【作者】 陈晓杰；

【导师】 胡银岗；

【作者基本信息】 西北农林科技大学 ， 作物遗传育种， 2013， 博士

【摘要】 小麦（Triticum aestivum L.）是人类最早栽培的作物之一，也是世界上分布最广的作物之一；在中国，小麦是仅次于水稻、玉米的第三大作物，主要种植在半湿润、半干旱地区。在我国干旱、半干旱地区约占国土面积的二分之一；即使在半湿润、甚至湿润地区也常会发生临时性、季节性或周期性的干旱。干旱是小麦增产的主要限制因子，因此，研究小麦抗旱性的鉴定指标，挖掘抗旱种质资源、同时利用数量性状基因位点QTL作图技术对控制小麦重要性状的基因进行定位、发掘优良等位变异，并将优异变异跟踪利用到新品种选育，对提高小麦的抗旱性和产量水平有着非常重要的作用。本研究以90份具有广泛代表性的中国冬小麦种质为材料，通过连续两年的抗旱棚水、旱处理试验，筛选抗旱种质材料、对抗旱相关性状进行了评价；并以其中12份育成品种为材料，对水、旱处理下碳同位素分辨率、籽粒产量、光合性状、旗叶气孔密度及矿质元素之间的相互关系进行了深入研究；同时利用269对小麦基因组上均匀分布的SSR标记对我国冬小麦的遗传多样性、群体结构和连锁不平衡进行分析，并对小麦主要农艺性状、光合性状和根系性状的QTL位点进行关联分析。取得的主要研究结果如下：1、通过两年的抗旱棚水、旱处理试验，采用抗旱系数隶属函数法筛选抗旱小麦种质，共筛选出抗旱性较好的材料5份。对各性状在干旱胁迫下的协同变化分析表明：蒸腾速率和气孔导度、单株籽粒产量和单株生物产量对干旱胁迫存在极强的协同变化趋势，株高、穗长、小穗数、穗粒数、单株籽粒产量和单株生物产量6个性状相互间也存在较强或极强的协同变化效应。以材料的抗旱隶属函数值为响应变量，各性状的抗旱系数为回归变量进行了逐步多元回归分析，最终单株籽粒产量、蒸腾速率（或气孔导度）、株高、穗下节长和穗长5个性状的抗旱系数被选入模型，并可以解释超过91%的抗旱隶属函数值的变异，为小麦种质大规模抗旱性鉴定与评价提供一种可行的方法，有助于提高选择效率。2、以12份育成品种为材料，系统研究了碳同位素分辨率（Δ13C）、籽粒产量、光合性状、旗叶气孔密度及矿质元素在水、旱两组水分处理下的表现以及它们之间的相互关系。光合速率、Δ13C、气孔导度、旗叶叶绿素含量、籽粒产量、籽粒灰分含量、籽粒K含量、籽粒Mn含量8个性状在干旱胁迫下表现为下降趋势，且与充分灌溉相比达到极显著水平（P<0.01）；籽粒Fe含量在干旱胁迫条件下表现为上升趋势，且与灌溉下相比达到极显著水平（P<0.01）。相关分析表明，籽粒Δ13C值与籽粒产量在干旱胁迫和充分灌溉条件下均呈现正相关，籽粒Δ13C值与灌溉条件下的旗叶下表皮气孔密度和干旱条件下的Ci/Ca值在P<0.05水平下相关。气孔导度、胞间CO₂浓度、蒸腾速率和Ci/Ca4个光合性状相互间在两种水分条件下均呈现极高的正相关。在干旱胁迫条件下，籽粒灰分含量与籽粒Fe含量、Zn含量、Mn含量和K含量表现为极显著的正相关（P<0.01）；在灌溉条件下，籽粒灰分含量与籽粒Fe含量、Zn含量、Mn含量、K含量和Mg含量均表现为正相关；在两种水分条件下，籽粒Fe含量、Zn含量、Mn含量和K含量4个指标相互间均呈明显或极显著正相关。通过多元回归分析建立多指标共同解释Δ13C的模型，在干旱条件下本研究所用性状不符合构建多元回归模型；而在灌溉条件下旗叶下表皮气孔密度和光合速率入选回归模型，并能解释60.9%的Δ13C变异。3、以90份广泛代表性的小麦种质为材料，对我国冬小麦21个主要性状的遗传变异分析表明，除穗长、小穗数、光合速率、胞间CO₂浓度和叶绿素含量5个性状外，其他16个性状均具有比较丰富的遗传变异。广义遗传力分析表明，以形态性状为主的简单性状（穗叶距、穗下节长、株高、胚芽鞘长、根直径、千粒重、穗长、叶绿素含量、穗粒数、小穗数、根长、旗叶长）具有较高的遗传力，适宜早代选择；产量性状（单株籽粒产量和单株生物产量）遗传力中等，但考虑其是育种改良的核心目标，同样建议作为一个可靠的早代选择指标；光合生理性状及复合性状（萌发期根表面积、根体积、胞间CO₂浓度、气孔导度、蒸腾速率和光合速率）遗传力最低，尤其是光合生理性状容易受外界环境的影响且不容易准确的测量，不适宜作为早代选择的指标。4、利用269对均匀分布于小麦21条染色体的SSR标记对90份中国冬小麦种质基因组进行扫描，对我国冬小麦的遗传多样性、群体结构和连锁不平衡的分析表明，中国冬小麦遗传多样性较差；不同基因组间差异显著，其中B基因组多样性最丰富，D基因组多样性最差。利用Structure聚类、UPGMA聚类和主坐标分析均表明中国冬小麦可以分为3个亚群，且亚群的划分在一定程度上与群体地理生态类型相关。中国冬小麦的连锁不平衡衰减距离较短，全基因组LD平均衰减距离约2.2cM，A、B和D三个基因组的LD最大衰减距离分别为2.2、0.6和8.6cM；表明利用中国冬小麦进一步关联作图时需要更高密度的标记。5、利用均匀覆盖小麦21条染色体的269对SSR标记，对我国冬小麦群体（90份具有广泛代表性材料）进行全基因组扫描，采用TASSEL软件的MLM模型对两年水、旱2种处理下的农艺性状、光合生理性状、萌发期根系及胚芽鞘等共19个性状进行关联分析。检测结果发现，在138个SSR位点共检测到274次与19个性状在P<0.01水平下极显著关联，与株高、穗下节长、穗叶距、旗叶长、旗叶宽、穗长、小穗数、穗粒数和千粒重9个形态性状关联的SSR位点数分别为20、18、18、13、8、13、35、14和20个；与光合速率、蒸腾速率、气孔导度、胞间CO₂浓度和叶绿素含量5个光合生理性状关联的SSR位点数分别为16、16、13、27和18个；与胚芽鞘长、根长、根直径、根表面积和跟体积5个萌发期性状关联的SSR位点数分别为7、4、5、3和6个；其中7个SSR位点同时在4种条件下共检测到14次与4个性状关联，表现出位点的一致性和稳定性；研究还发现了67个SSR标记（138的48.55%）同时与2个或多个性状相关联，这可能是性状本身存在相关或者一因多效；关联分析结果的部分SSR位点与前人连锁分析或关联分析定位的QTL位点一致，进一步验证了实验结果的真实性。研究结果对重要区段QTL精细定位及分子标记辅助育种具有重要的参考价值。更多还原

【Abstract】 Wheat (Triticum aestivum L.) is one of the first plants cultivated by mankind; it has beenextensively planted worldwide owing to its adaptation to a wide range of climates. Wheat isthe third most-produced cereal crop after maize and rice in China; it is mainly planted insemi-humid and semi-arid regions. The arid and semiarid area covers about half of the totalland area in China; and periodic drought, seasonal drought or temporary drought oftenoccurred in semi-humid area, even wet area; drought stress has been one of the mostimportant abiotic constraints greatly limiting wheat production. Therefore, the study ofdrought resistance indicators, the excavation of drought tolerance germplasms, QTL mappingfor important traits, exploitation of allelic variations and maker-assisted selection (MAS) inwheat breeding will play an important role in increasing crop production study. This studyregards90broadly representative Chinese winter wheat accessions as research objects,through normal and drought treatment experiments for two years, screening drought tolerancegermplasms, evaluating drought tolerance of different traits; second, the study regards12bredvarieties as research objects, through normal and drought treatment experiment, analysing therelationships among carbon isotope discrimination, grain yield, photosynthetic traits, stomataldensity of flag leaf, ash content and mineral elements; analysing the genetic variation andbroad sense heritability of the investigated traits for90accessions; to estimate the geneticdiversity, population structure and linkage disequilibrium in Chinese winter wheat with269SSR loci distributed over all21chromosomes; to analyze the association loci with mainagronomic, physiological and root traits using269SSR loci distributed over all21chromosomes. The main results are included as follows:1. Through normal and drought treatment experiments for two years,5accessions withhighly drought tolerance were screened according to the membership function value ofdrought tolerance (MFVD), which combined the drought-tolerant coefficient (DC) of14traitsthat could be easily estimated; these5accessions could be used as materials for droughttolerance improvement in wheat breeding. The analysis, consistency of traits variation todrought stress, indicated that there was a very strong consistency variation between transpiration rate (E) and stomatal conductance (Gs) to drought stress, also a very strongconsistency variation between biological yield per plant (BYPP) and grain yield per plant(GYPP); there were strong consistency variations among plant height (PH), spike length (SL),spikelet number (SN), grain number per spike (GMPS), biological yield per plant (BYPP) andgrain yield per plant (GYPP) under drought stress. The multi-regression analysis was donebetween the membership function value of drought tolerance (MFVD) and thedrought-tolerant coefficient (DC) of each trait. The results revealed that DC of GYPP, E (orGs), PH, UIL and SL could explain more than91%of the total variations in MFVD.Therefore, those five variables could be used as a combination to screen wheat germplasm orlines for drought tolerance in wheat breeding program.2. Through normal and drought treatment experiments in the2011-2012growing seasons,using12bred varieties as materials, the relationship among carbon isotope discrimination,grain yield, photosynthetic traits, stomatal density of flag leaf, ash content and mineralelements was studied. The mean values of8traits, including photosynthetic rate (Pn), carbonisotope discrimination (Δ13C), stomatal conductance (Gs), SPAD value of the flag leafchlorophyll content (SPAD), grain yield (GY), ash content (AC), K content (K) and Mncontent (Mn), significantly decreased under water stressed (WS) condition compared with thewell-watered (WW)(at least P <0.01); on the contrary, Fe content (Fe) significantly increased(P<0.01) under WS. Results of correlation analysis showed that Δ13C was positivelycorrelated with GY under two water conditions (WS and WW); Δ13C was significantlypositively correlated with Ci/Ca under WS (P<0.05), while Δ13C showed significantlynegative correlation with stomatal density of lower epidermis (SDLE) under WW (P<0.05).There were significant positive correlations among Gs, Ci, E and Ci/Ca with each other undertwo water conditions (at least P <0.05); AC was positively correlated with Fe, Zn, Mn and Kcontents under two water conditions; there were positive correlations among Fe, Zn, Mn andK contents with each other under two water conditions. The multiple regression model basedon Δ13C indicated that SDLE and Pn could explain about60.9%of total variations underWW, but multiple regression model could not be built under WS.3. Analysing the genetic variations for the21investigated traits with90Chinese winterwheat accessions, the result showed that the genetic variation of5traits of SL, SN, Pn, Ci andSPAD had lower variability (CVg<10%); other16traits contained abundant genetic variations(CVg>10%). The broad sense heritability for21investigated traits was estimated by geneticvariance and phenotypic variance. The simple traits mainly composed of some morphologicaltraits (SDL, UIL, PH, CL, RD, TKW, SL, SPAD, GNPS, SN, RL and FLL) had relatively highheritability; these traits are available to be selected in early generations. Yield traits (BYPP and GYPP) were of medium heritability; as these traits are the core goal in wheat breeding, sothese traits also are considered to be reliable indexes in early generations. Photosynthetic andother complex traits (Pn, E, Gs, Ci, RS and RV) were of low heritability; especiallyphotosynthetic traits were easily influenced by the environment thus could not be evaluatedaccurately; it is unsuitable for selecting these traits in early generation.4. Ninety winter wheat accessions were analyzed with269SSR markers distributedthroughout the wheat genome. Compared with previous reports, the genetic diversity of the90Chinese winter wheat accessions in this study was at a lower level. Significant difference ingenetic diversity among the three genomes was observed; the B genome showed the highestdiversity and the D genome the lowest. The90Chinese winter wheat accessions could bedivided into three subgroups based on STRUCTURE, UPGMA cluster and principalcoordinate analyses. The population structure derived from STRUCTURE clustering waspositively correlated to some extent with geographic eco-type. LD analysis revealed that therewas a shorter LD decay distance in Chinese winter wheat compared with other wheatgermplasm collections. The whole genome LD decay distance was approximately2.2cM(r~2>0.1, P<0.001), and the LD decay distance in A, B and D genomes of wheat was about2.2,0.6and8.6cM, respectively. Evidence from genetic diversity analyses suggested that wheatgermplasm from other countries should be introduced into Chinese winter wheat and distanthybridization should be adopted to create new wheat germplasm with increased geneticdiversity. The results of this study were expected to provide valuable information for futureassociation mapping by using this Chinese winter wheat collection.5. The marker-trait associations for19traits were studied with90elite wheat genotypesusing269SSR markers distributed throughout the wheat genome. The marker-traitassociation analysis was tested for each water condition in each year with the softwareprogram TASSEL v2.1by using the mixed linear model based on the Q-matrix and thekinship-Matrix. The results showed that a total of138SSR loci were associated with19traits,and274high significant marker-trait associations were detected at the P<0.01level in4environment (11SW,11WW,12SW and12WW). The numbers of SSR association loci with9agronomic traits (PH, UIL, SDL, FLL, FLW, SL, SN, GNPS and TKW) were20,18,18,13,8,13,35,14and20, respectively. The numbers of SSR association loci with5physiologicaltraits (Pn, Gs, E, Ci and SPAD) were16,16,13,27and18, respectively. The numbers of SSRassociation loci with5root traits (CL, RL, RD, RS and RV) were7,4,5,3and6, respectively.Seven SSR loci were associated with4traits, and14high significance marker-traitassociations were simultaneously detected in all4environment. Among138SSR loci,67SSR loci (48.55%of138) were associated with at least two traits and further referred as multi-trait QTL. There were a few loci associated with more traits simultaneously, whichmight be the genetic reason of correlation among traits or pleiotropic phenomena. Some SSRloci were in agreement with mapped QTLs from family-based linkage mapping procedure orassociation analysis, and verified the factcity of results. The results were expected to provideimportant practical value for the fine mapping of QTL and molecular marker for assistedbreeding.更多还原