Sequence variation of non-coding regions of chloroplast DNA of soybean and related wild species and its implications for the evolution of different chloroplast haplotypes

Soybean chloroplast DNAs (cpDNAs) are classified into three types (I, II and III) based on RFLP profiles. Type I is mainly observed in cultivated soybean (Glycine max), while type II and type III are frequently found in both cultivated and wild soybean (Glycine soja), although type III is predominant in wild soybean. In order to evaluate the diversity of cpDNA and to determine the phylogenetic relationship among different chloroplast types, we sequenced nine non-coding regions of cpDNA for seven cultivated and 12 wild soybean accessions with different cpDNA types. Eleven single-base substitutions and a deletion of five bases were detected in a total of 3849 bases identified. Five mutations distinguished the accessions with types I and II from those with type III, and seven were found in the accessions with type III, independently of their taxa. Four species of the subgenus Glycine shared bases that were identical to those with types I and II at two of the five mutation sites and shared bases that were identical to those with type III at the remaining three sites. Therefore, the different cpDNA types may not have originated monophyletically, but rather may have differentiated from a common ancestor in different evolutionary directions. A neighbor-joining tree resulting from the sequence data revealed that the subgenus Soja connected with Glycine microphylla which formed a distinct clade from Clycine clandestina and the tetraploid cytotypes of Glycine tabacina and Glycine tomentella. Several informative length mutations of 54 to 202 bases, due to insertions or deletions, were also detected among the species of the genus Glycine. Received: 16 December 1999 / Accepted: 12 February 2000     

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.     

CHLOROPLAST DNA POLYMORPHISM SUGGESTS NIGERIAN CENTER OF DOMESTICATION FOR THE COWPEA,VIGNA UNGUICULATA (LEGUMINOSAE)

Twenty-one independent chloroplast DNA polymorphisms were identified in Vigna unguiculata defining 19 different chloroplast DNA molecules (plastome types). Two plastome types, differing by a single character, were found among 32 accessions of cultivated cowpea (Vigna unguiculata ssp. unguiculata). Eighteen different plastome types were found among 26 accessions of wild cowpea (V. unguiculata ssp. dekindtiana). The very low level of chloroplast DNA diversity found in cultivated accessions relative to wild cowpea suggests that 1) the domesticated form was derived from a narrow selection of the wild germplasm and 2) chloroplast gene flow between wild and cultivated types has been very limited. Cladistic analysis of the cpDNA data generated a robust tree completely lacking homoplasy. Three wild accessions from Nigeria possessed a plastome type indistinguishable from one present in cultivated accessions, suggesting that Nigeria represents one center of domestication of the cowpea. The other plastome type within the cultivated germplasm was not found among wild accessions.     

Chloroplast DNA phylogeny of Lens (Leguminosae): origin and diversity of the cultivated lentil

A restriction-site analysis of chloroplast DNA (cpDNA) variation in Lens was conducted to: (1) assess the levels of variation in Lens culinaris ssp. culinaris (the domesticated lentil), (2) identify the wild progenitor of the domesticated lentil, and (3) construct a cpDNA phylogeny of the genus. We analyzed 399 restriction sites in 114 cultivated accessions and 11 wild accessions. All but three accessions of the cultivar had identical cpDNAs. Two accessions exhibited a single shared restriction-site loss, and a small insertion was observed in the cpDNA of a third accession. We detected 19 restriction-site mutations and two length mutations among accessions of the wild taxa. Three of the four accessions of L. culinaris ssp. orientalis were identical to the cultivars at every restriction site, clearly identifying ssp. orientalis as the progenitor of the cultivated lentil. Because of its limited cpDNA diversity, we conclude that either the cultivated lentil has passed through a genetic bottleneck during domestication and lost most of its cytoplasmic variability or else was domesticated from an ancestor that was naturally depauperate in cpDNA restriction-site variation. However, because we had access to only a small number of populations of the wild taxa, the levels of variation present in ssp. orientalis can only be estimated, and the extent of such a domestication bottleneck, if applicable, cannot be evaluated. The cpDNA-based phylogeny portrays Lens as quite distinct from its putative closest relative, Vicia montbretii. L. culinaris ssp. odemensis is the sister of L. nigricans; L. culinaris is therefore paraphyletic given the current taxonomic placement of ssp. odemensis. Lens nigricans ssp. nigricans is by far the most divergent taxon of the genus, exhibiting ten autapomorphic restriction-site mutations.     

Study on chloroplast DNA diversity of cultivated and wild pears (<Emphasis Type="Italic">Pyrus</Emphasis> L.) in Northern China

Eight pairs of chloroplast DNA (cpDNA) universal primers selected from 34 pairs were used to assess the genetic diversity of 132 pear accessions in Northern China. Among them, six amplified cpDNA fragments showed genetic diversity. A total of 24 variable sites, including 1 singleton variable site and 23 parsimony informative sites, as well as 21 insertion-deletion fragments, were obtained from the combined cpDNA sequences (5309–5535 bp). Two trnL-trnF-487 haplotypes, five trnL-trnF-413 haplotypes, five rbcL haplotypes, six trnS-psbC haplotypes, eight accD-psaI haplotypes and 12 rps16-trnQ haplotypes were identified among the individuals. Twenty-one haplotypes were identified based on the combined fragments. The values of nucleotide diversity (P_i), average number of nucleotide differences (k) and haplotype diversity (H_d) were 0.00070, 3.56408 and 0.7960, respectively. No statistical significance was detected in Tajima’s D test. Remarkably, the important cpDNA haplotypes and their representing accessions were identified clearly in this study. H_19 was considered as one of the ancient haplotypes and was a divergent centre. H_16 was the most common haplotype of the wild accessions. H_2 was the haplotype representing the most pear germplasm resources (46 cultivars and two wild Ussurian Pear accessions), followed by haplotype H_5 (30 cultivars, two wild Ussurian Pear accessions and four sand pears in outgroups) representing the cultivars ‘Dangshan Suli’ and ‘Yali’, which harbour the largest and the second largest cultivation areas in China. More importantly, this study demonstrated, for the first time, the supposed evolution routes of Pyrus based on cpDNA divergence in the background of pear phylogeny in Northern China.     

Introgression between wild and cultivated soybeans of Japan revealed by RFLP analysis for chloroplast DNAs

Wild soybeans collected in Japan were surveyed for RFLPs of chloroplast DNA. Three haplotypes were detected in RFLPs with a cpDNA clone which contains a LSC region adjacent to the left member of IR. Most of the plants tested possessed haplotype III, and a few plants, collected mostly in southern Japan, had haplotype II. Haplotype I, which is the predominant form in modern cultivars, was detected at six sites from four widely separated regions. Our results indicate that haplotype III is predominant in wild soybean of Japan. Some of the plants having haplotype I were phenotypically intermediate between wild and cultivated soybeans, while the others possessed a seed morphology and plant architecture typical of ordinary wild soybean. The plants having haplotype I appear to be either derivatives of hybridization between wild and cultivated soybeans or relics of a direct progenitor of soybean cultivars with the haplotype I chloroplast genome.     

RFLP analysis of cpDNA in the genus <Emphasis Type="Italic">Hypericum</Emphasis>

The chloroplast DNA of 43 species including 16 sections from the genus Hypericum was studied by PCR-RFLP analysis. The PCR-amplified products of four cpDNA regions, trnC-trnD, psbC-trnS, trnL-trnF and rbcL were digested with four restriction endonucleases. A high level of interspecific variation was detected while intraspecific diversity was not observed. The resulting parsimony analysis indicated the monophyletic assemblage of the sections Androsaemum, Olympia, Drosocarpium and Trigynobrathys. Monophyly of Hypericum is weakly supported, but close relationships of H. perforatum and H. maculatum are indicated. The members of Ascyreia are weakly resolved, but clustering of H. kouytchense and H. oblongifolium is well supported, however, H. reptans is nested with Olympia. CpDNA profiles and the positions on the parsimony tree indicate that the chloroplast donor among the putative parents of the hybrid species H. ×inodorum is H. androsaemum.     

The use of a non-coding region of chloroplast DNA in phylogenetic studies of the subtribeSonchinae (Asteraceae:Lactuceae)

The systematic utility of sequences from a non-coding region of chloroplast DNA (cpDNA) betweenpsbA andtrnH^(GUG) was examined by assessing phylogenetic relationships in subtribeSonchinae (Asteraceae:Lactuceae). Primers constructed against highly conserved regions of tRNA genes were used for PCR amplification and sequencing. ThepsbA-trnH intergenic spacer contains several insertions and deletions (indels) inSonchinae with the length varying from 385 to 450 bp. Sequence divergence ranges from 0.00% to 7.54% withinSonchinae, with an average of 2.4%. Average sequence divergence inSonchus subg.Sonchus is 2.0%, while the mean for subg.Dendrosonchus and its close relatives in Macaronesia (the woodySonchus alliance) is 1.0%. Our results suggest that this region does not evolve rapidly enough to resolve relationships among closely related genera or insular endemics in theAsteraceae. The phylogenetic utility ofpsbA-trnH sequences of the non-coding cpDNA was compared to sequences from the ITS region of nuclear ribosomal DNA. The results suggest that ITS sequences evolve nearly four times faster thanpsbA-trnH intergenic spacer sequences. Furthermore, the ITS sequences provide more variable and phylogenetically informative sites and generate more highly resolved trees with more strongly supported clades, and thus are more suitable for phylogenetic comparisons at lower taxonomic levels than thepsbA-trnH intergenic chloroplast sequences.     

Molecular phylogeny of mangroves VII. PCR-RFLP of trnS-psbC and rbcL gene regions in 24 mangrove and mangrove-associate species

Chloroplast DNA (cpDNA) regions, trnS-psbC and rbcL, from 120 individuals of 24 mangrove and mangrove associate species belonging to 11 orders, 13 families and 17 genera of Angiospermae were amplified by the polymerase chain reaction (PCR) and restriction-digested with HaeIII. Analysis of polymorphism in the restriction fragments (PCR-RFLP) revealed 18 classes of restriction banding pattern in trnS-psbC region. This has provided molecular evidence for diversity in the mangrove floral component at the above-species level. Intra-generic variations were observed in three genera, viz. Rhizophora, Avicennia and Suaeda. Species-specific restriction patterns were found in the genera Rhizophora and Suaeda. A natural hybrid belonging to the genus Rhizophora was also analysed, and its restriction pattern was the same as that of a putative parental species.PCR-RFLP analysis of rbcL gene region was less differentiating. However, it showed 13 different classes of restriction patterns and revealed the usefulness of these investigations for genome analysis at a higher taxonomic level. Intra-specific variation was not observed in any of the species in either of the cpDNA regions analysed. This is the first report which describes variations in the chloroplast genome of mangrove species. Received: 20 April 1999 / Accepted: 12 May 1999     

Phylogenetic analysis of the highland papayas (<Emphasis Type="Italic">Vasconcellea</Emphasis>) and allied genera (Caricaceae) using PCR-RFLP

The chloroplast and mitochondrial DNA diversity of 61 genotypes belonging to 18 Vasconcellea species, the so-called highland papayas, was studied by PCR-RFLP analysis of two non-coding cpDNA regions (trnM-rbcL and trnK1-trnK2) and one non-coding mtDNA region (nad4/1-nad4/2). This sample set was supplemented with six genotypes belonging to three other Caricaceae genera: the monotypic genus Carica, including only the cultivated papaya, and the genera Jacaratia and Cylicomorpha. Moringa ovalifolia was added as an outgroup species. The PCR-amplified cpDNA regions were digested with 18 restriction endonucleases, the mtDNA region with 11. A total of 22 point mutations and four insertion/deletions were scored in the sample. A higher level of interspecific variation was detected in the two cpDNA regions in comparison to the analysis of the mtDNA. Wagner parsimony and Neighbor-Joining analysis resulted in dendrograms with similar topologies. PCR-RFLP analysis supported the monophyly of Caricaceae, but among the 26 mutations scored, an insufficient number of markers discriminated between the different Caricaceae genera included in this study. Hence the inference of the intergeneric relationships within Caricaceae was impossible. However, some conclusions can be noted at a lower taxonomic level. The Caricaceae species were divided into two lineages. One group included only Vasconcellea spp., whereas the second included the remaining Vasconcellea spp., together with the papaya genotypes and those from the other Caricaceae genera. This may indicate a higher level of inter-fertility for the Vasconcellea species from the latter clade in interspecific crossings with papaya. The putative progenitors of the natural sterile hybrid V. × heilbornii, i.e. V. stipulata and V. cundinamarcensis, were only distantly related to V. × heilbornii. This indicates that probably none of these species was involved as the maternal progenitor in the origin of V. × heilbornii. Surprisingly, V. × heilbornii had organellar genome patterns identical with V. weberbaueri, suggesting a possible involvement of this species in the origin of V. × heilbornii. On the basis of discrepancy between morphological traits and the cpDNA profiles of some pairs of Vasconcellea species, we believe that besides V. × heilbornii, some other species have originated through interspecific hybridization. A reticulate evolution for Vasconcellea has therefore been suggested. Finally, intraspecific cpDNA variation was detected in V. microcarpa, thus providing molecular evidence for the high diversity previously indicated by morphological observations.Communicated by H. Nybom     

Genetic diversity and relationships of sweetpotato and its wild relatives in Ipomoea series Batatas (Convolvulaceae) as revealed by inter-simple sequence repeat (ISSR) and restriction analysis of chloroplast DNA

Genetic diversity and relationships of 40 accessions of Ipomoea, representing ten species of series Batatas, were examined using ISSR markers and restriction-site variation in four non-coding regions of chloroplast DNA. A total of 2071 ISSR fragments were generated with 15 primers in these accessions and, on average, 52 bands per accession were amplified. Most of the primers contained dinucleotide repeats. The ISSR fragments were highly polymorphic (62.2%) among the 40 accessions studied. Restriction analysis of chloroplast (cp) DNA revealed 47 informative restriction-site and length mutations. Phylogenetic analyses of ISSR and cpDNA datasets generally revealed similar relationships at the interspecific level, but the high polymorphism of ISSRs resulted in a better separation of intraspecific accessions. However, the combined ISSR and cpDNA dataset appeared to be appropriate in resolving both intra- and interspecific relationships. Of the species examined, I. trifida was found to be the most closely related to cultivated sweetpotato, the hexaploid I. batatas, while I. ramosissima and I. umbraticola were the most distantly related to I. batatas within the series. Ipomoea triloba, hitherto considered to be one of the ancestors of sweetpotato, was only distantly related to sweetpotato based on ISSR similarity index. Received: 4 January 1999 / Accepted: 27 September 1999     

Diversity and evolution of chloroplast DNA in Triticum and Aegilops as revealed by restriction fragment analysis

Summary Restriction fragment analysis of chloroplast (cp) DNAs from 35 wheat (Triticum) and Aegilops species, including their 42 accessions, was carried out with the use of 13 restriction enzymes to clarify variation in their cpDNAs. Fourteen fragment size mutations (deletions/insertions) and 33 recognition site changes were detected among 209 restriction sites sampled. Based on these results, the 42 accessions of wheat-Aegilops could be classified into 16 chloroplast genome types. Most polyploids and their related diploids showed identical restriction fragment patterns, indicating the conservatism of the chloroplast genome during speciation, and maternal lineages of most polyploids were disclosed. This classification of cpDNAs was principally in agreement with that of the plasma types assigned according to phenotypes arising from nucleus-cytoplasm interactions. These mutations detected by restriction fragment analysis were mapped on the physical map of common wheat cpDNA, which was constructed with 13 restriction endonucleases. Length mutations were more frequently observed in some regions than in others: in a 16.0 kilo base pairs (kbp) of DNA region, including rbcL and petA genes, 6 of 14 length mutations were concentrated. This indicates that hot spot regions exist for deletions/insertions in chloroplast genome. On the other hand, 33 recognition site mutations seemed to be distributed equally throughout the genome, except in the inverted repeat region where only one recognition site change was observed. Base substitution rate (p) of cpDNA was similar to that of other plants, such as Brassica, pea and Lycopersicon, showing constant base substitution rates among related taxa and slow evolution of cpDNA compared with animal mitochondrial DNA. Phylogenetic relationships among Triticum and Aegilops species were discussed, based on the present data.Contributions no. 45 and no. 490 from the Kihara Institute for Biological Research, Yokohama City University and the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, respectively.     

Comparative analysis of chloroplast DNA variability in wild and cultivated<Emphasis Type="Italic"> Citrullus</Emphasis> species

PCR amplification and restriction site analysis of chloroplast (cp) DNA regions was used to detect inter- and intraspecific differences in the genus Citrullus. More than 55 C. lanatus and 15 C. colocynthis accessions collected from diverse geographical areas, C. ecirrhosus and C. rehmii were used. Most of the cpDNA variation within Citrullus was the result of large indels and transitions and transversions. Indels at the ndhA, trnS-trnfM and trnC-trnD regions and several substitutions at restriction enzyme sites can be used to separate C. colocynthis from the other Citrullus species. A nucleotide substitution at a restriction enzyme site at the 3 flanking region of ndhF provided a diagnostic haplotype for C. lanatus var. lanatus, the cultivated watermelon. Similarly, a nucleotide substitution at an intergenic spacer region of the trnC-trnD region resulted in a diagnostic haplotype for citron, C. lanatus var. citroides. Several C. lanatus var. citroides accessions showed the var. lanatus haplotype. C. rehmii showed almost the same haplotype as C. lanatus var. citroides with the exception of a unique insertion at a cpSSR site. Since C. ecirrhosus lacks the derived diagnostic nucleotide substitutions of C. lanatus, it is probably the progenitor of the cultivated watermelon. Intraspecific haplotypes detected within C. colocynthis were associated with geographic origin.Communicated by J. Dvorak     

Chloroplast DNA variability in the genus Helianthus: restriction analysis and S1 nuclease mapping of DNA-DNA heteroduplexes

Chloroplast DNA (cpDNA) from 36 wild species of the genus Helianthus has been analysed with three restriction endonucleases (Bam HI, Hind III and Sst I). Out of the 71 restriction sites described on the reference cpDNA (sunflower cpDNA), three insertions/deletions and seven site modifications were detected during the survey of the other cpDNAs.Since restriction mapping showed only a very limited fraction of the DNA variability, we chose to adapt the S1 nuclease mapping technique to detect fine variations between chloroplast genomes. For this purpose, DNA-DNA heteroduplexes obtained between sunflower and wild-species DNAs were digested by S1 nuclease and the resulting mismatches were detected by classical endonuclease restriction and hybridization methods. The S1 nuclease mapping results were confirmed by sequencing one S1 nuclease-sensitive region detected between cultivated sunflower and two perennial wild-type species.As a result of these analyses, it appeared that the combination of restriction mapping and S1 nuclease mapping might be helpful to differentiate taxonomically close cytoplasms.     

Chloroplast DNA Diversity in Populations of Wild and Cultivated Barley

Chloroplast DNA (cpDNA) diversity was found within and among populations (245 accessions total) of wild barley, Hordeum vulgare L. ssp. spontaneum Koch from Israel and Iran. Three polymorphic restriction sites (HindIII, EcoRI, BclI) which define three distinct cpDNA lineages were detected. One lineage is common to populations in the Hule Valley and Kinneret of northern Israel, and in Iran. The second lineage is found predominantly in the Lower Jordan Valley and Negev. The distribution of the third lineage is scattered but widespread throughout Israel. Sixty two accessions of cultivated barleys, H. vulgare L., were found, with two exceptions, to belong to just one cpDNA lineage of wild barley, indicating that the cpDNA of cultivated barley is less variable than its wild ancestor. These results demonstrate the need for assessing intraspecific cpDNA variability prior to choosing single accessions for phylogenetic constructions at the species level and higher.     

Chloroplast DNA variation in the genusSecale (Poaceae)

The chloroplast genomes of 44 accessions ofSecale were surveyed for restriction site polymorphisms. The accessions were chosen to represent the geographic as well as taxonomic range of the genus. Using 12 restriction enzymes a total of 348 sites were detected. Twenty-nine mutation sites were phylogenetically informative and used in a cladistic analysis. Further, a 0.1 kb insertion separatedSecale from the outgroup species. Only the annual speciesS. sylvestre was distinct from the rest of the taxa. Cultivated rye together with both wild annual and wild perennial accessions were mixed among each other. Sequence divergence (p) among taxa ofSecale was low, varying from 0.000 to 0.005, suggesting a rather recent origin of the genus.     

Chloroplast DNA variation and evolution in pisum: patterns of change and phylogenetic analysis

Variation in 30 chloroplast DNAs, representing 22 wild and cultivated accessions in the genus Pisum, was analyzed by comparing fragment patterns produced by 16 restriction endonucleases. Three types of mutations were detected. First, an inversion of between 2.2 kilobase pairs (kb) and 5.2 kb distinguished a population of P. humile from all other Pisum accessions examined. Second, deletions and insertions of between 50 and 1200 base pairs produced small restriction fragment length variations in four regions of the 120-kb chloroplast genome. Two of these regions—one of which is located within the sequence that is inverted in P. humile—showed a high degree of size polymorphism, to the extent that size differences were detected between individuals from the same accession. Finally, a total of only 11 restriction site mutations were detected among the 165 restriction sites sampled in the 30 DNAs. Based on these results and previous data, we conclude that the chloroplast genome is evolving very slowly relative to nuclear and mitochondrial DNAs. The Pisum chloroplast DNA restriction site mutations define two major lineages: One includes all tested accessions of P. fulvum, which is known to be cytogenetically quite distinct from all other Pisum taxa. The second includes 12 of 13 cultivated lines of the garden pea (P. sativum) and a wild population of P. humile from northern Israel. These observations strongly reinforce an earlier conclusion that the cultivated pea was domesticated primarily from northern populations of P. humile. A 13th P. sativum cultivar has a chloroplast genome that is significantly different from those of the aforementioned lines and somewhat more similar to those of P. elatius and southern populations of P. humile. This observation indicates that secondary hybridization may have occurred during the domestication of the garden pea.     

Induction of multiple shoots in a monopodial orchid hybrid (Aerides vandarum Reichb.f × Vanda stangeana Reichb.f) using thidiazuron and analysis of their genetic stability

The effect of thidiazuron (TDZ) on direct multiple shoot induction in axenic seedlings of a monopodial orchid hybrid Aerides vandarum × Vanda stangeana, using a dual phase culture medium was studied. The culture system consisted of a basal agar solidified half-strength Murashige and Skoog medium overlaid by a liquid fraction of the same composition. Highest regeneration of multiple shoots (15.8 shoots per seedling) was obtained in the medium containing 2% sucrose (w/v) supplemented with 2 mgl⁻¹ TDZ. Genetic stability of the regenerated shoots was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), and restriction fragment length polymorphism of the PCR amplified (PCR-RFLP) nrITS region, as well as those of the coding (matK) and non-coding (trnL-F) regions of the cpDNA. Across the randomly selected mother plant and nine of its regenerated shoots, 2,680 bands were generated by 19 RAPD and 12 ISSR primers, exhibiting monomorphic banding profiles. Homogenous PCR-RFLP profiles were generated for nrITS using four restriction enzymes (REs), matK using five REs, and trnL-F using six REs. These molecular analyses showed no genomic alterations in all regenerated shoots obtained on medium containing 2 mgl⁻¹ TDZ.     

Utility of a multidisciplinary approach for genome diagnostics of cultivated and wild germplasm resources of medicinal Withania somnifera, and the status of new species, W. ashwagandha, in the cultivated taxon

Realizing the inconsistencies that exist in the extent and nature of differentiation in the Withania somnifera genetic resources in India, the 21 cultivated and wild accessions, and the two hybrids (cultivated?×?wild accessions and vice versa) were investigated for morphological, cytogenetical, chemical profiling, and crossability features. Their nuclear and chloroplast genomes were also assayed at the nucleotide sequence level, and by use of DNA markers. Chloroplast DNA diversity and somatic chromosome number (2n?=?48) were not helpful in identifying the differences. Other approaches, on the other hand, especially restriction endonuclease digests, types and sequence length composition of ITS 1 and ITS 2 of nuclear ribosomal DNA, AFLP fingerprinting, and crossability barriers unambiguously provided startling discrete differences between the cultivated and wild accessions, indicating a clear division of W. somnifera into two distinct lineages. These data, therefore, are indicative of the fact that because of the unique characteristics of its nuclear genome, and strong crossability barriers vis-à-vis wild accessions of W. somnifera, the cultivated accessions should be relegated to the rank of the separate species, W. ashwagandha.     

Chloroplast DNA diversity in Vicia faba and its close wild relatives: implications for reassessment

To obtain new information on phylogenetic relationships between wild and cultivated broad bean, restriction fragment length polymorphism (RFLP) analysis of chloroplast (cp) DNAs from Vicia faba and eight subspecies/species of its close wild relatives grouped together in the Narbonensis complex was carried out using 14 restriction endonucleases. The molecular sizes of the cpDNAs obtained were similar (122.6–123.4 kbp), indicating that they had all lost one of inverted repeats. Among the more than 300 sites surveyed, the three subspecies within V. narbonensis, which exhibit just as many types of karyotypes, were shown to have identical cp fragment patterns. Genetic distances between all of the pairs of species were calculated from RFLP data. The cpDNA diversity within the Narbonensis complex was found to be more extensive than expected, except for the genetic relationship between V. hyaeniscyamus and V. johannis in which a total of three mutations were detected among the 300 sites sampled, thereby showing their close relatedness. The cpDNA of V. faba vis-a-vis its wild relatives also exhibited startling differences, indicating a clear division of Vicia species into two distinct lineages. This analysis unambiguously provides new evidence that the wild species grouped in the complex did not contribute their plastomes to the evolution of V. faba, and hence none of the species can be considered to be putative allies of broad bean. The present study also demonstrates profound cpDNA diversity among closely related species that have lost one of inverted repeats.