【作者】 陈浩东；

【导师】 王坤波；

【作者基本信息】 中国农业科学院 ， 作物种质资源学， 2013， 博士

【摘要】 棉花是重要的经济作物，在我国国民经济中具有重要的意义。但由于占世界棉花产量90%的陆地棉栽培种的遗传基础过于狭窄，导致目前棉花无论在产量、品质还是抗逆育种上都徘徊不前。因此必须采取多种途径，尤其是从棉属野生种中发掘优良种质资源基因来丰富我国棉花种质资源的遗传多样性是非常必要的。达尔文氏棉属于四倍体野生棉种，它具有纤维细度好、耐干旱及抗枯黄萎病等优点。但是目前针对该棉种的诸多优良特性展开的基础和应用研究还未见任何报道。本文开展了达尔文氏棉旱胁迫转录组测序，EST-SSR功能标记开发及陆达种间高密度遗传图谱构建的研究。研究结果如下：1.达尔文氏棉幼苗干旱胁迫条件下Solexa转录组测序应用新一代高通量测序技术对达尔文氏棉苗期干旱胁迫后及对照的叶片进行转录组测序(RNA-Seq)。测序后共获得平均长度为95.79bp的有效读长（Valid reads）8.67千万条，经de novo拼接去重复获得58960条序列长度≥500bp的转录本（Transcript），与蛋白质数据库比对后进行了COG、KEGG和GO功能分类。根据基因表达丰度值RPKM进行差异表达分析，检测出干旱胁迫后差异表达基因19165条（32.5%），其中上调基因7392条，下调基因11773条。通过高丰度表达基因分析，找到一些可能参与或介导棉属耐旱胁迫相关生物途径的主要基因。达尔文氏棉旱胁迫转录组测序的成功实施，一方面可以获得大量该棉种的EST序列，进一步充实NCBI数据库中棉属EST的序列信息，另一方面有助于达尔文氏棉耐旱基因的挖掘、规模化旱胁迫响应表达的分析及EST-SSR功能标记的开发。2.达尔文氏棉EST-SSR标记开发与利用基于达尔文氏棉≥300bp的63762条EST序列经去冗余后得到51049条非冗余序列，查找到SSR位点1388个，从中首次开发780对达尔文氏棉EST-SSR引物，详细分析了EST-SSR位点的特征、引物的多态性及转移性。利用新引物对A111的种性鉴定结果显示，A111是一个独立的新种，和G基因组关系最近。利用在中棉所12-4和达尔文氏棉5-7之间表现多态性的引物135对，对陆达F2群体扩增后获得了141个基因分型位点，其中139个位点被整合到遗传图谱上，使图谱中EST-SSR位点数由1180个增加至1319个，增加了11.8%，实现了对陆达遗传图谱的进一步饱和。3．陆地棉×达尔文氏棉种间高密度遗传图谱构建以棉花陆达种间（中棉所12-4×达尔文氏棉5-7）的188个F2单株为作图群体，构建了完全基于SSR标记的高密度遗传图谱。该图谱包含26个连锁群（染色体），2763个标记位点，位点间平均距离为1.51cM，共覆盖4176.7cM的遗传距离。染色体上的标记数在49-169之间，平均每条染色体包含106.3个标记；染色体的长度在84.7-238.5cM，平均长度160.6cM。At染色体组全长2160.7cM，平均距离1.6cM; Dt染色体组全长2016cM，平均距离1.4cM。连锁图谱上偏分离标记601个，位于At染色体组偏分离标记位点数明显少于Dt染色体组上的偏分离标记位点数。据我们所知该图谱是目前棉花上最密的三张图谱之一，且是第一张完全基于SSR标记的陆地棉和野生棉种间高密度遗传图谱，不仅在达尔文氏棉基因组研究上具有重要的作用，同时在棉花基因组结构和功能、比较基因组学、野生棉抗逆等优良性状的QTL定位、图位克隆以及标记辅助选择等研究上都具有重要意义。更多还原

【Abstract】 Cotton is an important economic crop and has great significance in our national economy.Cultivated allotetraploid species Gossypium hirsutum account for90%of the world cotton production.But because of the genetic basis of the cultivated species is too narrow, resulting in cotton in terms ofyield, quality or stress resistance breeding stagnant. So it is very necessary to adopt a variety ofapproaches, in particular explore good genes from wild species of cotton to enrich the genetic diversityof germplasm resources of cotton in China. G. darwinii belongs to tetraploid wild cotton, which hasmany good characteristics like good fibre fineness, drought resistance and verticillium wilt resistance.But there is not any basic and applied research reported which focus on such excellent characteristicsof this cotton species. This paper conducted the transcriptome sequencing under drought stress,EST-SSR function markers development and high density interspecific genetic map construction of G.darwinii. The results are as follows:1. Solexa sequencing the transcriptome of G. darwinii seedlings under drought stressIn this study, we employed RNA-seq to analyse the transcriptomes from leaves of G. darwiniiseedlings with and without drought stress. A total of86.7million valid reads with average lengths of95.79bp were generated from the two samples and58960transcripts with length more than500bpwere assembled. Based on sequence similarity search with known proteins these transcripts wereannotated with COG, KEGG and GO functional categories. According to gene expression abundanceRPKM value we conducted differential expression analysis, obtained19,165differentially expressedgenes (32.5%) between two samples, including7,392up regulated genes and11,773downregulated genes. Through the analysis of the high abundance of gene expression, we found somemajor gene may be involved in mediating Cotton Drought stress resistence biological pathway.Successful implementation of G. darwinii drought stress transcriptome sequencing, on the one handwe can obtain a large amount of the cotton seed EST sequence to further enrich the EST sequenceinformation in NCBI database, on the other hand the data will facilitate genes discovery, large scaledrought stress response genes expression analysis and EST-SSR function markers development.2. Development and applicaton of G. darwinii EST-SSR markersA total of51,049non-redundant ESTs were obtained after removed redundant from the lengthmore than300bp EST sets of G. darwinii. From these non-redundant ESTs we detected1,388SSRloci, and first time developed780EST-SSR primers from G. darwinii species. Then the characteristicsof EST-SSR loci, polymorphism and transferability among different species of EST-SSR primers wereanalyzed in detail. UPGMA Cluster analysis using new primers revealed that A111is an independentnew species and has a close genetic relationship with G genome. Using135pairs of polymorphismprimers between G. hirsutum CRI12-4and G. darwinii to screen F2population including188individuals,141genotyping loci data were obtained, of which139loci were integrated into the genetic map. The map of EST-SSR Numbers increase from1,180to1,319, increased by11.8%, andimplements the further saturation of our interspecific genetic map.3. Construction a high density genetic linkage map in interspecific population of G. hirsutum×G. darwiniiIn this study, an interspecific high density linkage map of allotetraploid cotton was constuctedin F2population (G. hirsutum×G. darwinii). This map was based entirely on genome-wide simplesequence repeat (SSR) markers. A total of2,763markers were maped in26linkage groups(chromosomes) covering a genetic length of4,176.7cM, with the average interval distance betweenloci was1.5cM. The number of markers varied from49to169per chromosome, with the average106.3markers in each chromosome. The length of the chromosomes ranged from84.7to238.5cM,with average160.6cM each chromosome. At genome length was2,160.7cM, with the averagedistance of1.6cM; Dt genome length was2,016cM, with the average distance of1.4cM. Therewere601segregation distortion SSR loci in this map and the numbers of segregation distortion locilocated in At chromosomes was obviously less than the numbers in Dt chromosomes. To ourknowledge, this map is one of the three most dense linkage maps in cotton, and also is the firstinterspecific high density genetic map between upland cotton and wild cotton completely based onSSR markers. This map not only plays an important role on G. darwinii genome research, but alsolays groundwork for further genetic analysis of important stress resistance trait, map-based cloning,marker-assisted selection in wild cotton, and genome organization architecture in cotton as well asfor comparative genomics between cotton and other species.更多还原