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指纹标记的亚洲栽培稻与野生稻遗传多样性分析

为评价野生稻与亚洲栽培稻的遗传多样性及其变异关系,21对SSR引物被用于研究广泛地理分布的17份野生稻(其中11份O. rufipogon和6份O. nivara)和22份亚洲栽培稻(13份indica和9份japonica)样本.271条多态性片段被扩增出,占总扩增片段的96.10%.野生稻多态性片段的百分比(平均达64.30%)及Nei's遗传多样性值(GD)明显高于亚洲栽培稻,表明野生稻比亚洲栽培稻具更丰富的遗传多样性.UPGMA聚类分析显示野生稻的两个类群(O. rufipogon和O. nivara)关系密切,但在遗传上存在明显的分化,支持其作为两个独立物种的分类观点.野生稻中籼粳分化不很明显,亚洲栽培稻的籼粳亚种分化明显.亚洲栽培稻与多年生普通野生稻(O. rufipogon)关系更为密切,符合异源起源的遗传分化模式.     

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.     

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.     

一个药用野生稻异源单体附加系在减数分裂时期的GISH分析鉴定

异源单体附加系是从亲缘关系较远或属间的一个物种单条染色体附加到另一个物种中．栽培稻珍籼97B与药用野生稻Hy18杂交与连续回交，在BC2后代中得到一个药用野生稻单体附加系．生物素标屺的药用野生稻总DNA作为探针，未标记的栽培稻总DNA封阻，对其异源单体系减数分裂染色体进行基因组原位杂交．FISH结果表明，在栽培稻（AA，2n=24）基因组中附加了一条药用野生稻染色体，并鉴定为第8号染色体．研究表明，药用野生稻异源单体附加系的建立为药用野生稻的基因组学和遗传学的研究提供一个新的操作平台．而GISH技术在水稻远缘杂交育种中是最准确有效的染色体鉴定方法．在水稻育种改良中具有重要应用前景．     

禾本科同源染色体对趋同进化的比较基因组学

多个禾本科物种全基因组测序的相继完成为禾本科植物基因组物理和遗传结构进化历史的研究提供了前所未有的良好机遇。以五个禾本科物种为研究对象,利用基因同源共线性方法对其基因组进行了比对分析,获得了物种的同源信息,并根据同源信息结合基因组同源结构分析,确定了物种基因组内和基因组间的同源染色体片段。比较分析同源染色体对上重复DNA片段之间的分子距离,初步揭示了禾本科植物同源染色体对间趋同进化规律,研究结果有助于理解染色体结构受非正常遗传重组影响的进化机制。     

基于叶绿体基因组序列对50种菊科植物亲缘关系分析

菊科为双子叶植物纲的第一大科,其科内等级划分和系统学研究存在巨大挑战。叶绿体基因组具有高度的保守性和较慢的进化速率,其在植物系统发育和物种进化研究中应用广泛。对菊科20个属50种植物叶绿体基因组序列进行亲缘关系分析,基于叶绿体基因组编码序列(coding sequence, CDS)、rbcL基因序列、rbcL+matK基因序列分别构建系统发育树,并对其中8个物种进行密码子偏好性分析和IR(Inverted Repeat)边界分析。比较3种方法构建的系统发育树,发现利用单个基因序列或两个基因序列构建的系统发育树与CDS序列构建的系统发育树在属间结果不一致,且CDS构建的系统发育树自展支持率高于其余两种,表明基于单基因序列或基因串联序列适用于属内物种的研究,而CDS序列适用于属间物种的研究。密码子偏好性研究显示选取的8个物种都对A/U结尾的密码子有偏好性。边界序列分析结果显示4个物种菊科植物的IR边界序列ycfI基因有缺失。研究结果为菊科植物的遗传进化和系统发育研究提供了理论支撑。     

地黄居群遗传多样性的ISSR分析

采用ISSR分子标记,对地黄（Rehnwmnia glutinosa）14个自然居群、1个栽培居群和属内近缘种5个居群的遗传多样性与遗传结构进行了研究．结果表明：地黄不同居群的平均多态位点百分率（PPB）为40．53％,平均Shannon多样性指数为0．1915,平均Nei’s基因多样性指数h为0．1253,遗传多样性水平较低．AMOVA分析结果和基因分化系数显示遗传变异主要发生在居群内,居群间的遗传分化显著,其遗传分化水平介于异交物种和自交物种之间．地黄居群间的基因交流程度有限（基因流为0．3784）．Mantel检测显示自然居群间遗传距离与地理距离之间没有明显相关性．推断营养繁殖占优势和受人类栽培活动的长期影响可能是导致地黄遗传多样性水平低、居群间遗传分化显著的主要原因．     

基于ITS序列的竹亚科植物分子鉴定研究

利用ITS序列对竹亚科13个属76个竹种进行DNA条形码分子鉴定.该研究提取慈竹属、刚竹属、箬竹属、唐竹属、矢竹属、大明竹属、赤竹属、巴山木竹属、倭竹属、短穗竹属、簕竹属、酸竹属及少穗竹属共13个属62个竹种DNA,对其内转录间隔区ITS序列进行PCR扩增和双向测序,所得序列与GenBank数据库下载的14条ITS序列共76个竹种序列用Clustal X软件比对,再利用MEGA6.06软件构建K2P距离法的NJ系统发育树.结果显示,种内遗传距离为0~3.907,平均遗传距离为0.370;种间遗传距离为0~4.394,平均遗传距离为2.287,种内遗传距离远小于种间遗传距离.ITS序列可用于竹类资源的物种鉴定研究.     

籼-粳稻特异插入／缺失分子标记揭示的稻属植物遗传分化

籼-粳分化在亚洲栽培稻(Oryza sativa L.)的驯化过程中非常重要,但其分化机制仍不清楚.有的学者认为水稻籼-粳分化是亚洲栽培稻在驯化过程中适应不同生境的结果,也有人认为籼-粳分化在水稻的野生祖先种中就已经存在.为了研究普通野生稻的籼-粳分化,并解析稻属(Oryza)植物的籼-粳遗传变异,利用34对籼-粳特异插入/缺失分子标记(InDel)引物,研究了50份典型籼稻(O.sativa L.subsp.Indica Kato)和粳稻(O.sativa L.subsp.japonica Kato)样本以及来源于35个国家的348份稻属其他种材料.结果表明,亚洲栽培稻存在明显的籼-粳分化,普通野生稻复合体(O.rufipogon complex)中存在"偏籼"和"偏粳"类型,而稻属的其他种均未出现籼-粳分化,普通野生稻复合体中"偏籼"和"偏粳"类型的地理分布格局与籼稻和粳稻的地理分布格局相吻合.考虑到大部分普通野生稻复合体的样本取自邻近有栽培稻种植的普通野生稻群体,推测得出部分普通野生稻样本中表现出的"偏籼"和"偏粳"类型可能是栽培稻的籼稻品种和粳稻品种在普通野生稻的长期协同进化过程中基因交流的结果.     

Can transgenic rice cause ecological risks through transgene escape?

Alien transgene escape from genetically engineered rice to non-transgenic varieties or close wild relatives (including weedy rice) may lead to unpredictable ecological risks. However, for transgene escape to occur three conditions need to be met: (i) spatially, transgenic rice and its non-transgenic counterparts or wild relatives should have sympatric distributions; (ii) temporally, the flowering time of transgenic rice and the non-transgenic varieties or wild relatives should overlap; and (iii) biologically, transgenic rice and its wild relative species should have such a sufficiently close relationship that their interspecific hybrids can have normal generative reproduction. This paper presents research data on the geographic distribution, flowering habits, interspecific hybridization, and gene flow of cultivated rice (Oryza sativa) and its closely related wild relatives containing the AA genome. The objective is to estimate the possibility of transgene escape to non-transgenic rice varieties and wild relatives of rice, which may result in unpredictable ecological risks.     

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.     

Production of bacterial blight resistant lines from somatic hybridization between Oryza sativa L. and Oryza meyeriana L

Novel bacterial blight (BB) resistance gene(s) for rice was (were) introduced into a cultivated japonica rice variety Oryza sativa (cv. 8411), via somatic hybridization using the wild rice Oryza meyeriana as the donor of the resistance gene(s). Twenty-nine progenies of somatically hybridized plants were obtained. Seven somatically hybridized plants and their parents were used for AFLP (amplified fragment length polymorphism) analysis using 8 primer pairs. Results confirmed that these plants were somatic hybrids containing the characteristic bands of both parents. The morphology of the regenerated rice showed characters of both O.sativa and O.meyeriana. Two somatic hybrids showed highest BB resistance and the other 8 plants showed moderate resistance. The new germplasms with highest resistance have been used in the rice breeding program for the improvement of bacterial blight resistance.     

转基因水稻与野生稻（Oryza nivara Sharma et Shastry）杂交后代种子萌发率

种子休眠性是野生植物重要的适应性状,通过种子萌发实验可以分析种子休眠性强弱.通过人工杂交获得转抗虫基因栽培稻与一年生普通野生稻三种组合的杂种,对杂种的F3代和F4代种子采用直接萌发、打破休眠后萌发、埋土15 d和30 d 4种不同处理来检测种子活力和萌发率.结果显示转基因杂种后代种子表现出较强的休眠性,转基因对种子活力和休眠性没有明显的影响,种子休眠性有随种子世代的增加而逐渐减弱的趋势,提示水稻转基因逃逸后有在野生稻群体中宿存和扩散的可能性,但这种可能性可能会随世代增加而下降.这为进一步研究水稻转基因逃逸风险提供了参考.     

Spacer length variation in rice 5S rRNA genes revealed by polymerase chain reaction

Polymerase chain reaction (PCR) was used to amplify 5S rRNA spacer from wild rice (Oryza rufipogon and O. nivara) and cultivated rice (indica and japonica varieties of O. sativa L). The results show that there is spacer length variation within and between species, and the typical indica and japonica varieties have their unique banding patterns of amplified 5S rRNA spacers, whereas intermediate showed no specific amplification profile of spacer regions. The 5S rRNA genes in intermediate are either identical with that of indica variety or that of japonica variety. These data suggest that the spacer length polymorphisms can be used to distinguish between closely ralated species and subspecies. Supported by the National Natural Science Foundation of China Yi Qingming, born in Apr. 1938, Professor     

水稻的起源与驯化——来自基因组学的证据

 水稻是世界上最重要的粮食作物, 约1 万年前开始被驯化。由于水稻与其祖先野生种存在一定的遗传分化, 水稻的起源和驯化问题长期存在争议。本文综述了水稻起源和驯化方面的研究成果, 特别是近年来基因组学方面的证据, 认为水稻2 个亚种独立起源于野生祖先种内很早就分化的不同类群, 但一些驯化基因--控制重要农艺性状的基因, 可能首先在一个亚种中被驯化, 然后通过基因渐渗, 扩散到另一个亚种中。因此, 水稻驯化的关键是研究驯化基因的起源和扩散方式。随着大规模基因组测序技术的发展和相应数据分析方法的建立, 在全基因组水平对水稻及其祖先进行大规模分析, 已成为揭示水稻起源与驯化之谜的必由之路。     

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.     

Identification and mapping of genes for improving yield from Chinese common wild rice (O. rufipogon Griff.) using advanced backcross QTL analysis

To identify useful genes from wild rice which have been lost or weakened in cultivated rice has become more and more important for modern breeding strategy. In this study, a BC4 population derived from 94W1, an acces-sion of common wild rice (Oryza rufipogon Griff.) from Dongxiang in Jiangxi Province of China, as the donor, and a high-yielding Indica cultivar (O. sativa L.), "Guichao 2", as the recipient, was used to identify quantitative trait loci (QTL) associated with yield and its components. Based on the analysis for the genotype of BC4F1 population with 87 SSR markers distributed throughout the genome and investigation of the plant height, yield and yield components of BC4F2, a total of 52 QTLs, were detected. Of 7 QTLs associated with grain yield per plant, 2 QTLs on chro-mosome 2 and chromosome 11 for grain yield, explaining 16% and 11% of the phenotypic variance respectively, were identified. The alleles from Dongxiang common wild rice in those two loci could increase the yield of "Guichao 2" by 25.9% and 23.2% respectively. The QTL on chromosome 2 increasing grain yield of cultivar is actually a major gene, which did not coincide with any previously published QTLs in rice.