【作者】 赵勇；

【导师】 王海滨；

【作者基本信息】 南京农业大学 ， 观赏园艺学， 2023， 硕士

【摘要】 菊属(Chrysanthemum)属于菊科(Asteraceae)春黄菊族(Anthemideae),有40余个种,包括菊花(C.morifolium),甘菊(C.lavandulifolium),菊花脑(C.nankingense),委陵菊(C.potentilloides)以及各种野菊(C.indicum)等。菊属植物在我国分布广泛,其染色体基数为9,属种间和种内都存在丰富的染色体数目变化,但是菊属植物不同种的染色体核型并不清晰,染色体结构变异及种间进化关系等情况仍然有待进一步明确,而利用传统细胞学手段难以进行深度的研究与分析,为此本研究构建了甘菊的BAC文库,筛选单、低拷贝序列,进而开发菊属植物染色体特异的细胞学标记,为菊属植物的核型研究及进化分析提供参考,主要研究成果如下:1.利用栽培菊花近缘种甘菊构建了Hind III和Bam H I两种酶切位点的BAC文库。其中Hind III酶切文库共挑取单克隆151,680个,保存于395块384孔培养板;Bam H I酶切文库共挑取单克隆149,760个,保存于390块384孔培养板。文库克隆大小平均108 kb,约覆盖12倍甘菊完整基因组。2.利用菊花脑45S rDNA序列设计引物进行PCR扩增,结合随机选择法筛选甘菊Hind III酶切BAC文库,将获得的阳性克隆进行质粒提取和标记,并进行BAC-FISH分析。最终筛选到8个在甘菊中产生特异信号的BAC克隆,包括6个BACs在甘菊染色体末端产生信号(其中1个BAC克隆在染色体臂上也存在信号位点)和2个BACs在甘菊全部染色体着丝粒区域产生明显信号。3.利用上述分别在着丝粒区域和端部区域产生特异信号的BAC-H338P8和BAC-H340P10为探针对6种菊属植物根尖有丝分裂中期染色体进行了BAC-FISH及核型分析,结果发现甘菊、‘钟山紫桂’单倍体和六倍体‘神马’与菊花脑有着较近的亲缘关系;利用BAC探针套构建了甘菊更高分辨率的核型模式图。4.对2个特异BAC克隆进行全长测序及序列分析发现,着丝粒区域BAC-H338P8中含有一些重要基因,如乙烯信号转导途径的核转录因子EIN3,与拟南芥着丝粒结构和功能相关的AT5G13560.1和AT2G24550.1的同源基因;端部区域BAC-H340P10中含有一个菊花微卫星序列JH-2074。重复序列分析发现2个BACs在简单串联重复序列中整体上都存在重复单元越长拷贝数越低的趋势,但在BAC-H340P10中AACT/AGTT类型的微卫星基序明显富集,达到75.6%。在BAC-H338P8正反链上共注释到8个LTR,只有Cl-H-LTR4拥有完整结构,且其他LTR之间存在重叠区域。本研究构建的两种甘菊BAC文库为菊属植物的深入研究提供了宝贵材料,利用筛选的特异BAC克隆对部分菊属植物的染色体核型进行BAC-FISH分析,为菊属植物核型研究及进化分析提供了参考和帮助,此外通过对2个特异BAC克隆的全长测序与分析,为菊属植物染色体特殊区域的序列组成等研究提供了参考。更多还原

【Abstract】 Chrysanthemum belongs to Anthemideae in Asteraceae family,and comprises over 40species,including C.morifolium,C.lavandulifolium,C.nankingense,C.potentioides,and various C.indicum.They are widely distributed throughout China,with a chromosome base of 9.Chromosome number varies greatly both within and between species,however,the karyotype of different species of Chrysanthemum is not clear,and the chromosome structure variation and interspecific evolutionary relationships still need to be further clarified.Traditional cytological methods have limitations in studying these complex variations.Therefore,this study constructed a BAC library of C.lavandulifolium,screened for single and low copy sequences,and developed chromosome-specific cytological markers of Chrysanthemum,providing a valuable resource for future karyotyping research and evolutionary analysis.The main research findings are summarized as follows:1.Two BAC libraries were constructed using C.lavandulifolium,a species closely related to the cultivated chrysanthemum,with Hind III and Bam H I restriction sites.A total of 151,680 clones were picked from the Hind III library and stored in 395 384-well plates,while 149,760 clones were picked from the Bam H I library and stored in 390 384-well plates.The average size of the two libraries clones was 108 kb,providing coverage of approximately twelve times the complete genome of C.lavandulifolium.2.Using primers designed based on the 45S rDNA sequence of C.nankingense,PCR amplification and random selection were performed to screen Hind III BAC library.Positive clones were obtained and subjected to plasmid extraction and labeling for BAC-FISH analysis.Eventually,eight BAC clones were identified that produced specific signals in C.lavandulifolium,including six BACs with signals at the ends of chromosomes(one BAC clone also had signals on the chromosome arm)and two BACs with clear signals in the centromere regions of all chromosomes in C.lavandulifolium.3.The BAC-H338P8 and BAC-H340P10,which produced specific signals in the centromere and end regions,respectively,were used for BAC-FISH and karyotype analysis of the six species in the Chrysanthemum during mitotic metaphase.The results showed that C.lavandulifolium,C.morifolium cv.‘Zhongshanzigui’haploid and C.morifolium‘Jinba’,were closely related to C.nankingense.A higher resolution karyotype model for C.lavandulifolium was constructed using a set of BAC probes.4.Full-length sequencing and sequence analysis of the 2 specific BAC clones revealed that the centromere region BAC-H338P8 contained some important genes,such as EIN3,a nuclear transcription factor of the ethylene signaling pathway;homologous genes of AT5G13560.1 and AT2G24550.1,which related to the structure and function of the centromere in Arabidopsis thaliana.The terminal region BAC-H340P10 contained a microsatellite sequence JH-2074.Repeat sequence analysis showed that both BACs had an overall trend of lower copy number with longer repeat units in the simple tandem repeat sequence.However,the AACT/AGTT type of microsatellite sequence was significantly enriched in BAC-H340P10,accounting for 75.6%.Eight LTRs were annotated on both the forward and reverse strands of BAC-H338P8,but only Cl-H-LTR4 possessed the complete structure,and there are overlapping regions between the other LTRs.The two BAC libraries constructed in this study provide valuable materials for the in-depth study of Chrysanthemum plants.The BAC-FISH analysis of karyotypes of six Chrysanthemum plants using the screened specific BAC clones provides a reference and help for the karyotype research and evolutionary analysis of Chrysanthemum plants.In addition,the full-length sequencing and analysis of two specific BAC clones provided a reference for the study of the sequence composition of the special regions of the chromosomes of Chrysanthemum.更多还原