Isolation and characterization of polymorphic microsatellites in olive (Olea europaea L.) and their transferability to other genera in the Oleaceae

Seven polymorphic microsatellites were developed in olive. Six of them came from a genomic library enriched for GA and CA repeat sequences. They showed single locus polymorphism in a set of 23 olive cultivars (from six to nine alleles per locus). Three different pairs of loci were sufficient to discriminate all cultivars. The other polymorphic primer pair was designed from a published sequence for olive lupeol sgutase and revealed just two alleles. The seven primer pairs were tested on two accessions of five other species of the Oleaceae and three, EMO2, EMO13 and EMO90, revealed polymorphism in two, four and three species, respectively.     

Phenotypic plasticity and integration across the canopy of Olea europaea subsp. guanchica (Oleaceae) in populations with different wind exposures

Woody plants, as sessile and long-lived organisms, are expected to have effective mechanisms for dealing with recurrent environmental stresses. In the present study, we hypothesized that phenotypic plasticity (the ability to express alternative phenotypes) and integration (covariation among functionally related traits) are elicited in plants under stressful wind speed conditions. We investigated the within-crown variation of nine vegetative traits of a tree species (Olea europaea subsp. guanchica) in six populations that represented a gradient of wind speed exposures. Wind-exposed twigs in outer-canopy layers had smaller leaves; thinner, lighter, and shorter internodes; and a larger internode cross-sectional area to leaf area ratio. Comparison between field and greenhouse trials revealed that field differences among populations were mediated by phenotypic plasticity. Outer-canopy twigs expressed plastic responses in populations exposed to high wind speeds, whereas inner-canopy twigs displayed high phenotypic convergence among populations. In addition, phenotypic integration increased with wind exposure (outer canopy > inner canopy > greenhouse) and was consequently affected by canopy openness. We conclude that exposure to wind above a certain speed threshold in this woody species elicits a plastic response that is associated with increased integration among traits and involves mechanical and hydraulic rearrangements in more exposed parts of the trees.     

Chloroplast-DNA variation in cultivated and wild olive (Olea europaea L.)

Polymorphism in the lengths of restriction fragments of the whole cpDNA molecule was studied in cultivated olive and in oleaster (wild olive) over the whole Mediterranean Basin. Seventy two olive cultivars, 89 very old trees cultivated locally, and 101 oleasters were scored for ten endonucleases. Moreover, maternal inheritance of cpDNA in olive was shown by analysing the progeny of a controlled cross between two parents which differed in their cpDNA haplotypes. In the whole species, three site- and three length-mutations were observed, corresponding to five distinct chlorotypes. The same chlorotype (I) was predominant in both oleasters and cultivated olive trees, confirming that these are closely related maternally. Three other chlorotypes (II, III and IV) were observed exclusively in oleaster material and were restricted either to isolated forest populations or to a few individuals growing in mixture with olive trees possessing the majority chlorotype. An additional chlorotype (V) was characterised by three mutations located in distinct parts the cpDNA molecule but which were never observed to occur separately. This chlorotype, more widely distributed than the other three, in both cultivated and wild olive, and occurring even in distant populations, was observed exclusively in male-sterile trees showing the same specific pollen anomaly. However, in the present study, no evidence was provided for a direct relationship between the occurrence of the cpDNA mutations and male sterility. It is suggested that the large geographic distribution of chlorotype V may be related to the high fruit production usually observed on male-sterile trees. These may be very attractive for birds which are fond of olive fruit and spread the stones efficiently. Probably for the same reason, people preserved male-sterile oleasters for long periods and, in several places, used male-sterile cultivars over large areas. Received: 25 November 1998 / Accepted: 19 December 1998     

中国北亚热带油橄榄(城固32)开花生物学特性研究

为研究北亚热带内陆地区油橄榄（Olea europaea L.）开花物候、开花样式及花器官特征,探讨其有性繁殖系统的特点。试验对西秦岭南坡地区油橄榄（城固32）的开花生物学进行研究,统计开花进程,雄花和两性花在花序上的着生位置和数量,测定雄花和两性花形态指标并切片观察,电镜扫描分析雄花与两性花花粉粒。结果表明：（1）在北亚热带北缘内陆气候条件下油橄榄花期集中在5月,单株花期15~20 d,盛花期持续4~6 d;（2）油橄榄具有雄全同株的性系统且花器官较小,雄花及两性花都具有正常发育的雄蕊。通过制作石蜡切片观察,发现两性花的雌蕊正常发育,而雄花只有较小的子房和败育的柱头;（3）雌蕊是两种花型花器官生物量差异的主要部位,两性花雌蕊的生物量明显比雄花大（P<0.01）,树体分化出雄花投入的资源更少;（4）雄花与两性花在花粉量、花粉粒大小上差异不显著,在着生位置上,花序轴顶花全部为两性花,雄花出现在花序轴的中部和基部的几率分别是21.13%和30.77%。油橄榄品种城固32具有典型的雄全同株现象,雄花更倾向于在花序上出现的位置在行使它雄性功能方面并无优势可言,而且雄花花粉粒在数量、活力方面也没有优势,但是雄花的出现增加了花粉数量和P/O值,提高了植株的雄性适合度,雄花的出现也降低了两性花落花导致的树体资源浪费,保障其在资源有限的环境中能够繁殖最大化。     

The role of staminate flowers in the breeding system of Olea europaea (Oleaceae): an andromonoecious, wind-pollinated taxon

BACKGROUND AND AIMS: Andromonoecy, as a breeding system, has generated a considerable body of theory in terms of sexual selection, but extended records comparing the performance of pollen grains from staminate versus hermaphrodite flowers are still sparse. The objective in this study was to elucidate the role of staminate flowers in the andromonoecious breeding system of olive (Olea europaea). METHODS: To determine the meaning of staminate flowers, an evaluation was made of resource allocation to, and phenology of, staminate and hermaphrodite flowers in the cultivar 'Mission', and a comparison was made of the male function between both kinds of flowers. KEY RESULTS: Dry weight of hermaphrodite flowers was 19 % greater than dry weight of staminate flowers arising in comparable positions of the panicle. This difference was mainly due to pistil and petal weight; there were no significant differences in stamen weight. There were no significant differences between staminate and hermaphrodite flowers in either amount of pollen per anther, or pollen quality as determined by pollen viability, germinability or ability to fertilize other flowers. There was no significant link between gender and time of anthesis. However, position of the flower within the panicle correlated with time of anthesis and gender. Flowers at the apex and at primary pedicels tended to be hermaphrodite and open earlier, whereas flowers arising in secondary pedicels were mainly staminate and were commonly the last to reach anthesis. CONCLUSIONS: It is proposed that the main advantage provided by production of staminate flowers in olive is to enhance male fitness by increasing pollen output at the whole plant level, although a relict function of attracting pollinators cannot be completely discarded.     

High genetic diversity and clonal growth in relict populations of Olea europaea subsp. laperrinei (Oleaceae) from Hoggar, Algeria

BACKGROUND AND AIMS: The Laperrine's olive (Olea europaea subsp. laperrinei) is an endemic tree from Saharan massifs. Its populations have substantially regressed since the Pleistocene and are presently distributed in a fragmented habitat. Long-term persistence of this taxon is uncertain and programmes of preservation have to be urgently implemented. To define a conservation strategy, the genetic diversity and breeding system of this tree have to be investigated. METHODS: One hundred and eleven ramets were prospected in the laperrinei populations from the Tamanrasset region, southern Algeria. Genetic polymorphism was revealed at nuclear and chloroplast DNA (cpDNA) microsatellite loci allowing a comparative assessment of the genetic diversity of laperrinei and Mediterranean populations based on bi-parental and maternal markers. Additionally, nuclear microsatellite markers enabled the genotypes to be identified unambiguously. KEY RESULTS: Based on nuclear microsatellite data, the total diversity was high (Ht=0.61) in laperrinei populations and similar to that observed in western Mediterranean populations. A substantial cpDNA diversity (Ht=0.19) was also observed. Genetically identical ramets originated from the same stump (which can cover >80 m2) were identified in each population. Sixteen per cent of genets exhibited more than one ramet. In addition, several cases of somatic mutations were unambiguously revealed in distinct ramets stemming from the same stump. CONCLUSIONS: These data show that highly isolated and small laperrinei populations are able to maintain a high genetic diversity. This supports the existence of relict trees persisting for a very long time (probably since the last humid transition, 3000 years ago). It is proposed that the very long persistence associated with an asexual multiplication of highly adapted trees could be a strategy of survival in extreme conditions avoiding a mutational meltdown due to reproduction in reduced populations.     

Cryptic self‐incompatibility and distyly in Hedyotis acutangula Champ. (Rubiaceae)

Distyly, floral polymorphism frequently associated with reciprocal herkogamy, self‐ and intramorph incompatibility and secondary dimorphism, constitutes an important sexual system in the Rubiaceae. Here we report an unusual kind of distyly associated with self‐ and/or intramorph compatibility in a perennial herb, Hedyotis acutangula. Floral morphology, ancillary dimorphisms and compatibility of the two morphs were studied. H. acutangula did not exhibit precise reciprocal herkogamy, but this did not affect the equality of floral morphs in the population, as usually found in distylous plants. Both pin and thrum pollen retained relatively high viability for 8 h. The pollen to ovule ratio was 72.5 in pin flowers and 54.4 in thrum flowers. Pistils of pin flowers remained receptive for longer than those of thrum flowers. No apparent difference in the germination rate of pin and thrum pollen grains was observed when cultured in vitro, although growth of thrum pollen tubes was much faster than that of pin pollen tubes. Artificial pollination revealed that pollen tube growth in legitimate intermorph crosses was faster than in either intramorph crosses or self‐pollination, suggesting the occurrence of cryptic self‐incompatibility in this species. Cryptic self‐incompatibility functioned differently in the two morphs, with pollen tube growth rates after legitimate and illegitimate pollination much more highly differentiated in pin flowers than in thrum flowers. No fruit was produced in emasculated netted flowers, suggesting the absence of apomixis. Our results indicate that H. acutangula is distylous, with a cryptic self‐incompatibility breeding system.     

Pollen tube growth in the self‐compatible sweet cherry genotype, ‘Cristobalina’, is slowed down after self‐pollination

Sweet cherry is a self‐incompatible fruit tree species in the Rosaceae. As other species in the family, sweet cherry exhibits S‐RNase‐based gametophytic self‐incompatibility. This mechanism is genetically determined by the S‐locus that encodes the pollen and pistil determinants, SFB and S‐RNase, respectively. Several self‐compatible sweet cherry genotypes have been described and most of them have mutations at the S‐locus leading to self‐compatibility. However, ‘Cristobalina’ sweet cherry is self‐compatible due to a mutation in a pollen function modifier that is not linked to the S‐locus. To investigate the physiology of self‐compatibility in this cultivar, S‐locus segregation in crosses involving ‘Cristobalina’ pollen, and pollen tube growth in self‐ and cross‐pollinations, were studied. In the crosses with genotypes sharing only one S‐haplotype, the non‐self S‐haplotype was inherited more frequently than the self S‐haplotype. Pollen tube growth studies revealed that the time to travel the whole length of the style was longer for self‐pollen tubes than for cross‐pollen tubes. Together, these results suggest that ‘Cristobalina’ pollen tube growth is slower after self‐pollination than after cross‐pollination. This reproductive strategy would allow self‐fertilisation in the absence of compatible pollen but would promote cross‐fertilisation if cross‐compatible pollen is available, a possible case of cryptic self‐incompatibility. This bet‐hedging strategy might be advantageous for an ecotype that is native to the mountains of the Spanish Mediterranean coast, in the geographical limits of the distribution of this species. ‘Cristobalina’ blooming takes place very early in the season when mating possibilities are scarce and, consequently, self‐compatibility may be the only possibility for this genotype to produce offspring.     

Distribution, shape and clonal growth of the rare endemic tree Olea europaea subsp. laperrinei (Oleaceae) in the Saharan mountains of Niger

The populations of the Laperrine’s olive (Olea europaea subsp. laperrinei) are located in three main areas corresponding to the mountains of northern Niger (Aïr), southern Algeria (Hoggar), and north-western Sudan (western Darfur). The populations native of Niger were found to occur in very fragmented patches from 1550 to 1850 m in five isolated mountains with population sizes not exceeding 100 trees each, except in the Tamgak. Samples in the Tamgak and the Bagzane mountains were studied by combining field observations with identification of genets (using highly variable DNA marker analyzes). Trees were relatively small and multi-stemmed like those in the Hoggar, and were associated with some tropical species as in the western Darfur. They were found on borders of “wadis”, i.e., temporary water courses, in ravines and on hillsides. They were unable to rely on sexual reproduction, which was found to be ineffective. In contrast, 28% of the genetic profiles found (n _genet = 98) were represented with two or more trees without aboveground connections. Molecular and morphological data both demonstrated that the populations used clonal growth (CG) to survive in the current unfavorable period of hyper-aridity, and thus are likely to be remnant populations. Signs of human-related disturbances, recorded on 43% of the sampled trees, should be a determining factor of distribution as well. In the Bagzane mountains especially, where human activities are more intense than in the Tamgak mountains, trees developed preferentially on hillsides and ravines with low access rather than on wadi borders with better edaphic conditions but easier access for livestock/people. CG may be therefore, a survival strategy both against aridity and human-related disturbances. Sexual reproduction may be triggered during a less arid period, following the model of multiple demographic strategies, widespread in arid environments. However, survival would be affected by browsing and cuttings. Lastly, the absence of an efficient sexual strategy coupled with the high fragmentation of very small populations and a narrow altitudinal range of distribution indicates that today the populations of O. e. laperrinei in the Aïr mountain range are more endangered than those from southern Algeria and north-western Sudan.     

A set of primers for length and nucleotide‐substitution polymorphism in chloroplastic DNA of Olea europaea L. (Oleaceae)

Chloroplastic DNA (cpDNA) variation at five microsatellite motifs, two insertion‐deletion sites, and eight nucleotide substitution sites was investigated in the Olea europaea complex. Primers were designed for flanking regions of these sites to amplify short cpDNA regions. They provided polymorphism when polymerase chain reaction (PCR) products from a representative sample of 128 O. europaea individuals were either resolved by size into polyacrylamide gels (length polymorphism) or digested with restriction enzymes (nucleotide‐substitution polymorphism). These polymorphisms serve to distinguish most of the cytoplasmic haplotypes previously recognized. Potential application of these markers in O. europaea includes phylogeography, conservation and germplasm identification, even when using poorly preserved material from herbarium specimens or forensic and archaeological materials.     

A possible role for flowering locus T‐encoding genes in interpreting environmental and internal cues affecting olive (Olea europaea L.) flower induction

Olive (Olea europaea L.) inflorescences, formed in lateral buds, flower in spring. However, there is some debate regarding time of flower induction and inflorescence initiation. Olive juvenility and seasonality of flowering were altered by overexpressing genes encoding flowering locus T (FT). OeFT1 and OeFT2 caused early flowering under short days when expressed in Arabidopsis. Expression of OeFT1/2 in olive leaves and OeFT2 in buds increased in winter, while initiation of inflorescences occurred i n late winter. Trees exposed to an artificial warm winter expressed low levels of OeFT1/2 in leaves and did not flower. Olive flower induction thus seems to be mediated by an increase in FT levels in response to cold winters. Olive flowering is dependent on additional internal factors. It was severely reduced in trees that carried a heavy fruit load the previous season (harvested in November) and in trees without fruit to which cold temperatures were artificially applied in summer. Expression analysis suggested that these internal factors work either by reducing the increase in OeFT1/2 expression or through putative flowering repressors such as TFL1. With expected warmer winters, future consumption of olive oil, as part of a healthy Mediterranean diet, should benefit from better understanding these factors.     

Breeding systems in Cyrtanthus (Amaryllidaceae): variation in self‐sterility and potential for ovule discounting

• Breeding systems of plants determine their reliance on pollinators and ability to produce seeds following self‐pollination. Self‐sterility, where ovules that are penetrated by self‐pollen tubes that do not develop into seeds, is usually considered to represent either a system of late‐acting self‐incompatibility or strong early inbreeding depression. Importantly, it can lead to impaired female function through ovule or seed discounting when stigmas receive mixtures of self and cross pollen, unless cross pollen is able to reach the ovary ahead of self pollen (‘prepotency’). Self‐sterility associated with ovule penetration by self‐pollen tubes appears to be widespread among the Amaryllidaceae.
• We tested for self‐sterility in three Cyrtanthus species – C. contractus, C. ventricosus and C. mackenii – by means of controlled hand‐pollination experiments. To determine the growth rates and frequency of ovule penetration by self‐ versus cross‐pollen tubes, we used fluorescence microscopy to examine flowers of C. contractus harvested 24, 48 and 72 h after pollination, in conjunction with a novel method of processing these images digitally. To test the potential for ovule discounting (loss of cross‐fertilisation opportunities when ovules are disabled by self‐pollination), we pollinated flowers of C. contractus and C. mackenii with mixtures of self‐ and cross pollen.
• We recorded full self‐sterility for C. contractus and C. ventricosus, and partial self‐sterility for C. mackenii. In C. contractus, we found no differences in the growth rates of self‐ and cross‐pollen tubes, nor in the proportions of ovules penetrated by self‐ and cross‐pollen tubes. In this species, seed set was depressed (relative to cross‐pollinated controls) when flowers received a mixture of self and cross pollen, but this was not the case for C. mackenii.
• These results reveal variation in breeding systems among Cyrtanthus species and highlight the potential for gender conflict in self‐sterile species in which ovules are penetrated and disabled by pollen tubes from self pollen.

     

Homomorphic Self‐Incompatibility,with Stylar Rejection Site,in Luehea grandiflora Mart. and Zucc. (Tiliaceae‐Malvaceae s.l.)

Abstract: The breeding system of Luehea grandiflora (Tiliaceae‐Malvaceae s.l.) was investigated using hand pollinations and fluorescence microscopy studies of pollen tube growth. Although selfed flowers persisted for some 10 days, our study indicates that L. grandiflora is self‐incompatible, with self pollen tube inhibition in the upper style, as occurs in many taxa with homomorphic, gametophytic self‐incompatibility (GSI). L. grandiflora is only the second species reported within the Malvales with homomorphic stylar inhibition. This result is discussed within the context of a report for self‐compatibility in this species, and we also consider the phylogenetic implications for the occurrence of GSI in the family Malvaceae s.l.     

Genetic diversity and gene flow between the wild olive (oleaster, Olea europaea L.) and the olive: several Plio-Pleistocene refuge zones in the Mediterranean basin suggested by simple sequence repeats analysis

Aim The oleaster is believed to have originated in the eastern Mediterranean, implying that those in the western Mediterranean basin could be feral. Several studies with different molecular markers (isozymes, random amplified polymorphic DNA, amplified fragment length polymorphism) have shown a cline between the eastern and the western populations, which supports this hypothesis. To reconstruct the post‐glacial colonization history and establish a relationship between olive and oleaster populations in the Mediterranean basin, analyses were carried out on the genetic variation of chloroplast DNA (chlorotype) and at 12 unlinked simple sequence repeat (SSR) loci, sampling a total of 20 oleaster groves. Location This is the first known large‐scale molecular study of SSR loci based on samples of both oleasters and cultivars from the entire Mediterranean basin. Methods Samples were taken from 166 oleasters in 20 groves of modern populations, and 40 cultivars to represent molecular diversity in the cultivated olive. The Bayesian method and admixture analysis were used to construct the ancestral populations (RPOP; reconstructed panmictic oleaster populations) and to estimate the proportion of each RPOP in each tree. If one tree can be assigned to two or more RPOPs, it can be regarded as a product of hybridization between trees from different populations (i.e. admix origin). Results On this first examination of the SSR genetic diversity in the olive and oleaster, it was found to be structured in seven RPOPs in both eastern and western populations. Based on different population genetic methods, it was shown that: (1) oleasters are equally present in the eastern and the western Mediterranean, (2) are native, and (3) are not derived from cultivars. Chlorotypes (one and three in the eastern and western Mediterranean, respectively) revealed fruit displacement for the oleasters. Main conclusions Oleaster genetic diversity is divided into seven regions that could overlay glacial refuges. The gradient, or cline, of genetic diversity revealed by chloroplast and SSR molecular markers was explained by oleaster recolonization of the Mediterranean basin from refuges after the last glacial event, located in both eastern and western regions. It is likely that gene flow has occurred in oleasters mediated by cultivars spread by human migration or through trade. Animals may have helped spread oleasters locally, but humans have probably transported olives but not oleaster fruits over long distances. We found that cultivars may have originated in several RPOPs, and thus, some may have a more complex origin than expected initially.     

The colonization history of Olea europaea L. in Macaronesia based on internal transcribed spacer 1 (ITS-1) sequences, randomly amplified polymorphic DNAs (RAPD), and intersimple sequence repeats (ISSR)

Phylogenetic relationships in the Olea europaea complex and the phylogeography of 24 populations of the Macaronesian olive (O. europaea ssp. cerasiformis) were assessed by using three molecular markers: nuclear ribosomal internal transcribed spacer 1 (ITS-1) sequences, randomly amplified polymorphic DNAs (RAPD), and intersimple sequence repeats (ISSR). Parsimony analysis of the ITS-1 sequences and Neighbour-joining (NJ) analyses of RAPD and ISSR banding variation revealed four major lineages in the O. europaea complex: (1) ssp. cuspidata; (2) ssp. cerasiformis from Madeira; (3) ssp. laperrinei; and (4) ssp. cerasiformis from the Canary Islands plus ssp. europaea. These results provide unequivocal support for two independent dispersal events of Olea to the Madeira and Canary Islands. Molecular and morphological evidence led to recognition of two separate olive taxa in Macaronesia, to date included in ssp. cerasiformis. NJ analyses of the combined RAPD and ISSR data suggest that the colonization of the Canaries by O. europaea may have followed an east to west stepping-stone model. An interisland dispersal sequence can be recognized, starting from the continent to Fuerteventura, Gran Canaria, Tenerife, La Gomera, and finally La Palma. High dispersal activity of the lipid-rich Olea fruits by birds in the Mediterranean region is congruent with multiple dispersal of olives to Macaronesia and successive colonization of the archipelagos. The observation of strong genetic isolation between populations of different islands of the Canary Islands suggests, however, that subsequent interisland dispersal and establishment has been very rare or may not have occurred at all.