【作者】 朱新霞；

【导师】 张天真；

【作者基本信息】 南京农业大学 ， 遗传学， 2009， 博士

【摘要】 棉花是世界上主要的经济作物,在80多个国家种植,不仅是世界上主要的天然纤维来源,也是仅次于大豆的重要油料和蛋白作物。棉花作为我国的一个重要经济作物,近十余年来,杂交棉,特别是抗虫杂交棉,在长江流域和黄河流域已大面积推广,杂交棉的推广及应用在提高棉花产量方面起着决定作用。中棉所12是我国首次攻克抗病、高产、优质三者不易结合而培育成的优质、抗病、高产品种,具有较强的遗传力、配合力和遗传稳定性。作为棉花育种的种质材料,显示出骨干亲本巨大的应用价值。目前关于中棉所12的大量研究主要集中在遗传育种学和生理生化方面,而从分子水平探讨骨干亲本的研究还没见报道。随着分子生物学研究手段的日益提高和完善,有必要对骨干品种(亲本)中棉所12从分子水平进行深入剖析,了解复杂性状的遗传基础,揭示优良基因型的遗传本质,为骨干亲本在杂种优势中所起重要作用提供理论依据,在理论和生产实践上都具有十分重大的意义。本研究以中棉所12及其2个选系为亲本组配的4个杂交棉中棉所28、中棉所29、湘杂棉2号、冀棉18不同生长部位(根和叶),不同生育期(苗期、蕾期、花期)为研究材料,采用cDNA-AFLP技术分析杂交棉与亲本基因差异表达,从分子水平探讨骨干亲本在杂种优势中所起重要作用；同时采用MSAP方法从基因表达调控角度探讨杂交棉与亲本的胞嘧啶甲基化水平和遗传传递模式。结果如下：1、杂种优势表现的时空性通过对中棉所12及其选系配制的四个杂交棉苗期不同组织部位营养生长杂种优势的表现特性分析发现地上部分叶和地下部分根存在不同的杂种优势,说明杂种优势表现存在组织差异性。通过对中棉所12及其选系配制的四个杂交棉的不同生育时期营养生长参数干物质重量的杂种优势分析发现,在苗期,冀棉18、湘杂棉2号、中棉所29三个杂交棉表现出正向超中亲优势,中棉所28杂交棉表现为超亲优势；在蕾期,杂交棉冀棉18和中棉所28表现出正向超中亲优势,中棉所29和湘杂棉2号表现为超亲优势；在花期,杂交棉湘杂棉2号表现出正向中亲优势,冀棉18、中棉所28、中棉所29三个杂交棉表现为超亲优势。表明杂交棉在不同生育时期存在营养生长杂种优势差异；在产量上,冀棉18、中棉所28和中棉所29三个杂交棉表现出超亲优势,杂交棉湘杂棉2号表现为中亲优势,说明中棉所12选系配制的四个杂交棉在产量上可以表现出不同的杂种优势；四个杂交棉在纤维品质性状上都存在一定的优势现象,但是优势率比较低,其中纤维品质的主要指标比强度只有长江流域棉区品种湘杂棉2号有超亲优势,其余三个组合均无。说明四个杂交棉在纤维品质上的优势表现不是太明显。总之,杂种优势可以表现在棉花生长和发育的不同阶段、不同部位和不同性状上,尤其主要表现在营养生长和产量性状上,且不同杂交组合因其选配亲本和环境的影响,杂种优势表现不尽相同。2、基因差异表达和杂种优势采用cDNA-AFLP技术研究中棉所12及其选系组配的4个杂交棉中棉所28、中棉所29、湘杂棉2号、冀棉18苗期基因差异表达。基因差异表达分为4种类型：Ⅰ.杂交种上调表达型；Ⅱ.单亲显性表达型；Ⅲ.单亲下调表达型；Ⅳ.杂交种下调表达型。在根和叶的基因差异表达丰富程度上,叶部差异表达基因数目和比例(29.20%-46.09%)要比根部(15.65%-22.49%)高的多,说明根和叶的基因表达上存在相异性,叶中基因差异表达可能比根中基因差异表达对杂种优势形成作用更大。采用cDNA-AFLP技术研究中棉所12选系及其组配的4个杂交棉中棉所28、中棉所29、湘杂棉2号、冀棉18三个主要生育期—苗期、蕾期和花期的基因表达动态。从三个时期各差异表达类型的累计次数分布看,单亲显性表达型比例最高,约为37.26%(其中母本中棉所12显性表达型占19.37%,父本显性表达型占17.89%),其次为单亲下调表达型,约为34.18%(其中母本中棉所12下调表达型约占12.43%,父本下调表达型约占21.75%),杂交种F1上调表达型位居第三(15.03%),杂交种F1下调表达型比例最低(13.53%),后二者相差不大。在生长发育的不同时期,杂交种相对于亲本的各种基因差异类型所占比例有所不同,表明基因的差异表达具有时序表达的特征。分析基因表达差异与营养生长和产量性状优势之间的关系表明：在苗期杂交种上调表达有利于营养生长杂种优势的发挥；在蕾期杂交种上调表达和母本下调表达有助于营养生长杂种优势的发挥；在花期杂交种上调表达有利于产量杂种优势发挥,母本下调表达不利于杂交种高产。多种基因差异表达模式的并存说明杂种优势可能是多基因共同作用产生多种效应的结果,杂交种上调表达型与营养生长和产量杂种优势呈显著正相关,推测超显性效应在杂种优势产生中可能起主导作用；母本下调表达不利于产量杂种优势发挥和杂交种高产,暗示了亲本基因尤其是母本基因在杂交种中的抑制不利于杂交种表现杂种优势。只有有了好的亲本,才能育成好的杂交棉。中棉所12选系在苗期和蕾期都是冀棉18、中棉所29、中棉所28的高值亲本,干物质积累的营养生长优势,为杂交棉的高产奠定了物质基础。花期基因表达差异与产量性状优势之间的关系表明,母本中棉所12下调表达不利于杂交棉高产,从分子水平说明了中棉所12选系在杂交棉冀棉18、中棉所29和中棉所28的杂种优势产生中起重要作用。3、棉花DNA胞嘧啶甲基化水平和遗传模式研究采用MSAP技术研究了中棉所12配制的两个杂交棉(湘杂棉2号、中棉所28)苗期、蕾期、花铃期的基因组DNA 5’-CCGG位点胞嘧啶的甲基化水平和甲基化遗传传递模式。研究发现在苗期杂交棉湘杂棉2号组合和中棉所28号组合胞嘧啶甲基化水平为12.41%-18.34%；在蕾期,杂交棉湘杂棉2号组合和中棉所28号组合胞嘧啶甲基化水平为18.35%-20.05%；在花铃期,杂交棉湘杂棉2号组合和中棉所28号组合胞嘧啶甲基化水平为15.94%-17.07%。说明杂交棉不同发育时期的胞嘧啶甲基化水平不同,随着生育期的逐步推进,杂交棉与亲本甲基化水平出现两头低而中间高现象。DNA甲基化具有生育期特异性,这从一定程度可解释不同时期基因的差异表达。比较几种不同的甲基化水平可以发现,内侧胞嘧啶的全甲基化的水平(平均值为9.37%)高于外侧胞嘧啶的半甲基化水平(平均值为6.05%)和外侧或内外侧胞嘧啶全(双链)甲基化水平(平均值为1.85%),说明棉花基因组的DNA甲基化模式主要以内侧胞嘧啶的全甲基化为主。在各种胞嘧啶甲基化模式中,杂交棉湘杂棉2号组合和中棉所28组合花铃期的外侧或内外侧胞嘧啶全甲基化水平明显少于苗期和蕾期,说明在花铃期发生了显著的去甲基化,花铃期在DNA的总体甲基化水平比蕾期降低2.77%。棉花中绝大多数(96.61%-98.86%) CCGG胞嘧啶甲基化位点是由亲本稳定遗传给杂交种的,杂交棉只有1.14%-3.39%的位点显示了变异,其变异频率在不同亲本组合之间和不同发育时期都存在差异。通过对甲基化差异条带测序分析发现,其功能涉及到富含亮氨酸重复、类PDR的ABC转运蛋白、GTP结合蛋白、病程相关蛋白、磷酸激酶、功能未知的蛋白质和反转录酶等,部分差异序列没有产生有意义的匹配。比较杂交种与其亲本之间的胞嘧啶甲基化水平发现,三个亲本平均甲基化水平为17.29%,两个杂交棉平均甲基化水平为17.08%,即杂交棉平均甲基化水平略低于亲本平均甲基化水平。结合杂交棉在苗期、蕾期、花期营养生长优势和产量优势表现,发现杂交棉甲基化水平接近于中亲值(双亲平均值)有利于超亲优势发挥。总体上棉花甲基化程度与水稻一样与杂种优势相关性不高。更多还原

【Abstract】 As a major source of fibers, important oil and albumen plants, cotton is considered to be a high value crop and plays an important role in the global economy. During the past decades, the hybrid cotton, especially pest-resistant hybrid cotton was greatly planted by the Yellow River Valley and the Yangtze River Valley, The extension and application of cotton hybrids have played a decisive role in cotton productionCRI-12, a Chinese elite Upland cotton variety with high yield, elite fiber quality and disease resistance, is characterized by its high heritability, combining ability and genetic stability. As the germ plasm of cotton breeding, it displays enormous appliance values of Foundation parental. At present, the study of hybrid cotton and CRI-12 is mostly focus on breeding, physiology and biochemistry. It is urgent to go deeply into the genetic mechanism study of CRI-12 as Foundation parent from the molecule level, with molecular biological methods increasingly improvement, it will offer theoretical evidence to study the important role of CRI-12.In this research, CRI-12 and its pedigree-derived lines were used to develop high heterosis cotton hybrids such as CRI-28, CRI-29, XZM2 and Jimian18, Leaves and root at seedling stage, leaves at budding stages and flowering stages were picked from these hybrids and their corresponding parents sampled for cDNA-AFLP and MSAP analysis, the gene differential expression patterns and DNA methylation variation level and pattern between hybrids cotton and their parents. The results were listed as the following:1、Temporal Spatial expression of heterosisThe roots and leaves at seedling stage of four hybrids and their corresponding parents were sampled for vegetative growth heterosis analysis. It was found various heterosis at roots and leaves, It suggested vegetative growth heterosis has tissue specification.Heterosis analysis was undergone through the dry weight measure of four hybrid and their corresponding parents at three-leaf stage, budding and flowering stages, It was found Jimian 18、CRI-29 and XZM 2 exhibited the positive over-mid-parent heterosis, and CRI-28 exhibited over-parent heterosis at three-leaf stage; CRI-29 and XZM 2 exhibited the positive over-mid-parent heterosis, Jimian 18 and CRI-28 exhibited over-parent heterosis at budding stage; XZM 2 exhibited the positive over-mid-parent heterosis, Jimian 18、CRI-28 and CRI-29 exhibited over-parent heterosis at flower stage. It suggested vegetative growth heterosis has timeliness.Yield of four hybrid and their corresponding parents were detected for yield heterosis analysis. It was found Jimian 18, CRI-28 and CRI-29 exhibited over-parent heterosis, XZM 2 exhibited the positive over-mid-parent heterosis. It suggested hybrid cotton by CRI-12 lines could exhibit various heterosis.Fiber quality traits have small heterosis of four hybrid cotton, but it is very lower relatively, only XZM 2 exhibited over-parent heterosis in fibre strength. It suggested hybrid cotton heterosis aren’t distinctness in fiber quality traits.Integrated above results we presumed that heterosis had different temporal and spatial exhibition in various organ and various growth periods with various characterization, It mainly exhibited in vegetative growth and yield aspect.2、Gene differential expression and heterosisThe roots and leaves at seedling stage of four hybrids and their corresponding parents were sampled for cDNA-AFLP analysis. The gene differential expression patterns were detected between the hybrid and its parents and were described as follows:I. Up expression in hybrid showed the strong bands expressed only in hybrid but not in both parents; II. Dominant expression in single parent showed the strong bands expressed in one of the parents and hybrid but not in another parent, including the expression pattern in female parent and hybrid without in male parent and the expression pattern in male parent and hybrid without in female parent; III. Down expression in single parent showed the strong bands in one of the parents, including the expression pattern in male parent without in hybrid and female parent and the expression pattern in female parent without in hybrid and male parent; IV. Down expression in hybrids showed the strong bands in both parents but not in F1. Differential expression bands in leaves and roots accounted for 29.20%-46.09% and 15.65%-22.49% in total numbers of detected bands, respectively. The differential expression bands in leaves were more than those in roots, that explained some useful genes associated with heterosis maybe begin to express in seedling leaves.The gene differential expression patterns were detected between four hybrid and their parents at three-leaf stage, budding and flowering stages, The result as follows:The percentages of domain expression in single-parent were highest than other patterns, down expression genes in single-parent were higher than up expression in hybrids, down expression in hybrids were lowest of four patterns from three growth stage accumulative total number. The gene differential expression ratio of hybrid and parents was various in three growing stage, It suggested gene differential expression had timeliness.Further analysis of the gene differential expression ratio and vegetative growth heterosis and yield correlation revealed that the type of bands expressed only in F1 contributed to heterosis of vegetative growth at seedling and budding stage and it played an important role in yield heterosis occurrence at flowering stage; the type of bands expressed only in Female parent contributed to heterosis of vegetative growth at budding stage, but it possible decreased hybrid yield at flowering stage.Differentially expressed genes patterns in the four hybrids were detected, It revealed that many possible modes of gene action were involved in hybrids, with additivity、dominance、over-dominance、under-dominance and low-parent dominance. All possible modes of gene action coexisted supported the hypothesis of multiple molecular mechanisms contributed to heterosis. The up-regulated expression genes in hybrid were showed positive correlation with heterosis of vegetative growth and yield heterosis, over-dominance may play a vital role. The down-regulated expression pattern in female parent was harmful for yield heterosis and high yield in hybrids. It suggested that the inhibition of parental genes, especially female parental genes, was not beneficial to the occurrence of heterosis and formation of hybrid yield.Good hybrid cotton was breeding from good parents. CRI-12 was the high value parent of Jimian 18, CRI-29 and CRI-28 at the seedling and budding stage, high-yield of hybrid cotton based on dry weight accumulated heterosis of vegetative growth. Correlation analysis of differentially expression gene and yield heterosis revealed the type of bands expressed only in Female parent possible decreased yield of hybrid at flowering stage, which also provided confident evidence for CIR-12 played a predominant role in the expression of genes responsible for heterosis in CRI-28、CRI-29 and Jimian18 at molecular level.3、Variation of DNA methylation level and pattern in Hybrid CottonMS AP (methylation-sensitive Amplified fragment length polymorphism) was used in this study to detect the DNA methylation patterns in the 5’-CCGG sites of two cotton hybrid by CRI-12 and their parents.This study was used to understand the developmental stability and inheritance of cytosine methylation. It was found MSAP ratios in the two cotton hybrids were 12.41%-18.34% at seedling stage,18.35%-20.05% at budding, and 15.94%-17.07% at flowering stage, respectively. cytosine methylation profiles were variable during plant growth and development, DNA methylation experienced from increases to decrease progress throughout cotton development, which can partly explain differential gene expression in different growing stages.Compared the level of various DNA methylation,the Full methylation of internal cytosine (6.90%-11.47%) was highest than hemi-methylation of external cytosine and Full methylation of the internal Cs or external Cs. It suggested full methylation of internal cytosine was the dominant in two cotton hybrids. Full methylation of the internal Cs or external Cs at flowering stage distinct less than seedling stage and budding, that showed have demethylation occured at flowering stage, DNA methylation level of flowering stage decreased 2.77% than it at budding stage.Meanwhile, The MSAP profiles enable monitoring of inheritance or variation of parental methylation patterns in hybrid progenies. It was found that a great majority (from 96.61% to 98.86%, depending on crosses) of the methylation profiles in cotton inbred lines transmitted to the inter-strain hybrids; however, the otrer sites from 1.14% to 3.39% of the profiles in the hybrids exhibited variation from the expected parental additivity. Both inherited and altered methylation profiles can be divided into distinct groups, and their frequencies were variable among the cross-combinations, and during plant growth and development. In addition, sequencing of differentially methylated fragments and subsequent homology analysis of isolated bands that showed variation in hybrids indicated that diverse sequences were involved, including known-function cellular genes and mobile elements, Such as leucine-rich repeat family protein, PDR-like ABC-transporter, putative oligopeptide transporter, GTP-binding protein, Similar to pathogenesis-related protein, DOMON domain-containing protein, putative adenosine phosphosulfate kinase, putative protein, RNA-directe DNA polymerase et al. The remaining 14 bands showed no homology to the database sequences.Compared DNA methylation level of hybrid and parent, the methylation average level of hybrid (17.08%) was lower than methylation average level of parents (17.29%) Combined heterosis of vegetative growth and yield of hybrid at seedling stage, budding and flowering stage, methylation level near mid-parent value may be contributed to over-parent heterosis occurrence. In general, the correlation between methylation level and heterosis of cotton was lowness, like rice.更多还原