利用C基因组DNA和C0t-1 DNA对药用野生稻、大颖野生稻基因组的比较分析

采用药用野生稻C基因组DNA及C0t-1 DNA为探针,分别对药用野生稻(CC)自身和大颖野生稻(CCDD)体细胞染色体进行了基因组原位杂交(GISH)和荧光原位杂交(FISH)分析。利用C基因组DNA探针进行分析显示,药用野生稻24条染色体都被杂交信号覆盖;而在大颖野生稻中可区分为24条CC型染色体(杂交信号较强)和24条DD型染色体(杂交信号较弱)。以C基因组C0t-1 DNA探针进行分析显示,在药用野生稻染色体的端粒、着丝粒、近着丝粒区域有很强的杂交信号,而大颖野生稻中也有24条染色体在这些区域红色杂交信号较强,另24条染色体的杂交信号很弱。表明利用C基因组DNA和C0t-1 DNA为探针的GISH和FISH技术,都能很好地将大颖野生稻C、D染色体组区分开,C和D基因组亲缘关系较远,二者具有不同起源。与药用野生稻C基因组相比,大颖野生稻C基因组出现了一些分化。这些都为研究大颖野生稻C和D染色体组起源,探讨异源四倍体进化机制奠定了基础。     

利用栽培稻基因组DNA对非洲野生稻进行染色体荧光原位杂交分析

以地高辛标记的栽培稻基因组(基因组为AA)DNA为探针,对非洲野生稻(基因组为BBCC)的体细胞染色体进行荧光原位杂交分析,研究AA染色体组和BBCC染色体组之间的关系,同时对杂交后的染色体进行同源染色体配对。结果表明:栽培稻A基因组和非洲野生稻基因组有较高的同源性,其中高度重复DNA序列在栽培稻和非洲野生稻间具有保守性。     

利用C基因组对高杆野生稻和宽叶野生稻基因组的比较分析

[目的]采用基因组原位杂交（GISH）技术研究稻属2种CCDD基因组型的野生稻宽叶野生稻和高杆野生稻基因组之间的关系。[方法]利用药用野生稻C基因组总DNA为探针,分别对高杆野生稻和宽叶野生稻中期染色体进行基因组原住杂交。[结果]杂交结果显示,在一定的洗脱严谨度下,可以把CCDD染色体组中的C、D基因组染色体分开,并且发现高杆野生稻的CCDD基因组中的某些属于CC基因组的染色体与宽叶野生稻和药用野生稻中的CC基因组染色体存在较大差异,宽叶野生稻的基因组更加原始。[结论]对稻属中有相同基因组型的种进行比较分析,将有助于深入阐明植物基因组进化和物种进化及可能的途径。     

利用水稻C0t-1 DNA和基因组DNA对栽培稻、药用野生稻和疣粒野生稻基因组的比较分析

【目的】研究中高度重复序列在稻属不同物种基因组进化中的作用。【方法】用栽培稻C0t-1 DNA和基因组DNA(gDNA)作为探针，分别对栽培稻、药用野生稻和疣粒野生稻进行荧光原位杂交(FISH)和比较基因组杂交(CGH)。【结果】C0t-1 DNA覆盖栽培稻、药用野生稻和疣粒野生稻基因组比例(%)和大小(Mb)分别为47.10±0.16，38.61±0.13，44.38±0.13和212.33±1.21，269.42±0.89以及532.56±1.68。栽培稻gDNA在药用野生稻和疣粒野生稻基因组中的覆盖率约为91.0%和93.6%，含量分别约为634 Mb和1123 Mb，各有365 Mb和591 Mb不属于源自栽培稻基因组的中高度重复序列，未被栽培稻gDNA所覆盖的部分，分别为64 Mb和78 Mb左右。此外，以C0t-1 DNA的组成为依据，对这3个种核型进行了同源性聚类。【结论】稻属中度和高度重复序列和功能基因一样，在不同种中也存在着高度同源性和保守性，并在进化过程中得以保存下来。药用野生稻和疣粒野生稻基因组增大的重要原因之一，可能是基因组中度和高度重复序列加倍的结果，药用野生稻这种序列扩增相对疣粒野生稻要缓和得多。另外，这两个野生种在长期进化过程中，由于存在加倍、重排和基因选择性丢失等现象，形成了具有自己种的特异性的基因组成分。     

云南药用野生稻BIBAC文库的构建及分析

云南药用野生稻具有许多优良性状和有利基因,其CC基因组的优良基因很难通过有性杂交转移到栽培稻(AA基因组)上,但可以通过双元细菌人工染色体(BIBAC)以大片段的形式转化到栽培稻中.利用BIBAC2载体构建了云南药用野生稻基因组DNA文库,该文库包含53 760个克隆,平均插入片段76 kb,保存在140块384孔板中...     

栽培稻与Oryza officinalis相似群野生稻基因组VDAC基因的比较

根据水稻基因组文库日本晴的基因组VDAC基因的序列,设计引物分别扩增6种不同基因组的野生稻(BB、CC、BBCC及CCDD)和2种栽培稻(AA)的基因组DNA的VDAC基因片段.所有分别代表8个VDAC基因的8对引物中,除引物VDAC3外,其它7对引物均能扩增出预期大小的特异条带,其中一些片段是所有试验材料共有的,而另一些则具有明显的基因组或种的特异性.AA、BB、CC基因组均具有VDAC1、VDAC4、VDAC7和VDAC8引物的扩增位点,而DD基因组则不能确定.VDAC6的引物位点是AA基因组所特有.VDAC2引物的扩增位点存在于基因组AA、BB、DD,而CC基因组中则无此位点.而VDAC5则可区分同为CCDD的宽叶野生稻和高秆野生稻.栽培稻种与野生稻种基因组相比能扩增出更多的VDAC基因片段的试验结果,为进一步研究稻属中VDAC基因的起源及进化关系提供了基础.     

利用水稻BAC克隆对Gm-2和Gm-6在药用野生稻中的FISH定位

 采用栽培稻遗传图第 4连锁群中与抗稻瘿蚊基因 ,Gm- 6和 Gm- 2等位的 RFL P标记 RG2 14和 RZ5 6 9筛选出来的两个 BAC克隆为探针 ,对药用野生稻进行了荧光原位杂交物理定位。两个 BAC克隆的大小分别为 5 0 kb和86 kb,在 Cot- 1DNA封阻的情况下它们均被定位于药用野生稻第 4染色体长臂 ,与着丝粒百分距为 72 .33± 4.40、77.10± 2 .40 ,信号检出率分别为 6 1.2 %和 5 9.5 %。与此同时 ,用 RG2 14和 RZ5 6 9对药用野生稻进行了杂交 ,它们也被定位于药用野生稻第 4染色体长臂 ,与着丝粒百分距分别为 74.18± 2 .6 2和 78.2 3± 2 .31,信号检出率分别为 8.3%和 9.4%。 BAC克隆的 RFL P标记探针杂交位置几乎一致 ,这表明在栽培稻和野生稻中 RFL P标记 RG2 14和RZ5 6 9都在同一 BAC克隆的大插入片段中 ,药用野生稻与抗性基因 Gm- 6和 Gm- 2同源顺序就在第 4染色体信号出现的相应位置。药用野生稻第 4染色体的确定是根据 Jena等 (1994)和本研究的 RFL P的杂交结果进行的。文中讨论了利用栽培稻 BAC克隆对药用野生稻进行原位杂交物理作图的可行性等问题。     

药用野生稻抗源对褐稻虱的抗性遗传及利用研究

利用广谱高抗褐稻虱的药用野生稻（CC染色体组）与感虫栽培稻（AA染色体组）品种进行远缘杂交，通过幼胚培养获得绿苗，经过4代回交和4代自交，成功地将抗性基因转移到栽培稻中，获得了高世代B4F4株系。对其亲本和后代的鉴定结果表明，药用野生稻抗性受1对显性主效基因控制；其抗性基因与药用野生稻主要农艺性状没有连锁遗传关系。     

利用45S rDNA探针对宽叶野生稻和高秆野生稻基因组的FISH分析

利用45S rDNA作为探针,通过荧光原位杂交(FISH)技术对同样含有CCDD基因组的高秆野生稻(Oryza alta)和宽叶野生稻(O.latifolia)进行rDNA的荧光原位杂交定位分析和核型分析。结果显示:宽叶野生稻中45S rDNA信号分布于多条染色体上,位点数目为10~16;高秆野生稻中有6个信号点,分布于3对同源染色体上,其中2对信号位点位于染色体短臂,1对位于染色体长臂。研究结果表明,高秆野生稻45S rDNA在基因组中位点数目稳定,宽叶野生稻中45S rDNA位点数在不同个体中呈现一定的动态变化,显示这2种野生稻基因组存在一定差异;核型分析结果也表明二者基因组存在较大的差异。由此推测,高秆野生稻分化较早而趋向稳定,宽叶野生稻可能形成较晚,还处于进化过程之中。鉴于二者在基因组结构上的明显差异和进化上的不平衡性,建议把这2种野生稻划分为不同野生稻种,可能会更加符合二者的进化特性。同时,讨论了45S rDNA在染色体中分布特点与机制。     

野生稻细胞核DNA提取的研究

药用野生稻是中国最具利用价值的野生稻资源之一,具有许多优良的性状和有利基因,是改良栽培稻培育新品种的宝贵基因库,而其CC基因组的优良基因有望以大片段形式通过BIBAC等载体转化到栽培稻中.BIBAC文库不仅能作为大片段基因组文库的载体,而且能通过根癌农杆菌介导将克隆片段导入植物基因组直接进行转化,可用于筛选分离基因等研究.如何获得基因组DNA大片段非常关键,本研究采取 琼脂糖包埋基因组的方法获得大片段DNA,并对其中的一些步骤进行了优化,为构建药用野生稻BIBAC文库打下了很好的基础,值得在其它植物类似研究中参考.     

Comparative Analysis of Genomes in Oryza sativa, O.officinalis, and O. meyeriana with C0t-1 DNA and Genomic DNA of Cultivated Rice

Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were applied to somatic chromosomes preparations of Oryza sativa, O. officinalis, and O. meyeriana with labeled probes of C0t-1 DNA and genomic DNA from the cultivated rice. The coverage percentage (%) and size (Mb) of C0t-1 DNA in O. sativa, O. officinalis, and O. meyeriana were 47.1 ±0.16, 38.61 ±0.13, 44.38±0.13, and 212.33± 1.21,269.42 ± 0.89, 532.56± 1.68 Mb, respectively. The coverage percentage and size of genomic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0, 93.6% and 634, 1 123 Mb, respectively, in which 365 and 591 Mb in O. officinalis and O. meyeriana were from O. sativa genomic DNA, but not from repetitive sequences of O. sativa, and the uncoverage genome size in O. officinalis and O. meyeriana were 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of C0t-1 DNA in O. sativa, O. officinalis, and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during evolution. The repetitive sequences reduplication may be one of the important causes of the genome enlargement of O. officinalis and O. meyeriana, and O. officinalis genome enlarged more slowly when compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana were formed by reduplication, rearrangement, and gene selective loss during the evolution process.     

Comparative analysis of genomes in Oryza sativa, O. officinalis and O. meyeriana with C 0 t-1 DNA and genomic DNA of cultivated rice

Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were applied to somatic chromosome preparations of Oryza sativa, O. officinalis and O. meyeriana with labeled probes of C ₀ t-1 DNA and genomic DNA from cultivated rice. The coverage percentage (%) and size (Mb) of C ₀ t-1 DNA in O. sativa, O. officinalis and O. meyeriana were 47.1 ± 0.16, 38.61 ± 0.13, 44.38 ± 0.13 and 212.33 ± 1.21, 269.42 ± 0.89, 532.56 ± 1.68, respectively. The coverage percentage and size of probe signals with genomic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0%, 93.6% and 634 Mb, 1 123 Mb respectively, in which there were 365 and 591 Mb in O. officinalis and O. meyeriana which came from O. sativa genomic DNA not from repetitive sequences of O. sativa, and the uncovered genome size in O. officinalis and O. meyeriana was 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of C ₀ t-1 DNA in O. sativa, O. officinalis and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during the evolution process. The repetitive sequence reduplication might be one of the important causes of genome enlargement in O. officinalis and O. meyeriana; the O. officinalis genome enlarged more slowly compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana formed by reduplication, rearrangement and gene selective loss during the evolution process. Translated from Scientia Agricultura Sinica, 2006, 39(6): 1083–1090 [译自: 中国农业科学]     

Comparative karyotypic analysis of A and C genomes in the genus Oryza with C 0 t-1 DNA and RFLP

Fluorescence in situ hybridization (FISH) was applied to somatic chromosomes preparations of Oryza sativa L. (AA), O. glaberrima (AA), and O. officinalis Wall. (CC) with a labeled probe of C ₀ t-1 DNA. Genomic in situ hybridization to its own chromosomes (self-GISH) was conducted in a control experiment. The homologous chromosomes showed similar signal bands probed by C ₀ t-1 DNA, while karyotypic analysis of chromosomes between A genome in the two cultivated species and C genome in O. officinalis were conducted based on the band patterns. The ideograms with C ₀ t-1 DNA signal bands were also built. The nonuniform distribution of hybridization signals of C ₀ t-1 DNA from O. sativa and that on its own chromosome of O. officinalis were observed. However, the similarity and correspondence between C ₀ t-1 DNA signal patterns and genomic DNA signal patterns indicated that the self-GISH signals actually resulted from the hybridization of genomic repetitive sequences to the chromosomes. The restriction fragment length polymorphism (RFLP) marker, R2676, from the chromosome 8 of O. sativa and O. officinalis, was used as a probe to somatic hybrid on chromosomes for comparative karyotypic analysis between O. glaberrima and O. officinalis. The results showed that R2676 was located on the short arm of chromosome 7 in O. officinalis and chromosome 4 in O. glaberrima. The percentage distances from the centromere to hybridization sites were 91.56±5.62 and 86.20±3.17. Our results revealed that the relative length of O. officinalis chromosome 8 does not follow conventional chromosome length in descending order of number. C ₀ t-1 DNA of A genome signals were detected in the end of the short arm of O. officinalis chromosome 8, indicating that the highly and moderately repetitive DNA sequences in this region were considerably similar between C and A genomes. However, the fluorescence intensity on the chromosomes of C ₀ t-1 DNA of A genome was less than that of its own C genome from O. officinalis, which would be one of the causes for the fact that highly and moderately repetitive DNA sequences were amplified in O. officinalis. No homology signal of C ₀ t-1 DNA from O. sativa was detected in the end of the long arm of O. glaberrima, indicating that repetitive DNA sequences of A genome in two cultivated rice were lost in the evolutional history. In this paper, using comparative karyotypic analysis of RFLP combined C ₀ t-1 DNA signal bands, the evolutionary mechanism of genome in genus Oryza was also discussed.     

基于顺序GISH-FISH花生栽培种的染色体分析

【目的】针对花生染色体较小,染色体细胞学标记少,细胞遗传研究相对滞后,染色体分类识别困难的问题,建立能够准确区分栽培花生（Arachis hypogaea L.,2n=4x=40,AABB）A、B染色体组的新核型,提高染色体识别准确率,以揭示栽培花生和野生供体亲本的染色体对应关系,鉴定栽培种花生染色体结构变异体。【方法】以花生栽培种（Arachis hypogaea L.,2n=4x=40,AABB）的2个可能供体亲本即花生野生种Arachis duranensis（2n=2x=20,BB）和Arachis ipaënsis（2n=2x=20,AA）全基因组DNA及5S rDNA和45S rDNA为探针,利用顺序基因组荧光原位杂交（GISH）和多色荧光原位杂交（McFISH）技术（简称顺序GISH-FISH）结合DAPI染色,在准确区分花生栽培种A、B染色体组的基础上,对花生栽培品种Z5163及其供体亲本染色体进行分析,建立花生栽培种新核型,并利用该核型对其他栽培品种的染色体进行分析,以探讨该核型的应用潜力和栽培花生染色体组成特点。【结果】以A. ipaënsis和A.duranensis全基因组DNA为探针的GISH分析表明,以A. ipaënsis为探针在花生栽培种20条B组染色体上能够产生清晰稳定的杂交信号,在A组染色体上没有信号,而以A.duranensis为探针,只在18条A组染色体能产生信号,但1对A组的小染色体“A染色体”不易被区分,因此,以A. ipaënsis为探针可以准确区分花生栽培种A、B染色体组;综合5S rDNA和45S rDNA Mc-FISH和DAPI染色分析,发现花生栽培种A、B染色体组DAPI带纹、5S rDNA和45S rDNA的分布分别与A.duranensis和A. ipaënsis一致,此结果支持A.duranensis和A.ipaënsis是花生栽培种的供体亲本。DAPI染色结果显示,A. ipaënsis及花生栽培种的B组染色体均有14条染色体显示着丝粒带纹,明显多于前人报道,表明仅利用DAPI染色来区分花生栽培种A、B组染色体的方法具有局限性。综合DAPI染色、rDNA、A.duranensis和A. ipaënsis基因组探针进行顺序GISH-FISH分析,建立了可以准确识别花生栽培种A、B染色体组新核型。然后利用该核型对3个栽培种品种的染色体组成进行了分析,首次发现一个自发的花生染色体代换系MS B1（A1）,揭示了栽培花生染色体B1与A1之间存在部分同源关系。【结论】野生花生A. duranensis和A. ipaënsis分别与栽培花生A和B基因组染色体间具有很好的对应关系;研究建立的基于GISH-FISH和DAPI染色的栽培花生新核型,不但可以准确区分大部分A、B组染色体,而且还能识别栽培花生在多倍体化和人工进化过程中可能存在的自发的染色体变异,揭示A、B组染色体间的部分同源性。     

利用C基因组对高杆野生稻和宽叶野生稻基因组的比较分析(英文)

[Objective] Genomic in situ hybridization (GISH) was used to study the relationship between the two CCDD genomes of Oryza alta and Oryza latifolia. [Method] Total DNA of Oryza officinalis (C-genome) was used as a probe for genomic in situ hybridization on metaphase chromosomes from Oryza alta and Oryza latifolia, respectively. [Result] Under certain post-hybridization washing stringencies, C- and D-genome could be distinguished in CCDD genome type; there were huge differences in some CC chromosomes of Oryza alta, Oryza latifolia, and Oryza officinalis. The genome of Oryza latifolia was more original. [Conclusion] Comparative analysis of the Oryza species with identical genome type may facilitate to elucidate the possible approaches to plant genome evolution and species evolution.     

The Physical Location of Gm-2 and Gm-6 in O.officinalis with BAC-FISH Based on Comparative RFLP Map of Wild Rice and Cultivated Rice

Rice BAC library is used widely in rice genome research due to its distinctive advantages over other library systems. In this study, two rice BAC clones closely linked to rice gall midge resistance, Gm-2 and Gm-6, were in situ hybridized to Oryza officinalis chromosomes. They were located on the long arm of chromosome 4 with FL 72.33% and 77.10% respectively and their FL was consistent with the selective marker of rice, RG214 and RZ569. The frequency of signal detection was 61.2% and 59.5% respectively.Our study was based on comparative RFLP map of wild rice, O. officinalis, and cultivated rice, O. sativa.     

利用C基因组对高杆野生稻和宽叶野生稻基因组的比较分析

1材料与方法1．1植物材料和染色体制片药用野生稻稻株1589由广东省国家野生稻圃提供,宽叶野生稻IRW6和高杆野生稻IRW41由华南农业大学卢永根院士提供,试验材料情况见表1。染色体制片分别参照Yan等和Ren等的方法。