【作者】 朱华玉；

【导师】 郭旺珍；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2010， 博士

【摘要】 棉花是世界性重要的经济作物。在植物分类学上,所有野生和栽培的棉种都属于被子植物的锦葵目(Malvales)、锦葵科(Malvaceae)、棉族Gossypieae)、棉属(Gossypium)。棉属植物的分类研究已有近百年的历史。最有权威的棉属分类结果由美国植物学家Fryxell在1992年提出,主要包括4个亚属,8个组,9个亚组,50个种,其中45个二倍体种和5个异源四倍体种。异源四倍体种是由二倍体A染色体组棉种和D染色体组棉种种间杂交而成的,其中海岛棉和陆地棉已被驯化为栽培种。陆地棉的产量高,适应性广,纤维品质中等,因其高产及对环境和生产的普遍适应性,目前世界上97%的棉纤维都产自陆地棉；而海岛棉的特点是纤维品质优良,说明与优质纤维品质相关的基因在海岛棉中高效表达,但海岛棉产量低、适应性差,因而限制了其种植范围。陆地棉、海岛棉两者杂交的F1部分可育,但生育期长,且两者杂交的后代表现典型的种间杂种向两极疯狂分离。目前普遍认为异源四倍体棉种的A染色体组供体种是草棉(G. herbaceum)。而对于异源四倍体棉种的D染色体组供体种目前并没有统一的认识。尽管海岛棉和陆地棉是两大四倍体栽培棉种,但这两个栽培种的农艺性状,特别是纤维品质差异很大。因此,在本研究中,基于EST-SSR标记和已克隆的与棉纤维发育相关基因信息进行四倍体棉种D染色体亚组供体种来源,陆地棉和海岛棉棉纤维发育相关基因的结构,表达特征等方面系统分析,为进一步阐明海陆种间分化的机理奠定基础。研究结果如下：Guo等(2007a)在利用EST-SSR引物加密本实验室的遗传图谱时,发现来源于雷蒙德氏棉的EST-SSR引物在陆地棉和海岛棉之间的多态性远高于来源于亚洲棉和陆地棉的EST-SSR引物所产生的多态性。本研究基于上述研究结果,选择14对来源于雷蒙德氏棉的EST-SSR引物和23个供试棉种(2个二倍体A染色体组棉种、13个二倍体D染色体组野生种和8个异源四倍体材料)来研究棉属二倍体D染色体组棉种之间的亲缘关系,以及异源四倍体棉种D染色体亚组的供体种。来源于雷蒙德氏棉的14对EST-SSR引物在23个棉种中共获得了438个扩增条带,每个条带都被克隆、测序并分别对测序结果的SSR重复区和侧翼保守序列进行分析,然后将所有引物的侧翼序列进行重组,重组后的序列用于碱基替换率分析和构建进化树。对染色体亚组与其祖先种之间的SSR区及其侧翼序列进行比较,结果都是D染色体组和D染色体亚组的变异程度高于A染色体组和A染色体亚组。说明在进化过程中,A染色体组和A染色体亚组之间比D染色体组和D染色体亚组更保守。进一步我们分别对13个二倍体D染色体组棉种、8个异源四倍体棉种及所有23个供试棉种的侧翼重组序列进行了进化树的构建,结果显示,13个D染色体组棉种之间的关系与Fryxell (1992)的亚组分类相同。而D染色体组棉种与异源四倍体棉种D染色体亚组之间的关系表明,雷蒙德氏棉与所有异源四倍体棉种D染色体亚组的亲缘关系最近。为了进一步了解棉纤维发育的分子机制,从而揭示海陆栽培种间纤维品质差异的本质,本研究选取NCBI上已登录的18个与棉纤维发育相关基因和本实验克隆的一个新的蔗糖合酶基因(SusA1),以阿非利加棉、雷蒙德氏棉、陆地棉遗传标准系TM-1和海岛棉7124的基因组DNA为模板,获得基因的全长序列,对序列及结构进行分析,并根据各部分同源基因在海岛棉和陆地棉之间的PCR扩增多态性或SNP位点,利用本实验[(TM-l×Hai7124)×TM-1] BC1群体,对19对部分同源基因进行了染色体定位研究,根据前人研究中已定位的QTLs与本研究中的基因的染色体定位结果,进行这些基因表达特点与纤维品质的关联分析。进一步根据TM-1和海7124序列之间的SNP位点设计亚组特异定量PCR引物,研究19个棉纤维发育相关基因在TM-1和海7124纤维发育中的表达谱,以及其部分同源基因的表达谱；推测这些棉纤维发育相关基因在海陆栽培种间分化中的作用,为纤维品质改良的分子育种研究奠定基础。在本研究的19个基因中,68.42%的基因序列及结构在各棉种及染色体组间比较保守,而31.58%的基因序列及结构在各棉种及染色体组间变异比较大。对各基因在4个棉种中获得的序列进行聚类,结果显示在异源四倍体形成后,16个基因在四倍体的A染色体亚组和D染色体亚组之间是独立进化的,而3个基因在两染色体亚组之间发生了不同程度的殖民化。通过比较同一棉种内不同染色体亚组之间及同一染色体亚组在不同棉种之间的进化速率,发现无论在TM-1还是海7124中都是D染色体亚组的进化速率大于A染色体亚组。同时无论是A染色体亚组还是D染色体亚组,TM-1的进化速率都大于海7124。说明在漫长的进化历史中,四倍体的D染色体亚组经历了较快的进化速率,而与TM-1相比,海7124与其祖先种的亲缘关系更近一些。根据基因在TM-1和海7124之间的序列差异,26个重复基因被定位在本实验室的遗传图谱上,并且在其定位区间内,检测发现至少存在一个已报道的纤维品质相关的QTLs。基因的表达谱分析显示,在纤维起始及伸长早期,大部分基因在海7124中的表达量高于TM-1；在纤维伸长期,基因的表达模式显示多样性,有的在陆地棉中的表达优势,有的在海岛棉中表达优势,有的在陆地棉和海岛棉中表达水平相同,但无论哪种表达模式,大部分基因的表达显示8DPA是个例外,表现为在海岛棉中的表达显著高于陆地棉；在纤维发育初生壁到次生壁转换期,大部分基因在陆地棉中的表达高峰早于海岛棉中的表达高峰,暗示海岛棉的纤维伸长期比陆地棉长,可能是导致其纤维品质好于陆地棉的原因之一。进一步从亚组特异表达谱来看,大部分基因在陆地棉和海岛棉之间具有相同的亚组表达模式,A或D亚组优势表达或两亚组同等表达,与异源四倍体形成后亚组功能分化有关。一些基因在纤维发育的同一时期,在陆地棉和海岛棉之间具有不同的亚组偏向性,说明自然选择和人工驯化可能对海岛棉和陆地棉纤维品质的差异形成起了很大作用。通过综合分析各重复基因在海陆栽培种间结构和表达特征的差异、以及与纤维品质QTLs的关系,推测重要农艺性状的选择可能对陆地棉和海岛棉之间纤维发育相关基因的分子进化起重要作用。更多还原

【Abstract】 Cotton from the genus Gossypium is the world’s most important fiber crop plant. For nearly 100 years, systematists have been interested in the classification of the genus Gossypium. A wide variety of data, including morphologic, meiotic, karyotypic, genetic and molecular have been generated to address relationships among members of the genus. Currently, the most widely accepted classification for Gossypium follows Fryxell (1992), who divided Gossypium into four subgenera, eight sections, nine subsections and approximately 50 species. Forty-five of the species are diploid and differentiated cytogenetically into eight genome groups (A-G and K), and five are tetraploid. The five tetraploid species are of allopolyploid origins, originated from interspecific hybridization between diploid A- and D-genome species, two of them, G. hirsutum and G. barbadense, have been independently domesticated for their fiber. G. hirsutum has broad adaptation, moderate fiber quality and high yield. Because of its yield potential and adaptation to diverse environmental conditions and production systems, about 97% of the world’s cotton fiber derives from G. hirsutum (NCCoA,2006). On the other hand, G. barbadense is characterized by superior fiber quality, indicated G. barbadense contains novel alleles for superior fibre quality, but the narrow range of adaptation and low yield limited its planted regions. The two species are sexually compatible, although partial sterility, longer maturity, and hybrid breakdown are often observed in later generation hybrids.Now, the best extant model of the ancestral A-genome parent is G. herbaceum, while the D-subgenome donor in tetraploid cotton species remained elusive. Though G. hirsutum and G. barbadense are cultivated cotton species, they have very different agronomic and fiber quality characters. So, in this study, based on the EST-SSR sequence and the structures and expression partterns of fiber development genes, which had been cloned, we revealed the D-genome donor of tetraploid species, and put a solid foundation to further understand the genetic basis of cotton fiber development. In a previous study, we used EST-SSR to screen for polymorphisms to enhance our backbone geneticmap of allotetraploid cotton. Using G. raimondii derived-eSSR, a high polymorphismrate (47.9%) between G. hirsutum and G. barbadense was observed, which was much higher than that observed by G. arboreum and G. hirsutum derived-eSSR (Guo et al.,2007a). We questioned the role of the D-genome cotton species in the evolution of tetraploid cotton. In this study, we used EST-SSR sequences to reveale the relationship of D-genome in diploid in Gossypium and the D-subgenome donor in tetraploid species.14 EST-SSR primer pairs from G. raimondii were used to amplify 23 species in Gossypium. In total,438 amplicons were cloned, sequenced and analyzed. From the analysis of SSR motifs and the rates of base substitutions in the flanking region based on the combined data set, the D-genome and D-subgenome exhibited higher levels of polymorphism than the A-genome and A-subgenome, both in the microsatellite domains and the flanking regions. Suggest that in the process of evolution, the A-genome and A-subgenome have experienced lower divergence rates and are therefore more conserved than D-genome and D-subgenome. The phylogenetic trees of 13 D-genome diploid species,8 AD-genome tetraploid species and all the 23 species were constructed based on the combined SSR flanking sequence data, respectively. The results showed that 13 D-genome species were congruent with Fryxel’s subsection taxonomy. The relationship between D-subgenome tetraploid species and D-genome diploid species indicated that G. raimondii is the sole D-genome donor of all tetraploid species.To understand the genetic basis of cotton fiber development, further to reveal the essence of fiber quality difference between upland cotton and sea-island cotton, we selected 19 fiber development genes, including 18 accessioned to NCBI and a new sucrose synthase gene (SusA1) cloned by our lab, to study their structure and expression difference in the two cultivated tetraploid cotton. First, we cloned these genes in the genome DNA of G. hirsutum ace. TM-1, G barbadense cv. Hai7124 and their two putative diploid progenitor cottons, G. herbaceum and G. raimondii, to investigate their frame and sequence divergence. Then, the chromosomal locations of each homeolog of several studied genes, having effective SNP or amplification polymorphism loci between TM-1 and Hai7124, were carried out using our [(TM-1×Hai7124)×TM-1] inter-specific BC1 mapping population in allotetraploid cotton. Genes’distribution on chromosomes and their distance from QTLs, which were located by privous studies, were analysis. Further, expression patterns of each gene and each homeolog (duplicate genes) were explored to evaluate the expression difference on fiber development between G. hirsutum and G. barbadense. Synthesizing the analysis of genes frames, expression patterns and chromosomal location results, the interspecific divergence of fiber development genes in cultivated tetraploid cotton species was further elucidated. The results will also put a solid foundation for mining the key genes with important fiber quality contribution, further utilizing them to improve the fiber quality in cotton molecular breeding.In this study, in the orthologous and homoelogous loci of 19 studied genes, the sequence and structure of 68.42% were conservative and 31.58%were diverse. Gene tree topologies showed that 16 genes were independent evolution between A- and D-subgenome in the allopolyploid after polyploid formation, while 3 evolved different degrees of colonization. The evolution rates between A (D)-genome and A (D)-subgenome revealed that D-subgenome of allotetraploid had rapid differentiation in the evolution history, and compared with TM-1, Hai7124 may have a closer relationship with their primogenitor. Based on the sequence divergence between TM-1 and Hai7124,26 duplicate genes were located on our backbone genetic map, in which at least one fiber quality QTL reported previously was detected in the interval. The genes expression profiles showed that at fiber initiation and early elongation period, most genes had higher transcripts in Hai7124 than in TM-1, however, at fiber elongation period, most genes transcripts, except for CelA3 and HOX3, were the same or higher in TM-1 than in Hai7124 with an exception at 8DPA, and at primary-secondary transition period, expression peak of transcripts in most genes was earlier in TM-1 than in Hai7124. The genome-specific expression profiles showed that the same A- or D-biased or equal expression profile in the two cultivated cotton species were related with functional partitioning of genomic contributions during cellular development after the allopolyploid formation, however, significant alternation of homoelog A/D ratio at the same fiber developmental time points between G. hirsutum and G. barbadense indicated that domestication for fiber qualities may play an important role in fiber quality divergence of G. hirsutum and G. barbadense. The combined analysis of the structure and expression patterns of these studied genes further indicated that agronomic selection play important role in altering the molecular evolutionary patterns of genes related with fiber development between G. hirsutum and G. barbadense.更多还原