利用GISH和C0t-1DNA-FISH对稻属B，C，G基因组的比较分析

为探究稻属B,C,G基因组之间的关系以及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,利用药用野生稻和斑点野生稻的中度和高度重序列C0t-1 DNA和总基因组作为探针,对疣粒野生稻进行了比较染色体原位杂交分析.该2种野生稻的总基因组和C0t-1 DNA在疣粒野生稻染色体上信号覆盖率分别为(72.39±0.11) %,(75.60±0.18) %,(47.93±0.16) %,(55.47±0.12) %. 此外,以C0t-1 DNA的杂交信号组成为依据,对疣粒野生稻染色体组进行了核型分析.结果表明:G基因组和B,C基因组之间的关系都比较远,其原因可能是G基因组要早于B,C基因组从稻属的祖先中发生分化,并在进化过程中发生加倍、重排和基因选择性丢失等现象,形成了各自种的特异基因组成分.稻属基因组中度和高度重复序列与功能基因一样,在不同种中也存在着相当的同源性和保守性,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.     

利用C_ot-1 DNA FISH对3种异源四倍体野生稻的比较分析

利用来源于药用野生稻(CC基因组)的中高度重复序列C_ot-1 DNA作为探针,在相同的洗脱严谨度下,通过荧光原位杂交(FISH)对3种基因组同为CCDD的异源多倍体野生稻,宽叶野生稻(O.latifolia)、高杆野生稻(O.alta)、大颖野生稻(O.grandiglumis)进行基因组比较分析.结果发现:在80%的洗脱严谨度下,杂交信号在宽叶野生稻的C、D基因组上的分布差异较为明显,可以区分24条来源于C基因组的染色体和24条来自于D基因组的染色体;相同洗脱度下的高杆野生稻和大颖野生稻的C、D基因组则区别不明显,表明此2种野生稻中的C、D基因组亲缘关系比宽叶野生稻中的C、D基因组关系要近.在此基础上,分别利用来源于栽培稻(AA基因组)和药用野生稻(CC基因组)的C_ot-1DNA作为探针,在不同洗脱严谨度下,对C、D基因组关系相对最远的宽叶野生稻进行FISH分析,进一步研究宽叶野生稻染色体中C、D基因组的进化关系.随着洗脱严谨度的调整,杂交信号呈现出较高的特异性,主要分布在着丝粒、近着丝粒及端粒区域.结果表明:以不同洗脱严谨度下的FISH结果为基础进行的基因组分析.可进一步提高野生稻染色体识别的准确性,为研究异源多倍体的起源及进化提供依据.     

利用粳稻基因组DNA和C_ot-1 DNA探针对普通野生稻和亚洲栽培稻的比较分析

以粳稻日本晴基因组DNA和Cot-1 DNA为探针,分别对日本晴、籼稻广陆矮4号和普通野生稻的染色体组进行了基因组原位杂交(GISH)和Cot-1 DNA荧光原位杂交(FISH)分析,并对3种染色体组进行了同源聚类和比较研究.结果表明:粳稻基因组DNA和Cot-1 DNA探针信号在3种水稻染色体组中的分布状况和覆盖率相似,Cot-1 DNA的覆盖率分别为(47.13±0.18)%、(45.89±0.22)%、(44.24±0.21)%,3种水稻基因组同源性高,亲缘关系接近.Cot-1 DNA在3种水稻染色体上的杂交信号分布各有特点,中高度重复序列的变异在普通野生稻向栽培稻进化和亚洲栽培稻籼、粳分化过程中具有重要意义,中高度重复序列含量较低的2、5、8号染色体是水稻染色体组进化过程中相对活跃的成分.     

野生稻种质资源利用研究

文章综述了近年来野生稻遗传基础方面的研究及其利用的伟大成就．野生稻是水稻育种的重要基因库．与栽培稻亲缘关系较近的 Ａ Ａ 染色体组型野生稻,其优异基因的转移和利用已取得辉煌成就．非 Ａ Ａ 型野生稻与栽培稻远缘杂交不亲和是阻碍其优异基因利用的障碍．常规杂交和现代生物技术（胚拯救技术和细胞工程）相结合是克服种间屏障的有效方法     

籼稻和粳稻的高效分子鉴定方法及其在水稻育种和进化研究中的意义

为了建立准确高效的籼稻和粳稻鉴定方法,对基于籼稻（93—11）和粳稻（日本睛）的全基因组DNA序列比对而获得的45个特异插入／缺失（InDel）位点进行了实验验证．以包括93—11和日本晴在内的44个典型籼稻和典型粳稻品种为实验群体,用45对InDel引物对上述水稻品种的DNA样品进行了PCR扩增和聚丙烯酰胺凝胶电泳,获得了多态的电泳条带．对获得的各InDel位点的基因型数据矩阵进行了中性检测（neutrality test）,确定了与栽培稻的籼、粳遗传分化密切相关的34个InDel位点．进一步对来自亚洲11个国家的栽培稻品种和来源不同12个野生稻物种的PCR产物和电泳结果的读取和分析,计算这些栽培稻品种和野生稻DNA样品在这34个InDel位点上的籼型或粳型基因频率,最终确定了不同样品的籼、粳特性．该籼稻和粳稻鉴定方法被称为“InDel分子指数法99与传统基于形态特征鉴定栽培稻籼、粳特性的“程氏指数法”相比,该方法不仅能够准确鉴定籼稻和粳稻,而且还具有更快捷、简便和高效的特点．另外,InDel分子指数法还可以用于野生稻样品的籼、粳特性鉴定,扩大了被检测样品的范围,具有广阔的应用前景．InDel分子指数法的建立为栽培稻育种过程中正确选用籼稻或粳稻种质资源提供了新的技术方法,也为栽培稻的起源、籼-粳遗传分化、以及籼稻和粳稻在驯化过程中如何适应地理环境变化提供了新的研究思路．     

籼稻93-11和粳稻日本晴DNA插入缺失差异片段揭示的水稻籼-粳分化

碱基的插入/缺失(InDel)引起DNA序列变化并形成了DNA片段长度多态,可以用作遗传标记.为了评价基于籼稻93-11和粳稻日本晴全基因组序列比对获得的差异片段而设计的InDel引物在鉴定籼稻和粳稻两种生态型以及研究稻属不同物种之间亲缘关系的意义,采用45对InDel引物,对来自亚洲10个国家的49份籼稻、43份粳稻品种和24份野生稻进行了检验.结果表明,其中41对InDel引物鉴定籼稻或粳稻品种的准确率高于80%.主成分分析散点图显示:籼稻与粳稻存在明显的遗传分化;含AA基因组的野生稻物种与籼稻品种存在较近的亲缘关系;非AA基因组的野生稻物种不存在明显的籼-粳分化.并且证明了基于籼稻93-11和粳稻日本晴全基因组序列比对获得的InDel差异片段设计的引物可以用于栽培稻籼稻和粳稻品种的鉴定以及籼-粳分化问题的研究,及探索稻属不同物种间的亲缘关系.     

基于ISSR标记的稻属AA基因组遗传多样性分析

为了确定稻属AA基因组物种间的遗传差异和系统进化关系,62份来自广泛地理分布的水稻品系被用于ISSR标记分析。这些品系包含有6个野生稻种(O.nivara,O.rufipogon、O.barthi,O.longistaminata,O.glumaepatula,和O.meridionalis)和2个栽培稻种(O.sativa和O.glaberrima)。21条能产生良好重复性条带模式的ISSR引物被筛选出,并在62个水稻品系中揭示出非常好的多态性。全部样品的基因多样性为0.527,同时显示出ISSR标记在稻属物种遗传多样性研究中具有强大的作用。根据ISSR条带模式,利用Jaccard配对相似系数构建的一致性树状图,显示出具有良好自展支持率的稻属AA基因组遗传多样性关系。结果表明,来自不同大陆的稻属物种具有较近的亲缘关系,尤其是亚洲野生稻物种与Vaughan1989年建立的稻属分类系统具有良好的一致性。研究结果将对稻属AA基因组野生稻在水稻育种实践中的有效利用具有非常重要的意义。     

籼稻和粳稻的高效分子鉴定方法及其在水稻育种和进化研究中的意义

为了建立准确高效的籼稻和粳稻鉴定方法,对基于籼稻(93-11)和粳稻(日本晴)的全基因组DNA序列比对而获得的45个特异插入/缺失(InDcl)位点进行了实验验证.以包括93-11和日本睛在内的44个典型籼稻和典型粳稻品种为实验群体,用45对InDel引物对上述水稻品种的DNA样品进行了PCR扩增和聚丙烯酰胺凝胶电泳,获得了多态的电泳条带.对获得的各InDel位点的基因型数据矩阵进行了中性检测(neutrality test),确定了与栽培稻的籼、粳遗传分化密切相关的34个InDel位点.进一步对来自亚洲11个国家的栽培稻品种和来源不同12个野生稻物种的PCR产物和电泳结果的读取和分析,计算这些栽培稻品种和野生稻DNA样品在这34个InDel位点上的籼型或粳型基因频率,最终确定了不同样品的籼、粳特性.该籼稻和粳稻鉴定方法被称为"InDel分子指数法".与传统基于形态特征鉴定栽培稻籼、粳特性的"程氏指数法"相比,该方法不仅能够准确鉴定籼稻和粳稻,而且还具有更快捷、简便和高效的特点.另外,InDel分子指数法还可以用于野生稻样品的籼、粳特性鉴定,扩大了被检测样品的范围,具有广阔的应用前景.InDel分子指数法的建立为栽培稻育种过程中正确选用籼稻或粳稻种质资源提供了新的技术方法,也为栽培稻的起源、籼-粳遗传分化、以及籼稻和粳稻在驯化过程中如何适应地理环境变化提供了新的研究思路.     

着丝粒串联重复序列RCS2对亚洲栽培稻和非洲栽培稻的比较分析

为了揭示中高度重复序列在同为AA基因组的亚洲栽培稻和非洲栽培稻基因组中的差异以及重复序列在.栽培稻种的分化过程中可能起到的作用,利用水稻着丝粒串联重复序列RCS2作为探针分别对籼稻广陆矮4号、粳稻日本晴和非洲栽培稻的体细胞染色体进行荧光原位杂交(FISH)实验,并对其核型进行同源性聚类和比较分析,杂交结果显示:RCS2序列位于在3种栽培稻染色体组中,RCS2序列位于每条染色体的着丝粒位置,但有不同的分布特点,表明该3种栽培稻基因组的RCS2序列有不同的进化方向.探讨了RCS2序列结合Cot-1 DNA FISH方法对水稻染色体组进行核型分析的可行性和优势.     

小黑麦杂种后代的Giemsa C带研究

采用Ｇｉｅｍｓａ Ｃ显带技术，对小黑麦杂种后代的染色体组成进行了分析鉴定。从中鉴定出附加１Ｒ、４Ｒ的二体异附加系和附加７Ｒ的单体异附加系。     

Morphological and molecular characterization of two G. somalense monosomic alien addition lines （MAALs）

Two G. somalense monosomic alien addition lines were identified from the derived backcross progenies of allohexaploid between G. hirsutum and G. somalense through cytological and morphological observation. Furthermore, the alien addition chromosome was identified and distinguishedby RAPD analysis. A total of 160 RAPD primers were usedfor PCR amplification. Primer SBSG11 could produce a specific molecular marker (600 bp) for monosomic alien addition line Ⅰ (MAAL Ⅰ ). Primer SBSC03 could produce aspecific molecular marker (700 bp) for monosomic alien ad-dition line Ⅱ (MAAL Ⅱ). SBSE07 and SBSE08 could re-spectively produce common molecular marker for mono-somic alien addition lines Ⅰ and Ⅱ. G. somalense alienaddition lines could be important for cotton improvement.     

Physical location of riceGm-6, Pi-5(t) genes inO. officinalis with BAC-FISH

A fluorescence in situ hybridization (FISH) procedure was adopted to physically map two rice BAC clones 24E21 and 4F22 linked to Gm-6 and Pi-5(t) in O. offi-cinalis. FISH results showed that the two BAC clones were located at 4L. The percentage distance from the centromere to the hybridization sites was 72 + 2.62 for 24E21 and 54+ 5.43 for 4F22, the detection rates were 52.70% and 61.2%. The results obtained from the BAC and plasmid clones, RG214 and RZ565 of cultivated rice and O. officinalis were the same. This suggested that the markers, RG214 and RZ565 of cultivated rice and O. officinalis were in the same BAC clones. The homologous sequences of Gm-6 and Pi-5(t) in O. officinalis were positions that signals existed on the 4L. Many signals were observed when no Cot-1 DNA blocked. This also showed that repetitive sequences were some ho-molgous between cultivated rice and O. officinalis. The identification of chromosome 4 of O. officinalis is based on Jena et al. (1994). In our study, we discussed the possibility of physical map in O. officinalis with rice BAC clones.     

控制水稻剑叶形态相关性状的数量基因位点(QTL)的定位

水稻籽粒中一半以上的碳水化合物来自剑叶的光合作用，剑叶形态改良一直是水稻株型育种的一个重要目标．利用一个日本主要种植的粳稻品种越光（轮回亲本）和一个印度的籼稻品种Kasalath杂交产生的回交重组自交系群体（backcross recombinant inbred lines，BILs）对剑叶形态中的3个主要性状（剑叶长、叶宽以及其叶面积）进行了相关分析及其数量基因位点（quantitative trait loci，QTL）的定位．研究表明，控制剑叶形态的3个主要性状间存在极显著的正相关，并检测到影响3个性状的8个QTL，分布在第1，3，4，6条染色体上，贡献率介于4．94％～22．07％，其中第4染色体上C1016标记和第6染色体上C556标记附近的共有6个QTL，其两侧的紧密分子标记在水稻株型分子育种上具有一定应用价值．     

Comparative physical mapping of rice BAC clones linked to resistance genes Glh,Bph-3 and xa-5 in Oryza sativa L. and O. granulata Nees et Arn. ex Watt.

Oryza granulata Nees et Arn. ex Watt. is one of the three wild relatives of rice,which are the most valuable for study and utilization in China.In this study,the homology and physical locations of three rice resistance genes,Glh,Bph-3 and xa-5 are comparatively analyzed between O.sativa and O. granulata by Southern blotting and fluorescence in situ hybridization (FISH).The results of Southern blotting indicate that there exist homologous sequences of the tested RFLP markers in O. granulata.By using three bacterial artificial chromosome (BAC) clones scanned by the tested RFLP as probes, FISH signals are detected on both mitotic and pachytene chromosomes in O.sativa and O.granulata.Dual-color FISH demonstrates that two of the three BAC clones (14E16 and 38J9) are located on the short arm of the same chromosome pair in O. granulata. Additionally, colinearity is shown for the two clones between O.sativa and O.granulata. Another BAC clone 44B4 is located on the end of the short arm of other chromosome pair in these two species.Although the phylogenetic relationship between O.sativa and O.granulata is the most distinct in Oryza and these two species have evidently different biological features and ecological habits, the relative lengths and arm ratios of the detected chromosomes and the relative positions of the tested clone signals on chromosomes in O. granulata are quite similar to those in O. sativa.     

Sequence and analysis of rice chromosome 4

Rice is the principal food for over half of the population of the world. With its genome size of 430 megabase pairs (Mb), the cultivated rice species Oryza sativa is a model plant for genome research. Here we report the sequence analysis of chromosome 4 of O. sativa, one of the first two rice chromosomes to be sequenced completely. The finished sequence spans 34.6 Mb and represents 97.3% of the chromosome. In addition, we report the longest known sequence for a plant centromere, a completely sequenced contig of 1.16 Mb corresponding to the centromeric region of chromosome 4. We predict 4,658 protein coding genes and 70 transfer RNA genes. A total of 1,681 predicted genes match available unique rice expressed sequence tags. Transposable elements have a pronounced bias towards the euchromatic regions, indicating a close correlation of their distributions to genes along the chromosome. Comparative genome analysis between cultivated rice subspecies shows that there is an overall syntenic relationship between the chromosomes and divergence at the level of single-nucleotide polymorphisms and insertions and deletions. By contrast, there is little conservation in gene order between rice and Arabidopsis.     

Development and characterization of interspecific hybrids between Oryza sativa and O. latifolia by in situ hybridization

Oryza sativa and O. latifolia belong to the AA and CCDD genomes of Oryza, respectively. In this study, interspecific hybrids of these species were obtained using the embryo rescue technique. Hybrid panicle traits, such as long awns, small grain, exoteric large purple stigma, grain shattering and dispersed panicles, resemble that of the paternal parent, O. latifolia, whereas there is obvious heterosis in such respects as plant height, tillering ability and vegetative vigor. Chromosome pairing and the genomic components of the hybrid were subsequently investigated using genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) analysis. Based on the mitotic metaphase chromosome numbers of the root tips investigated, the hybrid is a triploid with 36 chromosomes. The genomic constitution of the hybrid is ACD. In the meiotic metaphase I of the hybrid pollen mother cell, poor chro- mosome pairing was identified and most of the chromosomes were univalent, which resulted in com- plete male sterility in the hybrid.     

Mapping of a new gene for brown planthopper resistance in cultivated rice introgressed fromOryza eichingeri

Wild rice species is an important source of useful genes for cultivated rice improvement. Some accessions of Oryza eichingeri (2n = 24, CC) from Africa confer strong resistance to brown planthopper (BPH), whitebacked planthopper (WBPH) and bacterial blight (BB). In the present study, restriction fragments length polymorphism (RFLP) and simple sequence repeats (SSR) analysis were performed on disomic backcross plants between Oryza sativa (2n = 24, AA) and O. eichingeri in order to identify the presence of O. eichingeri segments and further to localize BPH-resistant gene. In the introgression lines, 1—6 O. eichingeri segments were detected on rice chromosomes 1, 2, 6, or/and 10. The dominant BPH resistant gene, tentatively named Bph13(t), was mapped to chromosome 2, being 6.1 and 5.5 cM away from two microsatellite markers RM240 and RM250, respectively. The transfer and localization of this gene from O. eichingeri will contribute to the improvement of BPH resistance in cultivated rice.     

稻属基因组间相关性的AFLP分析

应用AFLP（amplified restriction fragment length polymorphism)－银染法对2种栽培稻和21种野生稻的基因组多态性进行了检测,共计使用了9对选择性引物组合,扩增出602个遗传位点,通过Jacard的方法将电泳带矩阵转化为遗传相似性系数矩阵,并进行了聚类分析和主成分分析,结果表明,稻属植物按其亲缘关系的远近被分为三个大的类群：O.sativa群,O.officinalis群和其它远缘混合群,本文还对两种栽培稻的起源途径以及稻属物种的不同类型基因组间的缘关系进行了初步的探讨,为深入研究稻属物种间的进化关系提供参考。     

Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH （genome in situ hybridization） and AFLP （amplified fragment length polymorphism） on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism （MSAP） analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines （CN12, 20.15%; CN17, 20.91%; CN18, 22.42%）, but the ratios of hemimethylated sites were significantly lowered （CN12, 21.41%; CN17, 23.43%; CN18, 22.42%）, whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns （33.74%）, 9 demethylation patterns （22.76%） and 7 uncertain patterns （4.07%）. In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.