RAPD polymorphisms in spring wheat cultivars and lines with different level of Fusarium resistance

Random amplified polymorphic DNA (RAPD) markers have been used to characterize the genetic diversity among 35 spring wheat cultivars and lines with different levels of Fusarium resistance. The objectives of this study were to determine RAPD-based genetic similarity between accessions and to derive associations between Fusarium head blight (FHB) and RAPD markers. Two bulked DNA from either highly resistant lines or susceptible lines were used to screen polymorphic primers. Out of 160 screened primers, 17 primers generated reproducible and polymorphic fragments. Genetic similarity calculated from the RAPD data ranged from 0.64 to 0.98. A dendrogram was prepared on the basis of a similarity matrix using the UPGMA algorithm, which corresponded well with the results of principal component analysis and separated the 35 genotypes into two groups. Association analysis between RAPD markers and the FHB index detected three RAPD markers, H19(1000), F2(500) and B1(2400), significantly associated with FHB-resistant genotypes. These results suggest that a collection of unrelated genotypes can be used to identify markers linked to an agronomically important quantitative trait like FHB. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.     

Detection of genetic diversity using RAPD-PCR and sugar analysis in watermelon [Citrullus lanantus (Thunb.) Mansf.] germplasm

RAPD (random amplified polymorphic DNA) markers generated by 15 arbitrary decamers were used to determine the frequency of DNA polymorphism in 39 watermelon [Citrullus lanantus (Thunb.) Mansf.] germplasms. Of the 15 primers tested, all except 1 (primer 275) directed the amplification of polymorphic products. A total of 162 amplification products were generated across all 39 genotypes. Among the 162 fragments, 35 (21%) appeared to be reliable polymorphic markers. The mean value by marker difference in this comparison was 0.24, and the highest, 0.69. Eight RAPD markers could be utilized in the unique variety discrimination 8 watermelon genotypes. From the phenograms constructed by UPGMA based on the comparison of RAPD markers, four clusters were resolved. Each group was also characterized and identified with morphological and genetic characteristics for each genotype. The free sugars of the edible parts of watermelons were analyzed by HPLC (high-performance liquid chromatography). Results from the phylogenetic analysis of band sharing data were consistent with sweetness as measured by HPLC. In conclusion, RAPD assays can be used for providing alternative markers for identifying genotypes and quantitative characteristics in watermelon.     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphicDNA)分子标记技术,寻找谭清苏铁(Cycas tanqingii)中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465(CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域(SCAR)分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

Detection of section-specific random amplified polymorphic DNA (RAPD) markers in Lilium

Random amplified polymorphic DNA (RAPD) markers were utilized for the identification of Lilium species and inter-specific hybrids. The optimum annealing temperature of the polymerase chain reaction (PCR) for the RAPD assay in Lilium was 54 °C, which is relatively higher than the temperature used for other genera reported by previous researchers. Among 76 primers used to amplify genomic DNA by PCR, 18 primers (24%) generated polymorphic DNA fragments in Lilium species and hybrids. Cultivars were also identified by RAPD markers. Some amplified fragments were unique to species of each section and to hybrids derived from these species; that is, they were the section-specific DNA markers. Sections, Sinomartagon, Leucolirion b, Leucolirion a and Archelirion could be identified by 6 section-specific markers amplified with five primers. Seven inter-section hybrids showed the section-specific bands of both parental sections, indicating that these markers would be useful for identifying the parental sections of inter-section hybrids.     

DNA polymorphism in various goose lines by RAPD-PCR

RAPD markers often primers were used to assess the polymorphism among pooled DNA of eight goose lines. The number of bands amplified by each primer ranged from 1 to 8, within a mean of 2.86. Some bands appeared specific for the line or genetic background. RAPD technique is an effective method for generating the polymorphic DNA marker in the goose. RAPD patterns from mixed DNA samples can reflect the genetic information of populations. The present study indicated that 10 generations selected for egg production and body weight under various pressure, resulted in genetic variation among goose lines as detected by RAPD. Selection for meat traits caused greater genetic diversity than selection for egg production.     

Molecular detection of a bacterial contaminant Bacillus pumilus in symptomless potato plant tissue cultures

An aberrant random amplified polymorphic DNA (RAPD) marker in genomic DNA of tissue culture plantlets was frequently observed during a comparison of DNA fingerprints derived from potato germplasm grown in tissue culture and the field. The RAPD marker was cloned, sequenced and determined to be of bacterial origin. A bacterial contaminant was isolated from the tissue culture plants and identified as a Bacillus pumilus. A set of sequence characterised amplified region (SCAR) primers were designed from the sequence of the cloned fragment and tested for the specific detection of B. pumilus. Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) were also used to generate B. pumilus profiles specific to our isolate in order to test and confirm the sequence homology of amplified markers generated from a range of DNA samples isolated from tissue culture plants and pure isolates of B. pumilus-like bacteria.     

谭清苏铁性别连锁的RAPD和SCAR分子标记

利用RAPD(Random amplified polymorphic DNA)分子标记技术,寻找谭清苏铁（Cycas tanqingii）中与性别相关的分子标记,筛选了160个10bp的随机引物,产生了2500多个RAPD条带。只有引物S0465 (CCCCGGTAAC)产生了一条大约500bp的雌性特异RAPD标记,该分子标记出现在所有的供试雌性植株中,而所有的供试雄性植株都不具有该标记。对该特异片段进行了克隆和序列测定,并根据序列分析结果将RAPD标记转化为重复性和特异性更好的特异特征序列扩增区域（SCAR）分子标记,并命名为STQC-S465-483。分子标记的建立可用于谭清苏铁幼苗性别的早期鉴定,为谭清苏铁就地保护和迁地保护提供技术支持。     

Genetic diversity in North American ginseng (Panax quinquefolius L.) grown in Ontario detected by RAPD analysis

Genetic diversity within North American ginseng (Panax quinquefolius L.) grown in Ontario was investigated at the DNA level using the randomly amplified polymorphic DNA (RAPD) method via the polymerase chain reaction (PCR). A total of 420 random decamers were initially screened against DNA from four ginseng plants and 78.8% of them generated RAPD fragments. Thirty-six of the decamers that generated highly repeatable polymorphic RAPD markers were selected for further RAPD analysis of the ginseng population. With these primers, 352 discernible DNA fragments were produced from DNA of 48 ginseng plants, corresponding to an average of 9.8 fragments per primer, of which over 45% were polymorphic. The similarity coefficients among the DNA of ginseng plants analyzed were low, ranging from 0.149 to 0.605 with a mean of 0.412, indicating that a high degree of genetic diversity exists in the ginseng population. Lower levels of genetic diversity were detected among 3-year-old ginseng plants selected on the basis of greater plant height than among the plants randomly selected from the same subpopulation or over the whole population, suggesting that genetic factors at least partly contribute to morphological variation within the ginseng population and that visual selection can be effective in identifying the genetic differences. The significance of a high degree of genetic variation in the ginseng population on its potential for improvement by breeding is also discussed.     

Identification of Bactrian camel cell lines using genetic markers

Iranian Bactrian camel population is less than 100 animals. Iranian biological resource center produced more than 50 Bactrian camel fibroblast cell lines as a somatic cell bank for conservation animal genetic resources. We compared two type markers performance, including 14 random amplified polymorphic DNA (RAPDs) (dominant) and eight microsatellite (co-dominant) for cell line identification, individual identification and investigation genetic structure of these samples. Based on clarity, polymorphism, and repeatability, four RAPD primers were selected for future analysis. Four RAPD primers and eight microsatellite markers have generated a total of 21 fragments and 45 alleles, respectively. RAPD primers revealed fragment size between 150 to 2000 bp and gene diversity since 0.27 (IBRD) to 0.46 (GC10), with an average of 0.37. Microsatellite markers generated number of alleles per locus ranged from 3 to 11, with an average of 5.62 alleles. The observed heterozygosity ranged from 0.359 (IBRC02) to 0.978 (YWLL08), and expected heterozygosity ranged from 0.449 (IBRC02) to 0.879 (YWLL08). Bottleneck analysis and curve showed that Bactrian camel population did not experience a low diversity. RAPD profiles were especially suitable for investigation population genetics. All primers generated novel and polymorphic fragments. Briefly, our results show that a multiplex PCR based on these markers can still be valuable and suitable for authentication of cell lines, investigating gene diversity and conservation genetic resources in Bactrian camel, while new technologies are continuously developed.     

Inheritance of RAPD markers in channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and their F1, F2 and backcross hybrids

The channel catfish ( Ictalurus punctatus ) has become the most important aquaculture species in the USA. A genetic linkage map in catfish is needed to improve efficiency of breeding by marker-assisted selection (MAS) and for identification of economically important genes such as disease resistance genes. To identify DNA-based genetic polymorphism, the present authors tested 42 randomly amplified polymorphic DNA (RAPD) primers for their utility in identifying genetic polymorphism in catfish. Out of these primers, 22 generated 171 highly reproducible RAPD markers, producing almost eight polymorphic bands per primer. The remaining 20 primers produced an additional 20 polymorphic bands. The RAPD markers were highly reproducible, transmitted to F1 hybrids, and segregated in F2 or backcross progeny in ratios that did not differ from Mendelian expectations. Because the interspecific hybrids of channel catfish and blue catfish are fertile, RAPD markers using the interspecific hybrid system will be useful for rapid construction of genetic linkage maps of catfish and for analysis of important quantitative trait loci.     

Genome relationship among nine species of Millettieae (Leguminosae: Papilionoideae) based on random amplified polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) marker was used to establish intergeneric classification and phylogeny of the tribe Millettieae sensu Geesink (1984) (Leguminosae: Papilionoideae) and to assess genetic relationship between 9 constituent species belonging to 5 traditionally recognized genera under the tribe. DNA from pooled leaf samples was isolated and RAPD analysis performed using 25 decamer primers. The genetic similarities were derived from the dendrogram constructed by the pooled RAPD data using a similarity index, which supported clear grouping of species under their respective genera, inter- and intra-generic classification and phylogeny and also merger of Pongamia with Millettia. Elevation of Tephrosia purpurea var. pumila to the rank of a species (T. pumila) based on morphological characteristics is also supported through this study of molecular markers.     

草鱼基因组DNA一些RAPD位点的遗传分析及分子标记筛选

RAPD技术的实验结果很容易因实验条件和反应参数的不同而造成差异。为了建立能通用的草鱼基因组多态性分析RAPD分子标记体系,需要利用RAPD位点按照孟德尔共显性规律遗传的特点,用不同遗传背景的材料对多态性的RAPD位点进行统计遗传学比较分析以判断其真实性。为此,本实验选择具有遗传多态性的湘江流域草鱼群体和经连续两代人工诱导雌核发育获得的雌核发育草鱼品系,对一些可能作为草鱼基因组DNA分子标记的RAPD位点进行了遗传学比较分析。所用的10条多态性随机引物共检测到30个多态性RAPD位点。两个不同遗传背景群体的遗传统计对比分析结果表明:这30个多态位性位点在湘江流域草鱼群体和雌核发育草鱼群体中的分布符合孟德尔遗传规律,可以作为草鱼基因组DNA分析的可靠分子标记。本实验的观察结果还表明:在人工诱导雌核发育过程中,存在RAPD位点的快速丢失现象,两次人工诱导雌核发育过程中共丢失了17个多态性位点。因此,加强对自然水体中草鱼种质资源多样性的保护和利用各种现代生物学技术纯化、筛选和组合优良性状基因,是草鱼遗传育种中同样重要和不可或缺的两个方面。       

Assessment of genetic variation withinBrassica campestris cultivars using amplified fragment length polymorphism and random amplification of polymorphic DNA markers

Genetic relationships were evaluated among nine cultivars ofBrassica campestris by employing random amplification of polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers. RAPDs generated a total of 125 bands using 13 decamer primers (an average of 9.6 bands per assay) of which nearly 80% were polymorphic. The per cent polymorphism ranged from 60–100%. AFLP, on the other hand generated a total of 319 markers, an average of 64 bands per assay. Of these, 213 were polymorphic in nature (66.8%). AFLP methodology detected polymorphism more efficiently than RAPD approach due to a greater number of loci assayed per reaction. Cultivar-specific bands were identified, for some cultivars using RAPD, and for most cultivars with AFLP. Genetic similarity matrix, based on Jaccard’s index detected coefficients ranging from 0.42 to 0.73 for RAPD, and from 0.48 to 0.925 for AFLPs indicating a wide genetic base. Cluster analyses using data generated by both RAPD and AFLP markers, clearly separated the yellow seeded, self-compatible cultivars from the brown seeded, self-incompatible cultivars although AFLP markers were able to group the cultivars more accurately. The higher genetic variation detected by AFLP in comparison to RAPD was also reflected in the topography of the phenetic dendrograms obtained. These results have been discussed in light of other studies and the relative efficiency of the marker systems for germplasm evaluation.     

Identification of cultivars and validation of genetic relationships in Mangifera indica L. using RAPD markers

Twenty-five accessions of mango were examined for random amplified polymorphic DNA (RAPD) genetic markers with 80 10-mer random primers. Of the 80 primers screened, 33 did not amplify, 19 were monomorphic, and 28 gave reproducible, polymorphic DNA amplification patterns. Eleven primers were selected from the 28 for the study. The number of bands generated was primer- and genotype-dependent, and ranged from 1 to 10. No primer gave unique banding patterns for each of the 25 accessions; however, ten different combinations of 2 primer banding patterns produced unique fingerprints for each accession. A maternal half-sib (MHS) family was included among the 25 accessions to see if genetic relationships could be detected. RAPD data were used to generate simple matching coefficients, which were analyzed phenetically and by means of principal coordinate analysis (PCA). The MHS clustered together in both the phenetic and the PCA while the randomly selected accessions were scattered with no apparent pattern. The uses of RAPD analysis for Mangifera germ plasm classification and clonal identification are discussed.     

云南普通野生稻遗传多样性和亲缘关系

野生稻（Oryza rufipogon）是稻属的重要组成部分,具有许多优良性状,是水稻遗传改良的天然基因库。本研究通过对形态学性状的观测,及ISSR和RAPDUPGMA聚类分析,将云南普通野生稻划分为4个类型,即元江类型、景洪紫杆直立型、景洪绿杆直立型和景洪匍匐型。在供试材料中筛选到具有多态性的ISSR和RAPD引物各11个,ISSR引物扩增出多态带113条,多态性条带比率（PPB）为82．26％,RAPD引物共扩增出多态性条带76条,PPB值为76．77％,两种分子标记的分析结果呈极显著正相关（r=0.951）。此外UPGMA聚类结果表明,云南普通野生稻不同类型与其它地区普通野生稻之间的遗传亲缘关系差异明显。     

A new strategy employed for identification of sweet orange cultivars with RAPD markers

We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.     

云南普通野生稻遗传多样性和亲缘关系

野生稻(Oryza rufipogon)是稻属的重要组成部分, 具有许多优良性状, 是水稻遗传改良的天然基因库。本研究通过对形态学性状的观测, 及ISSR和RAPD UPGMA聚类分析, 将云南普通野生稻划分为4个类型, 即元江类型、景洪紫杆直立型、景洪绿杆直立型和景洪匍匐型。在供试材料中筛选到具有多态性的ISSR和RAPD引物各11个, ISSR引物扩增出多态带113条, 多态性条带比率(PPB)为82.26%, RAPD引物共扩增出多态性条带76条, PPB值为76.77%, 两种分子标记的分析结果呈极显著正相关(r = 0.951)。此外UPGMA聚类结果表明, 云南普通野生稻不同类型与其它地区普通野生稻之间的遗传亲缘关系差异明显。     

RAPD和ISSR标记对水稻化感种质资源遗传多态性的分析

运用RAPD和ISSR技术分析水稻化感种质资源的遗传多态性。从供试材料中筛选到具有多态性的RAPD引物12条,ISSR引物7条。RAPD引物共扩增到85条清晰的多态性条带,多态性条带比率为69．4％。ISSR引物共扩增到34条清晰的多态性条带,多态性条带比率为53．0％。对两种标记结果进行UPGMA聚类分析,结果极其类似,呈极显著的正相关(r=0．74)。聚类结果表明,地理位置相近的品种聚为一类。部分具有较强化感作用潜力的水稻品种亲缘关系很近,表明控制其化感作用性状的基因可能是等位的相同基因。而部分化感作用潜力差异显著的水稻品种聚为一类,这是由于人类在长期高产品种的定向选择过程中,水稻化感作用性状不被注意而丢失,遗传基础日益狭窄的原因。     

Genetic stability of three economically important micropropagated banana (Musa spp.) cultivars of lower Indo-Gangetic plains, as assessed by RAPD and ISSR markers

An efficient micropropagation protocol produced large number of plants of the three elite banana (Musa spp.) cultivars Robusta (AAA), Giant Governor (AAA) and Martaman (AAB) from shoot tip meristem. The genetic relationships and fidelity among the cultivars and micropropagated plants as assessed by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers, revealed three somaclonal variants from Robusta and three from Giant Governor. A total of 5330 RAPD and 2741 ISSR fragments were generated with 21 RAPD and 12 ISSR primers in micropropagated plants. The percentage of polymorphic loci by RAPD and ISSR were found to be 1.75, 5.08 in Robusta and 0.83, 5.0 in Giant Governor respectively. Among the two marker systems used, ISSR fingerprinting detected more polymorphism than RAPD in Robusta and Giant Governor with most of the primers showing similar fingerprinting profile, whereas Martaman revealed complete genetic stability.     

Optimization of RAPD‐PCR conditions in cattle

Abstract

Random Amplified Polymorphic DNA polymerase chain reaction (RAPD‐PCR) is a fast and easy way of identifying DNA polymorphisms generated from several regions of the genome. This could expedite the process of identifying informative polymorphic markers that may be linked to important genes controlling economic traits. In cattle, failure to obtain consistent amplification patterns in RAPD‐PCR has been a cause for concern. This has been attributed to the fact that decamer primers that are used in RAPD‐PCR reactions are likely to amplify regions of DNA where the primer‐template base pairing has some degree of mismatch and that these mismatches fail to repeat from reaction to reaction. This paper describes the use of tricine buffer along with changes in reaction components and thermal cycling conditions that has yielded consistent and reproducible RAPD‐PCR amplifications using single primers and double primer combinations on bovine DNA.