花生原产于南美洲，现在已经成为全球第四大油料作物。全球的花生年产量接近35.5 百万吨。中国，印度和美国为全球前三大花生种植国家。花生可以直接食用，还可用来榨油和制作花生酱。除此之外，花生还可用来制作化妆品，药品和织物材料。基于花生的重要性和普遍性，对花生的科学研究是很有必要的。 / 栽培花生Arachis hypogaea (AABB, 2n = 40) 是异源四倍体。栽培花生是由两个二倍体祖先Arachis. duranensis (A genome) 和Arachis. ipaensis (B genome) 杂交后又经过染色体加倍形成的。对花生育种来说，野生的品种是很重要的种质资源，因为其对生物和非生物胁迫产生的优良基因型。 但是，由于栽培花生和野生花生之间的遗传，细胞遗传和进化关系知之甚少，阻碍了对野生花生的开发利用。染色体组型分析有利于上述问题的解决。但是，DNA 序列资源的不足和花生染色体本身形态差异不大的特征，阻碍了精细染色体图谱的构建。 / 在我们的研究中，四倍体花生和二倍体（A genome）的Cot-1文库被用来获得高度和中度的重复序列，并对这些重复序列在染色体上的分布进行分析。获得的基因组和染色体特异的标记（Markers）可以区分四倍体中的A genome 和B genome,同时这些还被用来构建精细的染色体图谱。A genome 和B genome 的着丝粒序列也被分离到，并通过细胞免疫组化的验证。A genome的着丝粒序列的卫星序列重复单元为 115 bp，B genome 的着丝粒序列的卫星序列重复单元为 317 bp。这两个着丝粒序列，还有rDNA( 5S, 45S), 拟南芥的端粒序列，三个染色体特异的细菌人工染色体序列一起用来构建成了精细的花生染色体图谱。在这个图谱中，20对染色体可以通过这些标记区分开来。通过三者的染色体图谱分析，，都支持关于A. duranensis (A genome) 和A. ipaensis (B genome) 是花生Arachis hypogaea的祖先的假说。同时，还发现染色体的倒位可能导致了端粒序列在栽培花生的染色体非末端的累积。三者的着丝粒序列和基因CenH3的序列比较表明着丝粒序列在进化过程变化较大而基因CenH3 高度保守。 / 花生的染色体图谱分析有利于构建花生的物理图谱，并可以为基因组序列拼接提供参考。另外，它也使花生的染色体结构和遗传研究成为可能，有助于花生的进化研究，并可推动野生品种在花生育种上的应用。着丝粒表观遗传标记基因CenH3的克隆会加速花生功能着丝粒的鉴定和着丝粒的进化研究。 / Peanut is native to southern America, but now it becomes the fourth-largest oil-seed crop. Total global peanut production is close to 35.5 million tons each year with China as the largest producer, followed by India and USA. Peanut can be directly consumed or made into vegetable oil and peanut butter. Additionally, peanut can be used to produce make-up, medicines and textile materials. The importance and popularity of peanut make it necessary for scientific studies. / The cultivar peanut, Arachis hypogaea (AABB, 2n = 40), is an allotetraploid. It was originated from hybridization of Arachis. duranensis (AA) and Arachis. ipaensis (BB) followed by chromosome doubling. The wild species in the Arachis section are useful genetic resources due to presence of genes that confer biotic and abiotic stress resistance. However, the resource is not well exploited because little comparative genome studies between cultivated peanut and its wild relatives were performed. Characterization of its chromosome components will contribute to comparative genome studies. But the paucity of information on the DNA sequence and the presence of morphologically similar chromosomes make it difficult to construct a detailed karyotype for peanut chromosome identification. / Tetraploid peanut and an A genome Cot-1 library were constructed to isolate highly and moderately repetitive sequences. Chromosomal distributions of these repeats were also investigated. Both genome and chromosome specific markers were identified. They can distiguish A and B genomes in tetraploid peanut and be used to construct a complete karyotyping of peanut chromosomes by FISH. In particular, a 115-bp tandem repetitive sequence was identified to be a B genome centromere repetitive DNA by immunofluoresence, mainly localized in the centromeres of B chromosomes, and a partial retrotransposable element was also identified at the centromeres of B chromosomes. Another 317 tandem repetitive sequence overlapped with A genome specific heterochromatin band in FISH, which was also found to be an A genome centromere repetitive DNA by immunofluoresence. These two tandem repetitive sequences, together with rDNA (5S, 45S), Arabidopsis telomere and three chromosome specific BAC clones were made probe cocktail, which can identify all 20 pairs of chromosomes in peanut and construct a complete karyotype. Karyotype analysis among three species support proveious report that A. duranensis and A. ipaënsis are ancestor of A. hypogaea. It also suggested that occurance of chromosome inversion might lead to accumulation of Arabidopsis telomere in the intercalary region of chromosome in A. hypogaea during evolution. Comparison of centromere DNA and CenH3 gene among three species suggested that centromere DNA show high variation and CenH3 gene highly conserved during evolution. / The karyotyping of peanut is the first step for the construction of peanut physical map, and may provide a reference tool for the genome sequence assembly. It also makes it possible to study peanut chromosome structure and behavior, the evolution of peanut, and the use of wild species in peanut breeding. Additionally, the isolation of CenH3 gene which is an epigenetic centromere marker will facilitate identification of fuctional centromere and centromere evolution in peanut. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Laining. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 120-131). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese. / List of figure --- p.VI / List of table --- p.IX / Table of abbreviation --- p.XI / Acknowledgement --- p.XIII / Abstact --- p.XIV / 摘要 --- p.XVI / Chapter Chapter 1 --- literature review --- p.1 / Chapter 1.1 --- Plant genome and chromosomes --- p.1 / Chapter 1.1.1 --- Single copy gene and repetitive DNA sequences --- p.1 / Chapter 1.1.2 --- Euchromatin and heterochromatin --- p.3 / Chapter 1.1.3 --- Centromeres, Telomeres and Ribosomal DNAs --- p.4 / Chapter 1.1.3.1 --- Centromeres --- p.4 / Chapter 1.2 --- Peanut cytogenetics and evolution --- p.8 / Chapter 1.2.1 --- Cytogenetics, FISH and karyotyping --- p.8 / Chapter 1.2.2 --- Chromosome Evolution --- p.10 / Chapter 1.2.3 --- Peanut cytogenetics and evolution --- p.12 / Chapter 1.2.4 --- Strategy to Construct Peanut FISH Karyotype --- p.14 / Chapter 1.3 --- Objectives and Significance of this study --- p.16 / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- Plant materials --- p.17 / Chapter 2.2 --- Cloning of previous reported markers --- p.17 / Chapter 2.3 --- Isolation of Cot-1 DNA --- p.18 / Chapter 2.4 --- Construction of a peanut Cot-1 library --- p.19 / Chapter 2.5 --- Characterization of the peanut Cot-1 library --- p.19 / Chapter 2.6 --- DNA probe preparation --- p.19 / Chapter 2.7 --- Somatic chromosome preparation --- p.20 / Chapter 2.8 --- FISH procedure --- p.20 / Chapter 2.9 --- Image capture and data processing of FISH images --- p.21 / Chapter 2.10 --- Cloning of full length of repetitive elements --- p.21 / Chapter 2.11 --- Screening BACs with low repeat sequence --- p.21 / Chapter 2.12 --- Isolation of BAC DNAs --- p.25 / Chapter 2.13 --- Probe preparation --- p.25 / Chapter 2.14 --- FISH karyotyping and image processing --- p.26 / Chapter 2.15 --- Isolation of CenH3 --- p.27 / Chapter 2.16 --- Peanut CenH3 antibody production --- p.32 / Chapter 2.17 --- Immunofluorescence detection of peanut CenH3 --- p.33 / Chapter Chapter 3 --- Results --- p.34 / Chapter 3.1 --- Peanut FISH markers --- p.34 / Chapter 3.1.1 --- Peanut Cot-1 DNA library --- p.34 / Chapter 3.1.1.1 --- Peanut Cot-1 DNA isolation --- p.34 / Chapter 3.1.1.2 --- Construction of peanut Cot-1 DNA library --- p.36 / Chapter 3.1.1.3 --- Chromosomal distribution of selected repeat sequences --- p.41 / Chapter 3.1.1.4 --- Cloning of B genome centromeric repeat --- p.45 / Chapter 3.1.1.5 --- Chromosomal Distribution of Clone 117 and the Centromere Satellite Repeat in Other Peanuts --- p.47 / Chapter 3.2 --- Cot-1 DNA library from an A genome diploid species (A. duranensis) --- p.49 / Chapter 3.2.1 --- Cot-1 DNA isolation --- p.49 / Chapter 3.2.2 --- Construction of an A genome Cot-1 DNA library --- p.51 / Chapter 3.2.3 --- Chromosomal distribution of selected clones --- p.56 / Chapter 3.2.4 --- Cloning of the A genome centromeric repetitive element --- p.59 / Chapter 3.3 --- Telomere repeat --- p.60 / Chapter 4.4 --- 5S and 45S (18S-26S) rDNAs --- p.63 / Chapter 3.5 --- Summary of repetitive FISH markers --- p.66 / Chapter 3.6 --- BAC clones --- p.69 / Chapter 3.6.1 --- BAC clone screening --- p.69 / Chapter 3.6.2 --- FISH screening of BAC clones --- p.74 / Chapter 3.6.3 --- Selection of BAC clones for karyotyping --- p.82 / Chapter 3.7 --- Peanut karyotyping --- p.84 / Chapter 3.7.1 --- Karyotyping of tetraploid peanut --- p.84 / Chapter 3.7.2 --- Karyotyping of diploid species --- p.91 / Chapter 3.8 --- Comparison of Centromere sequence --- p.98 / Chapter 3.9 --- Cloning of peanut centromere histone H3 (CenH3) --- p.100 / Chapter 3.9.1 --- Peanut CenH3 --- p.100 / Chapter 3.9.2 --- Peanut CenH3 antibody --- p.104 / Chapter 3.9.3 --- Western blot --- p.105 / Chapter 3.9.4 --- Immunofluorescence detection of peanut centromeres --- p.106 / Chapter Chapter 4 --- Discussions --- p.107 / Chapter 4.1 --- Cot-1 DNA library --- p.107 / Chapter 4.1.1 --- Retrotransposons in Cot-1DNA library: FIDEL and a centromere retrotransposon --- p.107 / Chapter 4.1.2 --- Chloroplast DNA --- p.108 / Chapter 4.1.3 --- Satellite DNA: rDNA and centromere satellite --- p.109 / Chapter 4.1.4 --- Chromosome identification, karyotyping, and comparative genomics with FISH markers --- p.110 / Chapter 4.2 --- Telomere --- p.111 / Chapter 4.3 --- rDNAs --- p.112 / Chapter 4.4 --- BAC clones --- p.113 / Chapter 4.5 --- Karyotyping --- p.114 / Chapter 4.6 --- Centromere identification in peanut --- p.116 / Chapter Chapter 5 --- Conclusion and perspective --- p.118 / References --- p.120
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328276 |
| Date | January 2013 |
| Contributors | Zhang, Laining., Chinese University of Hong Kong Graduate School. Division of Life Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xvii, 131 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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