本文選題：甜橙 + 細(xì)胞　； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：甜橙[Citrus sinensis(L.)Osbeck]是世界上栽培最廣的柑橘品種類群。因其風(fēng)味獨(dú)特,營(yíng)養(yǎng)價(jià)值高,品種豐富等優(yōu)勢(shì)而具有重大的經(jīng)濟(jì)價(jià)值。甜橙來(lái)源于橘和柚的種間雜交,染色體高度雜合。伏令夏甜橙染色體相互易位,極晚熟,少籽,商品性及豐產(chǎn)性良好,是柑橘無(wú)籽育種的優(yōu)良親本。隨著測(cè)序技術(shù)的發(fā)展,細(xì)胞遺傳圖譜作為數(shù)量性狀定位,圖位克隆及分子標(biāo)記輔助育種的重要工具,對(duì)育種和遺傳理論研究都具有重要的意義。前人通過(guò)花藥離體培養(yǎng),獲得雙單倍體,并進(jìn)行了全基因組測(cè)序。本研究在前人基礎(chǔ)上采用熒光原位雜交結(jié)合雙熒光染色技術(shù),創(chuàng)建雜種甜橙及其雙單倍體的分子細(xì)胞遺傳學(xué)研究平臺(tái),并拓展至柑橘屬及其近緣屬植物的細(xì)胞遺傳學(xué)研究,主要結(jié)果如下:1、甜橙脆性位點(diǎn)的特征。甜橙根尖分生組織細(xì)胞常規(guī)染色體制片時(shí)發(fā)現(xiàn)染色體片段化現(xiàn)象,結(jié)合端粒FISH和CMA/DAPI證實(shí)甜橙染色體不易被熒光染料染色的區(qū)域類似于人類和黑麥草上發(fā)現(xiàn)的脆性位點(diǎn)。本研究首次發(fā)現(xiàn)并證實(shí)了甜橙染色體上脆性位點(diǎn)的穩(wěn)定存在,位于B型染色體的近中部和D型染色體的近末端的CMA帶型處。Ag-NOR顯示脆性位點(diǎn)定位于具有轉(zhuǎn)錄活性的45S rDNA區(qū)域,5-m C免疫檢測(cè)到脆性位點(diǎn)在有絲分裂中期呈高度甲基化狀態(tài),說(shuō)明脆性位點(diǎn)的表達(dá)是表觀調(diào)控的。2、柑橘屬及其近緣屬核型分析和脆性位點(diǎn)分布特征分析。本研究結(jié)合前人對(duì)柑橘屬及其近緣屬植物的核型特征,補(bǔ)充山金柑等植物的核型分析,發(fā)現(xiàn)脆性位點(diǎn)廣泛地穩(wěn)定地存在于柑橘屬及其近緣屬植物中。盡管不同種植物的45S rDNA位點(diǎn)的數(shù)目和位置各不相同,脆性位點(diǎn)的位置也具有多態(tài)性,但各種二倍體植物中脆性位點(diǎn)的數(shù)目恒定,除了柚類具有3個(gè)脆性位點(diǎn)以外,其它種均具有2個(gè)脆性位點(diǎn)。脆性位點(diǎn)在雙單倍體和體細(xì)胞雜種形成過(guò)程中穩(wěn)定遺傳。脆性位點(diǎn)可能和染色體變異及物種進(jìn)化有關(guān)。3、伏令夏甜橙染色體易位。對(duì)栽培甜橙及其雙單倍體的核型特征分析發(fā)現(xiàn),栽培甜橙中兩個(gè)B型染色體為非同源染色體,他們分別與兩個(gè)D型染色體互為同源染色體。該結(jié)論修正了前人對(duì)甜橙染色體特征的描述,同時(shí)從細(xì)胞遺傳學(xué)水平進(jìn)一步證實(shí)了伏令夏甜橙染色體高度的雜合性。本研究推測(cè)這種b型染色體水平上的雜合性來(lái)源于發(fā)生在脆性位點(diǎn)處的染色體相互易位,并結(jié)合花粉母細(xì)胞減數(shù)分裂染色體行為加以佐證。4、甜橙細(xì)胞遺傳學(xué)圖譜的構(gòu)建。通過(guò)基因組學(xué)分析的高度串聯(lián)重復(fù)序列cl,中度重復(fù)rdna和單拷貝bac為探針,結(jié)合cma/dapi的熒光原位雜交技術(shù)將甜橙的6個(gè)染色體確定,剩下的3個(gè)f型染色體根據(jù)染色體的大小和連鎖群的長(zhǎng)度加以識(shí)別,進(jìn)而構(gòu)建了甜橙的細(xì)胞遺傳學(xué)圖譜。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S666.4

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