【作者】 陈丹；

【导师】 李立会；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2012， 博士

【摘要】 穗粒数是小麦产量三因素之一，通过远缘杂交创造具有多粒特性的新种质对于提高产量具有重要意义。本研究以从普通小麦–冰草（Agropyron cristatum （L.） Gaertn.,2n=4x=28, PPPP）远缘杂交后代中获得的具有突出多粒特性的“普冰3504”为材料，通过多年多点种植、遗传效应分析、QTL定位以及差异表达谱分析等方法，试图阐明“普冰3504”多粒特性的生态稳定性，分析与不同穗粒数小麦亲本组配的遗传效应，发掘控制穗粒数等产量相关性状QTL、基因组区段以及幼穗分化过程中差异表达基因/蛋白，为“普冰3504”在育种中的有效利用和候选基因克隆奠定理论基础。1、生态稳定性评价。通过在北京、陕西杨凌、四川成都、江苏南京、河北石家庄五个生态区2006-2007及2007-2008连续两个年度的种植和穗粒数性状调查，发现“普冰3504”在五个生态区均表现出多粒特性，穗粒数为82.7–145，平均为102.66，显著高于当地对照品种（41.7–63.26粒/穗）的49.81%–163.39%，说明“普冰3504”的多粒特性具有广泛和稳定的生态适应性。2、不同穗粒数小麦背景下的遗传效应分析。采用不完全双列杂交试验设计，将“普冰3504”与9个不同穗粒数的小麦品种（高组>52粒，中组40-52粒，低组<40粒）进行杂交，并分别将F1、F2种植于北京、四川成都、陕西杨凌试验区进行连续三年（2008-2009,2009-2010和2010-2011）的穗粒数和千粒重性状调查，采用QGAStation1.0软件进行遗传分析。结果表明，“普冰3504”与不同小麦亲本组配的F2群体的穗粒数平均数，均显著高于小麦亲本，平均提高46.07%；F2群体穗粒数性状均以加性遗传方差为主，狭义遗传率平均值为31.16%；遗传变异系数平均值为25.19%；在5%和1%选择强度下，群体选择响应（遗传进度）平均值分别为27.86%和36.25%，相对遗传进度分别达44.20%和57.53%；所有亲本中，“普冰3504”的加性效应最为显著，预测值为27.417；同一组合内，“普冰3504”作为父本对穗粒数提高的育种效应优于其作为母本的遗传表现。穗粒数与千粒重负相关程度较低（R²=0.0024），表明利用“普冰3504”改良小麦时，比较容易从杂交后代中选择出兼具高穗粒数和高千粒重的单株。3、“普冰3504”与京4839F_2:3群体表型分析。构建“普冰3504”与京4839F_2:3群体，并将F2群体在2008-2009年度种植于北京，F₃家系在2009-2010年度分别种植于北京、四川成都、陕西杨凌试验区，调查穗粒数、穗长、小穗粒数、每穗小穗数、千粒重、有效分蘖数、株高等7个重要农艺性状。结果表明，F2:3群体所有调查性状均具有丰富变异，广义遗传力均高于70%。其中，穗粒数性状在四个不同环境中的变异范围达16.78%-26.43%；广义遗传力为87%。并且，穗粒数与小穗粒数，每穗小穗数和穗长均正相关，尤其与小穗粒数正相关程度最高（R²=0.2809-0.7225）；同时，穗粒数与千粒重不相关；穗粒数与有效分蘖数（除2009年北京环境外）存在较低负相关（R²=0.1521-0.3721）。4、遗传连锁图谱构建。利用190对SSR、EST-SSR及STS分子标记，对“普冰3504”与京4839F2代的282个单株进行基因型分析，构建了一张覆盖（除2B染色体外）小麦所有染色体的遗传图谱。总遗传距离为3171.60cM，标记间平均遗传距离为16.69cM。5、穗粒数及产量相关性状的QTL定位。利用Network2.0软件，结合构建的遗传图谱，共检测到穗粒数等上述7个产量相关性状的QTL位点37个（单环境分析）和25个（联合分析）。其中，位于1A染色体短臂Xmag834标记附近的QGnps.cd-1A是控制穗粒数的主效QTL，四个环境下可重复检测到，对表型贡献率达13.01%-64.47%；QKps.cd-1A.1，QSns.cd-1A和QSl.cd-2D分别是控制小穗粒数，每穗小穗数和穗长的主效QTL，其中，QKps.cd-1A.1，QSns.cd-1A与QGnps.cd-1A位置相同，条件QTL分析表明，QGnps.cd-1A位点是控制穗粒数、小穗粒数和每穗小穗数3个性状的“一因多效”位点，穗粒数与小穗粒数和每穗小穗数，尤其是与小穗粒数存在较强的遗传相关性。并且也检测到1A染色体上存在控制有效分蘖数的主效QTL QSnp.cd-1A，4B染色体上存在控制千粒重和株高的主效QTL QTgw.cd-4B，QHt.cd-4B。同时，穗粒数、小穗粒数、穗长、有效分蘖数及株高存在QTL与环境互作效应，互作贡献率为0.21%-7.89%。所有性状中70.3%（单环境分析）和57.6%（联合环境分析）QTL位点的加性增效效应由“普冰3504”所提供。6、重要基因组区段分析。研究共发现5个与多粒性状相关的重要基因组区段，分布于1A、4A、4B、2D和4D染色体上。其中，1A染色体短臂Xmag834-Xbarc83标记区间是一个新发现的可以显著改良穗部性状，并且具骨干亲本遗传特性的重要基因组区段，在今后改良小麦穗粒数中可能发挥关键作用。此区段内存在控制穗粒数、小穗粒数、每穗小穗数穗部相关性状的主效QTL位点，表型贡献率为1.57%-64.47%；同时还涉及千粒重及有效分蘖数产量相关性状的QTL位点；位点具有良好的环境重复性。除有效分蘖数外，所有性状QTL位点的加性增效基因均来自“普冰3504”。7、P基因组遗传成分检测。利用本实验室开发的3362对小麦Fukuho背景下P基因组特异引物，对“普冰3504”进行检测。结果发现，2对特异引物能够在“普冰3504”背景下显示特异扩增，利用“普冰3504”与京4839的F2群体将这2对特异引物分别定位于3A染色体和4A染色体。8、基因与蛋白差异表达谱分析。以多粒种质“普冰3504”、“普冰3228”、小麦–冰草附加系“4844-12”及其小麦亲本Fukuho为材料，利用Affymetrix小麦全基因组芯片技术及iTRAQ-MS/MS技术，从转录和翻译水平进行差异比较分析。共检测到差异表达探针组611个（上调表达为272个，下调表达为339个）及差异表达蛋白282个（上调表达为123个，下调表达为159个）。其中，抑制开花因子GASA5及ZCCT2, ZCCT1的上调表达和促进开花因子FCA的下调表达，可能导致茎尖分生组织到花分生组织转变过程延迟，从而利于小穗分化而增加小穗数；控制花器官分化基因包括具有AP2结构域的蛋白激酶及HUA2上调表达，则利于小花分化以增加小花数，上述基因可能参与多粒性状形成的表达。并根据芯片差异表达EST序列开发可追踪小麦Fukuho背景中的P基因组特异标记143对及可能与多粒性状相关的小麦STS分子标记132对，并将其中5对定位于染色体1A、3A、6A和7B上。更多还原

【Abstract】 Grain number per spike (GNPS) is one of the three factors of wheat yield. High GNPS germplasm iskey genetic resources with high yielding potential. Pubing3504derived from progeny of the crossbetween common wheat and Agropyron cristatum (L.) Gaertn, showed prominent GNPS. In this study,by investigating the ecological stability of Pubing3504in different wheat ecological regions andanalyzing genetic effect of high GNPS under different wheat background, uncovering major QTLs andthe genomic regions of GNPS and yield-related traits, comparing differentially expressed genes andproteins of young panicle in Pubing3504, the genetic nature of high GNPS in Pubing3504wereexplained.1. The ecological adaptation was evaluated. Pubing3504were grown in five ecological areasincluding Beijing, Yangling in Shaanxi, Chengdou in Sichuan, Nanjing in Jiangsu and Shijiazhuang inHebei in2006-2007and2007-2008growth seasons and investigated GNPS. The results indicated thatPubing3504showed high GNPS in all ecological areas and the GNPS range from82.7to145with anaverage value of102.66, which was significant higher than control (41.7-63.26). The results indicatedthat Pubing3504had a stable and wide adaptability to different ecological regions.2. The genetic effect of GNPS under different genetic background was analyzed. An incompletediallel cross design was explored using Pubing3504as a common parent crossed with nine wheatvarieties (high GNPS level>52, mid GNPS level40-52, low GNPS level <40). The GNPS of F1hybridsand F2populations were investigated in Beijing, Sichuan and Shaanxi in2008-2009,2009-2010and2010-2011growth seasons, respectively. The results showed that the average relative increase ratio ofGNPS in populations was46.07%. Genetic analysis by QGAStation1.0software suggested that GNPSwas mainly controlled by additive genetic effects, and the average narrow-sense heritability was31.16%.Genetic variation coefficient was25.19%; and the genetic advance (selection response) under5%and1%selection intensity was27.86%and36.25%, respectively. The relative genetic advance was44.20%and57.53%, respectively. And additive effect of Pubing3504was the highest in parents with the value of27.417. In the same cross, the breeding effect of Pubing3504as the male parent was superior to itself asthe female parent. Correlation between GNPS and thousand grain weight (TGW) of F2populations waslow (R2=0.0024). The results showed that more individuals with both high GNPS and high TGW couldbe readily screened out from F2segregation populations using Pubing3504as parent.3. The F2:3population was developed from the cross between Pubing3504and Jing4839, and theseven yield traits including GNPS, spike length (SL), kernel number per spikelet (KPS), spikelet numberper spike (SNS), TGW, spike number per plant (SNP), plant height (PH) from F2population in Beijing(2008-2009) and F3family lines in Beijing, Sichuan, Shaanxi (2009-2010) were investigated,respectively. The results showed that the broad-sense heritability of all traits were up to70%. Correlationanalysis indicated that the significant positive correlations between GNPS and spike component factorsincluding KPS, SNS and SL, especially KPS (R2=0.2809-0.7225). While GNPS had insignificant correlation with TGW and weak correlation with SNP (R2=0.1521-0.3721).4. The genetic linkage map was constructed. A total of190SSR, EST-SSR and STS molecularmarkers were used to genotype the282individuals of F2population.The genetic map involving all thewheat chromosomes except2B, covered the genetic distance of3171.60cM with an average markerinterval of16.69cM.5. Based on the constructed genetic map, the QTL analysis by Network2.0for the yield traits wasconducted. As a result, a total of37QTLs (in a single environment) and25QTLs (in joint environment)were identified. A major QTL QGnps.cd-1A, were detected by four environments and explained13.01%-64.47%of the phenotypic variation. QKps.cd-1A.1, QSns.cd-1A and QSl.cd-2D were threemajor QTLs controlling KPS, SNS and SL, respectively. Among them, QKps.cd-1A.1and QSns.cd-1Awere in the same site with QGnps.cd-1A. Conditional QTL analysis showed that QGnps.cd-1A was apleiotropic QTL with GNPS, KPS and SNS. In addition, major QTL QSnp.cd-1A controlling SNP wasdetected, major QTLs including QTgw.cd-4B and QHt.cd-4B were detected, which controlled TGW andHT, respectively. The interaction of QTL×environment were detected with GNPS, KPS, SL, SNP andHT in joint environment analysis, and the contribution rate from QTL×environment interaction was0.21%-7.89%. Additive effects of70.3%(in a single environment) and57.6%（in joint environment）QTLs were positive contributed by Pubing3504alleles.6. QTL mapping results show that five important genomic regions on chromosomes1A，4A，4B，2D and4D were detected. Among them, the marker interval Xmag834-Xbarc83on the short arm ofchromosome1A was a novel important genomic region, which included the QTLs that controlled GNPS,KPS, SNS, TGW and SNP with good environmental repeatability. All these QTLs were major QTLsexcepting the TGW and all these QTLs additive effects came from Pubing3504excepting the SNP. Theregions showed the same genetic characteristics as founder parents and might play a very key role inimproving wheat yield in future.7. Fragments of P genome in Pubing3504were detected and tracked. There were3362P genomespecific markers in common wheat Fukuho background were used to identify alien P chromosome in“Pubing3504”. The results showed that two markers could be detected in “Pubing3504”, and mappingedon3A and4A chromosomes, respectively.8. Gene and protein expression profiling analysis were carried out. Compared to the control parentFukuho, the gene and protein expression profiling were studied in young spike of high GNPS wheatgermplasm (Pubing3504, Pubing3228and4844-12) using Affymetrix wheat chip and iTRAQ-MS/MStechnology. There were611transcripts (272up-regulated and339down-regulated transcripts) and282proteins (123up-regulated and159down-regulated proteins) expressed differentially in high GNPSgermplasm totally. Among them, flowering repressor GASA5, ZCCT2, ZCCT1were up-regulated, andflowering promotive factor FCA was down-regulated, implying that the conversion of the SAM into thefloral meristem was delayed, which was beneficial to forming more spikelets; Meanwhile, protein kinasewith AP2domain and HUA2were up-regulated, implying that expression of genes controlling thenumbers of floral organs were enhanced, which were beneficial to the floret formation in high GNPS germplasm. Both of the two reasons above might involve the formation of high GNPS characteristics.Furthermore, the143pairs P genome-special STS markers and132pairs the STS markers of wheatrelated to high GNPS were developed by differentially expressed EST sequences in genechips and fiveof them were mapped on1A,3A,6A and7B chromosomes, respectively.更多还原