Development of microsatellite markers in Lupinus luteus (Fabaceae) and cross-species amplification in other lupine species

? Premise of the study: Microsatellite primers were developed in Lupinus luteus L., an emerging temperate protein crop, to investigate genetic diversity, population structure, and to facilitate the generation of better yellow lupine varieties. ? Methods and Results: Thirteen polymorphic primer sets were evaluated in a European and Eastern European accession collection of L. luteus. The primers amplified di-, tri-, and tetranucleotide repeats with 2-4 alleles per locus. These revealed a moderate to low level of genetic variation, as indicated by an average observed heterozygosity of 0.0126. Select loci also amplified successfully in the closely related species L. hispanicus Boiss. & Reut. and in the New World species L. mutabilis Sweet. ? Conclusions: These results indicate the utility of primers for the study of genetic diversity across L. luteus populations and related lupine species. The use of these microsatellite markers will facilitate the implementation of several molecular breeding strategies in yellow lupine.     

Screening of cytoplasmic DNA diversity between and within Lupinus mutabilis Sweet and Lupinus albus sensu lato by restriction fragment length polymorphism (RFLP)

Seven populations and five mutant lines of the Andean lupin and four species from the section Albus were screened for their mitochondrial and chloroplast polymorphisms. For this purpose the RFLP method with EcoRI as a restriction enzyme was used. Lupinus luteus, Lupinus albus and Phaseolus vulgaris organellar clones as well as amplified fragments were used as probes. We found that mitochondrial probes were more suitable than chloroplast probes for identification of inter- and intra-specific variations within the examined material. Most mitochondrial probes differentiate the two species investigated. A high level of mitochondrial polymorphism was observed among the populations of L. mutabilis in contrast to monomorphism among the species in the section Albus. A limited polymorphism was detected between the mutant lines of L. mutabilis. We conclude from this study that the mitochondrial RFLP analysis is a valuable tool for identification of variability among Andean lupin populations.     

EFFECTS OF DIFFERENTIAL POLLEN-TUBE GROWTH ON HYBRIDIZATION IN THE LOUISIANA IRISES

To elucidate the importance of hybridization in evolution, it is necessary to understand the processes that affect hybridization frequency in nature. Here we focus on postpollination, prefertilization isolating mechanisms using two hybridizing species of Louisiana iris as a study system. We compared the effects of differential pollen-tube growth on the frequency of F₁ hybrid formation in experimental crosses between Iris fulva and Iris hexagona. Analyses of seed production in fruits from pure conspecific and heterospecific pollinations revealed that more seeds were produced in the top half than the bottom half of fruits for all four crosses. Heterospecific pollen was applied to flowers of each species at zero to 24 h prior to conspecific pollen, thereby giving a head start to the foreign pollen. Using diagnostic isozyme markers, the frequency of hybrid progeny was examined at the level of the whole fruit and separately for the top and bottom halves of fruits. In both species, the proportion of hybrid seeds per fruit increased significantly with increasing head starts, suggesting that differences in pollen-tube growth rates affect the frequency of hybridization. In I. fulva fruits, the increase in hybrid seeds occurred in both halves of the fruits, but in I. hexagona an increase was only detected in the top half of fruits. These findings are consistent with a model that assumes attrition of pollen tubes due to the greater length of I. hexagona styles. While pollen-tube growth rate appears to be the most important factor affecting hybridization frequency in I. fulva, both pollen-tube growth rate and pollen-tube attrition appear to be important in I. hexagona.     

Interspecific Pollen-Pistil Interaction in Eucalyptus L'Her. (Myrtaceae): The Effect of Taxonomic Distance

The pollen-pistil interaction was investigated in three intraspecific,57 interspecific and six intergeneric crosses using three speciesof Eucalyptus (Myrtaceae) subgenus Symphyomyrtus, section Bisectariaas female parents Interspecific prefertilization isolation occursin the pistil and manifests as a number of pollen-tube abnormalitiesin the style and ovary associated with a lowered probabilityof ovule penetration The major selection points in the pistilare the upper style and the ovary The seventy of abnormalitiesand the probability of pollen-tube arrest in the pistil wasproportional to the taxonomic distance between parent speciesOvule penetrations were seen mainly in crosses within the sectionBisectaria or between the sections Bisectaria and Adnataria Pollen storage, style length and mean maximum temperature duringthe flowering period of the male parent had no significant effecton pollen-tube growth in the crosses used Mechanisms of reproductiveisolation are discussed in relation to evolutionary relationshipsand the implications for taxonomic groupings Eucalyptus L'Hér, pollen-pistil interaction, incongruity, interspecific hybridization, pollen-tube growth, breeding system, taxonomy     

CONTRIBUTION OF CRYPTIC INCOMPATIBILITY TO THE MATING SYSTEM OF EICHHORNIA PANICULA TA (PONTEDERIACEAE)

Tristylous populations of the annual aquatic Eichhornia paniculata have high levels of outcrossing and intermorph mating despite being fully self- and intramorph compatible. Experimental studies of pollen germination, ???pollen-tube growth, and pollinations with mixtures of genetically marked pollen were used to determine whether postpollination processes contribute to the observed mating patterns. Differences in pollen germination were small and did not contribute to differences in pollen siring ability. The fraction of pollen tubes first entering the ovary, however, was greater for legitimate outcross pollen than for either of the other two pollen types (self or outcross illegitimate pollen) in all recipient morphs. Moreover, legitimate pollen had higher siring success when in competition with illegitimate pollen types (self or outcross) in each recipient style morph. The ranking of pollen performance for different pollen-style combinations was the same for both the pollen-tube growth and marker-gene experiments indicating that differences in pollen-tube growth rate are the principal cause of differences in pollen siring ability. Cryptic incompatibility in E. paniculata may represent a weak heteromorphic incompatibility system because the observed patterns of pollen-tube growth parallel pollen-tube growth and seed-set patterns that occur in related species with strong trimorphic incompatibility. The ability to produce mostly outcrossed progeny when pollinators are abundant, but to reliably produce seed under a variety of environmental and demographic conditions may be favored in E. paniculata because of its colonizing life history and occurrence in ephemeral habitats. Cryptic incompatibility may be more likely to occur in species subject to wide fluctuations in population size and levels of pollinator service.     

Relationships of pollen size,pistil length and pollen tube growth rates in Rhododendron and their influence on hybridization

Summary Pollen size and pistil length data have been collected for 93 species of Rhododendron (Ericaceae) belonging to a number of different subgeneric taxa. For a sample of eight species in section Vireya, pollen tube growth in the style after selfor interspecific pollination has been quantified. Pollen volume and the time taken for pollen tubes to reach the ovary were both related to pistil length. Pollen-tube growth rates were generally greater for species with longer pistils and larger pollen. Increasing temperature increased the rate of pollen-tube growth. There was no detectable effect of pollen tube density on tube growth rate in the style. After interspecific pollinations tube growth rates in foreign styles could be faster or slower than in self styles. A semisterile individual with two viable pollen grains per tetrad and a plant grafted as scion to a longer-styled stock both showed more rapid pollen-tube growth than expected on the basis of pistil size. Data collected for 26 species in section Vireya showed that where extreme disparity of pollen/pistil size causes failure of interspecific crosses, one or more bridging species with intermediate pollen/pistil size can generally be selected.     

Comparison of gene flow among species that occur within the same geographic locations versus gene flow among populations within species reveals introgression among several Elymus species

One of the challenges in evolutionary biology is to understand the evolution of speciation with incomplete reproductive isolation as many taxa have continued gene flow both during and after speciation. Comparison of population structure between sympatric and allopatric populations can reveal specific introgression and determine if introgression occurs in a unidirectional or bidirectional manner. Simple sequence repeat markers were used to characterize sympatric and allopatric population structure of plant species, Elymus alaskanus (Scribn. and Merr.) Löve, E. caninus L., E. fibrosus (Schrenk) Tzvel., and E. mutabilis (Drobov) Tzvelev. Our results showed that genetic diversity (H_E) at species level is E. caninus (0.5355) > E. alaskanus (0.4511) > E. fibrosus (0.3924) > E. mutabilis (0.3764), suggesting that E. caninus and E. alaskanus are more variable than E. fibrosus and E. mutabilis. Gene flow between species that occurs within the same geographic locations versus gene flow between populations within species was compared to provide evidence of introgression. Our results indicated that gene flow between species that occur within the same geographic location is higher than that between populations within species, suggesting that gene flow resulting from introgressive hybridization might have occurred among the sympatric populations of these species, and may play an important role in partitioning of genetic diversity among and within populations. The migration rate from E. fibrosus to E. mutabilis is highest (0.2631) among the four species studied. Asymmetrical rates of gene flow among four species were also observed. The findings highlight the complex evolution of these four Elymus species.     

DIFFERENTIAL POLLEN-TUBE GROWTH RATES AND NONRANDOM FERTILIZATION IN HIBISCUS MOSCHEUTOS (MALVACEAE)

The prevalence of nonrandom fertilization due to postpollination events has rarely been studied in natural populations, despite important implications for outcrossing rates, mate choice, and plant fitness. Nonrandom paternity within fruits can be caused by both unequal fertilization and unequal embryo abortion. Using self-compatible Hibiscus moscheutos, we studied the potential for nonrandom fertilization by comparing growth rates of pollen-tubes from different donors. The branched style of Hibiscus allowed within-flower comparisons between pollen donors. Relative pollen-tube growth rates were determined by applying pollen from pairs of donors to different stigmas on adjacent stylar branches. We then measured the number of callose plugs per tube in cross-sectional transects across the style after 3 hr. We demonstrate that rates of callose plug formation can be used as a sensitive indicator of relative pollen-tube growth rate. Differences between pollen donors were common and repeatable. Self-pollen-tubes grew slower than outcross pollen-tubes in some crosses and faster in others. Allozyme variation in glucose phosphate isomerase was used to show that individuals with fast-growing pollen-tubes sired a disproportionate number of seeds following mixed pollinations (up to 72%). Since seed abortion was negligible, we conclude that variation in pollen-tube growth rates leads to nonrandom paternity within fruits.     

Ethylene response to pollen tube growth in Nicotiana tabacum flowers

In flowers of Nicotiana tabacum L., pollination induces a transient increase in ethylene production by the pistil. The characteristic dynamics of the increase in ethylene correspond to the main steps of the pollen-tube journey into the pistil: penetration into the stigma, growth through the style, entry into the ovary and fertilization. Ethylene is synthesized de novo in the pistil, and its production is reduced in the dark. Ethylene production was monitored in tobacco flowers after pollination with incongruous pollen from three different Nicotiana species, N. rustica, N. repanda and N. trigonophylla, and with pollen from Petunia hybrida. Pollen from all of these different sources can germinate on the stigma surface but each pollen type shows a different behavior and efficiency in penetrating the pistil tissues. Thus, these different crosses provided a model with which to study the response of the pistil to pollination and fertilization. Ethylene evolution upon pollination in tobacco differed in each cross, suggesting that ethylene is correlated with the response to pollen tube growth in the tobacco flower.     

European yellow lupine, Lupinus luteus, and narrow-leaf lupine, Lupinus angustifolius, as hosts for the pea aphid, Acyrthosiphon pisum

The pea aphid, Acyrthosiphon pisum Harris (Homoptera: Aphididae), fed, developed, and reproduced on yellow lupine, Lupinus luteus L. (Fabaceae: Genisteae). No clear preferences for any variety within L. luteus were found. Acyrthosiphon pisum showed negative values of relative growth rate and no aphid completed development on any variety of narrow-leaf lupine Lupinus angustifolius L. Aphids did not ingest phloem sap while probing on L. angustifolius and the probes were very short. All varieties of L. angustifolius were rejected by aphids during an early stage of probing in peripheral tissues, that is, epidermis or mesophyll. There were qualitative and quantitative differences in alkaloid and soluble sugar content between the two lupine species. Within species, the relative content of individual compounds differed among the varieties. Lupinus angustifolius contained four quinolizidine alkaloids (13-hydroxylupanine, dehydrolupanine, lupanine, and angustifoline), while L. luteus contained two (lupanine and sparteine). Lupanine occurred in all varieties of both lupine species. The total content of soluble carbohydrates was similar in L. luteus and L. angustifolius . The following cyclitols were found in both lupine species: myo -inositol, D-ononitol, and D-pinitol. Lupinus angustifolius also contained D- chiro -inositol. The study of aphid probing behaviour, development, and reproduction demonstrated that L. luteus is a suitable host plant for A. pisum while L. angustifolius is not. It is likely that the rejection of L. angustifolius by A. pisum was caused by chemical factors detected by aphids at the epidermis and mesophyll level.     

Self-incompatibility reactions and compatible pollen-tube growth are retained with in vitro pollinations of sugar beet,Beta vulgaris L.

Summary It is shown that in vitro pollination can be used in future studies of the time course of pollen-tube development and analysis of self-incompatibility in sugar beet, Beta vulgaris L. Upon selfng a self-incompatible genotype showed the same incompatibility response after both in vitro and in vivo pollinations. No differences between cross-compatible and self-compatible pollentube growth were observed. The pollen-tube rejection occurred whether or not the pollen was prehydrated. RNA staining with Acridine Orange showed that there was less cellular RNA in the pistil tissue from in vitro-pollinated flowers. Nevertheless, pollen-tube growth and the self-incompatibility response were similar after in vivo and in vitro pollinations.     

Polymorphic growth rates in myrmecophilous insects

A polymorphism in growth rates was recently described affecting the larval development of the myrmecophilous butterfly Maculinea rebeli, spanning different years in a single insect population. The close integration of M. rebeli into the host ant colonies, facilitated by adaptations in behaviour and chemical mimicry, make extended larval development a successful strategy. Here we present additional data for M. rebeli and new data for Maculinea alcon (another cuckoo-feeding lycaenid) and the two myrmecophilous predators Maculinea arion and Microdon mutabilis (Diptera: Syrphidae). As predicted, M. alcon shows the same growth pattern as M. rebeli with a proportion of caterpillars developing in one year and the remainder over two years. This pattern holds in both northern and southern European populations, where M. alcon exploits different species of host. Against expectation, the same bimodal distribution of pre-pupation body weights, indicating one and two year developers, was found for the larvae of M. arion and M. mutabilis. As predators, both species are less closely integrated in their host ant colonies, suggesting that the polymorphism in growth rates is a more general adaptation to a myrmecophilous life style, arrived at by convergent evolution between the Maculinea and Microdon species. For predatory species we suggest that biennialism is an adaptation to the migratory behaviour of the host made possible by the predators' ability to fast over extended periods. We also hypothesize that M. arion represents an ancestral strategy in Maculinea butterflies and that the growth polymorphism might have become genetically fixed in the cuckoo-feeding species.     

Pollen-tube hypertrophy in ovules of Solanum and Lycopersicon following interspecific crosses

Summary The failure of pollen-tube dissolution in the synergid region, with subsequent pollen-tube overgrowth well into the central cell, was observed within embryo sacs following interspecific crosses between Solanum melongena and S. sisymbriifolium and between Lycopersicon esculentum and L. peruvianum. Such pollen-tube hypertrophy (PTH) was examined under fluorescence using both light and confocal microscopy in order to characterize this anomalous pollen-tube behavior and to verify its occurrence within the embryo sac central cell. Three genetically unrelated L. esculentum cultivars (Peto 95, Apex 1000, Nagcarlang) were utilized as the female parents in interspecific pollinations with L. peruvianum accession LA 1708, and seed set was obtained from all crosses. It was observed that PTH was not a rare event in these crosses, with the proportion of PTH ranging from 2.6% to 6.5% of all ovules examined. Self-pollinated controls revealed zero (e.g., Nagcarlang) to extremely low (<0.01%) frequencies (Apex 1000 and Peto 95). The differences in frequencies suggests that the specific genotypes of the parents has an affect on the occurrence of successful pollen-tube dissolution and sperm-nuclei transfer. Pollen-tube hypertrophy represents a poorly understood, though easily identified, form of fertilization failure occurring within the ovule. Its conspicuous nature, along with a relatively high frequency of occurrence in certain crossing combinations, should provide a valuable opportunity to better study the nature of such fertilization failures in angiosperms.     

Production of intraspecific F1 hybrids between wild and cultivated accessions of cowpea (Vigna unguiculata (L.) walp.) using conventional methods

Cowpea (Vigna unguiculata (L.) Walp.) is an important food legume in the tropics. It belongs to the Phaseoleae (L.) tribe (Fabaceae family), it is diploid and its chromosome number is 22. Its gene pool includes the cultivated cowpea and its wild relatives, which are connected with Vigna subgenus, Catiang section. Cowpea has a great potential in increasing food legume production. The cowpea varieties, however, are susceptible to a number of insect pests, especially the pod borer Maruca testulalis and a pod sucking-bug complex (e.g.: Clavigralla tomentosicollis, Anoplocnemis curvipes and Riptortus dentipes), which cause severe damage. The crossing programme presented here exploits the variability existing in the wild African germplasm of V. unguiculata and cultivated cowpea. To incorporate the insect pest resistance into the cultivated cowpea economically, reciprocal crosses between wild forms and cowpea varieties were performed, using the stigmatic pollination methods at anthesis. Some barriers were found in these intraspecific crosses. In the majority of reciprocal crosses, the growth of the pollen tubes was arrested in the stigmatic tissue. Only 16.01% of the ovules were fertilised. In these ovules, embryo development was normal at about 20-25 days after pollination. The failure of the intraspecific crosses in about 80.7% of the cases is thus the result of the lack of fertilisation and the unfertilised ovules. There seems to exist considerable incompatibility within the primary cowpea gene pool. The breeding programme carried out under controlled conditions has proved to be less successful in developed cowpea intraspecific F1 hybrids. Further studies should concentrate on germplasm from Africa with documented resistance to major insect pests. In addition, the application of techniques for bypassing barriers to hybridisation of parent genotypes should enable these embryos to grow to plants.     

POLLEN-TUBE COMPETITION,SIRING SUCCESS,AND CONSISTENT ASYMMETRIC HYBRIDIZATION IN LOUISIANA IRISES

Postpollination mechanisms can play an important role in limiting natural hybridization in plants. Reciprocal hand pollination experiments were performed to study these mechanisms in two species of Louisiana iris: Iris brevicaulis and I. fulva. Relative pollen-tube growth rates changed significantly through time, with I. fulva tubes increasingly outperforming I. brevicaulis tubes in both conspecific and heterospecific styles. However, this pattern of change in relative performance was a poor predictor of siring success: the majority of seeds sired by both maternal species was conspecific rather than hybrid. Experimental crosses and field studies show consistent asymmetric hybridization in Louisiana irises, with I. fulva being a more successful father and a more selective mother than both I. brevicaulis and a third species, I. hexagona. The cause of this pattern is not yet clear, but the pattern itself is unusual. Typically, short-styled species tend to be less successful in reciprocal crosses than long-styled relatives, but I. fulva has shorter styles than either I. brevicaulis or I. hexagona. The effects of pollen-tube competition, differential fertilization, and selective abortion in causing this pattern of asymmetric hybridization is discussed.     

Maternal inheritance of the chloroplast genome in Eucalyptus globulus and interspecific hybrids.

The utility of chloroplast DNA (cpDNA) in Eucalyptus, either as a molecular marker for genetic studies or as a potential vehicle for genetic manipulation, is based on knowledge of its mode of inheritance. Chloroplast inheritance in angiosperms can vary among and within species, and anomalous inheritance has been reported in some interspecific-hybrid combinations. In Eucalyptus, abnormalities of pollen-tube growth occur in a number of interspecific-hybrid combinations, and this might increase the likelihood of anomalous chloroplast transmission. We used a rapid PCR technique to determine chloroplast heritability in 425 progeny of Eucalyptus, comprising 194 progeny of the premier pulpwood species E. globulus and 231 interspecific hybrids between E. globulus and E. nitens (F1, F2, and backcrosses). At this sampling intensity, no pollen-mediated transmission of cpDNA was found in any of the 40 families tested. The results are discussed with reference to chloroplast engineering and the use of cpDNA as a seed-specific marker in phylogeographic studies of Eucalyptus.     

Genetic basis of seed setting in alfalfa

Summary The genetic basis of seed setting was evaluated in seven clones of alfalfa selected under predominantly self-pollinating conditions. They were hand crossed in all possible combinations. Their compatibility was studied by the percentage of flowers forming pods and number of seeds per pod during crossing. The variances for GCA, SCA and reciprocal effects were significant for percentage of pod set with a narrow sense héritability of 64 %. This suggested maternal influence of clones on percent pod set, controlled primarily by additive genetic components. GCA was the only significant component for number of seeds per pod with a narrow sense heritability of 71%. There were wide differences between the clones in their relative magnitude of GCA, SCA and reciprocal effects for both traits used as compatibility indexes. Performance of the diallel crosses was judged by studying seed yield and its related characters, namely seeds per pod, dry matter per plant, frost resistance, plant vigor and plant height. Although GCA and SCA variances were significant for all characters, reciprocal differences in general were absent. The SCA values were very high as compared to GCA. Narrow sense heritability values were very low while broad sense heritability were much higher. This suggested that almost none of the variation was due to additive genetic components and all the variability is controlled by interactions of a digenic, trigenic and quadrigenic nature and heterzozygosity. Heterosis was evaluated by comparing the seed yield of single crosses with their mid-parent and high-parent, and very high values were observed. Thus selection of better genes may not be feasible and further improvement in selected clones may have to be brought about by utilization of various interactions and heterosis. An attempt was made to find combinations of characters that may be used for the selection of seed yield but none were found to be satisfactory.     

P Nutrition during Seed Development : Leaf Senescence, Pod Retention, and Seed Weight of Soybean

Soybean (Glycine max [L.] Merrill) leaf senescence, which may partially result from mineral redistribution, appears to limit grain yield. Two experiments were designed to test the effects of supplemental inorganic phosphate (Pi), K, malate, and methionine (Met) infusions on senescence and yield. A novel stem infusion technique using pediatric intravenous kits was developed to supply these nutrients throughout seed growth. An average of 48.4 milliliters per plant was successfully infused into lower stem internodes during a 4 to 6 week period. Senescence was unaffected by K or malate infusions, but was delayed by Pi infusions (up to 8 days) and by increased nutrient solution Pi levels (up to 21 days) in separate experiments. Treatments which delayed senescence also improved yield as much as 3-fold, due primarily to increased pod retention and secondarily to increased seed size. Met infusions further increased pod retention at the lower, infused nodes, and thus increased total plant yield also. The influence of higher Pi levels during reproductive growth on soybean pod retention and yield may have been the result of sustained sucrose export due to altered C partitioning in leaves. The role of Met in improving yield was not clear. However, these results clearly demonstrate the importance of adequate Pi for delaying senescence and improving pod retention and yield.