【作者】 杨光；

【导师】 谢甫绨；

【作者基本信息】 沈阳农业大学 ， 作物学， 2017， 博士

【摘要】 大豆为光周期反应敏感的短日照作物,光周期反应决定生育期长短,生育期与大豆产量、品种适宜种植范围密切相关。开花（始花）是植物从营养生长向生殖生长转变的典型特征,与生育期密切相关。分枝数是大豆重要农艺性状之一,其影响大豆冠层构型、群体受光、抗倒伏能力及种植密度等,与产量密切相关。因此大豆始花期和分枝数基因/QTL的挖掘与利用能够为大豆分子育种提供理论依据和技术指导。迄今为止,在大豆中已经定位到控制开花的基因/QTL有E1-E9和J。然而,在相同已知始花期基因群体中,仍存在始花期相差很大的现象,这表明还有其他未知基因影响大豆始花期。为定位始花期和分枝数QTL,研究其之间的关系,本试验利用遗传背景差异较大的日本栽培大豆品种Toyomusume（e1-nl,e2,E3-Mi,E4）和中国东北栽培大豆品种绥农10号（E1,e2,e3-T,E4）杂交构建F2代、F3代群体及其衍生群体。本试验利用均匀分布在大豆20对染色体上的671对SSR标记,通过父母本（Toyomusume和绥农10号）及其F3代群体（N=141）间具有多态性的158对SSR标记（174对多态性引物中有16对冗余引物）构建遗传连锁图谱,包括27个连锁群。试验又利用 Illumina SoySNP8k iSelect BeadChip DNA 芯片（7189 对 SNP 引物）对亲本（Toyomusume ×绥农10号）及其F2代群体（N=100）进行基因分型,除去异常和无多态性及冗余的SNP标记,共有1306对多态性SNP标记用于构建遗传连锁图谱。该SNP遗传连锁图谱包括20个连锁群,一一对应大豆的20对染色体,SNP标记的遗传距离和物理位置（Gmax₂75_Wm82.a2.v1版本）高度一致,为QTL准确定位奠定了基础。3个始花期QTL（#T₂、qFT₆、qFT₁6）都在2张遗传连锁图谱上定位到,在SSR和SNP遗传连锁图谱上还分别定位到qFT₃和qFT₁9。对父母本及不同年代不同地点的8个F2代、F3代群体研究表明qFT₆、qFT₁9、qFT₁6为E1、E3、E9位点。对F4代群体研究表明qFT₂是真实存在调控始花期的微效QTL,到目前为止该位点尚未见报道。对不同年代不同地点的8个F2代和F3代群体及8种类型群体的E1、E3、E9、qFT₂位点进行研究,结果表明:不同环境条件下调控开花效应最大的为E1基因,其次为E9基因。E1、E3、E9基因等位变异不同组合的8种类型群体始花期变异规律在不同年份和不同地点皆表现一致,即始花期E1/E3-Mi/e9>E1/e3-T/e9>E1/E3-Mi/E9>E1/e3-T/E9>e1-nl/E3-Mi/e9>e1-nl/e3-T/e9>e1-nl/E3-Mi/E9>e1-nl/e3-T/E9。为研究始花期基因和分枝数之间的关系,对不同年代不同地点的8个F2代和F3代群体及8种类型群体的E1、E3、E9基因进行研究,结果表明:分枝数和E1基因呈极显著相关;分枝数和E9区域附近的差异位点呈显著连锁;分枝数和E3基因的关系没有达到显著水平。结合该群体始花期受E1、E9基因调控开花影响较大,E3基因调控开花影响较小,在遗传角度上推测开花调控效应较大的基因（E1、E9基因）对分枝数具有"一因多效"性。E1基因是调控开花最重要的基因,因此利用根瘤农杆菌介导法将E1基因转入野生型植株（东农50（e1-as））,并获得2株E1超表达转基因株系（E1#L16和E1#L18）。对E1超表达转基因株系和野生型植株的分枝数统计分析表明E1超表达转基因株系的分枝数显著多于野生型植株。本试验进一步推测E1基因具有对分枝数的"一因多效"性,但还需要在分子水平上进一步验证。除分枝数之外,在始花期基因（E1、E3、E9）附近还定位到其他重要农艺性状的QTL。研究表明,E1基因对株高、主茎节数、分枝荚数、分枝粒数作用明显。E3基因对株高、主茎节数、主茎荚数和主茎粒数有一定影响。由于E9基因是提早开花基因,其对株高和主茎节数影响不大。为排除始花期基因对重要农艺性状影响,挑选始花期基因相同（E1、E2、E3、E4和E9基因）且始花期相近的4个大豆品种（超高产大豆品种:沈农12、中黄35、辽豆14;普通大豆品种:辽豆11）,通过田间施肥处理和生理试验进一步研究大豆叶片生理与籽粒产量之间的关系。结果表明,在鼓粒关键时期超高产大豆品种叶片净光合速率和叶色值显著高于普通大豆品种;在鼓粒期叶片净光合速率日变化没有出现"光合午休"的现象,14点后超高产大豆品种叶片净光合速率显著高于普通大豆品种。在R1、R6、R7时期,超高产大豆品种叶片SOD活性均显著高于普通大豆品种,MDA含量均低于普通大豆品种。更多还原

【Abstract】 Soybean,as a short day crop,is photoperiodic sensitive which can determine growth duration.Growth period is related to the yield and the adaptability of a soybean cultivar.Flowering is a typical characteristic that represents the transition from the vegetative to the reproductive phase in plants.Flowering time is controlled by environmental factors and internal physiological and molecular process in plants.Branching is also an important agronomic trait related to yield,which often affects canopy architecture,light reception,lodging and density of plant.So,the identification and utilization of genes underlying flowering time and branching will help us to understand more about the molecular mechanism and to give technical advice for molecular breeding.To date,a series of E loci associated with flowering time and maturity have been identified in soybean,including E1-E9 and J.Among them,El,E2,E3 and E4 play important roles in flowering time.However,the ranges of time to flowering and maturity were quite large in some cultivar soybean with different genetic background,implying the presence of some other unknown QTL or genes that are related to control flowering time or maturity.In this study,we developed eight F2 populations,eight F2.3 populations,and eight specific sub-populations from a cross between a Japanese cultivar Toyomusume（el-nl,e2,E3-Mi,E4）and a Chinese cultivar Suinong 10（E1,e2,e3-T,E4）for identifying novel QTL controlling flowering time and studying the relationship between flowering time genes and branching numbers.The genetic linkage map was constructed by SSR markers in 141 plants of F3 population between Toyomusume and Suinong 10.A total of 671 pairs of SSR markers covering 20 chromosomes of soybean were screened.Of them,174 SSR primer pairs showed polymorphisms between the parents and among the F3 population.Apart from 16 unlinked markers,157 SSR markers were successfully mapped to 27 groups.SNP genotyping for parents（Toyomusume × Suinong 10）and F2 population（N=100）were performed using the Illumina Iscan platform（Illumina,Inc.San Diego,CA）.Illumina SoySNP8k iSelect BeadChip with 7189 SNPs was specifically manufactured at Infinium HD Ultra.The 2363 SNP polymorphic markers were analyzed using QTL IciMapping₄.0 software.After discarding the redundant or unmapped markers,1306 unique loci SNP markers were retained and mapped leading to the construction of 20 linkage groups corresponding to the 20 chromosomes of soybean.The typical S type curve represents the consistency between genetic map and the physical position in the chromosome（Current version Gmax₂75-Wm82.a2.v1）,which is a basis for identifying QTL accurately.Three QTL for flowering time were located on Chr 02,Chr 06,Chr 16 in both the F2 and F3 populations using SNPs and SSRs,and we verified the results.The results showed that qFT6 was a major QTL for delaying flowering time mapped onto Chr 06 nearby El gene in the F2 and F3 populations,which has the same effective of El.Judging from different El loci between parents,and genotyping F2 and F3 populations,we firmly conclude QTL of qFT6 was corresponding to E1.Similar to the qFT6 result,we conclude QTL of qFT19 and qFT16 were corresponding to E3 and E9 respectively.A novel QTL（qFT₂,new QTL,no reported）for flowering time was located on Chr 02 in both the F2 and F3 populations using SNPs and SSRs.We genotyped each individual in specific population using markers located in qFT₂ region,and made correlation analysis.We concluded that qFT₂ locus was real existence.At the same time,we also detect a novel minor QTL（qFT₃）in F3 population.In order to study the relationship among the QTL identified in the population,we genotyped each individual in eight F2 and F3 populations and eight specific-populations at El,E3,E9,qFT₂ locus and made correlation analysis with flowering time.The results indicated that El alleles have a major effect on flowering and maturity,E9 has a relative large effect compared to E3 in this population,and qFT₂ has a minor effect.Flowering variation regularity of eight type combinations in different El,E3 and E9 allelic has consistent performance in different environment,that is E1/E3-Mi/e9>E1/e3-T/e9>E1/E3-Mi/E9>E1/e3-T/E9>e1-nl/E3-Mi/e9>e1-n1/e3-T/e9>e1-nl/E3-Mi/E9>e1-nl/e3-T/E9.The QTL for branching number identified in both the F2 and F3 populations using SNPs and SSRs were all nearby QTL for flowering time.In order to study the relationship between E loci and branching number,we made correlation analysis between E1,E3 and branching in eight F2 and F3 populations and eight specific sub-populations.We also made correlation analysis between discrepant site nearby E9 and branching in eight F2 and F3 populations and eight specific sub-populations.The results showed that branching number was significantly positive correlation with E1 genotype,branching number links to the discrepant site nearby E9,and there is no significant correlation between E3 genotype and branching number.We speculated that the genes for flowering time have a pleiotropic effect on branching at the genetic level.Overexpression of E1 significantly enhanced branching number in transgenic lines,which further suggest E1 have a pleiotropic effect on branching,but this needs to be further confirmed by genetic studies and at the molecular level.QTL for other agronomic traits were also mapped nearby the flowering time genes（E1,E3,E9）except the QTL for branching number.Correlation analysis of E1,E3,E9 genotype with phenotype of plant height,nodes on the stem,pods on the stem,pods on the branching,total pods showed that E1 have a significant effect on plant height,nodes on the stem,pods on the branching,seeds on the branching,and had no significant effect on pods on the stem,seeds on the stem.E3 have certain influence on plant height,nodes,pods on the stem,and seeds on the stem.Because it is an early flowering gene,E9 has no significant effect on plant height and nodes.We used four soybean varieties（super high yield soybean cultivars Shennong 12,Zhonghuang 35,Liaodou 14 and common soybean cultivar Liaodou 11）with similar flowering time period and the same flowering time genes（E1,E2,E3 and E9 genes）which were identified for cloned flowering time genes.We studied the relationship among soybean yield and photosynthetic system,antioxidant enzyme activity via field experiment and physiological test.The results showed that the cultivars with the same flowering time genes did not show "noontime snooze" at R6 growth stage,and the photosynthesis of super-high-yield cultivars was higher than that of common cultivar after 14.O’clock.These indicated that super-high-yield cultivars had greater photosynthetic assimilation capacity than that of common cultivar at R6 growth stage,which was one of the important physiological reasons for super-high-yield cultivars greater yield excluding the influence of flowering time genes.In present study,the SOD activity of the four cultivars with the same flowering time genes declined with the developmental process and the content of MDA increased.At the same time,SOD activity and MDA content of the super-high-yield soybean varieties were higher and lower than that of common soybean variety at R1,R6,and R7 growth stage,respectively.The results also showed that POD activity of the super-high-yield soybean are higher than that of common soybean at R6,R7 stage under 225 kg/hm,300 kg/hm2,indicating that the super-high-yield soybean cultivars had the stronger active oxygen clearing ability.In this study,the nitrogen fertilizer could not only enhance the SOD and POD activities,but also decreased the MDA content,indicating that fertilizer treatment could delay leaves senescence effectively.In order to map novel QTL for flowering time and branching,we developed the population from Japan soybean cultivar（Toyomusume:e1-nl,e2,E3-Mi,E4,and e9）and China soybean cultivar（Suinong 10:E1,e2,e3-T,E4,and E9）,which have big genetic background differences between parents,and the population can provide good materials for studying interaction among flowering time genes.In this study,we revealed that flowering time genes have a pleiotropic effect on branching at the genetic level.Furthermore,the population in this study will be very useful for the fine-mapping of minor QTL on Chr 02 or Chr 03 for flowering time,and for future study of interactions among E1,E3,E9,and other QTL.Our understanding of the regulatory networks for flowering time and other agronomic traits will be extended and highly beneficial for molecular designer breeding.更多还原