104份欧洲黑杨基因型为材料,通过生长量和生理指标的测定,研究土壤施氮和不施氮条件下其氮素利用效率的差异。结果表明:不同供氮水平下,不同基因型欧洲黑杨生长差异显著。根据2个供氮水平的苗木年平均材积生长量将欧洲黑杨群体划分为4个类型:双高效型、高氮高效型、低氮高效型和双低效型。并结合氮反应指数在双高效型与高氮高效型中分别筛选出8个氮利用效率高、生长表现优良的基因型,具较高的育种价值。比较分析不同基因型氮利用效率相关指标,发现双高效型的净光合速率、硝酸还原酶和谷氨酰胺合成酶活性、根系干质量、体积、比表面积均高于高氮高效型,初步揭示了不同类型欧洲黑杨氮利用效率存在差异的机理。2、欧洲黑杨氮素吸收利用相关功能基因SNP位点的筛选和分析。利用PCR扩增、测序和比对技术,从3个基因与植物吸收铵态氮相关基因(AMT1;2、AMT1;3和AMT1;5),4个与植物吸收硝态氮相关基因(NRT1.2、NRT2.1、NRT2.3和NRT2.4),2个与植物氮素同化相关基因(GW1和GW2)中共筛选了205个核苷酸变异,其中包括164个SNP位点(平均64bp一个)和41Indel(平均250bp一个)。所测序DNA区域的核苷酸多样性为总核苷酸多样性θw、πT分别是0.00525和0.00380。,9个候选基因Ka/Ks值均小于1,因此说明这些基因受负向选择影响,核苷酸序列相对保守。进化的中性检测结果表明,除NRT1.2和AMT1;2外,其他基因符合中性进化,欧洲黑杨群体在基因NRT1.2序列区域存在搭载效应和负选择作用;基因AMT1;2序列存在负选择作用或者有害变异频率。通过9个候选基因的重组分析研究,欧洲黑杨的SNP间的最小重组事件为0.1951。对其中的5个基因进行连锁不平衡(LD)分析,表明随着核苷酸序列长度的增加,SNP连锁不平衡程度逐渐减弱。说明欧洲黑杨适合采用候选基因进行关联分析和功能SNP标记的开发。3、氮素利用效率性状与SNP标记的关联分析。利用基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术对与氮素吸收利用相关的AMT1;2等基因的17个位点进行分型检测。采用单因素方差分析(ANOVA)和一般线性模型(GLM)分析,对分型的SNP标记和施氮区/不施氮区的材积、苗高和地径这3个生长性状值进行了关联分析。结果表明有7个SNP位点与生长性状显著关联。其中SNP2和SNP7均与不施氮区/施氮区的生长性状达到极显著关联,因此,这2个标记与欧洲黑杨氮素吸收利用相关的功能性SNP分子标记,说明AMT1;2和AMT1;5基因可以作为与氮素利用效率相关的候选基因或与主基因相连锁。AMT1;2基因SNP2(C-T转换),引起编码蛋白质的氨基酸变化(Thr-Ile),CT基因型的材积值、树高和地径高于CC基因型;AMT1;5基因SNP7(A-G转换),该位点是同义突变,GA基因型的材积值和地径高于AA基因型; AMT1;3基因的SNP3、SNP4、SNP5和SNP6均位于编码区,除SNP5外,其他都属于非同义突变,SNP3的GG基因型、SNP4的TT基因型、SNP5的GA基因型和SNP6的CC基因型的材积值、树高和地径高于其他基因型。SNP14位点位于NRT2.4基因的非编码区,GA的基因型的地径高于AA基因型。更多还原

【Abstract】 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.更多还原