A Mesoamerican origin of cherimoya (Annona cherimola Mill.): Implications for the conservation of plant genetic resources.

Knowledge on the structure and distribution of genetic diversity is a key aspect to plan and execute an efficient conservation and utilization of the genetic resources of any crop as well as for determining historical demographic inferences. In this work, a large data set of 1,765 accessions of cherimoya (Annona cherimola Mill, Annonaceae), an underutilized fruit tree crop native to the Neotropics and used as a food source by pre-Columbian cultures, was collected from six different countries across the American continent and amplified with nine highly informative microsatellite markers. The structure analyses, fine representation of the genetic diversity and an ABC approach suggest a Mesoamerican origin of the crop, contrary to previous reports, with clear implications for the dispersion of plant germplasm between Central and South America in pre-Columbian times. These results together with the potential distribution of the species in a climatic change context using two different climate models provide new insights for the history and conservation of extant genetic resources of cherimoya that can be applied to other currently underutilized woody perennial crops.

Paternal-specific S-allele transmission in sweet cherry (Prunus avium L.): the potential for sexual selection.

Homomorphic self-incompatibility is a well-studied example of a physiological process that is thought to increase population diversity and reduce the expression of inbreeding depression. Whereas theoretical models predict the presence of a large number of S-haplotypes with equal frequencies at equilibrium, unequal allele frequencies have been repeatedly reported and attributed to sampling effects, population structure, demographic perturbation, sheltered deleterious mutations or selection pressure on linked genes. However, it is unclear to what extent unequal segregations are the results of gametophytic or sexual selection. Although these two forces are difficult to disentangle, testing S-alleles in the offspring of controlled crosses provides an opportunity to separate these two phenomena. In this work, segregation and transmission of S-alleles have been characterized in progenies of mixed donors and fully compatible pollinations under field conditions in Prunus avium. Seed set patterns and pollen performance have also been characterized. The results reveal paternal-specific distorted transmission of S-alleles in most of the crosses. Interestingly, S-allele segregation within any given paternal or maternal S-locus was random. Observations on pollen germination, pollen tube growth rate, pollen tube cohort size, seed set dynamics and transmission patterns strongly suggest post-pollination, prezygotic sexual selection, with male-male competition as the most likely mechanism. According to these results, post-pollination sexual selection takes precedence over frequency-dependent selection in explaining unequal S-haplotype frequencies.

Pollen performance, cell number, and physiological state in the early-divergent angiosperm Annona cherimola Mill. (Annonaceae) are related to environmental conditions during the final stages of pollen development.

Pollen performance is an important determinant for fertilization success, but high variability in pollen behavior both between and within species occurs in different years and under varying environmental conditions. Annona cherimola, an early-divergent angiosperm, is a species that releases a variable ratio of bicellular and tricellular hydrated pollen at anther dehiscence depending on temperature. The presence of both bi- and tricellular types of pollen is an uncommon characteristic in angiosperms and makes Annona cherimola an interesting model to study the effect of varying environmental conditions on subsequent pollen performance during the final stages of pollen development. In this work, we study the influence of changes in temperature and humidity during the final stages of pollen development on subsequent pollen performance, evaluating pollen germination, presence of carbohydrates, number of nuclei, and water content. At 25 °C, which is the average field temperature during the flowering period of this species, pollen had a viability of 60-70 %, starch hydrolyzed just prior to shedding, and pollen mitosis II was taking place, resulting in a mixture of bi- and tricellular pollen. This activity may be related to the pollen retaining 70 % water content at shedding. Temperatures above 30 °C resulted in a decrease in pollen germination, whereas lower temperatures did not have a clear influence on pollen germination, although they did have a clear effect on starch hydrolysis. On the other hand, slightly higher dehydration accelerated mitosis II, whereas strong dehydration arrested starch hydrolysis and reduced pollen germination. These results show a significant influence of environmental conditions on myriad pollen characteristics during the final stages of pollen development modifying subsequent pollen behavior and contributing to our understanding of the variability observed in pollen tube performance.

Self-compatibility in 'Cristobalina' sweet cherry is not associated with duplications or modified transcription levels of S-locus genes.

Sweet cherry shows S-RNase-based gametophytic self-incompatibility, which prevents self- and cross-fertilization between genetically related individuals. The specificity of the self-incompatible reaction is determined by two genes located in the S-locus. These encode a pistil-expressed ribonuclease (S-RNase) that inhibits self pollen tube growth, and a pollen-expressed F-box protein (SFB) that may be involved in the cytotoxicity of self-S-RNases. Initial genetic and pollination studies in a self-compatible sweet cherry cultivar, 'Cristobalina' (S (3) S (6)), showed that self-compatibility was caused by the loss of pollen function of both haplotypes (S (3) and S (6)). In this study, we further characterize self-compatibility in this genotype by molecular analysis of the S-locus. DNA blot analyses using S-RNase and SFB probes show no duplications of 'Cristobalina' S-locus genes or differences in the restriction patterns when compared with self-incompatible cultivars with the same S-genotype. Furthermore, reverse transcriptase-PCR of S-locus genes and quantitative reverse transcription-PCR of SFBs revealed no differences at the transcription level when compared with a self-incompatible genotype. The results of this study show that no differences at the S-locus can be correlated with self-compatibility, indicating the possible involvement of non-S-locus modifiers in self-incompatibility breakdown in this cultivar.

The progamic phase of an early-divergent angiosperm, Annona cherimola (Annonaceae).

BACKGROUND AND AIMS: Recent studies of reproductive biology in ancient angiosperm lineages are beginning to shed light on the early evolution of flowering plants, but comparative studies are restricted by fragmented and meagre species representation in these angiosperm clades. In the present study, the progamic phase, from pollination to fertilization, is characterized in Annona cherimola, which is a member of the Annonaceae, the largest extant family among early-divergent angiosperms. Beside interest due to its phylogenetic position, this species is also an ancient crop with a clear niche for expansion in subtropical climates. METHODS: The kinetics of the reproductive process was established following controlled pollinations and sequential fixation. Gynoecium anatomy, pollen tube pathway, embryo sac and early post-fertilization events were characterized histochemically. KEY RESULTS: A plesiomorphic gynoecium with a semi-open carpel shows a continuous secretory papillar surface along the carpel margins, which run from the stigma down to the obturator in the ovary. The pollen grains germinate in the stigma and compete in the stigma-style interface to reach the narrow secretory area that lines the margins of the semi-open stylar canal and is able to host just one to three pollen tubes. The embryo sac has eight nuclei and is well provisioned with large starch grains that are used during early cellular endosperm development. CONCLUSIONS: A plesiomorphic simple gynoecium hosts a simple pollen-pistil interaction, based on a support-control system of pollen tube growth. Support is provided through basipetal secretory activity in the cells that line the pollen tube pathway. Spatial constraints, favouring pollen tube competition, are mediated by a dramatic reduction in the secretory surface available for pollen tube growth at the stigma-style interface. This extramural pollen tube competition contrasts with the intrastylar competition predominant in more recently derived lineages of angiosperms.

Alternative food improves the combined effect of an omnivore and a predator on biological pest control. A case study in avocado orchards.

Ecological communities used in biological pest control are usually represented as three-trophic level food chains with top-down control. However, at least two factors complicate this simple way of characterizing agricultural communities. First, agro-ecosystems are composed of several interacting species forming complicated food webs. Second, the structure of agricultural communities may vary in time. Efficient pest management approaches need to integrate these two factors to generate better predictions for pest control. In this work, we identified the food web components of an avocado agro-ecosystem, and unravelled patterns of co-occurrence and interactions between these components through field and laboratory experiments. This allowed us to predict community changes that would improve the performance of the naturally occurring predators and to test these predictions in field population experiments. Field surveys revealed that the food-web structure and species composition of the avocado community changed in time. In spring, the community was characterized by a linear food chain of Euseius stipulatus, an omnivorous mite, feeding on pollen. In the summer, E. stipulatus and a predatory mite, Neoseiulus californicus, shared a herbivorous mite prey. Laboratory experiments confirmed these trophic interactions and revealed that N. californicus can feed inside the prey nests, whereas E. stipulatus cannot, which may further reduce competition among predators. Finally, we artificially increased the coexistence of the two communities via addition of the non-herbivore food source (pollen) for the omnivore. This led to an increase in predator numbers and reduced populations of the herbivore. Therefore, the presence of pollen is expected to improve pest control in this system.

How do Neoseiulus californicus (Acari: Phytoseiidae) females penetrate densely webbed spider mite nests?

The persea mite Oligonychus perseae is a pest of avocado trees that builds extremely dense webbed nests that protect them against natural enemies, including phytoseiid mites. Nests have one or two marginal entrances that are small and flattened. The predatory mite Neoseiulus californicus co-occurs with O. perseae in the avocado orchards of the south-east of Spain. Penetration inside nests through the entrances by this predator is thought to be hindered by its size and its globular-shaped body. However, in the field it has repeatedly been found inside nests that were clearly ripped. Perhaps penetration of the nests has been facilitated by nest wall ripping caused by some other species or by unfavourable abiotic factors. However, to assess whether N. californicus is also able to enter the nest of O. perseae by itself, we carried out laboratory experiments and made a short film. They show how this predator manages to overcome the webbed wall, and that it can penetrate and forage inside nests of O. perseae.

PERMANENT GENETIC RESOURCES: Development of 52 new polymorphic SSR markers from cherimoya (Annona cherimola Mill.): transferability to related taxa and selection of a reduced set for DNA fingerprinting and diversity studies.

Fifty-two single locus polymorphic microsatellites were developed using two genomic libraries digested with HaeIII and RsaI of cherimoya cv. Fino de Jete enriched in CT/AG repeats. A total of 222 alleles were detected with the selected simple sequence repeats (SSRs). The observed and expected heterozygosities ranged from 0.08 to 0.73 and from 0.20 to 0.84, respectively. Most of the SSRs were transferable to other species in the Annonaceae. A set of 20 microsatellites was selected to facilitate the exchange of data among laboratories.

Molecular characterization and genetic diversity in an avocado collection of cultivars and local Spanish genotypes using SSRs.

In this work, 75 avocado accessions maintained in an ex situ germplasm collection at the E.E. la Mayora in Málaga (Spain) were characterized with 16 microsatellites previously developed in this species. This avocado collection includes both local Spanish genotypes obtained through prospection and genotypes obtained by exchange with different countries. A total of 156 different amplification fragments were detected ranging from 4 to 16 per locus with an average of 9.75 alleles per locus. All the microsatellites were highly informative with an expected heterozygosity higher than 0.5 and a probability of identity below 0.36. The total probability of identity was 2.85x10(-14). Fifteen of the 16 loci studied showed a positive Wright's fixation index (F) indicating a deficit of heterozygotes with an average over all the SSRs of 0.18. A dendrogram was generated using UPGMA (unweighted pair group method with arithmetic averages) based on the Nei and Li similarity index. This dendrogram classified most of the genotypes analyzed into three major groups which mainly differed in racial origin although with low bootstrap support probably due to the presence of many interracial hybrids in the collection. All the genotypes studied could be unequivocally distinguished with the combination of SSRs used except some putative mutations of 'Hass' and an additional group of two cultivars. The results obtained indicate that the set of SSRs used is highly informative and are discussed in terms of their implications for avocado germplasm characterization and management.

Influence of genotype-temperature interaction on pollen performance.

Pollen competition and selection have significant evolutionary consequences, but very little is known about how they can be modulated. We have examined in cherry (Prunus avium L.) how pollen performance is affected by the genotype of the pollen and by the environmental conditions under which it grows, namely the pistilar tissue and temperature. The different pollen donor genotypes tested in this work differed in their behaviour both in vitro and in vivo and this behaviour was modulated depending on the female recipient they grew on. Furthermore, there was a significant temperature-genotype interaction that affected the pollen tube population census that succeeded in reaching the base of the style. The combination of these three factors, while enabling a capacity of response to variations in environmental pressures, could maintain variability in pollen performance avoiding the fixation of the genes that control pollen tube growth rate.

The effect of temperature on pollen germination, pollen tube growth, and stigmatic receptivity in peach.

Temperature is a major climatic factor that limits geographical distribution of plant species, and the reproductive phase has proven to be one of the most temperature-vulnerable stages. Here, we have used peach to evaluate the effect of temperature on some processes of the progamic phase, from pollination to the arrival of pollen tubes in the ovary. Within the range of temperatures studied, 20 degrees C in the laboratory and, on average, 5.7 degrees C in the field, the results show an accelerating effect of increasing temperature on pollen germination and pollen tube growth kinetics, as well as an increase in the number of pollen tubes that reach the style base. For the last two parameters, although the range of temperature registered in the field was much lower, the results obtained in the laboratory paralleled those obtained in the field. Increasing temperatures drastically reduced stigmatic receptivity. Reduction was sequential, with stigmas first losing the capacity to sustain pollen tube penetration to the transmitting tissue, then their capacity to offer support for pollen germination and, finally, their capacity to support pollen grain adhesion. Within a species-specific range of temperature, this apparent opposite effect of temperature on the male and female side could provide plants with the plasticity to withstand changing environmental effects, ensuring a good level of fertilization.

Cloning and characterization of genomic DNA sequences of four self-incompatibility alleles in sweet cherry ( Prunus avium L.).

Gametophytic self-incompatibility (GSI) in sweet cherry is determined by a locus S with multiple alleles. In the style, the S-locus codifies for an allele-specific ribonuclease ( S-RNase) that is involved in the rejection of pollen that carries the same S allele. In this work we report the cloning and genomic DNA sequence analysis including the 5' flanking regions of four S-RNases of sweet cherry ( Prunus avium L., Rosaceae). DNA from the cultivars Ferrovia, Pico Colorado, Taleguera Brillante and Vittoria was amplified through PCR using primers designed in the conserved sequences of sweet cherry S-RNases. Two alleles were amplified for each cultivar and three of them correspond to three new S-alleles named S23, S24 and S25 present in 'Pico Colorado', 'Vittoria' and 'Taleguera Brillante' respectively. To confirm the identity of the amplified fragments, the genomic DNA of these three putative S-RNases and the allele S12 amplified in the cultivar Ferrovia were cloned and sequenced. The nucleotide and deduced amino-acid sequences obtained contained the structural features of rosaceous S-RNases. The isolation of the 5'-flanking sequences of these four S-RNases revealed a conserved putative TATA box and high similarity among them downstream from that sequence. However, similarity was low compared with the 5'-flanking regions of S-RNases from the Maloideae. S6- and S24-RNase sequences are highly similar, and most amino-acid substitutions among these two RNases occur outside the rosaceous hypervariable region (RHV), but within another highly variable region. The confirmation of the different specificity of these two S-RNases would help elucidate which regions of the S-RNase sequences play a role in S-pollen specific recognition.

Development, characterization and variability analysis of microsatellites in lychee (Litchi chinensis Sonn., Sapindaceae).

We report 12 microsatellites enriched in CT repeats obtained from a genomic library of the lychee ( Litchi chinensis Sonn.) cultivar Mauritius. The polymorphisms revealed by these microsatellites were evaluated in a collection of 21 lychee cultivars. A total of 59 fragments were detected with these 12 SSRs, with an average of 4.9 bands/SSR. Three primer pairs seem to amplify more than a single locus. The mean expected and observed heterozygosities over the 9 single-locus SSRs averaged 0.571 (range: 0.137-0.864) and 0.558 (range: 0.169-0.779) respectively. The total value for the probability of identity was 7.53 x 10(-5). In addition, the selected SSRs were used to amplify DNA from four longan cultivars. Eleven of the 12 SSRs produced amplification fragments in longan, and eight of these fragments were polymorphic. All except two of the products amplified from longan were the same size as those amplified from lychee, suggesting a close genetic proximity between the two species. The SSRs studied produced 22 different patterns, allowing the unambiguous identification of 16 lychee and the 4 longan cultivars studied. Discrimination was possible with just four selected microsatellites. Two groups with two and three undistinguishable cultivars were obtained, reflecting probable synonymies. Unweighted pair-group method of artimetic averages (UPGMA) cluster analysis divided the lychee cultivars studied into two main groups, one consisting of ancient cultivars and the other with more diverse recent cultivars. This is the first report of microsatellite development in the Sapindaceae, and the results demonstrate the usefulness of microsatellites for identification, similarity studies and germplasm conservation in lychee and related species.

Effect of temperature on pollen tube kinetics and dynamics in sweet cherry, Prunus avium (Rosaceae).

Prevailing ambient temperature during the reproductive phase is one of several important factors for seed and fruit set in different plant species, and its consequences on reproductive success may increase with global warming. The effect of temperature on pollen performance was evaluated in sweet cherry (Prunus avium L.), comparing as pollen donors two cultivars that differ in their adaptation to temperature. 'Sunburst' is a cultivar that originated in Canada with a pedigree of cultivars from Northern Europe, while 'Cristobalina' is a cultivar native to southeast Spain, adapted to warmer conditions. Temperature effects were tested either in controlled-temperature chambers or in the field in a plastic cage. In both genotypes, an increase in temperature reduced pollen germination, but accelerated pollen tube growth. However, a different genotypic response, which reflected the overall adaptation of the pollen donor, was obtained for pollen tube dynamics, expressed as the census of the microgametophyte population that successfully reached the base of the style. While both cultivars performed similarly at 20°C, the microgametophyte population was reduced at 30°C for Sunburst and at 10°C for Cristobalina. These results indicate a differential genotypic response to temperature during the reproductive phase, which could be important in terms of the time needed for a plant species to adapt to rapid temperature changes.

Molecular characterisation of sweet cherry (Prunus avium L.) genotypes using peach [Prunus persica (L.) Batsch] SSR sequences.

A total of 76 sweet cherry genotypes were screened with 34 microsatellite primer pairs previously developed in peach. Amplification of SSR loci was obtained for 24 of the microsatellite primer pairs, and 14 of them produced polymorphic amplification patterns. On the basis of polymorphism and quality of amplification, a set of nine primer pairs and the resulting 27 informative alleles were used to identify 72 genotype profiles. Of these, 68 correspond to unique cultivar genotypes, and the remaining four correspond to three cultivars that could not be differentiated from the two original genotypes of which they are mutants, and two very closely related cultivars. The mean number of alleles per locus was 3.7 while the mean heterozygosity over the nine polymorphic loci averaged 0.49. The results demonstrate the usefulness of cross-species transferability of microsatellite sequences allowing the discrimination of different genotypes of a fruit tree species with sequences developed in other species of the same genus. UPGMA cluster analysis of the similarity data divided the ancient genotypes studied into two fairly well-defined groups that reflect their geographic origin, one with genotypes originating in southern Europe and the other with the genotypes from northern Europe and North America.

Molecular characterization and similarity relationships among apricot ( Prunus armeniaca L.) genotypes using simple sequence repeats.

A collection of 48 apricot genotypes, originated from diverse geographic areas, have been screened with 37 SSR primer pairs developed in different species of Prunus in order to identify and characterize the genotypes and establish their genetic relations. Thirty one of those primer pairs resulted in correct amplifications and 20 produced polymorphic repeatable amplification patterns with the 48 genotypes studied. A total of 82 alleles were detected for the 20 loci. All the genotypes studied could be unequivocally distinguished with the combination of SSRs used. The results obtained evidence for the cross-species transportability of microsatellite sequences, allowing the discrimination among different genotypes of a given fruit-tree species with sequences developed in other species. UPGMA cluster analysis of the similarity data grouped the genotypes studied according to their geographic origin and/or their pedigree information.

Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis.

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in the dioecious species, Pistacia vera. Progenies from two female parents pollinated by a common male parent were studied. Two bulks of DNA were made in each cross, one from males and one from females, by pooling an equal weight of fresh leaves from each individual contributing to the bulk prior to DNA extraction. DNA was extracted from each bulked sample and from each of the contributing individuals. DNA was also extracted from 14 cultivars of P. vera and from 94 open-pollinated, fewweeks-old P. vera seedlings of unknown sex. Seven hundred different decamer oligonucleotide primers were used to perform DNA amplification, with 1 of these (OPO08) producing a 945 bp amplification band that was present only in the bulked female samples and absent in the bulked male samples of the two crosses. The relationship between band presence and female sex expression was conserved in every individual obtained from the two crosses and in the 14 cultivars unrelated to the crosses. We propose that this band is tightly linked to the gene(s) that control sex determination in pistachio. The OPO08945 RAPD marker could be used in a breeding program to screen the gender of pistachio plants long before they reach reproductive maturity, resulting in considerable savings of time and economic resources. In order to verify that assumption we screened 94 additional seedlings with the OPO08 primer and obtained results consistent with a 1∶1 male:female ratio.

Pollen selection.

In recent years a large number of reports have been published hinting about the possible role of selection at the male gametophyte level in natural conditions and its possible use as a tool in plant breeding programs. In this work, the evidence that has so far emerged on gametophytic selection is reviewed, and its relationship to pollen competition under natural conditions is analyzed. Information supporting an overlap in the genetic expression between the gametophytic and sporophytic phases is also reported, mainly in terms of isozymes and RNA hybridization. The correspondence between both phases is reviewed through their common response to different external agents such as temperature, salinity, metals, herbicides, fungal toxins, and other factors. Finally, the implications that pollen selection may have in plant breeding are evaluated.