Variability and phylogenetic relationships of the Cucumis sativus L. species inferred from NBS profiling and RAPD analysis

Genetic variability of the Cucumis sativus species and its phylogenetic relationsips with other species of the genus were studied on the basis of RAPD marking and analysis of intra- and interspecific polymorphism of the nucleotide sequences of the NBS-LRR gene family in species of the genus Cucumis with the use of the NBS profiling method. According to RAPD analysis, cucumber cultivars from different geographic regions are highly similar, except for accessions k-3835 and k-3833 from Afghanistan. NBS profiling analysis revealed phylogenetically most distinct accessions expected to be characterized by specificity of resistance: k-3845 from Uzbekistan, k-3851 from Kyrgyzstan, line 701, k-3835 and k-3833 from Afghanistan, k-2757 and k-3079 from Netherlands, vr.k. 908 from Canada, k-2926 from Bulgaria, Russian cultivars Monastyrskii, Izyashchnyi, and Lel'. Three essentially different groups of species were distinguished, and the C. sativus species (subgenus Cucumis) was found to be distant from the species belonging to the subgenus Melo.     

Genetic relatedness among some wild cherry (<Emphasis Type="Italic">Prunus</Emphasis> subgenus <Emphasis Type="Italic">Cerasus</Emphasis>) genotypes native to Iran assayed by morphological traits and random amplified polymorphic DNA analysis

Subgenus Cerasus species are useful genetic resources for cherry breeding programs. A total of 17 morphological traits together with 19 random amplified polymorphic DNA (RAPD) primers were used to study 39 accessions including 34 wild Cerasus subgenus genotypes belonging to Prunus avium L., P. cerasus L., P. mahaleb L., P. microcarpa Boiss., P. incana Pall., and P. brachypetala Boiss. species, along with an unknown wild Cerasus sample, two advanced cherry cultivars (‘Lambert’ and ‘Bulgar’), and two rootstocks (‘Colt’ and ‘Gisela 6’). Genotypes were separated into different groups according to their species and collection sites using cluster analysis performed by Ward’s clustering method based on morphological data. Nineteen RAPD primers from 60 screened produced 304 polymorphic reproducible bands (98.15% polymorphism). According to the similarity matrix, the lowest similarity was obtained between P. avium and P. microcarpa samples. A dendrogram was prepared by the unweighted pair-group method with arithmetic average (UPGMA), and the accessions were separated according to their species and geographic origin. In both morphological and molecular results, the advanced cultivars and rootstocks were separated from wild genotypes, and the unknown genotype was grouped with P. mahaleb accessions. Grouping by morphological characteristics was compared with the results of RAPD analysis, with no significant correlations between morphological and molecular data being found. This is the first report of molecular (RAPD) genetic diversity study in wild Cerasus subgenus genotypes from Iran, and the results demonstrate the high potential of RAPD analysis for discrimination of Cerasus subgenus genotypes.     

The Genus Syringa: Molecular Markers of Species and Cultivars

RAPD analysis was carried out with 22 accessions of the genus Syringa, including six species, one interspecific hybrid, and 15 cultivars. In total, 512 polymorphic fragments were detected; species-specific and cultivar-specific markers were identified. For the first time, genetic polymorphism and genome similarity coefficients were estimated and phylogenetic relationships were established for the genus Syringa.     

Phylogenetic relationships among Cucumis species based on the ribosomal internal transcribed spacer sequence and microsatellite markers

The phylogenetic relationships within the genus Cucumis (a total of 25 accessions belonging to 17 species) were studied using the nuclear ribosomal DNA internal transcribed spacer (ITS) region. The analysis included commercially important species such as melon (C. melo L.) and cucumber (C. sativus). Two additional cucurbit species, watermelon and zucchini, were also included as outgroups. The data obtained reflected the clustering of Cucumis species in four main groups, comprising accessions from cucumber, melon, C. metuliferus and the wild African species. Some of the species clustered in different positions from those reported in classifications previously described by other authors. The data obtained clearly identify a division between the 2n=2x = 14 species (C. sativus) and the 2n = 2x = 24 ones (C. melo and wild species). Within the wild species we identified a subgroup that included C. sagittatus and C. globosus. Oreosyce africana, also classified as Cucumis membranifolius, was shown to be nested within Cucumis. Three accessions previously classified as independent species were shown to be genotypes of Cucumis melo. A set of melon and cucumber SSRs were also used to analyse the Cucumis species and the results were compared with the ITS data. The differential amplification of the SSRs among the accessions made it possible to distinguish three main groups: melon, cucumber and the wild species, though with less detail than applying ITS. Some SSRs were shown to be specific for melon, but other SSRs were useful for producing PCR fragments in all species of the genus.We are grateful to NCRPIS, IPK in Gatersleben, Semillas Fitó S.A., Michel Pitrat and Fernando Nuez for providing seeds. We would also like to thank Vanessa Alfaro, Trinidad Martínez and Núria Galofré for their excellent technical assistance. This work was financed by project AGL2000-0360 of Spains Ministerio de Ciencia y Tecnología (MCYT). AJMs work was supported by a postdoctoral contract from Spains MCYT.     

Relationships among <Emphasis Type="Italic">Leymus</Emphasis> species assessed by RAPD markers

The DNA genetic diversity of 40 accessions of genus Leymus was analyzed by random amplified polymorphic DNA (RAPD) markers. A total of 352 products were amplified by 34 10-mer arbitrary primers, among which 337 products (95.74 %) were found to be polymorphic. 5–14 polymorphic bands were amplified by each polymorphic primer, with an average of 9.91 bands. The data of 352 RAPD bands were used to generate Jaccard’s similarity coefficients and to construct a dendrogram by means of UPGMA. Great genetic diversity in genus Leymus was observed, the genetic diversity among the different species more abundant than that of the different accessions, and the different accessions in a species or the species from the same areas were clustered together.     

Development of novel chloroplast microsatellite markers for Cucumis from sequence database

The development of chloroplast microsatellite (cpSSR) markers in Cucumis species and analysis of their polymorphism and transferability were reported. Fifteen microsatellite markers, represented by mononucleotide repeats, were developed from the complete sequence of Cucumis sativus chloroplast genome. Intraspecific variation was successfully detected in C. sativus and C. melo and revealed mean 1.6 and 1.9 alleles per cpSSR locus, respectively. With the exception of two exon region-located cpSSR markers being monomorphic, each of the others amplified polymorphic fragments in C. sativus or C. melo. A total of 34 polymorphic loci were detected with these cpSSR markers in the two species. Transferability of the newly developed cpSSR markers was checked on an additional set of 41 Cucurbitaceae accessions (belonging to 12 different species), and except for two markers with no amplification in Cucurbita maxima, the others could be transferable to all the accessions tested. Of the 15 cpSSR markers, 14 markers generated fragments with expected band sizes and 13 markers detected interspecific polymorphism among the accessions. Intraspecific polymorphism was also observed within four Cucurbitaceae species excluding C. sativus and C. melo.     

Characterization of genetic diversity of nuclear and mitochondrial genomes in Daucus varieties by RAPD and AFLP

The genetic diversity of nuclear genomes of five Daucus species and seven Daucus carota L. subspecies involving 26 accessions was characterized with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP). AFLP produced more than four times as many discrete bands per reaction compared with RAPD analysis, while both AFLP and RAPD basically led to similar conclusions. The dendrograms constructed with both RAPD and AFLP revealed that all accessions of D. carota were grouped into a major cluster delimited from other Daucus species, in good agreement with the classification by morphological char-acteristics. All accessions of cultivated carrots [(D. carota ssp. sativus (Hoffm.) Arcang.] were clustered in the same group while the variation within D. carota was relatively extensive. Genetic diversity of mitochondrial genomes was also documented with RAPD for the same accessions. The mitochondrial dendrogram differed from that of the nuclear genome, suggesting that nuclear and mitochondrial genomes of some accessions had separate evolutionary histories. Received: 20 September 1997 / Revision received: 19 January 1998 / Accepted: 28 March 1998     

Phylogenetics of <Emphasis Type="Italic">Cucumis</Emphasis> (Cucurbitaceae): Cucumber (<Emphasis Type="Italic">C. sativus</Emphasis>) belongs in an Asian/Australian clade far from melon (<Emphasis Type="Italic">C. melo</Emphasis>)

Background  

Melon, Cucumis melo, and cucumber, C. sativus, are among the most widely cultivated crops worldwide. Cucumis, as traditionally conceived, is geographically centered in Africa, with C. sativus and C. hystrix thought to be the only Cucumis species in Asia. This taxonomy forms the basis for all ongoing Cucumis breeding and genomics efforts. We tested relationships among Cucumis and related genera based on DNA sequences from chloroplast gene, intron, and spacer regions (rbcL, matK, rpl20-rps12, trnL, and trnL-F), adding nuclear internal transcribed spacer sequences to resolve relationships within Cucumis.     

RAPD Analysis in Crocus sativus L. Accessions and Related Crocus Species

In the present paper a Random Amplified Polymorphic DNA (RAPD) investigation was carried out on DNAs from five Crocus sativus L. (saffron) accessions cultivated in different countries and on six closely related Crocus species. Aims of the study are to check whether cultivated saffron has maintained a constant genomic organisation and to clarify its relationships with possible ancestor species. For the fifteen primers, which produced positive results, DNAs of saffron corms from different accessions present the same amplification pattern, in accordance with the similar DNA content and base composition pointed out in previous studies. The amplification of the seven Crocus species DNAs with twenty-one primers provided 217 repeatable and interpretable fragments, which were scored for presence/absence and employed for a cluster analysis. Results indicated that C. sativus is very closely related to C. cartwrightianus and also similar to C. thomasii. This result, concurring with part of the previous evidence, would rule out the hypothesis of close relationships between C. sativus and C. pallasii.     

Reconstruction of ancestral karyotype illuminates chromosome evolution in the genus Cucumis

Karyotype dynamics driven by complex chromosome rearrangements constitute a fundamental issue in evolutionary genetics. The evolutionary events underlying karyotype diversity within plant genera, however, have rarely been reconstructed from a computed ancestral progenitor. Here, we developed a method to rapidly and accurately represent extant karyotypes with the genus, Cucumis, using highly customizable comparative oligo-painting (COP) allowing visualization of fine-scale genome structures of eight Cucumis species from both African-origin and Asian-origin clades. Based on COP data, an evolutionary framework containing a genus-level ancestral karyotype was reconstructed, allowing elucidation of the evolutionary events that account for the origin of these diverse genomes within Cucumis. Our results characterize the cryptic rearrangement hotspots on ancestral chromosomes, and demonstrate that the ancestral Cucumis karyotype (n = 12) evolved to extant Cucumis genomes by hybridizations and frequent lineage- and species-specific genome reshuffling. Relative to the African species, the Asian species, including melon (Cucumis melo, n = 12), Cucumis hystrix (n = 12) and cucumber (Cucumis sativus, n = 7), had highly shuffled genomes caused by large-scale inversions, centromere repositioning and chromothripsis-like rearrangement. The deduced reconstructed ancestral karyotype for the genus allowed us to propose evolutionary trajectories and specific events underlying the origin of these Cucumis species. Our findings highlight that the partitioned evolutionary plasticity of Cucumis karyotype is primarily located in the centromere-proximal regions marked by rearrangement hotspots, which can potentially serve as a reservoir for chromosome evolution due to their fragility.     

Comparison of RFLP and RAPD markers to estimating genetic relationships within and among cruciferous species

Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers are being used widely for evaluating genetic relationships of crop germplasm. Differences in the properties of these two markers could result in different estimates of genetic relationships among some accessions. Nuclear RFLP markers detected by genomic DNA and cDNA clones and RAPD markers were compared for evaluating genetic relationships among 18 accessions from six cultivated Brassica species and one accession from Raphanus sativus. Based on comparisons of genetic-similarity matrices and cophenetic values, RAPD markers were very similar to RFLP markers for estimating intraspecific genetic relationships; however, the two marker types gave different results for interspecific genetic relationships. The presence of amplified mitochondrial and chloroplast DNA fragments in the RAPD data set did not appear to account for differences in RAPD- and RFLP-based dendrograms. However, hybridization tests of RAPD fragments with similar molecular weights demonstrated that some fragments, scored as identical, were not homologous. In all these cases, the differences occurred at the interspecific level. Our results suggest that RAPD data may be less reliable than RFLP data when estimating genetic relationships of accessions from more than one species.     

Intra- and inter-specific variations in Lens revealed by RAPD markers

Randomly amplified polymorphic DNA (RAPD) markers were used to estimate intra- and interspecific variations in the genus Lens (lentil). Twenty cultivars of L. culinaris ssp. culinaris, including 11 microsperma (small-seeded) and nine macrosperma (large-seeded) types, and 16 wild relatives (four accessions each of L. culinaris ssp. orientalis, L. odemensis, L. nigricans and L. ervoides), were evaluated for genetic variability using a set of 40 random 10-mer primers. Fifty reproducibly scorable DNA bands were observed from ten of the primers, 90% of which were polymorphic. Genetic distances between each of the accessions were calculated from simple matching coefficients. A dendrogram showing genetic relationships between them was constructed by an unweighted pair-group method with arithmetical averages (UPGMA). This study revealed that (1) expect for L. ervoides, the level of intraspecific variation in cultivated lentil is lower than that in wild species, (2) L. culinaris ssp. orientalis is the most likely candidate for a progenitor of the cultivated species, and (3) microsperma and macrosperma cultivars were indistinguishable by the RAPD markers identified here.     

A comparative study of genetic relationships among the AA-genome<Emphasis Type="Italic"> Oryza</Emphasis> species using RAPD and SSR markers

In order to estimate genetic relationships of the AA-genome Oryza species, RAPD and SSR analyses were performed with 45 accessions, including 13 cultivated varieties (eight Oryza sativa and five Oryza glaberrima) and 32 wild accessions (nine Oryza rufipogon, seven Oryza nivara, three Oryza glumaepatula, four Oryza longistaminata, six Oryza barthii, and three Oryza meridionalis). A total of 181 clear and repeatable bands were amplified from 27 selected RAPD primers, and 101 alleles were detected from 29 SSR primer pairs. The dendrogram constructed using UPGMA from a genetic-similarity matrix based on the RAPD data supported the clustering of distinct five groups with a few exceptions: O. rufipogon/O. nivara/O. meridionalis, O. barthii/O. glaberrima, O. glumaepatula, O. sativa and O. longistaminata. The dendrogram based on the SSR analysis showed a more-complicated genetic variation pattern, but the O. longistaminata and O. barthii/O. glaberrima accessions were consistently separated from all other accessions, indicating significant differentiation of the African AA-genome Oryza species. For accessions in the O. rufipogon/O. nivara/O. sativa complex, it is apparent that geographical isolation has played an important role in differentiation of the Asian AA-genome Oryza taxa. It is also demonstrated from this study that both RAPD and SSR analyses are powerful methods for detecting polymorphisms among the different AA-genome Oryza accessions. However, the RAPD analysis provides a more-informative result in terms of the overall genetic relationships at the species level compared to the SSR analysis. The SSR analysis effectively reveals diminutive variation among accessions or individuals within the same species, given approximately the same number of primers or primer-pairs used in the studies.Communicated by Q. Zhang     

Evaluation of random amplified polymorphic DNA for species identification and phylogenetic analysis inStylosanthes (Fabaceae)

Random amplified polymorphic DNA (RAPD) was assessed for its suitability as a tool to be used in the identification of taxa from the genusStylosanthes (Fabaceae, Papilionoideae, Aeschynomeneae). Five random primers were used to fingerprint accessions from seven species in the genus, and generated RAPD profiles that were species-specific. Data were used to examine evolutionary relationships between taxa, employing both clustering and ordination techniques, and the results were compared with those from a previous cladistic analysis of chloroplast DNA (cpDNA) restriction fragments. Both multivariate approaches indicated relationships that were generally similar to those obtained by RFLP analysis of cpDNA. However, while cluster analysis grouped together all accessions within species, ordination placed certain accessions ofS. humilis, S. macrocephala andS. capitata into separate groups. Experiments to test the assumed homology of comigrating RAPDs estimated 85.7% homology for accessions within species, and 53.8% homology for accessions between species. The value of RAPD data in systematics is discussed.     

A comparative analysis of phloem exudate proteins from Cucumis melo,Cucumis sativus and Cucurbita maxima by polyacrylamide gel electrophoresis and isoelectric focusing

Summary Proteins in sieve tube exudate from Cucumis melo L., Cucumis sativus L. and Cucurbita maxima Duch. were analysed by gel electrophoresis and isoelectric focusing. Estimated molecular weights and isoelectric points for the major and minor proteins from each plant species are presented. Electrophoresis revealed striking differences between the protein complements of exudatc from the two genera investigated. Similarly, although a few exudate proteins from the two species of Cucumis possessed identical molecular weights, several major proteins were peculiar to each species. Isoelectric focusing of proteins in exudate samples from the three plants confirmed the marked differences in their protein complements. Furthermore, focusing also revealed differences between cultivars of Cucumis sativus. Both Cucumis sativus and Cucurbita maxima possessed relatively large amounts of basic proteins; these were absent in exudate from Cucumis melo. The implications of these results are discussed in relation to present concepts regarding the interrelationships and possible functional roles of P-proteins.Abbreviations DTT dithiothreitol - Bis N,N-methylenebisacrylamide - SDS Sodium dodecyl sulphate - TCA trichloroacetic acid     

RAPD重建的大豆属植物的亲缘关系

利用８种ＲＡＰＤ引物（ＯＰＨ－２、ＯＰＨ－３、ＯＰＨ－５、ＯＰＨ－１２、ＯＰＨ－１５、ＯＰＨ－１６、ＯＰＨ－１８和ＯＰＨ－２０）对大豆属的２１份植物材料,其中包括Ｇｌｙｃｉｎｅ亚属的１０个种和Ｓｏｊａ亚属的３个种,进行了基因组指纹图谱构建。通过对获得的基因组指纹图谱的量化分析,利用Ｕｎｗｅｉｇｈｔｅｄｐａｉｒｇｒｏｕｐｗｉｔｈｍａｔｈｅｍａｔｉｃａｖｅｒａｇｅ（ＵＰＧＭＡ）对大豆属中的各个种进行了亲缘关系重建。重建的亲缘关系表明：Ｇ．ｔｏｍｅｎｔｅｌｌａ种中存在３种不同的进化类型,其分化距离已大于某些种种间的分化距离,它们可能是被形态遮蔽的３个种。Ｓｏｊａ亚属内３个种的分化关系同前人的研究推断相同,其亲缘关系很近,这一结果支持将这３个种归并为一个种的观点。但是重建的亲缘关系未能显示出大豆属两个亚属的划分格局。     

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     

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.     

Molecular analysis of interspecific and intergeneric relationships of Banksia using RAPDs and non-coding chloroplast DNA sequences

 Random amplified polymorphic DNA (RAPD) and chloroplast DNA (cpDNA) markers were used to study interspecific and intergeneric relationships of Banksia (Proteaceae) to aid breeding of the genus for cut flower production. The accepted morphological phylogeny of Banksia, with two subgenera, two sections and 13 series, is unclear regarding the relationships of the commercial cut flower species B. coccinea. Fifteen RAPD primers and a non-coding cpDNA sequence between the trnL (UAA) and trnF (GAA) gene were applied to species of Banksia, the related genus Dryandra, and to Musgravea heterophylla as the outgroup, with cluster analysis applied to the results. The two methods were in broad agreement with each other, and with the accepted taxonomy, with closely related species pairs and groups clustering together, but RAPDs were not informative between distantly related species or species pairs. Banksia coccinea clustered with Dryandra and formed a polytomy with 2 Dryandra species and the two sections of subgenus Banksia. Subgenus Isostylis formed a polytomy with D. formosa, basal to subgenus Banksia, but with B. cuneata and B. illicifolia (both in subgenus Isostylis) polyphyletic. Dryandra did not separate as a clade and fell within Banksia, raising questions about the currently accepted view of the two as sister genera with parallel morphological development. The results indicate that interspecific and intergeneric hybridisation with genus Dryandra and subgenus Banksia may be possible routes for improvement of the commercial species B. coccinea. Received: 25 September 1996 / Accepted: 8 November 1996