【作者】 张新忠；

【导师】 许如根；

【作者基本信息】 扬州大学 ， 作物遗传育种， 2013， 博士

【摘要】 为进一步了解大麦杂种优势的机理及遗传规律,开发与大麦强优势相关的分子标记,促进大麦杂种优势的应用,本研究以扬州大学大麦研究所选育的10个核质互作不育系(A)及其相应保持系(B)和11个恢复系(R)为材料,于2009年与2010年分别按4×2和8×9配制F1杂种播种于扬州大学农学院试验田,2011年按8×6配制Fl杂种分别播种于方强农场、上海农场和扬州大学农学院试验田3个试验点,植株成熟后随机取竞争株考种,考种性状包括株高、穗下节间长、穗长、单株穗数、每穗粒数、千粒重、单株粒重和单株干重,分析各性状的杂种优势表现和稳定性及配合力效应。利用改进的大麦cDNA-AFLP技术分析4×2杂交种与其亲本间的基因表达差异,对可能与杂种优势相关的差异谱带进行回收测序分析；基于差异谱带的序列信息开发大麦杂种优势表现相关的分子标记,并利用8×9和8×6试验对分子标记的选择效果进行了评估。结果表明：(1)利用大麦cDNA-AFLP技术分析了4×2试验中8个杂种与其亲本间的基因表达差异性,256对引物共扩增出8538条带,其中杂种与亲本差异谱带为3971条,占46.52%；杂交大麦与亲本间基因表达主要有共同表达型(111)、P1表达型(100)、P2表达型(001)、F1表达型(010)、P2F1表达型(011)、P1F1表达型(110)和P1P2表达型(101)七种类型,其中后6种类型为杂种亲本差异表达类型,在不同组合间的比例具有明显差异。综合分析差异谱带类型与杂种优势后,对23个可能与大麦杂种优势相关的TDFs进行回收并BLAST分析,23个片段依据功能可分为三类：第一类为参与植株的生长发育调控的主要功能蛋白,第二类主要涉及信号传导和能量传输,第三类为未知功能或新发现的转录本片段。(2)杂交大麦8个性状普遍存在中亲优势,但超优亲优势仅存在少部分组合中。8×9和8×6两个试验的120个杂种8个性状的显著中亲优势的组合出现率为57.60%,其中正向显著的组合占88.24%；显著超优亲优势组合出现率为25.94%。组合间和性状间的杂种优势具有显著差异,其中株高、穗下节间长和穗长、每穗粒数和千粒重5个性状的显著中亲优势组合出现率较高,分别为94.2%、84.2%、66.7%、62.5%和67.5%,单株穗数、单株粒重和单株干重3个性状的显著中亲优势组合出现率较低,仅为17.5%、25.0%和37.5%,穗下节间长、穗长和千粒重3个性状的超优亲优势出现率较高,分别为59.2%、41.7%和59.2%,每穗粒数、单株穗数、单株粒重和单株干重4个产量性状的超优亲优势出现率均低于10%；株高性状没有出现显著的超优亲优势组合；大麦杂种的超优亲优势与组合棱型具有密切的关系,异棱型杂交种的显著超优亲优势组合出现率高于同棱型杂交种。综合各性状,A1×R3、A1×R10、A2×R9、A2×R10、A6×R3等组合为强优势组合。(3)8×6试验中的48个杂交大麦在方强农场、上海农场和扬州大学农学院试验田3个试验点共调查了株高、穗下节间长、穗长、单株穗数、每穗粒数、千粒重和单株粒重7个性状,7个性状在3个试验点的中亲优势和超优亲优势平均出现率分别为36.81%和8.23%；不同环境下杂种优势的出现率具有明显差异,方强农场、上海农场和扬州大学3个点的显著中亲优势出现率分别为12.76%、32.03%和51.04%,超优亲优势的出现率分别为2.86%、2.86%和15.89%。杂交大麦主要性状的超优亲优势在3个试验点之间也具有差异,部分组合仅在单个或两个试验点的超优势优势达显著水平。(4)21个亲本各性状上的GCA效应方差在8×6和8×9试验中均达到显著水平,大部分性状的SCA效应方差也达到显著水平；相同亲本不同性状的配合力效应不同；两个试验中相同亲本的GCA和相同组合间的SCA也存在差异。依据不同性状的GCA和SCA方差对亲本进行聚类分析,能有效地对亲本的利用价值进行评价。(5)基于回收谱带的序列或其同源序列信息,共设计开发了23对与杂种优势相关的分子标记。通过8×9和8×6两个试验进行验证,共有4对标记对产量性状的杂种优势的筛选效果较好,分别是TDF5-P、TDF6-P、TDF10-P和TDF12-P。4对标记的对千粒重性状强优势组合筛选的特异度变幅为43.48%～73.68%,敏感度变幅为50.00%～78.95%；对单株粒重性状强优势组合筛选的特异度变幅为5.26%～56.52%,敏感度变幅为33.330%～100%。更多还原

【Abstract】 In order to further understand mechanisms of barley heterosis and develop molecular markers related to barley heterosis, ten CMS sterile lines (A) and their respective maintainer lines (B) and eleven restore lines (R) were used as parent materials in this study. Hybrids produced by4×2and8×9were planted in experiment fields of Yangzhou University in2009and2010, respectively. And hybrids produced by8×6were planted in Fangqiang Farm, Shanghai Farm and experiment fields of Yangzhou University in2011. After maturity, competitive plants were choosed from every block randomly. And the traits height (PH), spike length excluding awns (SL), inter-node length (IL), spikes per plant (SP), kernels on main spike (KMS), kilo-grain weight (KW), kernel weight per plant (KWP) and dry matter weight per plant (DWP) were investigated. Then heterosis and its stability of hybrids and combining ability in every trait were analysed. The different express profiles between4×2hybrids and their parents were analysed by barley modified cDNA-AFLP technology. Then the transcript-derived fragments TDFs related to barley heterosis were recovered and sequenced. Based on the sequence information, the heterosis-relative molecular markers were developed. Then the effects of markers were estimated by8×9and8×6. The results showed that:(1) The different express profiles of eight hybrids and their parents in experiment I were analysed by barley cDNA-AFLP technology, total8538distinct bands were detected, among which3971(46.52%) bands was different between hybrids and their parents. Seven gene expression types were observed between hybrids and their parents, which included bands detected in hybrid and two parents (’111’type), band only detected in female parent (’100’type), band only detected in male parent (’001’type), band only detected in hybrid (’010’type), bands detected in female parent and hybrid (’110’type), bands detected in male parent and hybrid (’011’type) and bands detected in two parents (’101’type). Among the seven expression types, the last six types represented the differential gene expression between hybrid and its parents. After conjoint analyzing of different express fragments and barley heterosis, twenty three TDFs possibly related to barley heterosis was recovered and analyzed. Based of the exact or predicted function, the twenty-three TDFs were classified into three categories. First category was TDFs with functions to regulate barley growth and development. Second category was the TDFs involved in signal transduction and energy transfer. And the third category was unknown TDFs and no homology sequences TDFs.(2) Mid-parents heterosis was ubiquity among eight traits, but over-better-parent heterosis was only in some crosses. For all120hybrids in experiment II and III, the occurrence rate of significant mid-parents heterosis was57.60%. Among the significant mid-parents heterosis crosses, the ccurrence rate of positively significant Hm crosses was88.24%. However, the occurrence rate of significant over-better-parent heterosis was only25.94%. The heterosis was different between crosses and traits. Among the different traits, the occurrence rates of significant Hm corsses in PH, IL, SL, KMS and KW were higher, which were94.2%,84.2%,66.7%,62.5%and67.5%, respectively. While the occurrence rates of significant Hm corsses in SP, KWP and DWP were only17.5%,25.0%and37.5%. The occurrence rates of significant Hb crosses in IL, SL and KW were higher, which were59.2%,41.7%and59.2%, respectively. While the occurrence rates of significant Hb crosses in other triats were all lower than10%. The hybrids from the parents with different row type generated more significant heterosis over better parent than that from the parents with the same row type. For all traits, crosses such as A1×R3, A1×R10, A2×R9, A2×R10and A6×R3and so on were superior crosses.(3) Forty eight hybrids in experiment Ⅲ were planted three experiment fields Fangqiang Farm, Shanghai Farm and Yangzhou University, respectively. Then PH, SL, IL, SP, KMS, KW and KWP of hybrids and their parents were investigated and heterosis was analysed. For all traits, the occurrence rates of significant Hm and Hb in three sites were36.81%and8.23%, respectively. However, the occurrence rates were different between three sites. The occurrence rates of significant Hm were12.76%,32.03%and51.04%, respectively. And the occurrence rates of significant Hb were2.86%,2.86%and15.89%, respectively. Besides that, the degrees of heterosis were also different between three sites. The Hm of some corsses were only significant in one site or two sites. (4) For experiment Ⅱ and Ⅲ, the GCA variances in all traits and SCA variances of most traits were significant in twenty one parents. For one parent, the combining abilities were different among different traits. And the combining abilities were different betweent the two experiments. Based on GCA and SCA variances, the twenty one parents were clustered. And it is effective to estimate the use value of parents by this cluster method.(5) Based on the original sequence and homologous sequence information, twenty three molecular markers were developed. After estimation, four markers with better choosing effect were gained, which were TDF5-P, TDF6-P, TDF10-P and TDF12-P. The range of specificity of four markers to choose KW was43.48%～73.68%and the range of sensitivity was50.00%～78.95%. And the range of specificity of four markers to choose KWP was5.26%～56.52%and the range of sensitivity was33.33%～100%.更多还原