【作者】 褚延广；

【导师】 苏晓华；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2009， 博士

【摘要】 欧洲黑杨（Populus nigra L.）是世界主栽杨树品种重要的基因供体,也在我国杨树遗传改良中起重要作用。我国黑杨派基因资源严重不足,仅新疆北部有少量欧洲黑杨分布。近年来我国已陆续从欧洲15个国家引进了许多欧洲黑杨无性系,极大地丰富了我国欧洲黑杨育种资源。我国是严重缺水的国家,土壤干旱缺水严重限制了杨树的生长和木材生产,提高杨树对水分的利用效率、增强抗旱能力,培育节水抗旱的杨树新品种对木材生产和通过绿化改善环境都具有重要意义。光合作用是影响植物生物量积累的重要因素,充分利用环境的光能资源,提高杨树对光能资源的利用能力,培育具有较高光合利用效率的杨树新品种,是高产杨树人工林培育的重要方向。本研究以从国外引进和我国新疆的欧洲黑杨基因资源为材料,对光合利用效率进行了评价,结合之前对水分利用效率(碳稳定同位素比率,δ¹³C)的评价结果,从水分与光合利用相关功能基因中进行了单核苷酸多态性（SNP）标记的筛选与分析,通过SNP标记在基因资源中的分型并与水分利用效率、光合生理特征和生长性状进行关联分析,开发与目标性状相关联的功能SNP标记,为杨树水分、光合利用性状分子标记辅助育种提供参考。论文主要研究结果如下:欧洲黑杨水分及光合利用效率评价。由于之前已完成了水分利用效率的评价,本文着重对光合利用效率进行了评价。对气体交换、叶绿素荧光及生长性状的测定与分析证明,欧洲黑杨基因资源的光合生理和生长性状具有丰富的遗传变异;来自我国和英国的无性系具有较高的净光合速率（Pn）,来自匈牙利的无性系叶绿体光系统II（PSII）最大光化学效率（Fv/Fm）最高。各检测参数在欧洲黑杨中具有较高的广义遗传力（H2值在0.750⁰.962间）且变异系数较高。分析结果发现,Pn、胞间二氧化碳浓度（Ci）、气孔导度（Gs）和PSII电子传递速率（ETR）等参数指标与欧洲黑杨高生长性状显著相关,在光合利用效率评价中具有潜在的应用价值;起源于南欧和东欧的欧洲黑杨可在进一步的杨树高光效育种研究中作为优良基因型选择的重点。欧洲黑杨水分及光合利用相关功能基因SNP的筛选与分析。利用PCR扩增和序列测定,从7个水分利用效率相关基因（SuSY1、dhn、lea3、PIP、Expa1、ERF和GPX1）和2个光合利用效率相关基因（rubisco和Lhcb2）中共筛选出312个SNP位点,SNP频率为1/26 bp。所测序DNA区域的核苷酸多样为θ_W=0.01074和πT=0.00702,高于欧洲山杨（P. tremula）、毛果杨（P. trichocarpa）和多数针叶树种;进化的中性检测结果表明,目标基因DNA区域以低频率突变为主,其中多数非同义突变可能与欧洲黑杨的适应性进化过程有关;GPX1、dhn、lea3和Expa1基因不符合中性进化原则,可能在进化过程中受到产生遗传搭载效应的选择清除作用。不同基因内的连锁不平衡（LD）水平各不相同;欧洲黑杨总体的LD水平较低,r²值在300 bp的DNA物理距离范围内从0.45降低至0.20以下,该规律与其它多数林木相似,说明欧洲黑杨适合于采用目标基因进行关联分析和功能SNP标记的开发。水分利用效率性状(δ¹³C值)与SNP标记的关联分析。利用SNaPshot技术对SuSY1、lea3、PIP、Expa1和ERF等5个基因的25个SNP位点进行了分型。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标记与δ¹³C值进行了关联分析。结果显示,共有6个SNP标记与欧洲黑杨δ¹³C值显著关联。SNP2为T-C转换,遗传贡献率为7.705%,基因型为TT的欧洲黑杨具有较高的δ¹³C值。SNP15为G-A转换,并引起编码氨基酸的变化（Val-Ile）,遗传贡献率为7.570%;GG基因型的欧洲黑杨具有较高的δ¹³C值。SNP25、SNP26、SNP27和SNP31均位于Expa1基因内,遗传贡献率为6.436%⁶.900%;这4个位点的纯合、杂合基因型频率均具有相似的分布特征,其中SNP25、SNP27和SNP31的TT基因型所对应个体的δ¹³C值明显高于杂合基因型。光合利用效率性状与SNP标记的关联分析。利用SNaPshot技术对rubisco和Lhcb2基因的20个SNP位点进行了分型。采用ANOVA和GLM分析,对分型的SNP标记与光合特征及生长性状进行了关联分析。结果表明,共有4个SNP标记与光合特征及生长性状显著关联。其中SNP12、SNP36和SNP50位于rubisco基因内,SNP12和SNP36为非同义突变,分别导致天冬氨酸（Asn）-组氨酸（His）、酪氨酸（Tyr）-苯丙氨酸（Phe）间的变化;两标记均与ETR值显著关联,其AA基因型欧洲黑杨ETR值高于其它基因型;SNP50同时与Pn、ETR、Fv/Fm、树高和胸径显著关联,CT基因型欧洲黑杨具有较高的ETR值、Fv/Fm值、树高、胸径和较低的Pn值。Lhcb2基因的SNP44与树高性状显著关联,其GG基因型个体具有较大的树高生长量。更多还原

【Abstract】 Populus nigra L. is an important gene donor for major poplar varieties worldwide, and plays crucial roles in the genetic improvement programs of poplar in China. There is a serious shortage of gene resources of Aigeiros section in China, as P. nigra distributes only across a small area of Northern Xinjiang. In recent years, many P. nigra genotypes were introduced from 15 European countries to China successively, making gene resources of P. nigra substantially increased in our country. China is a nation with a shortage of fresh water, resulting in restrained growth and production of poplar plantations due to insufficient water supply for land soils. Thus, breeding water economic and drought resistance poplar variety by improving its water use efficiency （WUE） and the capacity of drought tolerance is important for poplar production and conservation of environments. Photosynthesis is the key factor that impact biomass accumulation of plants. This makes the breeding of poplar variety with higher photosynthetic-light use efficiency （PUE） is a key point in the establishment of high productive plantations. Such varieties should have high ability of the utility of light resource.In this paper, gene resources of P. nigra collected from Europe and Xinjiang were used as materials. PUE was evaluated and single nucleotide polymorphisms （SNPs） within water and photosynthesis-related genes were discovered and analyzed combined with the results of evaluation of WUE (δ¹³C) studied previously. Functional SNP markers were found by using association analyses between SNPs and related traits, proving a foundation for marker-assistant breeding on water-use and photosynthesis traits in poplar. The main results are described as following:Evaluation of WUE and PUE in P. nigra. Since the evaluation of WUE has been done in previous work, this paper focused on the evaluation of PUE. Gas exchange and chlorophyll fluorescence-related parameters were measured in gene resources of P. nigra. The results showed abundant genetic variation for traits of photosynthesis and growth in P. nigra. The genotypes from China and the U.K. have relatively higher values of photosynthesis rate （Pn）, while genotypes from Hungry have the highest values of quantum efficiency of chloroplast photosystem II （PSII） centers （Fv/Fm）. The measured traits were found to have generally high broad sense heritabilities （H2 ranges from 0.750 to 0.962） with considerable variation coefficients. The results showed that parameters including Pn, intercellular concentration of carbon dioxide （CO2） （Ci）, stomatal conductance （Gs） and electron transport rate （ETR） were significantly correlated with height growth, indicating potential applications for the evaluation of PUE in P. nigra. Focus should be on genotypes from counties of Southern and Eastern Europe in further breeding research based on PUE in poplar.Discovery and analysis of SNPs from genes that are involved in water and photosynthesis use. A total of 312 single nucleotide polymorphism （SNP） sites are found from seven water-use related （SuSY1, dhn, lea3, PIP, Expa1, ERF, and GPX1） and two photosynthetic-light use related （rubisco and Lhcb2） genes. The median SNP frequency is one site per 26 bp. The average nucleotide diversity for the sequenced regions was calculated to beθ_W=0.01074 andπ_T=0.00702, higher values than those observed in P. tremula, P. trichocarpa and most conifer species. Tests of neutrality for each gene reveal a general excess of low-frequency mutations, and many non-synonymous polymorphisms of these loci might be involved in local adaptation in P. nigra. The results reject neutral evolution at GPX1, dhn, lea3, and Expa1. These loci are likely to be undergoing a selective sweep which can cause hitchhiking effect. The level of linkage disequilibrium （LD） varies among loci. The overall LD in P. nigra is low, decaying rapidly from 0.45 to 0.20 or less within a distance of 300 bp. This is similar to the rate of decay reported in most other tree species, indicating that P. nigra is suitable for association analysis and mining of functional SNP markers based on candidate genes.Association analysis between SNPs and WUE trait (δ¹³C value). A total of 25 SNPs were genotyped using SNaPshot^? technology. These SNPs are from five genes, namely SuSY1, lea3, PIP, Expa1, and ERF. Association analysis between marker and trait were carried out using both One-way Analysis of Variance （ANOVA） and General Linear Model （GLM）. The results showed six associations between markers andδ¹³C value. SNP2 shows T-C transition with a genetic effect of 7.705%. The trees with genotype TT at SNP2 have higherδ¹³C values than that for other genotypes. SNP15 shows G-A transversion with a genetic effect of 7.570%. SNP15 causes an amino acid alteration between Val and Ile, and genotype GG has higherδ¹³C value. SNP25, SNP26, SNP27 and SNP31 are all located in Expa1, with a genetic effect ranging from 6.436% to 6.900%. One genotype was not detected for each of the four SNPs, and similar patterns of genotype frequency was found among them. For SNP25, SNP27 and SNP31, genotype TT has higherδ¹³C value than heterozygous genotype, implying similar mechanism responsible for the variation ofδ¹³C value in P. nigra.Association analysis between SNPs and PUE related traits. A total of 20 SNPs were genotyped using SNaPshot? technology. These SNPs are from genes of rubisco and Lhcb2. Association analysis between marker and traits were carried out using both ANOVA and GLM. The results showed that 4 SNPs are closely associated with photosynthesis and growth traits. SNP12, SNP36 and SNP50 are all located in rubisco. Among them, SNP12 and SNP36 are non-synonymous mutations, which result in amino acid alterations of Asn-His and Tyr-Phe, respectively. SNP12 and SNP36 are associated with ETR, and the genotype AA has higher ETR value than other genotypes. SNP50 is associated with Pn, ETR, Fv/Fm, height and diameter, and genotype CT has higher values of ETR, Fv/Fm, height and diameter and lower value of Pn. SNP44 of Lhcb2 is associated with height growth, and the genotype GG has higher height growth than other genotypes.更多还原