【作者】 徐珍珍；

【导师】 李付广；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2013， 博士

【摘要】 棉花（Gossypium spp）是世界上重要的纤维作物和油料作物之一，在我国国民经济发展中起着非常重要的作用。长期以来，传统的常规育种方法使棉花很多农艺性状都有了很大的提高，但是长期的人工选择也造成其遗传基础狭窄，种质资源匮乏等，最终导致棉花育种水平徘徊不前。近年来，随着植物转基因技术的研究和应用迅速发展，棉花也不可避免的成为了基因工程改造的目标。目前，农杆菌介导法是棉花转基因技术中应用最广泛的方法，它依赖于植物组织培养高效再生体系。棉花组织培养再生体系同时也存在很多问题：培养周期长，重复率比较低；畸形胚频率较高；愈伤组织再分化为胚性愈伤组织困难，严重受基因型限制。其中愈伤组织再分化胚性愈伤组织受基因型限制是亟待解决的重要问题之一。本研究通过转录组分析，转录组分子标记的开发及QTL定位的方法对棉花组织培养中的基因型限制进行了一系列的研究，并取得了以下一些研究结果：棉花姊妹系体细胞胚胎发生过程的转录组分析：1.为了更好的研究棉花体细胞胚胎发生过程的分子机制，本试验利用RNA-Seq的方法初步研究了W10（CCRI24）姊妹系在体细胞胚胎发生过程转录水平的动态变化。通过de novo拼接214977462Illumina reads最终获得了204349个unigenes。选取不同功能不同表达量的33个基因进行qRT-PCR验证，结果显示，除了个别的几个基因外，RNA-Seq结果与qRT-PCR验证结果呈现显著的正相关（相关系数R2=0.841，P≤0.01），数据表明了RNA-Seq结果的可靠性。2.在两个姊妹系的两个培养阶段，生长素和细胞分裂素在培养基中的比例不同，这就暗示了生长素和细胞分裂素在体细胞胚胎发生过程中可能起着非常重要的作用。通过全面分析植物激素相关基因，我们鉴定了大量与生长素、细胞分裂素合成和信号通路相关基因，这些基因分别注释到tryptophan metabolism，zeatin biosynthesis，AUX1，TIR1，Aux/IAA，ARF，SAUR，GH3gene family，AHK2₃₄，AHP，ARR-B和ARR-A。进一步分析，发现这些基因在姊妹系及不同阶段具有显著的差异。在以后的研究中，过表达或者基因沉默在不同姊妹系及不同阶段显著上调或者下调的某些基因将具有非常重要的意义。尽管生长素和细胞分裂素在体细胞胚胎发生过程中具有非常重要的作用，但是它们之间的相互作用及与其它影响因子的作用机制目前尚不清楚。3.利用本研究转录组数据得到的204349个大小不等的unigenes开发SSR标记，并将设计的引物所在的序列与CMD上已用于SSR开发的序列进行比对去重，共得到1091对新的EST-SSR引物。棉花叶柄愈伤组织分化率的遗传分析：通过研究发现，棉花愈伤组织分化率属于数量性状，受多基因控制。利用QTXNetwork软件的全基因组关联分析方法从F2分离世代中检测与棉花愈伤组织分化率有关的QTLs。共检测到7个加性（A）QTLs，10个显性（D）QTLs，5对愈伤组织分化率的AD上位性QTLs，8对愈伤组织分化率的DA上位性QTLs及7对愈伤组织分化率的DD上位性QTLs。这些QTLs分布在LG2，LG4，LG6，LG7，LG9，LG10，LG16，LG17，LG19，LG21，LG22，LG25，LG26，LG33和LG40连锁群上，所对应的染色体分别为Chr5，Chr6，Chr2，Chr14，Chr9，Chr23，Chr3，Chr1，Chr11，Chr2，Chr4，Chr20，Chr12，LG33和Chr11。愈伤组织分化率的DD上位性QTLs中，qRE-9-14与qRE-22-2之间DD上位性QTL效应的遗传力最高，达到了15.09%，qRE-6-2和qRE-33-1显性（D） QTL可解释的表型变异率次之，分别解释10.79%和10.29%的表型变异率。上述所有类型的QTLs的遗传力总和为92.95%。更多还原

【Abstract】 Cotton （Gossypium spp.） is the preeminent source of natural fiber as well as a significantsource of cooking oil in the world, and it has been playing a very important role in the economicdevelopment of China. Because of significant economic and social values of cotton, major effortsare being to improve the crop. Conventional breeding has made great contributions to improve theagronomic traits. Nevertheless, further improvement is being constrained by the limited geneticdiversity of cotton. In recent years, gene transfer has been used to augment conventional breedingof major economic crops including cotton. Agrobacterium-mediated transformation （via somaticembryogenesis, SE） has been a preferred method for genetic transformation, but it requires areproducible and highly efficient plant regeneration scheme. While many factors, including lowefficiency of tissue culture with excessive time and labor, high frequency of abnormal embryosand the genotype limitation of plant regeneration can affect somatic embryogenesis, and the keyfactor is the genetic background. Global analysis of transcriptome dynamics during cotton SE,primer design based on transcriptome data and QTLs location were performed to studygenotype-dependent response during cotton SE.Transcriptome dynamics during SE in different sister lines analysis:1. To get a broader view on the molecular mechanisms underlying SE in cotton, globalanalysis of cotton transcriptome dynamics during SE in different sister lines was performed usingRNA-Seq. A total of204349unigenes were detected by de novo assembly of the214977462Illumina reads. Quantitative real-time PCR （qRT-PCR） was applied to33genes with differentexpression levels and functional assignments to confirm the validity of RNA-Seq analysis. TheqRT-PCR measurements were positively correlated with the RNA-Seq results for almost all thetested genes with the exception of2genes （R2=0.841, correlation was significant at the0.01level）.2. Different phytohormone （auxin and cytokinin） concentration ratios in medium and theendogenous content changes of these two phytohormones at2stages in different sister linessuggested the roles of auxin and cytokinin during cotton SE. On the basis of global generegulation of phytohormone-related genes, numerous genes from all the differentially expressedtranscripts were involved in auxin and cytokinin biosynthesis and signal transduction pathwayswhich belonged to tryptophan metabolism, zeatin biosynthesis, AUX1, TIR1, Aux/IAA, ARF,SAUR, GH3gene family, AHK2₃₄, AHP, ARR-B and ARR-A. Analyses of differentiallyexpressed genes involved in these nodes of pathway revealed the substantial changes in gene typeand abundance between two sister lines. Isolation, cloning and silencing/over-expressing the geneswhich revealed remarkable up-or down-expression during cotton SE n were important. Furthermore, auxin and cytokinin play a primary role in SE, but potential cross-talk with eachother or other factors remains unclear.3. All the204349unigenes were used to design SSR primers.We got1091pairs of newEST-SSR primer after blasting CMD.Inheritance analysis of SE callus redifferentiation rate in cotton tissue culture:After a series of experiments in our study, we deduced that many genes may be involved inSE, not only one gene.37QTLs associated with regeneration frequency of calli were detectedwith software QTXNetwork by Genome-Wide Association Studies （GWAS） method in F2population, including7estimated dominant （D） of QTLs,10estimated additive （A） of QTLs,5estimated epistasis （AD） of QTLs,8estimated epistasis （DD） of QTLs and7estimated epistasis（DD） of QTLs for regeneration frequency of calli. The QTLs were anchored in LG2, LG4, LG6,LG7, LG9, LG10, LG16, LG17, LG19, LG21, LG22, LG25, LG26, LG33and LG40, and theirlocated chromosomes were Chr5，Chr6，Chr2，Chr14，Chr9，Chr23，Chr3，Chr1，Chr11，Chr2，Chr4，Chr20，Chr12，LG33and Chr11. The phenotype variation of estimated epistasis （DD）beteen qRE-9-14with qRE-22-2was the highest among all the detected QTLs, and explained15.09%phenotype variation in F2set. The qRE-6-2explained10.79%phenotype variation andqRE-33-1can explain10.29%phenotype variation. The sum of all the QTLs heritability were92.95%.更多还原