Diversity of Olea genotypes and the origin of cultivated olives

Tandem repeats belonging to three DNA sequence families (OeTaq80, OeTaq178, and OeGEM86) were isolated from the nuclear DNA of Olea europaea cv. Carolea and dot-hybridized to the genomic DNA of 14 hypothetically different Olea species, 78 olive cultivars, and 14 wild olives. The copy number per unreplicated haploid genome of OeTaq80- and OeTaq178-related sequences was in the 10⁷-10⁶ range and that of OeGEM86-related sequences was in the 10⁵ range in cultivars, wild olives and some Olea species. A large variation in the frequency of repeats belonging to each sequence family was observed within each group of plants. Positive correlations existed in each genome between the frequencies of repeats belonging to each family, and their overall frequency was positively correlated to the genome size. Duncan grouping showed that the frequency variation of tandem repeats within each group of plants was not continuous. Two main groups and several subgroups of genotypes could be separated within both the olive cultivars and the wild olives. Discrete areas in the Mediterranean Basin could be delimited by the geographic distribution of cultivated olives with different genotypes and the wild plants were associated with the cultivars in these areas according to genotypic similarity. The Olea species could be divided into four genotypic groups. Three of these, comprising accessions from Asia and North Africa, showed similarity with the genotypes of cultivars and wild olives. These results suggest a polyphyletic origin of cultivated olives from different wild Olea forms distributed throughout the Mediterranean Basin.     

Establishing the genetic relationships between the wild and cultivated olives using a nuclear intron from nitrate reductase (nia-i3)

In Oleaceae the most outstanding biological issue is to clarify the taxonomic relationships of cultivated and wild olives. To establish the genetic relationships between the wild (Olea europaea subsp. europaea var. sylvestris (Mill.) Lehr.), the cultivated olive (Olea europaea subsp. europaea var. europaea), and other taxa of the genus Olea (Olea europaea subsp. cuspidata (Wall. ex G. Don) Cif., Olea europaea subsp. cerasiformis G. Kunkel & Sunding, Olea paniculata R. Br.) and other Oleaceae (represented by Ligustrum vulgaris) we carried out the amplification by polymerase chain reaction (PCR) and the sequencing of the third nuclear intron of the nitrate reductase gene (nia-i3). Sequence analyses showed the presence of two different functional variants of the intron (nia1 and nia2) in the Oleaceae, in addition to a shorter non-functional one. Notably, while the shortest and the nia1 variants were present in all the taxa analysed, the nia2 variant was present only in the wild and the cultivated olive. These data confirm the close phylogenetic relationship between wild and cultivated olives and suggest that this gene could be duplicated in these two taxa after its divergence from the remaining Oleaceae. The presence of a target for AflII enzyme in nia2 and its absence in nia1 variant enables easy distinction by PCR-RFLP between, on the one hand, wild and cultivated olive, and on the other the remaining subspecies of the Olea europaea L. complex (O. e. subsp. cuspidata and O. e. subsp. cerasiformis) as well as other Oleaceae (O. paniculata, L. vulgaris L.). Additionally, nia1 sequences provide useful information about phylogeny of the wild and cultivated olives inside the genus Olea.     

Chloroplast DNA variation in the cultivated and wild olive taxa of the genus Olea L.

Polymorphism in the lengths of restriction fragments of the whole cpDNA molecule were studied in 15 taxa (species or subspecies) of the genus Olea. From restriction analysis using nine endonucleases, 28 site mutations and five length polymorphisms were identified, corresponding to 12 distinct chlorotypes. From a phenetic analysis based on a Nei’s dissimilarity matrix and a Dollo parsimony cladistic analysis using, as an outgroup, a species of the genus Phillyrea close to Olea, the ten taxa of section Olea were distinguished clearly from the five taxa of section Ligustroides which appear to posses more ancestral cpDNA variants. Within the section Ligustroides, the tropical species from central-western Africa, Olea hochtetteri, showed a chlorotype which differed substantially from those of the other four Olea taxa growing in southern Africa, supporting a previous assessment according to which O. hochtetteri may have been subjected to a long period of geographical isolation from the other Olea taxa. Within the Olea section, three phyla were identified corresponding to South and East Africa taxa, Asiatic taxa, and a group including Saharan, Macaronesian and Mediteranean taxa, respectively. On the basis of cpDNA variation, the closest Olea taxa to the single Mediterranean species, Olea europaea, represented by its very predominant chlorotype, observed in both wild and cultivated olive, were found to be Olea laperrinei (from the Sahara), Olea maroccana (from Maroccan High Atlas) and Olea cerasiformis (from Macaronesia). These three taxa, which all share the same chlorotype, may have a common maternal origin. Received: 5 December 1999 / Accepted: 30 December 1999     

Population genetics of Mediterranean and Saharan olives: geographic patterns of differentiation and evidence for early generations of admixture

Background and Aims

The olive (Olea europaea subsp. europaea) was domesticated in the Mediterranean area but its wild relatives are distributed over three continents, from the Mediterranean basin to South Africa and south-western Asia. Recent studies suggested that this crop originated in the Levant while a secondary diversification occurred in most westward areas. A possible contribution of the Saharan subspecies (subsp. laperrinei) has been highlighted, but the data available were too limited to draw definite conclusions. Here, patterns of genetic differentiation in the Mediterranean and Saharan olives are analysed to test for recent admixture between these taxa.

Methods

Nuclear microsatellite and plastid DNA (ptDNA) data were compiled from previous studies and completed for a sample of 470 cultivars, 390 wild Mediterranean trees and 270 Saharan olives. A network was reconstructed for the ptDNA haplotypes, while a Bayesian clustering method was applied to identify the main gene pools in the data set and then simulate and test for early generations of admixture between Mediterranean and Saharan olives.

Key Results

Four lineages of ptDNA haplotypes are recognized: three from the Mediterranean basin and one from the Sahara. Only one haplotype, primarily distributed in the Sahara, is shared between laperrinei and europaea. This haplotype is detected once in ‘Dhokar’, a cultivar from the Maghreb. Nuclear microsatellites show geographic patterns of genetic differentiation in the Mediterranean olive that reflect the primary origins of cultivars in the Levant, and indicate a high genetic differentiation between europaea and laperrinei. No first-generation hybrid between europaea and laperrinei is detected, but recent, reciprocal admixture between Mediterranean and Saharan subspecies is found in a few accessions, including ‘Dhokar’.

Conclusions

This study reports for the first time admixture between Mediterranean and Saharan olives. Although its contribution remains limited, Laperrine''s olive has been involved in the diversification of cultivated olives.     

Prospects for improving biological control of olive fruit fly,Bactrocera oleae (Diptera: Tephritidae), with introduced parasitoids (Hymenoptera)

Olive fruit fly is a key pest of olive and consequently a serious threat to olive fruit and oil production throughout the Mediterranean region. With the establishment of Bactrocera oleae in California a decade ago, interest was renewed in classical (introduction) biological control of the pest. Here we discuss the prospects of identifying natural enemies of B. oleae in Africa and Asia that may help reduce B. oleae populations in California and elsewhere. Based on the current understanding of Bactrocera phylogenetics, early opinions that B. oleae originated in Africa or western Asia rather than the Mediterranean region or the Near East are taxonomically and ecologically supportable. Closely related to cultivated olive, the wild olive Olea europaea cuspidata is widely distributed in southern and eastern Africa, the Arabian Peninsula, and eastwards into Asia as far as southwestern China. Little is known regarding the biology and ecology of B. oleae in Africa and eastern Asia, especially in wild olives. While the diversity of parasitoids of B. oleae in the Mediterranean region is low and unspecialized, a diverse assemblage of parasitoids is known from B. oleae in Africa. Conversely, regions in Asia have remained largely unexplored for B. oleae and its natural enemies.     

<Emphasis Type="Italic">Olea europaea</Emphasis> (Oleaceae) phylogeography based on chloroplast DNA polymorphism

Chloroplast DNA diversity in the olive (Olea europaea L.) complex was studied using PCR-RFLP and microsatellite markers. Fifteen chlorotypes were distinguished. We constructed a cpDNA phylogenetic tree in which five clades were recognised and located in distinct geographic areas: clade A in Central and Southern Africa, clade C in Asia, clade M in North-West Africa, clade E1 in the Mediterranean Basin and Sahara, and clade E2 in West Mediterranea. Cultivated olive clustered with Mediterranean and Saharan wild forms (clades E1 and E2). Strong genetic differentiation for cpDNA markers was observed between eastern and western Mediterranean olives, suggesting that these areas have represented different glacial refugia. Humans most likely spread one eastern chlorotype, preponderant in cultivars, across the western Mediterranean Basin. Its presence in O. e. subsp. laperrinei from the Sahara suggests a possible Mediterranean olive origin in an African population, which may have overlapped in the Southern Mediterranean during the Quaternary.     

西南地区6种魔芋属植物基于cpDNA序列的遗传多样性研究

研究野生作物资源的遗传变异及分化机制对种质资源的收集与改良具有重要意义。魔芋是我国西南地区的特色经济作物,但由于受到人为活动干扰,野生种群不断衰退。为评估西南地区魔芋属(Amorphophallus)野生群体的遗传多样性,探究代表性物种的系统发育地位,该研究利用3个叶绿体DNA(cpDNA)片段,分析了魔芋6个物种的遗传多样性,重建了种间系统发育关系。结果表明:(1)西南地区野生魔芋群体的遗传多样性普遍较低,虽然单倍型多样性(H_d)均值为0.428,但近一半群体只有1个单倍型,6个物种整体水平上的单倍型多样性在0.704到0.983之间。(2)在6个物种间检测到高水平的遗传分化,遗传分化系数(F_ST)值在0.481到0.967之间。(3)系统发育分析表明,选取的27个魔芋种主要聚成3个分支:非洲分支、东南亚分支和东亚大陆分支。疣柄魔芋(A. paeoniifolius)隶属于东南亚分支,而东亚大陆分支A包含花魔芋(A. konjac)和西盟魔芋(A. krausei),东亚大陆分支B由东亚魔芋(A. kiusianus)、滇魔芋(A. yunnanensis)和东京魔芋(A. tonkinensis)构成。生境隔离与人为干扰造成了西南地区野生魔芋群体较低的遗传多样性,魔芋属东亚大陆分支的分化可能与早期的快速扩张和生态适应有关。该研究为西南地区魔芋资源的合理保护、可持续利用和杂交育种提供了参考资料。     

Hybridization and phylogeography of the Mozambique tilapia <Emphasis Type="Italic">Oreochromis mossambicus</Emphasis> in southern Africa evidenced by mitochondrial and microsatellite DNA genotyping

Many Oreochromis species utilized in aquaculture were extensively introduced outside their native range in Africa. Given their recent evolutionary radiation, these species hybridize easily, posing a threat to the integrity of local adaptation. The objective of this work was to study the genetic diversity of the Mozambique tilapia (Oreochromis mossambicus) in its native range, southern Africa, and provide a method for identifying hybrids with genetic markers. We genotyped the mitochondrial DNA (mtDNA) control region (385 bp) of wild and farmed O. mossambicus, wild and farmed O. niloticus and morphologic wild hybrids. These data were complemented with published sequences of parapatric and sympatric Oreochromis taxa. Phylogeographic analysis showed the presence of two O. mossambicus lineages, the southernmost representing a recent Holocene radiation. Hybridization of O. mossambicus was indicated by the presence of O. niloticus and O. mortimeri–andersonii mtDNA specimens in the Limpopo basin and of O. karongae mtDNA in specimens from Malawi. We also genotyped seven suspected hybrid individuals from the Limpopo River, and 137 wild and farmed Mozambique and Nile tilapia samples with five microsatellite markers. Factorial Component Analysis, Bayesian clustering and assignment analyses consistently delineated an O. mossambicus and an O. niloticus group, with the putative hybrids positioned in between. Different levels of hybridization were detected by the Bayesian assignment. The complex nature of hybridization and introgression between cichlid species raises major concerns for the long-term integrity of Mozambique tilapia.     

Somatic embryogenesis induction in leaves and petioles of a mature wild olive

We describe a protocol for somatic embryogenesis (SE) induction from an adult wild olive tree (Olea europaea ssp. europaea var. sylvestris. The protocol used confirms for the first time that there is no need to use juvenile or rejuvenated material for SE induction. For SE induction, petiole and leaf (proximal, intermediary and distal zones) explants were grown on Murashige and Skoog (MS) or Olive Medium (OM) media with different combinations of plant growth regulators (PGR): α- naphthaleneacetic acid (NAA), Zeatin (Zea), indole-3-butyric acid (IBA), 2-isopentyl adenine (2iP), thidiazuron (TDZ) and 6-benzylaminopurine (BAP). All media had 30 g/l sucrose and 7 g/l agar, and the pH was adjusted to 5.8. Cultures were incubated in the dark and, after 3 months, they were transferred to MS medium without PGR for expression. Petiole explants gave the highest callus production, while for SE induction and expression distal blade leaf and petiole explants gave the highest rates. The best medium for SE induction was MS with 12.25 μM IBA plus 4.56 μM Zea. Histological analyses confirmed the individuality of globular somatic embryos. This is the first report of SE expression in explants without rejuvenation in Olea genus, and opens perspectives for using this strategy in SE protocols both for this wild genotype and for commercial genotypes.     

Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers

Micropropagated plants from two wild-olive species, Olea maderensis and O. europaea ssp. europaea var. sylvestris were screened for genetic stability. O. maderensis shoots were elongated/multiplied on OMG medium with zeatin (9.12 μM), and rooted on 1/2 OMG with NAA (3.22 μM). O. europaea var. sylvestris shoots were elongated/multiplied on OM medium with zeatin, and rooting was optimal after a hormonal shock (IBA 100 μM) followed by transfer to the same medium without growth regulators. In both species, acclimatization was successful and plants looked normal and morphologically identical to the donor field trees. Genetic variability was assessed at several stages of the micropropagation process using flow cytometry (FCM) and nuclear microsatellites (SSR). No changes in ploidy level were found among micropropagated plants, though small deviations, putatively due to the negative effects of cytosolic compounds on propidium iodide staining, between these and field plants were observed. In SSRs analyses, ten SSR markers were able to distinguish between genotypes. No mutations were found in these tested SSR loci among the donor tree and micropropagated plants, suggesting, for the tested markers, genetic uniformity throughout the process. The FCM and SSR results obtained do not exclude the occurrence of other changes in the nuclear genome but, considering the morphological stability of micropropagated plants, indicate that both protocols are suitable and efficient for large scale, true-to-type micropropagation of these two wild olive species.     

Microsatellite analysis of Latial Olea europaea L. cultivars

Olea europaea L. is one of the oldest domesticated tree species. O. europaea varieties cannot be confused because they are very different in morphology, genetics, and secondary metabolite content. It is important to study and establish the genetic structure of vegetal cultivars to better distinguish them, to solve past misclassification, to preserve plant biodiversity, and to increase their use, diffusion, selection, resistance to adversities, marketing, and scientific applications. Five simple sequence repeat loci (DCA-3, DCA-9, UDO99-9, UDO99-35, and EMO-3) were used to differentiate 39 individuals, representing 13 olive cultivars sampled in Latium (Central Italy). The markers showed a high discrimination power and were able to differentiate 39 alleles. Observed heterozygosity ranged from 0.538 at locus UDO99-9 to 1 at locus UDO99-35, with a mean value of 0.784. DCA loci were the most informative ones. Sample clustering, based on their genetic distance and similarity values, produced a phylogenetic network that has shown a unique major group of cultivars, composed of two sub-branches, and two independent taxa.     

AFLP based assessment of genetic relationships among shiitake (<Emphasis Type="Italic">Lentinula</Emphasis> ssp.) mushrooms

Despite the economical importance of shiitake (Lentinula ssp.) mushrooms, until the present date little information exists on cultivated and wild species in correlation with geographic origin applying molecular techniques. Use of a high resolution molecular tool like AFLP for assessing genetic similarity and geographical diversity would be an important step towards understanding of different Lentinula species. Thirteen wild and 17 cultivated accessions of 3 Lentinula species were analysed with 64 EcoRI–MseI primer combinations and finally 32 reproducible and polymorphic primer combinations were considered for the analysis. A total of 816 informative AFLP markers were generated and scored as binary data. These data were analysed using various method packages for cluster analysis, genetic diversity and genetic differentiation. Percentage polymorphism was high (62.99%) among the species studied. Different clustering analysis segregated the wild and the cultivated species into two major branches, with the wild samples being further grouped according to their geographic location. Overall polymorphisms among cultivated strains in the USA were higher than that of the cultivated strains in Japan (58.9%).     

Phylogenetic relationships among Oryza species revealed by AFLP markers

 The genus Oryza to which cultivated rice belongs has 22 wild species. Seventy-seven accessions of 23 Oryza species, five related genera, and three outgroup taxa were fingerprinted using amplified fragment length polymorphism (AFLP). A total of 1191 polymorphic markers were obtained using five AFLP primer combinations. AFLP data were analyzed to study species relationships using different clustering algorithms, and the resulting phenograms were tested for stability and robustness. The findings suggest a common ancestry to the genus Oryza. Moreover, the results demonstrate that: (1) evolution in Oryza has followed a polyphyletic path wherein multiple lineages underwent independent divergence after separation early in the evolution from a common ancestor/pool of related taxa; (2) newly assigned genomes, GG for O. meyeriana and HHJJ for O. ridleyi complexes, are among the most diverged in the genus; (3) CCDD tetraploids have a relatively ancient origin among the Officinalis complex; (4) O. malampuzhaensis, O. indandamanica, O. alta, and O. grandiglumis are diverged enough to deserve species status; (5) O. officinalis and O. eichingeri (CC) are putative progenitors of O. minuta ^* O. malampuzhaensis and tetraploid O. punctata, respectively, (6) O. brachyantha is most diverged species in the genus. AFLP is reliable molecular technique and provides one of the most informative approaches to ascertain genetic relationships in Oryza, which may also be true for other related species/organisms. Received: 1 July 1998 / Accepted: 2 November 1998     

Macrogeographic population structuring in the cosmopolitan agricultural pest Bactrocera cucurbitae (Diptera: Tephritidae)

The macrogeographic population structure of the agricultural pest Bactrocera cucurbitae (Diptera: Tephritidae) was investigated in order to identify the geographic origin of the species and reconstruct its range expansion. Individuals of B. cucurbitae were collected from 25 worldwide‐distributed localities (n = 570) and genotyped at 13 microsatellite loci. The Bayesian clustering reveals that B. cucurbitae can be subdivided into five main groups corresponding to populations from (i) the African continent, (ii) La Réunion, (iii) Central Asia, (iv) East Asia and (v) Hawaii. The proportions of inter‐regional assignments and the higher values of genetic diversity in populations from Pakistan, India and Bangladesh suggest that B. cucurbitae originated in Central Asia and expanded its range to East Asia and Hawaii on one hand and to Africa and the islands of the Indian Ocean on the other. A number of outliers (10–19 specimens according to different clustering algorithms) show high levels of admixture (Q > 0.70) with populations from different regions and reveal complex patterns of inter‐regional gene flow. Anthropogenic transport is the most plausible promoter of this large‐scale dispersal. The introduction of individuals from geographically distant sources did not have a relevant role in the most recent African invasions, which originated from the expansion of local populations. These results could provide a useful background to better evaluate invasion risks and establish priorities for the management of this cosmopolitan agricultural pest.     

Comparison of similarity coefficients used for cluster analysis based on RAPD markers in wild olives

Five different similarity coefficients (Jaccard, Sorensen-Dice, simple matching, Rogers and Tanimoto, and Russel and Rao) were evaluated and 10 wild olives analyzed with RAPD markers. The influence of the similarity coefficients on wild olives clustering was investigated. Forty-five primers were used on samples from 10 wild olives (Wild 1 and 2 obtained from Mugla province; Wild 3, 4, 5, 6, 7, and 8 from Manisa province and Wild 9 and 10 from Izmir province of Turkey). The similarity matrices obtained from RAPD markers were compared by the Mantel test. Cluster analysis was made with UPGMA dendrograms, and the consensus fork indexes between all pairs of dendrograms were calculated. The Jaccard and Sorensen-Dice coefficients gave the same results, due to the fact that both exclude negative co-occurrences. The dendrograms using the simple matching and Rogers and Tanimoto coefficients were similar; Wild 4 (Akhisar, Manisa) and Wild 9 (Bornova, Izmir) olives had the closest genetic similarities. This occurred because these coefficients include negative co-occurrences. The Russel and Rao coefficients produced different results, because they include negative co-occurrences in the denominator. We concluded that the coefficients that do not include negative co-occurrences are more efficient for studies of wild olives clustering based on RAPD markers.     

Identification of new polymorphic regions and differentiation of cultivated olives (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) through plastome sequence comparison

Background  

The cultivated olive (Olea europaea L.) is the most agriculturally important species of the Oleaceae family. Although many studies have been performed on plastid polymorphisms to evaluate taxonomy, phylogeny and phylogeography of Olea subspecies, only few polymorphic regions discriminating among the agronomically and economically important olive cultivars have been identified. The objective of this study was to sequence the entire plastome of olive and analyze many potential polymorphic regions to develop new inter-cultivar genetic markers.     

Transferability of olive microsatellite loci across the genus <Emphasis Type="Italic">Olea</Emphasis>

The transferability of microsatellite markers developed for olive cultivars (Olea europaea L.) has been tested and confirmed in the Olea complex. Thirty two genotypes, belonging to different taxa of the genus Olea, have been analyzed with four olive SSRs. Positive amplifications at all loci were obtained in 13 taxa (at least one accession per species). Sixty seven different alleles have been detected at the four loci analyzed. Polymorphic products have been observed at the inter- and intra-species level. Some SSR loci have shown multiple amplification products in some species. The high number of unique alleles has allowed the unambiguous discrimination of most accessions. Similarity coefficients and relationships among the Olea taxa have been calculated based on SSR amplification results. The reliability of SSRs as markers for intra-species variability evaluation has been confirmed while their use to explore relationships at the inter-species level is discussed, being dependent on the locus analyzed.Communicated by H.F. Linskens     

Patterns of morphological variation among populations of the widespread annual killifish Nothobranchius orthonotus are independent of genetic divergence and biogeography

Populations of annual killifish of the genus Nothobranchius occur in patchily distributed temporary pools in the East African savannah. Their fragmented distribution and low dispersal ability result in highly structured genetic clustering of their populations. In this study, we examined body shape variation in a widely distributed species, Nothobranchius orthonotus with known phylogeographic structure. We tested whether genetic divergence of major mitochondrial lineages forming two candidate species is congruent with phenotypic diversification, using linear and geometric morphometry analyses of body shape in 23 wild populations. We also conducted a common‐garden experiment with two wild‐derived populations to control for the effect of local environmental conditions on body shape. We identified different allometric trajectories for different mitochondrial lineages and candidate species in both sexes. However, in a principal components analysis of population‐level body shape, the separation among mitochondrial lineages was incomplete. Higher similarity of mitochondrial lineages belonging to different candidate species than that of same candidate species prevented distinction of the two candidate species on the basis of body shape. Analysis at the individual level demonstrated that N. orthonotus express high intrapopulation variability, with major overlap among individuals from all populations. In conclusion, we suggest that N. orthonotus be considered as a single species with an extensive geographic range, strong population genetic structure and high morphological variability.     

Genetic relationship of Porteresia coarctata Tateoka using molecular markers

ABSTRACT

Twenty-one species belonging to Oryza, including wild rices, were compared with a tetraploid (2n=48) halophytic wild rice relative, Porteresia coarctata Tateoka (=Oryza coarctata) for the genetic relatedness using AFLP and RAPD markers. Diploid and tetraploid groups were clearly separated except in the case of a few species where the clustering was unique and different. The molecular analysis has helped in positioning Porteresia in the vicinity of other wild rice species, and to better understand the pattern of species differentiation in Oryza. From our study, O. australiensis seems to be related to P. coarctata; thus, O. australiensis may be an effective “bridge” species in transferring genetic traits from P. coarctata to O. sativa. The usefulness of molecular marker systems for studying polymorphism and classification, and in clarifying genetic relationships between wild species has been confirmed.