甜橙细胞遗传学图谱构建和脆性位点分析

发布时间：2018-06-17 13:02

本文选题：甜橙 + 细胞　；参考：《华中农业大学》2016年博士论文

【摘要】：甜橙[Citrus sinensis(L.)Osbeck]是世界上栽培最广的柑橘品种类群。因其风味独特,营养价值高,品种丰富等优势而具有重大的经济价值。甜橙来源于橘和柚的种间杂交,染色体高度杂合。伏令夏甜橙染色体相互易位,极晚熟,少籽,商品性及丰产性良好,是柑橘无籽育种的优良亲本。随着测序技术的发展,细胞遗传图谱作为数量性状定位,图位克隆及分子标记辅助育种的重要工具,对育种和遗传理论研究都具有重要的意义。前人通过花药离体培养,获得双单倍体,并进行了全基因组测序。本研究在前人基础上采用荧光原位杂交结合双荧光染色技术,创建杂种甜橙及其双单倍体的分子细胞遗传学研究平台,并拓展至柑橘属及其近缘属植物的细胞遗传学研究,主要结果如下:1、甜橙脆性位点的特征。甜橙根尖分生组织细胞常规染色体制片时发现染色体片段化现象,结合端粒FISH和CMA/DAPI证实甜橙染色体不易被荧光染料染色的区域类似于人类和黑麦草上发现的脆性位点。本研究首次发现并证实了甜橙染色体上脆性位点的稳定存在,位于B型染色体的近中部和D型染色体的近末端的CMA带型处。Ag-NOR显示脆性位点定位于具有转录活性的45S rDNA区域,5-m C免疫检测到脆性位点在有丝分裂中期呈高度甲基化状态,说明脆性位点的表达是表观调控的。2、柑橘属及其近缘属核型分析和脆性位点分布特征分析。本研究结合前人对柑橘属及其近缘属植物的核型特征,补充山金柑等植物的核型分析,发现脆性位点广泛地稳定地存在于柑橘属及其近缘属植物中。尽管不同种植物的45S rDNA位点的数目和位置各不相同,脆性位点的位置也具有多态性,但各种二倍体植物中脆性位点的数目恒定,除了柚类具有3个脆性位点以外,其它种均具有2个脆性位点。脆性位点在双单倍体和体细胞杂种形成过程中稳定遗传。脆性位点可能和染色体变异及物种进化有关。3、伏令夏甜橙染色体易位。对栽培甜橙及其双单倍体的核型特征分析发现,栽培甜橙中两个B型染色体为非同源染色体,他们分别与两个D型染色体互为同源染色体。该结论修正了前人对甜橙染色体特征的描述,同时从细胞遗传学水平进一步证实了伏令夏甜橙染色体高度的杂合性。本研究推测这种b型染色体水平上的杂合性来源于发生在脆性位点处的染色体相互易位,并结合花粉母细胞减数分裂染色体行为加以佐证。4、甜橙细胞遗传学图谱的构建。通过基因组学分析的高度串联重复序列cl,中度重复rdna和单拷贝bac为探针,结合cma/dapi的荧光原位杂交技术将甜橙的6个染色体确定,剩下的3个f型染色体根据染色体的大小和连锁群的长度加以识别,进而构建了甜橙的细胞遗传学图谱。
[Abstract]:Sweet orange (Citrus sinensis L. Osbeck) is the most widely cultivated citrus variety group in the world. Because of its unique flavor, high nutritional value, rich variety and other advantages, it has great economic value. Sweet orange originated from the interspecific hybridization between orange and pomelo, and the chromosomes were highly heterozygous. Volingsia sweet orange is a good parent for seedless breeding because of its translocation of chromosomes, very late maturing, low seed, good commercial and high yield. With the development of sequencing technology, cell genetic map, as an important tool for quantitative trait mapping, map-cloning and molecular marker-assisted breeding, is of great significance for breeding and genetic theory research. Two haploids were obtained by anther culture in vitro, and the whole genome was sequenced. On the basis of previous studies, the molecular cytogenetic research platform of hybrid sweet orange and its dihaploid was established by fluorescence in situ hybridization and double fluorescence staining, and extended to the cytogenetic research of citrus and its relative genera. The main results are as follows: 1, the characteristics of fragile sites in sweet orange. Chromosomal fragmentation was found in the meristem cells of sweet orange root tips. Combining with telomere fish and CMA / DAPI, it was proved that the regions of sweet orange chromosomes not easily stained by fluorescent dyes were similar to the fragile sites found in human and ryegrass. In this study, the stable existence of fragile sites on sweet orange chromosomes was first discovered and confirmed. At the near middle of the B chromosome and the near end of the D chromosome, the. Ag-NOR showed that the fragile site was located in the 45s rDNA region with transcriptional activity, and 5-m C immunoassay showed that the fragile site was highly methylated in the metaphase of mitosis. The results showed that the expression of fragile sites was epithetically regulated. The karyotype and distribution of fragile sites in Citrus and its relative genera were analyzed. Based on the karyotypic characteristics of Citrus and its related genera, we found that fragile sites are widely and stably present in Citrus and its relative genera. Although the number and location of 45s rDNA sites in different species of plants are different, and the location of fragile sites is also polymorphic, the number of fragile sites in various diploid plants is constant, except for three fragile sites in pummelos. All other species have two fragile sites. Fragile sites were stably inherited during the formation of dihaploid and somatic hybrids. Fragile sites may be related to chromosome variation and species evolution. The karyotypic characteristics of cultivated sweet orange and its dihaploid were analyzed. It was found that the two B chromosomes in cultivated sweet orange were non-homologous chromosomes and they were homologous chromosomes with two D-type chromosomes respectively. This conclusion revises the previous description of the chromosomal characteristics of sweet orange and further confirms the heterozygosity of the chromosomal height of Volinghamia sweet orange from the cytogenetic level. In this study, we speculated that the heterozygosity at the level of b chromosome was derived from chromosome translocation at fragile sites, which was supported by the meiotic chromosome behavior of pollen mother cells, and the construction of cytogenetic map of sweet orange. Six chromosomes of Sweet Orange were identified by genomic analysis of high tandem repeats (cll), moderate repeated rdna and single copy bac as probes, and fluorescence in situ hybridization (Fish) of cma/dapi. The remaining 3 f chromosomes were identified according to the size of chromosome and the length of linkage group, and the cytogenetic map of sweet orange was constructed.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S666.4

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