The effects of non-homology in RAPD bands on similarity and multivariate statistical ordination in Brassica and Helianthus

 In order to estimate the impact of mis-coding non-homologous, co-migrating DNA bands as homologous, two sets of data were utilized. Analyses were conducted using three Helianthus species in which each co-migrating band had previously been confirmed. Comparisons of the similarities between these three Helianthus species using the original 177 RAPD bands and the corrected, homology verified, 197 RAPD band data set revealed that the triangular relationship among these three species was almost identical in both data sets. The non-homology errors in the Helianthanus data sets were found to be random. These random errors merely reduced the absolute similarities, but not the relative similarities nor the relationships among the taxa, in principal-coordinate-analysis ordination. Analyses of RAPDs for the classical Brassica U triangle were made by inserting random non-homologies for 5, 10, 15 and 20% of the original 220 RAPD bands. These analyses revealed a progressive decrease in similarities and less loading on the first two axes in principal coordinate analysis (PCO). However, the basic U triangle of relationships among these six Brassica species was maintained. It appears that if errors in homology of co-migrating DNA bands are random, this will have little effect on the relative similarities and on PCO ordination. This helps explain the successful use of RAPDs at the specific level. Received: 6 December 1997 / Accepted: 11 December 1997     

Comparative analysis of genetic relationship and diagnostic markers of several taxa of Guizotia Cass. (Asteraceae) as revealed by AFLPs and RAPDs

Amplified fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) were employed to examine the genetic relationship between Guizotia taxa, to suggest the taxonomic status of some of these taxa, and to identify their diagnostic markers. Results from AFLPs and RAPDs share some features in common, both revealing G. scabra ssp. schimperi as the most closely related taxon to G. abyssinica, and indicating that G. arborescens and G. zavattarii are the most divergent taxa. Most of the diagnostic markers revealed in this study were specific to G. arborescens and G. zavattarii. Our analysis suggests that G. scabra ssp. scabra, G. scabra ssp. schimperi, Chelelu and Ketcha are separate species. In this study, AFLP was found to be superior to RAPD in detecting genetic variation, in internal consistency of the data and in the fitness of its clusters to genetic similarity data. AFLPs revealed genetic relationship between Guizotia taxa that is more inline with the cytogenetic and hybridization studies than that revealed by RAPDs.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

Phytogeny of Brassica and allied genera based on variation in chloroplast and mitochondrial DNA patterns: molecular and taxonomic classifications are incongruous

Summary Chloroplast DNA (cpDNA) variability of 60 taxa of the genus Brassica and allied genera comprising 50 species was studied. RFLPs for seven enzymes were generated and F values were estimated from five frequently cutting enzymes. Phenetic clusterings indicated a clear division of Brassica coenospecies into two distinct lineages referred to as the Brassica and Sinapis lineages. Two unexplored genera, Diplotaxis and Erucastrum, also exhibited two lineages in addition to the genera Brassica and Sinapis. This finding is inconsistent with the existing taxonomic classification based on morphology. Mitochondrial DNA (mtDNA) variability studied from EcoRI RFLP patterns, by hybridizing total DNA with four cosmid clones containing non-overlapping mtDNA fragments, did not show any congruence with cpDNA variation patterns. However, at the cytodeme level, the patterns of genetic divergence suggested by the cpDNA data could be correlated with mtDNA variation. In the Brassica lineage, Diplotaxis viminea was identified as the female parent of the allotetraploid D. muralis. The chloroplast DNAs of Erucastrum strigosum and Er. abyssinicum were found to be very closely related. In the Sinapis lineage, Brassica maurorum was found to be the diploid progenitor of autotetraploid B. cossoneana. B. amplexicaulis showed a very different cpDNA pattern from other members of the subtribe. Brassica adpressa was closest to Erucastrum laevigatum and could be the diploid progenitor of autotetraploid Er. laevigatum. Based on the close similarity of the cpDNA pattern of Diplotaxis siifolia with that of D. assurgens, we have proposed the retention of this species in the genus Diplotaxis. The taxonomic positions of some other species have also been discussed.     

Relationships among cultivated and wild lentils revealed by RAPD analysis

RAPD markers were used to distinguish between six different Lens taxa, representing cultivated lentil and its wild relatives. Twenty-four arbitrary sequence 10-mer primers were identified which revealed robust and easily interpretable amplification-product profiles. These generated a total of 88 polymorphic bands in 54 accessions and were used to partition variation within and among Lens taxa. The data showed that, of the taxa examined, ssp. orientalis is most similar to cultivated lentil. L. ervoides was the most divergent wild taxon followed by L. nigricans. The genetic similarity between the latter two species was of the same magnitude as between ssp. orientalis and cultivated lentil. In addition, species-diagnostic amplification products specific to L. odemensis, L. ervoides and L. nigricans were identified. These results correspond well with previous isozyme and RFLP studies. RAPDs, however, appear to provide a greater degree of resolution at a sub-species level. The level of variation detected within cultivated lentils suggests that RAPD markers may be an appropriate technology for the construction of genetic linkage maps between closely related Lens accessions.On sabbatical leave from HP Agricultural University, Palampur 176 062, India     

A phylogenetic analysis of Pisum based on morphological characters, and allozyme and RAPD markers

Cladistic analyses of 17 wild and cultivated pea taxa were performed using morphological characters, and allozyme and RAPD (random amplified polymorphic DNA) markers. Both branch-and-bound and bootstrap searches produced cladograms that confirmed the close relationships among the wild species and cultivars of Pisum proposed by a variety of systematic studies. Intraspecific rankings were supported for northern P. humile, southern P. humile, P. elatius and P. sativum, which together comprise a single-species complex. P. fulvum, while clearly the most divergent of the pea taxa, could also be assigned to the same species complex without violating the hierarchial logic of the cladogram. Its inclusion or exclusion depends on whether the level of interfertility it displays with other pea taxa or its overall morphological and chromosomal distinction are emphasized. As suggested by previous studies, northern P. humile was the most likely sister taxon to cultivated P. sativum; although, rigorous phylogenetic evaluation revealed a close genealogical affinity among P. elatius, northern P. humile and P. sativum. Despite their limited number, the 16 morphological characters and allozyme markers used precisely organized the pea taxa into established taxonomic groupings, perhaps in part reflecting the role morphology has played historically in pea classification. The RAPD data also generally supported these same groupings and provided additional information regarding the relationships among the taxa. Given that RAPDs are relatively quick and easy to use, are refractory to many environmental influences, can be generated in large numbers, and can complement traditional characters that may be limited in availability, they provide a valuable new resource for phylogenetic studies.     

Qualitative and quantitative characterization of RAPD variation among snap bean (Phaseolus vulgaris) genotypes

Ten snap bean (Phaseolus vulgaris) genotypes were screened for polymorphism with 400 RAPD (random amplified polymorphic DNA) primers. Polymorphic RAPDs were scored and classified into three categories based on ethidium bromide staining intensity. An average of 5.19 RAPD bands were scored per primer for the 364 primers that gave scorable amplification products. An average of 2.15 polymorphic RAPDs were detected per primer. The results show that primer screening may reduce the number of RAPD reactions required for the analysis of genetic relationships among snap-bean genotypes by over 60%. Based on the analysis of the distribution of RAPD amplification, the same number of polymorphic RAPDs were amplified from different genotypes for all RAPD band intensity levels. A comparison of RAPD band amplification frequency among genotypes for the three categories of bands classified by amplification strength revealed a measurable difference in the frequencies of RAPDs classified as faint (weakly amplifying) compared to RAPD bands classified as bold (strongly amplifying) indicating a possible scoring error due to the underscoring of faint bands. Correlation analysis showed that RAPD bands amplified by the same primer are not more closely correlated then RAPD bands amplified by different primers but are more highly correlated then expected by chance. Pairwise comparisons of RAPD bands indicate that the distribution of RAPD amplification among genotypes will be a useful criterion for establishing RAPD band identity. For the average pairwise comparison of genotypes, 50% of primers tested and 15.8% of all scored RAPDs detected polymorphism. Based on RAPD data Nei's average gene diversity at a locus was 0.158 based on all scorable RAPD bands and 0.388 if only polymorphic RAPD loci were considered. RAPD-derived 1 relationships among genotypes are reported for the ten genotypes included in this study. The data presented here demonstrate that many informative, polymorphic RAPDs can be found among snap bean cultivars. These RAPDs may be useful for the unique identification of bean varieties, the organization of bean germplasm, and applications of molecular markers to bean breeding.     

DNA fingerprinting of Peronospora parasitica,a biotrophic fungal pathogen of crucifers

The fungus Peronospora parasitica (Pers. ex Fr.) Fr. is an obligate biotroph infecting a wide range of host species in the family Cruciferae. Isolates from different hosts are morphologically similar, and pathotypes are usually distinguished on the basis of host range. Random Amplified Polymorphic DNA (RAPD) fingerprints were generated from a range of P. parasitica isolates from different Brassica species. Reaction conditions, in particular DNA template, primer and Mg²⁺ concentrations, were optimized to ensure that amplifications were reproducible. Possible artefacts arising through host plant DNA were assessed by including such DNA in control reactions. Confirmation that diagnostic RAPD bands were generated from fungal DNA was also obtained by Southern hybridization of a RAPD band to genomic fungal DNA. By screening 20 decamer primers, 2 were found to detect sufficient genetic variation to allow complete differentiation between pathotypes. These results illustrate the potential value of RAPDs for detecting polymorphisms between isolates of a non-culturable plant pathogenic fungus.     

Genetic variation in two sympatric European populations of Bosmina spp. (Cladocera) tested with RAPD markers

We used RAPDs (Random Amplified Polymorphic DNA) to test genetic divergence between two populations of Bosmina spp. in Lake Östersjön, Sweden. Previous taxonomic studies on European species within the genus Bosmina have been based on morphological characters alone. RAPD markers distinguished the two populations and supported the specific status of B. coregoni and B. longispina based on morphological characters. Furthermore, juveniles with a long antennule and a mucro were classified as B. coregoni. RAPDs also revealed genetic differences among the tested individuals, suggesting several clones within each species.     

Comparison of four molecular markers in measuring relationships among the wild potato relatives Solanum section Etuberosum (subgenus Potatoe)

We evaluated chloroplast DNA (cpDNA), isozymes, single to low-copy nuclear DNA (RFLPs), and random amplified polymorphic DNAs (RAPDs) in terms of concordance for genetic distance of 15 accessions each of Solanum etuberosum and S. palustre, and 4 accessions of S. fernandezianum. These self-compatible, diploid (2n=24), and morphologically very similar taxa constitute all species in Solanum sect. Etuberosum, a group of non-tuber-bearing species closely related to Solanum sect. Petota (the potato and its wild relatives). Genetic distance and multidimentional scaling results show general concordance of isozymes, RFLPs and RAPDs between all three taxa; cpDNA shows S. etuberosum and S. palustre to be more similar to each other than to S. fernandezianum. Interspecific sampling variance shows a gradation of resolution from allozyme (low) to RAPD to RFLP (high); while intraspecific comparisons graded from RFLPs (low) to RAPDs (high; lack of sufficient allozyme variability within species precluded comparisons for allozymes). Experimental error was low in RFLPs and RAPDs.Names are necessary to report factually and available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable     

Giemsa C-banded karyotypes inCapsicum (Solanaceae)

Giemsa C-banding is applied for the first time inCapsicum, allowing preliminary karyotype differentiation of six diploid species. Comparison of interphase nuclei and heterochromatic C-bands reveals striking differences between taxa and contributes to their taxonomic grouping. Therefore, C-banding appears to be a powerful tool for the cytogenetics and karyosystematics of the genus. Banding patterns are characterized by the omnipresence of centromeric bands and a variable number of smaller to larger distal bands, with the addition of intercalary bands in some cases. Satellites are always C-positive. Relationships between species and possible trends of karyotype evolution are discussed, with special reference to the origin of x = 13 from x = 12 and the increase of heterochromatin, regarded as advanced features.Chromosome studies inCapsicum (Solanaceae), III. For the first and the second part seeMoscone (1990, 1993).     

The potential of AFLPs in biosystematics: A first application inSolanum taxonomy(Solanaceae)

Using the AFLP technique highly informative DNA fingerprints were generated from 19 taxa ofSolanum sect.Petota (potatoes) and three taxa ofSolanum sect.Lycopersicum (tomatoes). Both phenetic and cladistic analyses were conducted from the individual genotypic level to the species level. An AFLP fingerprint, using a combination of suitable AFLP primers, generated 12 to 71 scorable fragments per genotype which was sufficient for taxonomic interpretation. The classifications based on the molecular markers were generally in agreement with current taxonomic opinions. Unexpectedly,S. microdontum was associated with ser.Megistacroloba rather than with ser.Tuberosa, andS. demissum (ser.Demissa) and species of ser.Acaulia appeared closely affiliated. AFLP is an efficient and reliable technique to generate biosystematic data and therefore a promising tool for evolutionary studies.     

Characterization of terminal-repeat retrotransposon in miniature (TRIM) in Brassica relatives

We have newly identified five Terminal-repeat retrotransposon in miniature (TRIM) families, four from Brassica and one from Arabidopsis. A total of 146 elements, including three Arabidopsis families reported before, are extracted from genomics data of Brassica and Arabidopsis, and these are grouped into eight distinct lineages, Br1 to Br4 derived from Brassica and At1 to At4 derived from Arabidopsis. Based on the occurrence of TRIM elements in 434 Mb of B. oleracea shotgun sequences and 96 Mb of B. rapa BAC end sequences, total number of TRIM members of Br1, Br2, Br3, and Br4 families are roughly estimated to be present in 660 and 530 copies in B. oleracea and B. rapa genomes, respectively. Studies on insertion site polymorphisms of four elements across taxa in the tribe Brassiceae infer the taxonomic lineage and dating of the insertion time. Active roles of the TRIM elements for evolution of the duplicated genes are inferred in the highly replicated Brassica genome. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Tae-Jin Yang and Soo-Jin Kwon have equally contributed to this work.     

Electrophoretic seed albumin patterns and species relationships inVicia sect.Faba (Fabaceae)

Electrophoretic analysis of seed albumins (PAGE) covered 173 accessions representing nine species ofVicia sect.Faba. The number of albumin bands recorded in particular species varied from three inV. eristaloides to 23 inV. faba; in total, 38 bands were distinguished in the investigated material. The examined species, exceptV. eristalioides, showed intraspecific variation with respect to the number and relative staining intensity of albumin bands; individual variation was especially marked inV. faba and inV. narbonensis. Hierarchical clustering of the investigated taxa was based onBhattacharyya distances calculated from the electrophoretic data. The taxa grouped in three main clusters.Vicia faba and the rather remotely relatedV. kalakhensis formed one cluster. The second cluster was composed ofV. narbonensis distantly related toV. hyaeniscyamus. The third cluster comprised three subgroups: 1.V. johannis, V. galilaea andV. serratifolia, 2.V. eristalioides, and 3.V. bithynica. The obtained results are discussed with reference to taxonomic relationships inVicia sect.Faba.     

Identification of Prosopis juliflora and Prosopis pallida Accessions Using Molecular Markers

There has been much taxonomic confusion over the identification of Prosopis species, especially where introduced. Prosopis juliflora is the most widespread species in the arid and semi-arid tropics, although it has been confused with other species, particularly the closely related Prosopis pallida. In this study, RAPDs markers were used for the first time to distinguish between these species. Eighteen primers were used in amplification reactions, which yielded an average of 120 bands per accession. A dendrogram showing genetic similarities among accessions was constructed using UPGMA cluster analysis and the Nei and Li similarity coefficient. The genetic similarity observed between P. juliflora and P. pallida is similar to the value in sympatric Prosopis species in North America, and reconsideration of the series rank in section Algarobia is suggested. Species-specific markers confirmed that material in Burkina Faso is P. juliflora, but suggested that material collected in Brazil, Cape Verde and Senegal is P. pallida, whereas this has previously been identified as P. juliflora.     

Production of interspecific somatic hybrids between tuber mustard (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">juncea</Emphasis>) and red cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

In the present investigation, the interspecific somatic hybridization between tuber mustard and red cabbage was established in order to introduce valuable genes from red cabbage (Brassica oleracea) into Brassica juncea. Prior to fusion treatment, protoplasts of red cabbage were inactivated with 2 mM iodoacetamide to inhibit cell division. Micro-calluses were obtained at a frequency of 10.3% after approximately 5 weeks culture following protoplast fusion. Some of the fusion-derived calluses possessed red pigmented cells after being transferred to proliferation medium, and they were presumably considered to be somatic hybrid cell lines. Plantlets were regenerated from 12 cell lines, of which nine plantlets exhibited characteristics intermediate of both parents in terms of plant morphology. With the exception of common protein bands featured by two parents, there were unique banding patterns produced in the hybrids by using SDS-PAGE analysis. By chromosome countings, it was showed that they ranged approximately from 2n=30 to 42 in chromosome numbers. Their hybridity were further confirmed by RAPD analysis revealing that genes of both parents were partially incorporated into the hybrids. Positively, all these hybrids were capable of seed-setting. The pod-setting was 4.2 in somatic hybrid H₇ when backcrossed with tuber mustard.     

Species-specific primers discriminate schistosome intermediate hosts: unambiguous PCR diagnosis of Bulinus forskalii group taxa (Gastropoda: Planorbidae)

The morphological definition of taxa has proved difficult within the Bulinus forskalii group, which includes intermediate hosts of medically important Schistosoma species in West Africa. Although B. forskalii and B. senegalensis transmit different schistosome species they are conchologically similar and their distributions overlap. Randomly amplified polymorphic DNA (RAPD) allows differentiation of sibling species in the genus Bulinus, but RAPDs are difficult to standardize, impairing their value as a taxonomic tool. Hence, RAPD products diagnostic for either B. senegalensis or B. forskalii from West Africa were cloned, sequenced and a panel of species-specific primers designed. Sequencing of RAPD products identified a homology in two apparently independent RAPD loci, a problem where RAPDs are indiscriminately scored for phylogenetic analyses. Specificity of primers was confirmed by widespread sampling throughout each species' range. This approach produced a simple, robust, unambiguous PCR-based species identification strategy for this difficult group.     

淫羊藿属分类学研究:进展、问题与展望

淫羊藿是我国特有且传统的重要药用植物,逐渐步入大宗品种行列。物种的准确鉴定是药效保障和用药安全的前提,为促进其资源的合理开发和利用,该文对淫羊藿属分类学研究进行系统梳理,并对其中存在的问题及存疑类群进行阐述。淫羊藿属共发表68种,中国58种(85.3%),其中57种为特有分布,具有显著的资源优势。淫羊藿属中国类群的分类学研究较为特殊,共26种集中发表于1990s,共31种(53.4%)为国外研究者命名,且绝大多数依据少量栽培个体命名。由于缺乏广泛的形态调查和性状变异分析,导致大量类群的形态描述不准确或不全面,后续20个类群被归并或降级。依据栽培个体命名的类群是补充描述和分类修订的重点。花色、根茎类型、花茎叶的数量及着生方式等性状在中国类群中存在广泛变异。经分类修订后,该属目前包括46种、1亚种和2变种。淫羊藿属中国组类群仍处于活跃进化中,其形态变异复杂,种间关系无法得到解决,为该属分类的最大挑战。但种质资源的研究和利用需要建立在清晰的分类学基础上。未来研究应基于居群调查,完善各物种的形态描述;在此基础上,整合形态变异特征、地理分布格局和基因序列特征,检测自然种间杂交事件,从而揭示物种的分化和进化历程。     

Comparison of RAPD and RFLP genetic markers in determining genetic similarity among Brassica oleracea L. genotypes

Genetic similarity among 45 Brassica Oleracea genotypes was compared using two molecular markers, random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphisms (RFLPs). The genotypes included 37 broccolis (var. italica), five cauliflowers (var. botrytis) and three cabbages (var. capitata) which represented a wide range of commercially-available germplasm, and included open-pollinated cultivars, commercial hybrids, and inbred parents of hybrid cultivars. Fifty-six polymorphic RFLP bands and 181 polymorphic RAPD bands were generated using 15 random cDNA probes and 62 10-mer primers, respectively. The objectives were to compare RFLP and RAPD markers with regard to their (1) sampling variance, (2) rank correlations of genetic distance among sub-samples, and (3) inheritance. A bootstrap procedure was used to generate 200 random samples of size n (n=2,3,5,... 55) independently from the RAPD and RFLP data sets. The coefficient of variance (CV) was estimated for each sample. Pooled regressions of the coefficient of variance on bootstrap sample size indicated that the rate of decrease in CV with increasing sample size was the same for RFLPs and RAPDs. The rank correlation between the Nei-Li genetic similarity values for all pairs of genotypes (990) based on RFLP and RAPD data was 0.745. Differences were observed between the RFLP and RAPD dendrograms of the 45 genotypes. Overlap in the distributions of rank correlations between independent sub-samples from the RAPD data set, compared to correlations between RFLP and RAPD sub-samples, suggest that observed differences in estimation of genetic similarity between RAPDs and RFLPs is largely due to sampling error rather than due to DNA-based differences in how RAPDs and RFLPs reveal polymorphisms. A crossing algorithm was used to generate hypothetical banding patterns of hybrids based on the genotypes of the parents. The results of this study indicate that RAPDs provide a level of resolution equivalent to RFLPs for detemination of the genetic relationships among genotypes.     

Phytoextraction of selenium from soils irrigated with selenium-laden effluent

This two-part study compared the efficacy of different plant species to extract Se from soils irrigated with Se-laden effluent. The species used were: Brassica napus L. (canola), Brassica juncea Czern L. and Coss (Indian mustard), and Hordeum vulgare L. (barley). In Study 1 we irrigated the plants with a saline effluent containing 0.150 mg Se L^–1, while in Study 2, the same species were planted in a saline soil selenized with 2 mg Se L^–1. Plants were simultaneously harvested 120 days after planting. In Study 1, there were only slight effects of treatment on dry matter (DM) yield. Plant Se concentrations averaged 21 g Se g^–1DM for the Brassica species, and 4.0 g Se g^–1 DM for barley. Total Se added to soils via effluent decreased by 40% for Brassica species and by 20% for barley. In Study 2, total DM decreased for all species grown in saline soils containing Se. Plant Se concentrations averaged 75 g g^–1 DM for Brassica species and 12 g Se g^–1 DM for barley. Total Se added to soils prior to planting decreased by 40% for Brassica species and up to 12% for barley. In both studies, plant accumulation of Se accounted for at least 50% of the Se removed in soils planted to Brassica and up to 20% in soils planted to barley. Results show that although the tested Brassica species led to a significant reduction in Se added to soil via use of Se-laden effluent, additional plantings are necessary to further decrease Se content in the soil.