RFA法构建水稻DNA指纹图谱

水稻基因组DNA用Psf I酶切同时与人工接头连接后,使用选择性引物进行PCR扩增,琼脂糖凝胶电泳检测所构建的水稻DNA指纹图谱。结果表明在JXl7和ZYQ8问以及5种野生稻间均存在DNA多态性片段。     

中国杂交籼稻DNA甲基化多样性与遗传稳定性

水稻育种一直关注DNA序列多样性,但表观多样性(如DNA甲基化)也影响性状,不应被忽略.分析了20个杂交稻育种中广泛应用的籼稻亲本系间的DNA甲基化多样性及其遗传稳定性.结果表明,水稻育种系之间广泛存在DNA甲基化多样性;DNA甲基化与序列多样性之间存在正相关关系;DNA甲基化可以通过自交或杂交遗传给下一代.本研究揭示了DNA甲基化在水稻育种中应用的价值.     

重金属胁迫引起的水稻和小麦幼苗DNA损伤

供试浓度的重金属Cu2 、Cd2 和Hg2 能在不同程度上导致水稻和小麦DNA损伤。 (1)Hg2 ≥ 0 .0 2 5mmol L、Cu2 和Cd2 ≥ 0 .1mmol L处理使水稻幼苗期外DNA合成速率显著高于对照 ,且当Cu2 、Cd2 和Hg2 浓度进一步增高时 ,水稻幼苗期外DNA合成速率呈现先上升而后较最大值有所下降的规律。 (2 ) 0 .1～1.0mmol L的Cu2 及 0 .1mmol L的Cd2 、Hg2 能引起水稻、小麦叶内DNA和蛋白质交联 ,并且这种和DNA交联的蛋白质易被胰蛋白酶水解。 (3) 0 .1～ 1.0mmol L的Hg2 及 1.0mmol L的Cd2 还能使水稻、小麦叶片内DNA链间发生交联     

太湖稻区与国内部分香稻SSR指纹图谱构建及遗传多样性初析

以太湖稻区和国内其他地区的39个香稻品种,以及籼型恢复系2个对照品种为研究材料,利用SSR分子标记进行DNA指纹图谱构建和遗传多样性分析。从40对国标中公布的水稻SSR引物中筛选出13对核心引物,在41个水稻品种中共检测到36个多态性片段。据此建立41个水稻品种的DNA指纹图谱,进行聚类分析,发现供试41个品种的遗传相似系数在0.58以上,而供试香稻品种的遗传相似系数在0.64以上,基本反映了不同品种间的亲缘关系。     

环丁烷嘧啶二聚体累积与水稻UV—B敏感性的关系

利用单克隆抗体ELISA ,研究了UV_B对水稻DNA中环丁烷嘧啶二聚体 (CPD)的诱导形成及其光、暗修复 ,并对CPD累积与水稻UV_B敏感性的关系进行了分析。结果表明 ,我国南方的 5个水稻 (OryzasativaL .)品种经13.6kJ·m-2 ·d-1UV_B处理 15d后 ,在株高、生物量、光合作用等方面表现出明显的品种间差异。不同品种水稻的DNA中CPD累积比对照明显增加 ,且敏感品种CPD的累积比抗性品种显著提高。统计分析证实 ,CPD的累积与生物量的抑制呈显著正相关 (r2 =0 .6 2 2 )。UV_B诱导的水稻DNA中CPD的清除以光修复为主 ,不同品种CPD暗修复能力相似 ,而光修复能力存在明显差异。根据以上结果推测 ,不同水稻品种UV_B敏感性与CPD光修复能力的差异有关。     

转Xa21基因水稻中T-DNA整合的遗传定位

利用转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR方法扩增T－DNA整合的侧翼序列。从中筛选属于水稻基因组DNA的T－DNA整合的侧翼序列作为探针,将外源基因整合位点定位到窄叶青／京系17DH群体构建的水稻分子连锁图谱上。共获得属于水稻基因组DNA的T－DNA侧翼序列22个,其中的19个序列在定位群体的两个亲本之间显示RFLP多态性,分别定位在水稻基因组的第3,4,5,7,9,10,11和12染色体上。带有转基因Xa21的T－DNA整合的定位为研究外源基因在不同染色体位点的位置效应和稳定遗传打下基础。     

水稻中期染色体和DNA纤维的高效制备技术

水稻中期染色体和DNA纤维制备是水稻分子细胞遗传学研究中的关键技术。目前,这两个技术还有很多不足,该研究建立了高效制备水稻中期染色体和DNA纤维的方法。该方法制备的染色体,分裂相多、杂质少、背景清晰、染色体分散且形态好,水稻根尖分生组织细胞的分裂指数高达25％。植物细胞的细胞壁是制备DNA纤维的最大障碍,所以必须先提取细胞核,然后裂解细胞核释放出DNA纤维。在这个研究中,还建立了一个用刀切法分离细胞核,进而用SDS裂解核膜,用载玻片拖出DNA来制备水稻DNA纤维的方法。该方法制备的DNA纤维多呈平行的细线,背景清晰,伸展的程度均匀,适合于原位杂交。     

Shotgun法测序制备高纯度水稻基因组DNA的方法探讨(英)

在用散弹 (shotgun)法测定水稻 (OryzasativaL .ssp .indica)基因组全序列的过程中 ,叶绿体和线粒体DNA的污染问题非常严峻 .应用脉冲场电泳 (PFGE)技术对水稻基因组DNA进行纯化 ,结果表明它能够有效去除叶绿体和线粒体DNA ,使其污染率从 3%降低到 0 2 % .同时 ,比较了水稻绿苗和黄化苗的DNA得率 ,以及HB法和NIB法制备大分子质量(HMW)DNA的异同 .最后提出一套制备水稻基因组DNA的方法 ,包括黄化苗培养 ;细胞核的分离、包埋和裂解 ;脉冲场电泳纯化、回收聚集在低熔点 (LMP)胶中的水稻HMWDNA .用该方法所得的水稻基因组DNA ,纯度高 (无叶绿体和线粒体DNA污染 )、基因组完整 (机械剪切和降解少 )、回收率高 (操作过程中DNA损失少 ) .另外 ,首次报道在融化的低熔点(LMP)胶中对水稻HMWDNA于 38℃进行超声波处理 ,能够获得用于shotgun文库和梯度文库构建所需要的各种DNA片段(1 5～ 3kb ,3～ 12kb) ,并且效果优于在TE中进行操作     

RAPD标记鉴定水稻3种不同细胞质雄性不育类型的杂交种及其亲本

利用RAPD引物对3种不同细胞质雄性不育类型的杂交水稻组合及其亲本共21个材料进行了DNA多态性分析。从264个随机引物中筛选出具有非常明显多态性的引物25个, 对25个引物在3种不同细胞质不育类型的杂交组合及其亲本间的DNA扩增多态性差异进行比较,最终选出具有不同类型间特异性扩增带的引物7个,利用这些特异性扩增带能有效地区分和鉴定目前在生产上大面积种植和推广,或者是具有应用潜力的3种不同细胞质雄性不育类型—野败型(WA)、红莲型(HL)和包台型(BT))—的6种杂交水稻组合及其亲本。     

黑龙江省近年审定水稻品种基于SSR标记的遗传多样性分析

为评估黑龙江省水稻品种的遗传基础,利用24个用于水稻DNA指纹图谱构建的SSR标记以及其他均匀分布于水稻12条染色体的38个SSR标记,对黑龙江省近年审定的73个水稻常规稻品种进行遗传多样性分析。结果表明,在62个SSR标记位点中,共检测到142个等位基因,平均每个标记2.3个,多态性比率平均为71.0%,多态性频率变幅为0~0.775,平均值为0.246。供试品种间两两遗传相似系数的平均值为0.759,变幅为0.622~0.966,且96.4%的品种间遗传相似系数在0.66~0.86之间,表明供试的73个品种亲缘关系较近。通过SSR标记基因型聚类分析将这些品种划分为6个类群,与系谱分析趋势一致,类群间的差异主要表现在生育期和米质方面。综上所述,黑龙江省近年审定的水稻品种遗传基础狭窄,在育种中需要导入新的种质资源,加强种质资源创新,以期丰富水稻品种的遗传多样性,进一步提高水稻产量和抗性。     

Construction of Rice DNA Finger Print by SRFA

ＲＡＰＤ用于生物鉴定具有快速、简便、经济等优点。但是,由于该法所用引物通常为９～１０个寡聚核苷酸,与ＰＣＲ使用特异引物相比,其Ｔｍ值相对较低,扩增反应易受外界条件影响,重复性较差。ＳＲＦＡ技术采用设计有限制内切酶位点的人工合成接头与引物互补,弥补了ＲＡＰＤ法的上述不足。Ｆｉｇ．１为ＳＲＦＡ的技术路线。为提高连接效率,在同一试管内,用ＰｓｔＩ酶解基因组ＤＮＡ,并与人工接头连结,制备ＳＲＦＡ扩增的模板。合成与接头序列相应的引物。对南方５个野生稻品种和籼、粳稻各一个栽培品种进行ＳＲＦＡ扩增。Ｆｉｇ．２表示：每种材料均能获得约１０条以上的扩增条带。条带的大小在４００２０００ｂｐ之间。栽培稻品种中出现多态性片段：用引物１可得一条（１．３ｋｂ）片段,用引物２可得２条（１．１ｋｂ、０．７ｋｂ）片段。与ＦＡＰＤ法相比,ＳＲＦＡ法增加２０％的多态性。栽培稻与野生稻之间在多态性上没有差异,说明两者亲缘关系非常接近。五种野生稻的图谱可分为两类：江西、湖南、广西的与籼稻相近,广东、云南的与粳稻相近。当然,这还有待其它方面的研究来验证。接头的设计要求避免自身连结,与目的片段连结后不能再被ＰｓｔＩ切开。引物包括不变序列和选择序列两部     

Genetic variation detected by DNA fingerprinting with a rice minisatellite probe in Oryza sativa L.

A rice minisatellite probe detecting DNA fingerprints was used to assess genetic variation in cultivated rice (Oryza sativa L.). Fifty-seven cultivars of rice, including 40 closely related cultivars released in the US, were studied. Rice DNA fingerprinting revealed high levels of polymorphism among distantly related cultivars. The variability of fingerprinting pattern was reduced in the closely related cultivars. A genetic similarity index (S) was computed based on shared fragments between each pair of cultivars, and genetic distance (D) was used to construct the dendrograms depicting genetic relationships among rice cultivars. Cluster analysis of genetic distance tended to group rice cultivars into different units corresponding with their varietal types and breeding pedigrees. However, by comparison with the coefficients of parentage, the criterion of relatedness based on DNA fingerprints appeared to overestimate the genetic relationships between some of the closely related US cultivars. Although this may reduce the power of fingerprints for genetic analysis, we were able to demonstrate that DNA fingerprinting with minisatellite sequences is simpler and more sensitive than most other types of marker systems in detecting genetic variation in rice.This paper reports the results of research only. Mention of a proprietary product does not consititute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the US Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, and the University of Missouri Agricultural Experiment Station Journal Series No. 12178.     

High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis

We have developed an automated, high-throughput fingerprinting technique for large genomic DNA fragments suitable for the construction of physical maps of large genomes. In the technique described here, BAC DNA is isolated in a 96-well plate format and simultaneously digested with four 6-bp-recognizing restriction endonucleases that generate 3' recessed ends and one 4-bp-recognizing restriction endonuclease that generates a blunt end. Each of the four recessed 3' ends is labeled with a different fluorescent dye, and restriction fragments are sized on a capillary DNA analyzer. The resulting fingerprints are edited with a fingerprint-editing computer program and contigs are assembled with the FPC computer program. The technique was evaluated by repeated fingerprinting of several BACs included as controls in plates during routine fingerprinting of a BAC library and by reconstruction of contigs of rice BAC clones with known positions on rice chromosome 10.     

Towards rice genome scanning by map-based AFLP fingerprinting

Map-based DNA fingerprinting with AFLP markers provides a fast method for scanning the rice genome. Three hundred AFLP markers identified with ten primer combinations were mapped in two rice populations. The genetic maps were aligned and almost full coverage of the rice genome was obtained. The transferability of AFLP markers between indica × japonica and indica × indica crosses was tested. The chromosomes were divided into DNA Fingerprint Linkage Blocks (DFLBs) defined by specific AFLP markers. Using these blocks, the degree of similarity or divergence within specific chromosome regions was calculated for nine varieties. Applications of map-based fingerprinting for biodiversity studies and maker-assisted selection are discussed. Received: 6 June 1998 / Accepted: 11 November 1998     

Study on genetic diversity of Magnaporthe grisea using PCR and determination of the mating type alleles distribution in Mazandaran province, Iran

The population structure of Magnaporthe grisea, the causal agent of the rice blast, was analyzed in Mazandaran province, using DNA fingerprinting based on RAPD-PCR by means of three primers including "I", "D" and "H". Total DNA of 47 isolates was extracted and amplified according to a specific PCR program. As a result, variable length fragments were generated. Each isolate was subjected to DNA fingerprinting and clonal lineages were determined. Phenetic analysis differentiated three distinct fingerprint lineages. In order to study on fertility status and distribution of the mating type idiomorphs (alleles), 72 monoconidial isolates from Mazandaran province were paired with four standard fertile hermaphrodite isolates. The mating type of 36 isolates was determined as Mat 1-1. The others (36 isolates) did not form any perithecia in pairing with standard isolates     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

Genomic characterization of <Emphasis Type="Italic">Oryza</Emphasis> species-specific CACTA-like transposon element and its application for genomic fingerprinting of rice varieties

A repeated DNA fragment (pKRD) was isolated from the genomic library of weedy rice in Korea. The pKRD showed significant homology to Em/Spm CACTA-like transposon in whole genome sequences of rice released in the Blast rice sequence database of NCBI and was closely related to the TNP2 transposase group, including a TNP-like transposable element of rice. A Southern hybridization experiment demonstrated that the pKRD sequence is unique to the Oryza genome. The 126 sequences homologous to pKRD were evenly distributed in all 12 different chromosomes in rice genomes with multiple copy numbers. Different copy numbers ranging from 1,500 to 4,500 corresponding to rice species were detected by slot blot hybridization. In a DNA fingerprinting experiment, a pKRD probe was assessed to be the potential molecular marker for studying evolution and divergence, biodiversity and phylogenic analysis of rice species.     

Genetic mapping of hypervariable minisatellite sequences in rice (Oryza sativa L.)

Minisatellites, or DNA fingerprinting sequences, have been utilized in animal linkage studies for several years but have not been used as markers for plant genome mapping. In animal genome mapping they have resulted in limited success because they are evenly dispersed in some species but are often clustered near telomeric regions, as observed on human chromosomes. The purpose of the present study was to generate DNA fingerprints utilizing several rice-derived minisatellites containing different core sequences and numbers of repeat units, followed by assessing their potential for use as genetic markers when mapped to a rice recombinant inbred line (RIL) population. Sites of segregating minisatellite loci were mapped onto 11 of the 12 rice RIL linkage maps. The implications for the use of rice minisatellite core sequences as genetic markers on linkage maps in rice are discussed. Received: 1 March 1999 / Accepted: 22 June 1999     

DNA Polymorphism among Rice Somaclones

Molecular markers were used to detect the influence of high concentrations of 2,4 dichlorophenoxyacetic acid (2,4-D) in the callusing media on DNA variations in regenerated rice plants. Restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD) and polymerase chain reaction (PCR) based RFLP analysis were carried out on 12 somaclones of Oryza sativa L. cv. B-370. In vitro culture induced DNA variations were detected in the regenerated plants but the effect of high auxin concentration in the medium could not be revealed. In a second study, fingerprinting of 15 semi-dwarf, high yielding somaclones of B-370 was carried out using RAPD technique. Amplification using 20 random primers produced a total of 167 DNA bands out of which 97 bands were polymorphic. A total of 32 unique DNA bands were detected across all the somaclones and they could be grouped based on their similarity to B-370. RAPD analysis helped to reveal similarity or differences among the somaclones while fingerprinting using additional RAPD markers was not successful.     

用gFM31探针进行谷子品种的指纹分析

用高度多态性探针ｇＦＭ３１对５９个谷子（ＳｅｔａｒｉａｉｔａｒｌｉｃａＢｅａｕｖ．）品种（包括地方品种、育成品种及品系）进行ＤＮＡ指纹分析,共鉴别出５８种类型,而黑谷和黑粒１５１６给出完全相同的带型,二者有可能是同一材料。通过对ｇＦＭ３１ＤＮＡ序列分析,未发现其中有小卫星ＤＮＡ和微卫星ＤＮＡ序列。该探针虽在谷子品种中显示很高的多态性,但与禾谷类的其他物种的ＤＮＡ杂交信号微弱,表现出较强的谷子基因组特异性。