RAPD markers of mitochondrial origin exhibit lower population diversity and higher differentiation than RAPDs of nuclear origin in Douglas fir

We developed a method of screening RAPD markers for the presence of organelle DNA products using enriched organelle DNA probes, then used these markers to compare the structure of nuclear and mitochondrial RAPD diversity in Douglas fir. Of 237 screened RAPD fragments from 25 primers, 16% were identified as originating in the mitochondrial genome and 3% in the chloroplast genome. The mitochondrial DNA probe correctly distinguished fragments with known maternal inheritance (which is exclusive for the mitochondrial genome in the Pinaceae), and neither of the organelle probes hybridized to biparentally inherited fragments. Mitochondrial RAPD markers exhibited low diversity within populations compared to nuclear RAPD diversity ( H _S = 0.03 and 0.22, respectively), but were much more highly differentiated than were fragments of nuclear origin at both the population ( G _ST = 0.18 and 0.05, respectively) and racial levels ( G _ST = 0.72 and 0.25, respectively). Both nuclear and mitochondrial DNA based phylogenetic analyses identified the varieties as monophyletic groups; the nuclear RAPD markers further separated the north and south interior races.     

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.     

Use of RAPD markers for identifying Nelore bulls with early reproductive maturation onset

Random amplified polymorphic DNA (RAPD) markers were used for identifying animals with early (precocious) or late (non-precocious) reproductive maturation onset. Animals (n=34) were phenotyped according to spermatozoa appearance in ejaculates (group A, 20 animals) or to the expected progeny difference (EPD) values for scrotal circumference (group B, 14 animals). The RAPD markers were initially detected by amplifying two pooled samples of equimolar amounts of DNA from the eight precocious 12 and non-precocious animals of group A. Only 38 out of 320 random primers used for screening group A pooled samples detected polymorphisms. These polymorphic primers generated 443 distinguishable and reproducible bands, from which 174 were polymorphic and 269, monomorphic. These polymorphic primers were then used in RAPD reactions to amplify individual DNA samples from animals belonging to both groups, A and B. The dendrograms generated from RAPD patterns allowed phenotypic class differentiation in both cases. Therefore, RAPD markers can be used as a tool for identifying genotypes favoring early sexual maturation in Nelore breeding programs.     

Application of random amplified polymorphic DNA and inter-simple sequence repeat markers in the genus Crataegus

Hawthorn ( Crataegus spp.) has a long history as an ornamental and a source of medicine. We report the use of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers to determine genetic relationships in the genus Crataegus . Twenty-eight accessions, including eight species ( Crataegus pinnatifida , Crataegus bretschneideri , Crataegus maximowiczii , Crataegus kansuensis , Crataegus altaica , Crataegus songarica , Crataegus dahurica and Crataegus sanguinea ) and two botanical varieties ( C. pinnatifida var. major and C. maximowiczii var. ninganensis ) were analysed. Twelve RAPD primers reproducibly and strongly amplified 128 fragments of which 116 were polymorphic; similarly, 13 ISSR primers generated 127 products of which 119 were polymorphic. Dendrograms based on unweighted pair group method with arithmetic average analysis were constructed from both the RAPD and the ISSR data. Similarity coefficient based on RAPD and ISSR markers ranged from 0.22 to 0.98 and 0.23 to 0.98, respectively. The range in similarity coefficient indicated that the genus has a high level of genetic diversity. The Mantel test on the similarity matrices produced by RAPD and ISSR markers gave r  = 0.86, showing high correlation between RAPD and ISSR markers in their ability to detect genetic relationships between Crataegus accessions. RAPD and ISSR appear to be reliable methods for the analysis of genetic relationships among hawthorns.     

Analysis of tetraploid Elymus species using wheat microsatellite markers and RAPD markers.

An analysis of Amplification fragment polymorphism of DNA from 27 accessions of 19 tetraploid Elymus species was carried out using 18 wheat microsatellite (WMS) primer pairs and 10 decamer primers. Ten WMS primer pairs produced multiple polymorphism on all accessions tested. Two independent phenograms, one based on WMS-PCR and one on RAPDs, separated the 19 tetraploid species into two main groups, viz., the SH genome species group and the SY genome species group. The results coincide with the genomic classification of these species and hence support previous studies showing that Elymus is not a monophyletic genus. The assays indicated that accessions within a species cluster together, which concurs with the morphological classification. Interspecific and intraspecific polymorphisms were detected by the WMS-PCR and RAPD analyses. Variation was observed among accessions of Elymus caninus. The WMS-PCR detected a much higher level of polymorphism than the RAPD analysis. WMSs seem to be more efficient markers than RAPD markers for studying the population diversity of Elymus species. The potential of cross-species amplification of microsatellite markers as an additional source for genetic analysis and applications in Elymus is discussed in the context of these results.     

Assessment of the genetic diversity among strains of Xanthomonas cynarae by randomly amplified polymorphic DNA analysis and development of specific characterized amplified regions for the rapid identification of X. cynarae

The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.     

低等鲤科鱼类RAPD分析及系统发育研究

通过对鲤科鱼类３０个个体所代表的１８个种的随机扩增,作者得到了大量有系统发育信息的ＤＮＡ多态片段。通过Ｒａｐｄｐｌｏｔ程序将ＤＮＡ的多态片段转换成遗传距离（ｄ＝１－Ｓ,Ｓ＝２ＮｘＮｙ／Ｎｘ＋Ｎｙ）。该遗传距离的矩阵经ＰＨＹＬＩＰ软件包中的Ｎｅｉｇｈｂｏｒ（ｏｐｔｉｏｎ＝ＮＪ）程序处理后,生成了低等鲤科鱼类代表属种的分支系统图。从该系统图中以看出：ＲＡＰＤ分析方法在鲤科鱼类的系统发育研究中有一定的局     

SCAR markers for discriminating species of two genera of medicinal plants, Liriope and Ophiopogon

The development of DNA markers that can closely discriminate between Liriope and Ophiopogon species is vital for efficient and accurate identification of these species, and to ensure the quality, safety, and efficacy of medicines made from these plants. We developed species-specific molecular markers for these two genera. Forty RAPD primers were tested to detect polymorphism; species-specific RAPD bands were gel-purified, cloned, and sequenced. Primers for sequence-characterized amplified regions (SCARs) were then designed, based on nucleotide sequences of specific RAPD primers. SCAR markers SA06 and SB05, specific to Ophiopogon japonicus, amplified 460- and 553-bp DNA fragments, respectively. The marker SA12 amplified a 485-bp fragment specific to Liriope platyphylla. This is the first report of a species-specific SCAR marker for this group. These markers will be useful for rapid identification of closely related Liriope and Ophiopogon species.     

Sexing birds using random amplified polymorphic DNA (RAPD) markers

We used random amplified polymorphic DNA (RAPD) markers to sex birds from small tissue (usually blood) samples. Arbitrarily chosen 10-mer PCR primers were screened with DNA from known-sex individuals for the production of a bright female-specific band. Suitable primers were found for seven bird species after screening about 30 primers (range 2–63), and no primer was found for three other species after screening about 50 primers for each species. Investigations into the reliability of RAPD markers for sexing great tits Parus major and oystercatchers Haematopus ostralegus show that: (i) when PCR reaction conditions for great tit DNA are varied, either the presence of the female-specific band correctly predicts the individual's sex or no DNA amplification occurs; (ii) the female-specific band in great tits can be sequenced, and subsequently amplified using specific PCR primers; (iii) null alleles of the female-specific fragment occur at an estimated frequency of 0% ( n = 241 females) in great tits and 0.6% ( n > 290 females) in oystercatchers; (iv) the female-specific fragment in great tits occurs in individuals from a wide geographical range encompassing two subspecies; and (v) the relative intensity of bands in great tit RAPD banding profiles is consistent across individual birds and scorers. The RAPD primers that we have identified are generally species specific, and the consequent time cost of screening for primers is the chief disadvantage of using RAPD markers to sex birds. However, with large sample sizes this disadvantage is outweighed by the relative technical simplicity and low cost of the technique.     

Assessment of the Genetic Diversity among Strains of Xanthomonas cynarae by Randomly Amplified Polymorphic DNA Analysis and Development of Specific Characterized Amplified Regions for the Rapid Identification of X. cynarae

The randomly amplified polymorphic DNA (RAPD) method was used to investigate the genetic diversity in Xanthomonas cynarae, which causes bacterial bract spot disease of artichoke. This RAPD analysis was also intended to identify molecular markers characteristic of this species, in order to develop PCR-based markers which can be used to detect this pathogenic bacterium in artichoke fields. Among the 340 RAPD primers tested, 40 were selected on their ability to produce reproducible and reliable fingerprints in our genetic background. These 40 primers produced almost similar patterns for the 37 X. cynarae strains studied, different from the fingerprints obtained for other Xanthomonas species and other xanthomonad-like bacteria isolated from artichoke leaves. Therefore, X. cynarae strains form a homogeneous genetic group. However, a little DNA polymorphism within this species was observed and the collection of X. cynarae isolates was divided into two groups (one containing three strains, the second one including all other strains). Out of seven RAPD markers characteristic of X. cynarae that were cloned, four did not hybridize to the genomic DNA of strains belonging to other Xanthomonas species. These four RAPD markers were converted into PCR markers (specific characterized amplified regions [SCARs]); they were sequenced, and a PCR primer pair was designed for each of them. Three derived SCARs are good candidates to develop PCR-based tests to detect X. cynarae in artichoke fields.     

DNA fragments of organellar origin in random amplified polymorphic DNA (RAPD) patterns of sugar beet (Beta vulgaris L.)

The technique of random amplified polymorphic DNA (RAPD) offers a broad range of applications in the investigation of plant genomes. A promising prospect is the use of RAPD products as genetic markers. We have investigated a possible organellar source of fragments in RAPD patterns of total DNA. Two nearly-isogenic lines of cytoplasmic male-sterile and male-fertile sugar beet (Beta vulgaris L.) were subjected to RAPD analysis with six different primers. Total, nuclear, mitochondrial (mt), and chloroplast (cp), DNA from each line were investigated. Reproducible DNA fingerprints could be obtained from both organellar DNAs. Differences in band patterns of mtDNA between cytoplasmic male-sterile and -fertile lines were observed with five out of six primers, whereas different cpDNA patterns were generated by one of the primers. Consequently, the RAPD technique can be used to discriminate between different cytoplasms. Clear evidence is provided for the organellar origin of fragments in genomic (total DNA) RAPD patterns. The consequences of these results for the interpretation of RAPD analyses are discussed.     

Identification of random amplified polymorphic DNA (RAPD) marker for differentiating male from female and sporophytic thalli of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Rhodophyta)

There have been limited reports on molecular sex markers for macroalgae. We report the use of random amplified polymorphic DNA analysis (RAPD) to identify molecular sex markers for Gracilaria changii (Xia et Abbott) Abbott, Zhang et Xia. Two DNA extraction methods were used: a modified CTAB and phenol-chloroform combination method and the DNeasy Plant Mini Kit. The CTAB and phenol-chloroform method gave the best yield of DNA in quality and quantity and is suitable for larger-sized specimens like G. changii. Sixty-nine RAPD primers were screened to search for sex-linked DNA markers for G. changii, and only one sex-linked marker (716 bp) was identified using OPA 18. RAPD was also used to investigate the molecular characteristics of the three life-stages (male, female, tetrasporophyte) of G. changii. Seven (OPA7, OPA18, S14, S61, S64, S75 and S76) out of the 69 primers showed polymorphism and were selected for interpopulation analysis for DNA isolated from 23 samples collected from Morib and Sungai Pulai in Malaysia. The combination of data produced by the seven primers generated a dendrogram that grouped the specimens into different clades according to their sex and life-stage using the unweighted pair group and arithmetic averages (UPGMA) method. It showed that RAPD was able to differentiate tetrasporophytes, females, and males. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

两个地区东方田鼠基因组RAPD分析比较研究

目的　从DNA的水平分析比较两个地区东方田鼠的分子遗传特征,探讨以RAPD标记鉴别两个地区的东方田鼠。方法　筛选6条10bp的随机引物对洞庭湖和青铜峡地区的东方田鼠基因组进行了随机扩增多态DNA(RAPD)分析,并对这两个地区的东方田鼠的基因组DNA进行了比较。结果　①两个地区东方田鼠的所有受试个体中共有的片段数为20条,这是两个地区东方田鼠的共性所在;②两个地区东方田鼠各有其特异性扩增片段;③引物S17和S80可作为鉴别两个地区东方田鼠的特异性引物;④不同地区的东方田鼠其不同个体之间的共享度较低,且存在较大差异;两个地区东方田鼠的遗传背景均呈非均一性。结论　运用RAPD方法可以作为鉴别不同地区东方田鼠的基因多态性的标记。     

大豆灰斑病菌生理小种的RAPD标记*

运用随机扩增多态性DNA（RandomamplifiedpolymorphicDNA,RAPD）技术对发生于中国东北的大豆发斑病菌（Cercosporidiumsojinum）的10个生理小种进行基因组DNA多态性分析。用13个10-核苷酸随机引物共计获得了111个RAPD标记,其中86.5％具有多态性,通过聚类分析确定了供试小种间的亲缘关系。试验证明,RAPD技术分析大豆灰斑病菌遗传变异可提供大量分子标记,综合分析13个随机引物的扩增谱带可将供试菌株清楚分开。RAPD技术是一项操作简单、快速和灵敏的方法,极具对病菌群体遗传分析的潜力。     

大豆灰斑病菌生理小种的RAPD标记

运用随机扩增多态性DNA（RandomamplifiedpolymorphicDNA,RAPD）技术对发生于中国东北的大豆发斑病菌（Cercosporidiumsojinum）的10个生理小种进行基因组DNA多态性分析。用13个10-核苷酸随机引物共计获得了111个RAPD标记,其中86.5％具有多态性,通过聚类分析确定了供试小种间的亲缘关系。试验证明,RAPD技术分析大豆灰斑病菌遗传变异可提供大量分子标记,综合分析13个随机引物的扩增谱带可将供试菌株清楚分开。RAPD技术是一项操作简单、快速和灵敏的方法,极具对病菌群体遗传分析的潜力。     

Polymorphism and divergence of multilocus DNA markers in sibling species Chironomus riparius Meigen and Chironomus piger Strenzke (Diptera, Chironomidae)

Intra- and interspecific variation and divergence of multilocus markers for genomic DNA of the sibling species from the thimmi group, Chironomus riparius and C. piger, were studied by PCR with arbitrary primers (RAPD). A high level of RAPD polymorphism was determined in both laboratory and natural populations of these species. The genetic distances were estimated between the C. riparius populations and between the sibling species C. riparius and C. piger. The genetic distance between C. riparius and C. piger was 4 to 5 times higher than that between the C. riparius populations. A comparison of the variation and divergence for the RAPD markers with those for other genomic markers--enzyme-coding genes and chromosomes (linked gene groups)--showed that different components of the genome differed in their contribution to the genome divergence.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Use of RAPD and AFLP markers to identify inter- and intraspecific hybrids of Mentha

Three controlled crosses were carried out involving Mentha arvensis and Mentha spicata [M. spicata CIMAP/C30 x M. spicata CIMAP/C33 (cv. Neera); M. arvensis CIMAP/C18 x CIMAP/C17 (cv. Kalka); and M. arvensis CIMAP/C17 x M. spicata CIMAP/C33]. The parents were subjected to random amplified polymorphic DNA (RAPD) analysis with 80 primers, and polymorphic primers were tested for detecting coinherited RAPD profiles among the progeny of these crosses. Of 50 seedlings tested from each intraspecific cross, all demonstrated dominant profiles with the selected RAPD primers except the detected hybrid from respective crosses. Coinherited markers could be detected with the primers OPJ 01, MAP 06, OPT 08, and OPO 20 for M. arvensis; OPJ 05, OPJ 14, OPO 19, and OPT 09 for M. spicata; and OPJ 07, OPJ 10, OPJ 11, OPJ 14, and OPO 02 for the cross M. arvensis x M. spicata. In our amplified fragment length polymorphism (AFLP) analysis, 40 coinherited marker fragments were identified for the cross involving M. arvensis, 32 for the cross involving M. spicata, and 41 for the interspecific cross between M. arvensis and M. spicata. In all crosses, similarity values between the parents were less than those between the parents and the hybrids. Although RAPD markers are generally considered dominant, it is possible to identify a few codominant markers that behave like restriction fragment length polymorphism (RFLP) markers. This molecular marker system may be helpful in rapidly screening out hybrids in crops where cross-pollination is a problem.     

Linkage analysis of sex determination in the honey bee (Apis mellifera)

A colony-level phenotype was used to map the major sex determination locus (designatedX) in the honey bee (Apis mellifera). Individual queen bees (reproductive females) were mated to single drones (fertile males) by instrumental insemination. Haploid drone progeny of an F1 queen were each backcrossed to daughter queens from one of the parental lines. Ninety-eight of the resulting colonies containing backcross progeny were evaluated for the trait ‘low brood-viability’ resulting from the production of diploid drones that were homozygous atX. DNA samples from the haploid drone fathers of these colonies were used individually in polymerase chain reactions (PCR) with 10-base primers. These reactions generated random amplified polymorphic DNA (RAPD) markers that were analyzed for cosegregation with the colony-level phenotype. One RAPD marker allele was shared by 22 of 25 drones that fathered low brood-viability colonies. The RAPD marker fragment was cloned and partially sequenced. Two primers were designed that define a sequence-tagged site (STS) for this locus. The primers amplified DNA marker fragments that cosegregated with the original RAPD marker. In order to more precisely estimate the linkage betweenX and the STS locus, another group of bees consisting of progeny from one of the low-brood viability colonies was used in segregation analysis. Four diploid drones and 181 of their diploid sisters (workers, nonfertile females) were tested for segregation of the RAPD and STS markers. The cosegregating RAPD and STS markers were codominant due to the occurrence of fragment-length alleles. The four diploid drones were homozygous for these markers but only three of the 181 workers were homozygotes (recombinants). Therefore the distance betweenX and the STS locus was estimated at 1.6 cM. An additional linked marker was found that was 6.6 cM from the STS locus.     

The Use of RAPD Markers to Assess Catfish Hybridization

Taiwan's endemic catfish Clarias fuscus is gradually disappearing from its native habitat, and has been proposed for genebank preservation. Environmental pressures, including exotic species interference and habitat destruction, as well as possible competitive advantages of the hybrids over this species. In order to quickly and effectively provide a reliable DNA fingerprint for the pure strain of C. fuscus we used RAPD markers to assess C. fuscus, C. mossambicus, and C.batrachus. Of the 200 primers screened to prime PCR amplification of DNA from wild-caught C. fuscus, 16 yielded reproducible DNA bands. Unique RAPD markers generated from 3 PCR primers (#211, #245 and #287) are shown to be alleles present in the genomes of C. mossambicus but absent in the genome of C. fuscus. Hybrids of C. fuscus and C. mossambicus, therefore, could possibly be distinguished by the use of these specific molecular markers. Catfish caught from the Mingder Dam were then cautiously removed from the preserved stock because of the appearance of hybrid markers in their genomes.