Detection of Polymorphic DNA and Taxonomic Relationships Among 10 Wild Perennial Soybean Species Using Specific and Arbitrary Nucleotide Primers

Polymorphic DNA in complex genomes of agronomic crops can be detected using specific nucleotide and arbitrary primers and the polymerase chain reaction (PCR). Nineteen accessions representing 10 species of the wild perennial soybean were evaluated using 4 sets of specific primers and 3 sets of random amplified polymorphic DNAs (RAPD) primers. The potential of the RAPD assays was further increased by combining two primers in a single PCR. The fragments generated by the two assays discriminated 10 wild species by banding profiles. The size of the amplified DNA fragments ranged from 100 to 2100 base pairs. The resolved PCR products yielded highly characteristic and homogeneous DNA fingerprints. The fingerprints were useful not only for investigating genetic variability but also for further characterizing the wild soybean species by detecting inter- and intra-specific polymorphisms, constructing dendrograms defining the phylogenetic relationships among these species, and identifying molecular markers for the construction of genetic linkage maps. Furthermore, unique markers distinguishing particular species were also identified. Thus, it is expected that PCR will have great relevance for taxonomic studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic segregation of random amplified polymorphic DNA in diploid cultivated alfalfa.

Polymerase chain reaction was used, with single 10-mer primers of arbitrary sequence, to amplify random regions of genomic DNA from a diploid cultivated alfalfa backcross population. Segregation of the random amplified polymorphic DNA (RAPD) fragments was analysed to determine if RAPD markers are suitable for use as genetic markers. Of the 19 primers tested, 13 amplified a total of 37 polymorphic fragments, of which 28 (76%) segregated as dominant Mendelian traits. RAPD markers appear useful for the rapid development of genetic information in species like alfalfa where little information currently exists or is difficult to obtain.     

Random amplified polymorphic DNA analysis in Hordeum species.

Random amplified polymorphic DNA analysis was performed by applying a set of 13 arbitrary 10-mer primers to 19 Hordeum species and subspecies. High levels of variation in fragment pattern were observed both within and among species with most of the primers used. Genetic similarities between accessions and species were calculated from the fragment patterns. The resulting phenograms confirmed previous relationships among the Hordeum species.     

Identification of broccoli and cauliflower cultivars with RAPD markers

Summary RAPD (Random Amplified Polymorphic DNA) markers generated by 4 arbitrary 10-mer primers, discriminated 14 broccoli and 12 cauliflower cultivars (Brassica oleracea L.) by banding profiles. The size of the amplified DNA fragments ranged from 300 to 2600 base pairs. Twenty-eight percent of the markers were fixed in both broccoli and cauliflower, whereas 12.5% were specific to either crop. The rest were polymorphic in either or both crops. The markers generated by two and three primers were sufficient to distinguish each of the broccoli and cauliflower cultivars, respectively. The average difference in markers was 14.5 between broccoli and cauliflower markers, 5.8 between two broccoli cultivars and 7.9 between two cauliflower cultivars. Larger differences for each crop were found between cultivars from different seed companies than within the same company. RAPD markers provide a quick and reliable alternative to identify broccoli and cauliflower cultivars.     

Genetic relationships in Lens species and parentage determination of their interspecific hybrids using RAPD markers

Broadening of the genetic base and systematic exploitation of heterosis in cultivated lentils requires reliable information on genetic diversity in the germplasm. The ability of random amplified polymorphic DNA (RAPD) to distinguish among different taxa of Lens was evaluated for several geographically dispersed accessions/cultivars of four diploid Lens species. This study was carried out to assess whether RAPD data can provide additional evidence about the origin of the cultivated lentil and to measure genetic variability in lentil germplasm. Three cultivars of Lens culinaris ssp. culinaris, including one microsperma, and two macrosperma types, and four wild species (L. culinaris ssp. orientalis, L. odemensis and L. nigricans) were evaluated for genetic variability using a set of 1 11-mer and 14 random 10-mer primers. One hundred and fifty-eight reproducible and scorable DNA bands were observed from these primers. Genetic distances between each of the accessions were calculated from simple matching coefficients. Split decomposition analysis of the RAPD data allowed construction of an unrooted tree. This study revealed that (1) the level of intraspecific genetic variation in cultivated lentils is narrower than that in some wild species. (2) L. culinaris ssp. orientalis is the most likely candidate as a progenitor of the cultivated species, (3) L. nigricans accession W6 3222 (unknown) and L. c. ssp. orientalis W6 3244 (Turkey) can be reclassified as species of L. odemensis and (4) transmission of genetic material in Lens interspecific hybrids is genotypically specific, as identified by the RAPD markers in our study.     

RAPD技术在几个苋属植物遗传分类中的应用研究

利用RAPD技术结合聚类分析,研究了亚洲和中南美洲产的苋属植物7种14个品系遗传关系。从80个10碱基长度的随机引物中筛选出8个有效引物,共扩增出103个DNA片段,平均每个引物扩增12.9个片段,其中多态性片段95个,占92．2％,依据8个有效引物扩增的DNA片段对供试材料进行了UPGMA（非加权成组算术平均法）聚类分析,结果分为4类：（1）包括A.deflexus和A.dubius各1品系的杂草苋种;（2）祖先野生种A.hybridus 1个品系;（3）包括A.hypochondriacus6品系、A.caudatus 2品系的籽粒苋种;（4）包括A.tricolor2品系和A.mangostanus 1品系的蔬菜苋种。     

Analysis of genetic relationships among perennial and annual Cicer species growing in Turkey using RAPD markers

Random amplified polymorphic DNA (RAPD) fragments were used to assess genetic relationships among Cicer spp. growing in Turkey. Seven 10-mer primers selected from a 50 random oligonucleotide primer set, depending on their ability to amplify genomic DNA in all species, were used to detect RAPD variation in 43 wild and cultivated accessions representing ten species. These primers yielded 95 reproducible amplification products, 92 of which were polymorphic. Pairwise genetic distances of accessions estimated according to Nei and Li (1979) were used to produce a dendrogram using UPGMA. The dendrogram contained two main clusters, one of which comprised accessions of the four perennial species (Cicer montbretii, Cicer isauricum, Cicer anatolicum and Cicer incisum) together with the accessions of the three annual species (Cicer pinnatifidum, Cicer judaicum and Cicer bijugum), and the other cluster included the remaining three annual species (Cicer echinospermum, Cicer reticulatum and Cicer arietinum). Analysis of RAPD variation showed that C. incisum is the most similar perennial species to annuals, and C. reticulatum is the closest annual species to chickpea. These results generally agree with our allozyme study which was carried out using same Cicer collection and previous studies of relationships among annual species. The results also show that RAPD markers can be used to distinguish Cicer species and to survey genetic variation and relationships among taxonomic units in this genus.     

A PCR Assay to Identify and Distinguish Single Juveniles of Meloidogyne hapla and M. chitwoodi

Random amplified polymorphic DNA (RAPD) bands that distinguish Meloidogyne hapla and M. chitwoodi from each other, and from other root-knot nematode species, were identified using a series of random octamer primers. The species-specific amplified DNA fragments were cloned and sequenced, and then the sequences were used to design 20-mer primer pairs that specifically amplified a DNA fragment from each species. Using the primer pairs, successful amplifications from single juveniles were readily attained. A mixture of four primers in a single PCR reaction mixture was shown to identify single juveniles of M. hapla and M. chitwoodi. To confirm specificity, the primers were used to amplify DNA from several isolates of M. hapla that originated from different crops and locations in North America and also from isolates of M. chitwoodi that differed in host range. In characterizing the M. hapla isolates, it was noted that there was a mitochondrial DNA polymorphism among isolates for cleavage by the restriction endonuclease DraI.     

Species relationships in Fagopyrum revealed by PCR-based DNA fingerprinting

Random amplified polymorphic DNA (RAPD) markers were used to distinguish between 28 different accessions belonging to 14 species and two sub-species of Fagopyrum. Of the 75 random 10-mer primers tested, only 19 generated robust, easily interpretable amplification products. A total of 364 bands were observed with an average of 19.15 bands per primer, of which 99.45% were polymorphic. Primer OPN-08 produced the maximum number of fragments and UBC-183 produced the minimum number of fragments. The data were utilized to elucidate genetic relationships among 14 species and two sub-species of Fagopyrum. Cluster analysis using the unweighted paired group method of arithmetic means (UPGMA) showed four main clusters, two each of the cymosum and urophyllum groups. The results showed that Fagopyrum tataricum is closer to its wild ancestor F. tataricum ssp. potanini Batalin, closely followed by Fagopyrum giganteum. Cultivated common buckwheat ( Fagopyrum esculentum) showed affinity with its putative wild ancestor F. esculentum ssp. ancestrale and the other closely related diploid species Fagopyrum homotropicum. In the urophyllum group, Fagopyrum macrocarpum and Fagopyrum pleioramosum formed one cluster, whereas Fagopyrum capillatum, Fagopyrum gracilipes and Fagopyrum gilessii clustered separately. Except for a few cases, our results correspond with previously reported studies on Fagopyrum using the isozyme, RFLP and RAPD methods. Species-diagnostic amplification products specific to some species in the cymosum and urophyllum groups were identified. Our results show that RAPDs can be successfully used to analyze species relationships in Fagopyrum and also for constructing linkage maps.     

Identification of RAPD markers linked to a fertility restorer gene for theOgura radish cytoplasmic male sterility of rapeseed (Brassica napus L.)

Bulked segregant analysis was employed to identify random amplified polymorphic DNA (RAPD) markers linked to the restorer gene (Rfo) used in theOgura radish cytoplasmic male sterility of rapeseed. A total of 138 arbitrary 10-mer oligonucleotide primers were screened on the DNA of three pairs of bulks, each bulk corresponding to homozygous restored and male sterile plants of three segregating populations. Six primers produced repeatable polymorphisms between paired bulks. DNA from individual plants of each bulk was then used as a template for amplification with these six primers. DNA polymorphisms generated by four of these primers were found to be completely linked to the restorer gene with the polymorphic DNA fragments being associated either with the fertility restorer allele or with the sterility maintainer allele. Pairwise cross-hybridization demonstrated that the four polymorphic DNA fragments did not share any homology. Southern hybridization of labelled RAPD fragments on digested genomic DNA from the same three pairs of bulks revealed fragments specific to either the male sterile bulks or to the restored bulks and a few fragments common to all bulks, indicating that the amplified sequences are low copy. The four RAPD fragments that were completely linked to the restorer locus have been cloned and sequenced to develop sequence characterized amplified regions (SCARs). This will facilitate the construction of restorer lines used in breeding programs and is the first step towards map-based cloning of the fertility restorer allele.     

The use of RAPD markers in Hordeum phylogeny.

The phylogenetic relationships among 39 wild Hordeum species, subspecies, and cultivated barley were investigated using RAPD markers as discriminating characters. Seventy-six RAPD fragments were generated using 12 single decameric primers of arbitrary nucleotide sequences. Amplification reactions resulted in fragments ranging in length between 200 and 2000 bp. Clearly resolved bands were scored for their presence or absence in a binary matrix. Amplified products were treated as independent characters to generate a phenogram using the NTSYS-PC package. Tree topology was generally found to be consistent with those based on morphological treatments. However, a few species like H. erectifolium, H. jubatum and, to a lesser extent, H. bulbosum occupied a position different from previous classifications. The results demonstrated that RAPD technology represents a useful and reliable tool for detecting polymorphism for phylogenetic studies. Key words : RAPD analysis, molecular markers, phylogenetic studies, Hordeum species, barley.     

Application of RAPD analysis for identification of Lactococcus lactis subsp. cremoris strains isolated from artisanal cultures

Randomly amplified polymorphic DNA (RAPD) was used for identification of Lactococcus lactis subsp. cremoris strains isolated 40 years ago from various dairy homemade products. Total genomic DNAs from six randomly chosen isolates and the reference strain Lactococcus lactis subsp. cremoris NIZO B64 were amplified using four different 10-mer primers. Although most RAPD fragments were common to all six isolates, a sufficient number of polymorphic fragments were also detected that allowed clear distinction of the isolates and the reference strain. The results indicate that RAPD analysis could be a useful and efficient method to distinguish Lactococcus lactis subsp. cremoris at the strain level and to detect genetic diversity.     

随机扩增多态性DNA技术在鲍氏层孔菌菌株鉴别中的应用

用20个随机引物对7个不同来源的鲍氏层孔菌菌株进行了RAPD分析．结果表明120个随机引物中,有17个引物的扩增产物DNA条带表现出明显的多态性,不同引物对供试菌株扩增出现的DNA条带数目少则10条,多达33条．DNA片段从250bp到2000bp;采用17个引物对7个鲍氏层孔菌菌株共扩增出DNA片段带377条,不同引物扩增出的DNA片段谱带存在较大差异．采用UPGMA系统聚类法,将7个菌株聚类为两大类,能直观准确地揭示菌株间的差异并加以鉴别．     

微卫星(TATG)n基序在香菇菌种中的验证

以（TATG）4重复序列为引物对香菇属的3个种13个菌株的微卫星区DNA进行PCR扩增,15%的琼脂糖凝胶电泳,获得了25个条带,并且在供试菌株上表现出多态性,可以实现遗传分类研究。为了验证微卫星分子标记实验准确性,又用RAPD技术对13个供试菌株进行了实验。7个引物在13个菌株上共获得了102条多态性条带。通过聚类分析,RAPD获得的分类结果与微卫星分子标记获得的结果一致。此外,为了证明微卫星分子标记获得的条带不是假阳性,在实验中回收了No.10菌株的PCR扩增产物,进行克隆测序。测序结果显示有（TATG）n基序存在,并且达到了微卫星基序重复数量的最低限度。通过本实验可知,香菇中是存在微卫星（TATG）n基序的, 且基序的多态性可以用于香菇的遗传分类研究。     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

Genetic fingerprinting of pigeonpea [Cajanus cajan (L.) Millsp.] and its wild relatives using RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used for the identification of pigeonpea [Cajanus cajan (L.) Millsp.] cultivars and their related wild species. The use of single primers of arbitrary nucleotide sequence resulted in the selective amplification of DNA fragments that were unique to individual accessions. The level of polymorphism among the wild species was extremely high, while little polymorphism was detected within Cajanus cajan accessions. All of the cultivars and wild species under study could be easily distinguished with the help of different primers, thereby indicating the immense potential of RAPD in the genetic fingerprinting of pigeonpea. On the basis of our data the genetic relationship between pigeonpea cultivars and its wild species could be established.NCL Communication No. 6062     

利用RAPD分子标记对三组三系杂交水稻及亲本的遗传分析和鉴定

利用RAPD技术,从248个随机寡聚核苷酸（10bp）中筛选出13个引物能在供试的三组三系杂交水稻及亲本间扩增出43条稳定性较好的多态性片段,其中6个引物能在供试材料间扩增出20个强的多态性标记。利用这些标记能有效地区分各组合中不育系、保持系、恢复系和F1,并能看出各组合中不育系与保持系、不育系与恢复系、F1与亲本间的遗传关系。 Abstract:A total of 248 arbitrary 10-mer oligonucleotide primers were screened using RAPD (random amplified polymorphic DNA) techniques with the genome DNA of three groups of three-line hybrid rice and their parents.Thirteen primers produced 43 polymorphism fragments.Six primers of them produced 20 obviously repeatable polymorphic markers among rice lines tested.Using this RAPD markers,the hybrid rice combinations (sterile-line,maintainer-line,restorer-line and F1)can be effectively identified,and the genetic relationship among them can be shown.     

RAPD and ISSR fingerprints as useful genetic markers for analysis of genetic diversity, varietal identification, and phylogenetic relationships in peanut (Arachis hypogaea) cultivars and wild species.

Abstract: Twenty-one random and 29 SSR primers were used to assess genetic variation and interrelationships among subspecies and botanical varieties of cultivated peanut, Arachis hypogaea (2n = 4x = 40), and phylogenetic relationships among cultivated peanut and wild species of the genus Arachis. In contrast with the previous generalization that peanut accessions lack genetic variation, both random and SSR primers revealed 42.7 and 54.4% polymorphism, respectively, among 220 and 124 genetic loci amplified from 13 accessions. Moreover, the dendrograms based on RAPD, ISSR, and RAPD + ISSR data precisely organized the five botanical varieties of the two subspecies into five clusters. One SSR primer was identified that could distinguish all the accessions analysed within a variety. Although the polymorphic index content varied from 0.1 to 0.5 for both ISSR and RAPD markers, primer index values were substantially higher for RAPD primers (0.35-4.65) than for SSR primers (0.35-1.73). It was possible to identify accessions, particularly those of divergent origins, by RAPD and (or) ISSR fingerprints. Based on these results, marker-based genetic improvement in A. hypogaea appears possible. None of the 486 RAPD and 330 ISSR amplification products were found to be commonly shared among 13 species of section Arachis and one species each of sections Heteranthae, Rhizomatosae, and Procumbentes. Dendrograms constructed from RAPD, ISSR, and RAPD + ISSR data showed overall similar topologies. They could be resolved into four groups corresponding to the species grouped in four taxonomic sections. The present results strongly support the view that Arachis monticola (2n = 4x = 40) and A. hypogaea are very closely related, and indicate that A. villosa and A. ipaensis are the diploid wild progenitors of these tetraploid species.     

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.     

西瓜种质资源遗传亲缘关系的RAPD分析

利用 RAPD 技术对国内外32份西瓜主栽品种与其骨干亲本及野生类型的遗传亲缘关系进行了研究。从720个随机引物(10bp)中筛选出15个能产生稳定多态性的引物用于 RAPD 反应,共扩增出104条 DNA 带,其中多态性 DNA 条带43条,占41.35%,平均每个引物扩增的 DNA 条带的数目为7.0条。聚类分析将供试材料分为6个类群:1个东亚生态型类群、1个美国生态型类群、2个中间生态型类群和2个非洲野生型类群,与传统的西瓜生态型分类基本吻合。每个生态型类群都有其特有的扩增(缺失)条带,同时分析了同一生态型中各个品种之间的亲缘关系及其品种的特异条带。本实验结果不仅从分子水平验证了西瓜是遗传基础狭窄的作物,而且在分子水平对西瓜传统分类与地理生态型分类进行了分析。