Plants regenerated from embryo cultures of an apomictic clone of Kentucky bluegrass (Poa pratensis L. ‘Baron’) are not apomictic in origin

Plants were regenerated from tissue cultures of embryos dissected from seeds that were harvested from a self-pollinated clonal selection of Kentucky bluegrass (Poa pratensis L.) ‘Baron’, an apomictic cultivar. Plants were regenerated from 35 embryo-derived callus cultures of the 3280 embryos that were plated. Flow-cytometric (FCM) and RAPD-marker analyses were performed to determine if regenerants were or were not apomictic in origin. Fifteen regenerants with a 3c DNA content were classified as arising from 2n + n aberrant embryos, which was a higher frequency than expected, based on a chi-square analysis. Of the remaining 20 regenerants with a 2c DNA content, a chi-square test showed that all could have arisen from n + n sexually-derived embryos, based on the observed segregation of n + n regenerants, which fit the expected 3:1 ratio of dominant:recessive RAPD-marker phenotypes. The apparent lack of regenerants of apomictic origin, and implications for genetic transformation and breeding of Kentucky bluegrass are discussed.     

Evolution and adaptedness in a facultatively apomictic grass, Poa pratensis L.

Summary Facultative apomixis confers great adaptedness and evolutive potential on biotypes belonging to the Poa pratensis L. agamic complex. Pioneering and recent research have dealt with and sometimes elucidated different aspects of apomictic expression, such as cytological mechanisms, distribution patterns, genetic control and environmental effects, in this species. We carried out an investigation on the mode of reproduction expressed in Italian natural populations, cultivars and selected genotypes together with an extensive time (56 years) and space (six countries) review in order to obtain a comprehensive picture of apomixis expression in P. pratensis. Histograms of the estimations were prepared. They showed that, due to breeder and natural selection, both cultivars and natural populations very frequently expressed a high degree of apomixis. Variations in the degree of apomixis across generations, different pollen donors, environments and estimation levels were generally low. It would, therefore, seem that the tenet which holds that apomixis in P. pratensis is strongly influenced by external factors should be reconsidered. Despite the fact that little is known of the genetic, physiological and molecular control underlying apomixis, the overall picture that emerges from our study helps to explain how the balance between sexual and asexual reproduction confers extreme versatility, adaptedness and evolutive capacity on this remarkable grass.     

Regeneration and somaclonal variation in apomictic Paspalum dilatatum Poir

Summary In an attempt to incorporate variation into a uniform obligate apomict, plants of apomictic common dallisgrass, Paspalum dilatatum Poir., were regenerated from callus derived from immature inflorescences. Plants developed through both organogenesis and embryogenesis. A total of 682 regenerants were produced and more than 400 were transplanted into a field nursery and screened for somaclonal variation. Eventually 20 regenerants were selected, increased, and planted into a replicated nursery along with normal common dallisgrass. The characteristics examined were maturity date, plant height, number of racemes per inflorescence, number of spikelets per raceme, pubescence, stigma and anther color, ergot resistance, seed germination, seed set, pollen stainability, method of reproduction, and chromosome number. There were differences among the regenerants and between them and common dallisgrass for all traits except chromosome number, stigma and anther color, and ergot resistance. One of the more important regenerants had significantly higher seed set than common dallisgrass. All regenerants reproduced by aposporous apomixis but some exhibited a high degree of abortion while others had more aposporous embryo sacs per ovule than common dallisgrass. These findings demonstrate that common dallisgrass can be regenerated through tissue culture and that somaclonal variation is expressed in some of the regenerants, even though some of the altered traits are deleterious.     

Karyotype analysis and interspecific hybridisation in three perennial Sesbania species (Leguminosae)

Summary The somatic chromosome number in Sesbania sesban var. nubica, S. goetzei and S. keniensis (Leguminosae; Papilionoidae) was found to be 2n=12. These findings were in agreement with earlier reports on S. sesban and S. keniensis. The chromosome number 2n=12 is a new record for S. goetzei. Similarities in karyotypes were found in the three species. All species had one pair of long metacentric chromosomes; the second pair was submedian, followed by four smaller pairs of metacentric chromosomes. Nucleolar organiser regions in the form of satellites were found on the short arm of the fourth chromosome pair in S. sesban and S. keniensis. Interspecific crosses in all possible combinations were carried out, resulting in pod and viable seed formation for the crosses S. sesban x S. goetzei, S. sesban x S. keniensis, S. goetzei x S. sesban and S. goetzei x S. keniensis. The two crosses with S. keniensis as a female parent were unsuccessful. The hybrid plants established normally and produced viable seeds.     

Screening RAPD primers for molecular taxonomy and cultivar fingerprinting in the genus Actinidia

Summary Eighty ten-base long arbitrary primers were tested for PCR-based DNA amplification of three species of the genus Actinidia (A. deliciosa the kiwifruit, A. chinensis, and A. kolomikta), with the aim of screening species-specific and genotype-specific markers.Of the 80 primers tested, 30 gave an average of 3.5 bands which were monomorphic within one or two species and absent in the remaining one(s), thus resulting in useful markers for taxonomic and phylogenetic purposes. None of the primers tested produced bands linked to sex. Twenty primers out of the twenty-five selected from a preliminary screening showed high levels of polymorphism, producing two to eleven patterns each from the 13 kiwifruit cultivars examined.We found the Stoffel fragment and the Taq polymerase were both suitable for RAPD analysis, the most noticeable difference being the smaller size of fragments (0.4–1.2 kb) produced by the former in comparison to the latter (1.0–3.4 kb). We tested also three different annealing temperatures (35, 37, and 39° C) and found the intermediate one best for number of amplified bands and reproducibility of results.Abbreviations 2-BE 2-butoxyethanol - CTAB hexadecyltrimethylammonium bromide - MAS Marker-Assisted-Selection - PCR Polymerase Chain Reaction - RAPD Random Amplified Polymorphic DNA - RFLPs Restriction Fragment Length Polymorphisms     

Influence of photoperiod on the frequency of sexual embryo saes in facultative apomictic buffelgrass

Summary The frequency at which sexual embryo sacs occur in some facultatively apomictic species is influenced by photoperiod. This research was conducted to study changes in the frequency of sexual embryo sacs (SES) in field-grown accessions of buffelgrass [Pennisetum ciliare (L.) Link] and to determine the influence of photoperiod on the expression of sexuality under controlled environmental conditions. In a 3-yr field study, mean frequency of SES collected from two facultative buffelgrass accessions (PI 409266 and 409277) ranged from 2.5 to 8.6 percent. Within each year, the greatest number of SES were observed during spring and summer, while a reduction in SES occurred during the late summer and early fall. Under controlled environmental conditions equal numbers of SES were observed from plants grown at 8, 12, and 16 h. For the two facultative accessions of buffelgrass examined in this study, it was concluded that no relationship existed between photoperiod and frequency of SES under either field or controlled environmental conditions.     

Evaluating genetic variation and relationships among two bromegrass species and their hybrid using RAPD and AFLP markers

The two most widely grown bromegrass species in North America are smooth bromegrass (Bromus inermis Leyss.), a hay type grass, and meadow bromegrass (Bromus riparius Rehmann), a pasture type grass. Recently a hybrid bromegrass population between the two species has been produced as a dual-purpose hay-pasture grass. Molecular markers have the potential to improve selection procedures to enhance bromegrass breeding. The objective of this study was to use RAPD and AFLP markers to determine genetic relationships and variations among bromegrass populations. Forty-three RAPD markers from 21 primers and 83 AFLP markers from seven primer combinations were used. Both marker types were able to group the individuals into their respective populations. The relationships among the individuals within each of the populations were not similar between the two marker types. Analysis of molecular variance (AMOVA) detected greater within-population variation than among-population variation for both marker types. The highest variation was observed in the hybrid population followed by meadow and then smooth bromegrass. The inter-population distance from both markers indicated that the highest genetic distance was between meadow and smooth bromegrass and lowest between smooth and hybrid bromegrass, which reflect the breeding history of the hybrid population. This study showed that both markers are capable of differentiating bromegrass genotypes into their respective populations, detecting genetic variation and relationships of the populations. Results of this study suggest that these two markers can be used in the future to enhance the current breeding practices in bromegrass, however, AFLP markers would be the marker of choice due to the high number of polymorphic markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chemical induction of apomictic seed formation in maize

Summary Silks of 18 maize (Zea may L.) F₁ hybrids were treated with different combinations of 9 growth regulators, colchicine, and dimethyl sulfoxide (DMSO) for the purpose to induce apomixis (agamospermy) in 1988 and 1989. Hybrid K301 × K303 gave the highest (0.36%) average frequency of seed induction among the hybrids. The most effective treatments were DMSO, gibberellic acid plus 6-benzyl aminopurine (6-BA), and DMSO plus methanesulfonic acid. Individually, the highest frequency of seed induction was 1.4% for hybrid K731×K306 when treated with -naphthalene acetic acid (NAA)-zeatin mixture. The frequency of seed induction seemed to depend partially on the interaction between chemicals and hybrids. Cytological observation of root-tip cells indicated that the majority of the seeds obtained were diploid, some were mixoploid, and a few were haploid. Diploid plants from induced seeds from the same parent were morphologically uniform and resembled the parent. Variations in plant and ear heights were comparable to those of the hybrid parent. Cytological and morphological investigations suggested that the chemically induced seeds originated mainly from somatic tissue but occasionally came from reduced cells in the embryo sac, leading to haploids. The results showed that chemical induction of adventitious embryony in maize hybrids is possible, but the more effective chemicals, their concentrations, and ways of application for increasing the frequency of seed induction need to be explored for practical use.     

Assessment of genetic relationships in Mentha species

A set of 60 random primers was used to analyse 11accessions from six taxa of Mentha developed byCIMAP. These accessions were maintained in the nationalgene bank for medicinal and aromatic plants at CIMAP.A total of 630 bands could be detected as amplifiedproducts upon PCR amplification, out of which 589 werepolymorphic (93.5%). Further analysis of these RAPDprofiles for band similarity indices clearlydifferentiated five of the Mentha arvensis L.accessions from the rest. Among two accessions of Mentha spicata L. CIMAP/C33 could bedistinguished from CIMAP/C32. Mentha × gracilis Sole cv. cardiaca showed a muchhigher similarity with Mentha spicata L. as wellas Mentha arvensis L. which amongst themselvesshowed rather a greater distance indicating that Mentha × gracilis Sole cv. cardiaca might have evolved as a natural hybridbetween M arvensis L. and M. spicataL. In terms of uniqueness of amplified bands fordeveloping RAPD markers, it was observed that at taxalevel 298 bands were unique to one of the six taxa,singly amounting to 47.3% of total amplifiedfragments. Primers MAP 10 and 17 produced polymorphismonly in case of M. spicata L. and Menthaspicata L. cv. viridis while MAP 08 producedpolymorphic bands in all 4 other species than thesetwo. Similarly unique patterns were observeddifferentiating all six species and could be used asRAPD markers for differentiating Mentha species.     

Frequency distributions and linkage relationships of 2-tridecadone in interspecific segregating generations of tomato

Summary The levels of the naturally occurring insecticide 2-tridecadone (2TD) were measured in leaves of Lycopersicon hirsutum f. glabratum, L. esculentum, the interspecific F₁ hybrid, the F₂ and backeross generations. The wild species contains 50 times more 2TD than the cultivated tomato and the frequency distribution of the substance indicates that there is dominance for low levels of the compound. The genotype of F₂ plants was determined with respect to 6 codominant isozyme markers and 4 dominant/recessive markers. Significant associations were detected between 5 of the marker genes and the level of 2TD. We interpret these results as implying linkage between marker genes and genes that control the level of 2TD. The behavior of the gene for the determinant growth habit of the plants suggests that it has a pleiotropic effect on the level of 2TD.     

Species relationships in the subgenus Ceratotropis (genus Vigna) as revealed by RAPD analysis

Summary The genetic variation among 23 accessions of 5 species in the subgenus Ceratotropis, genus Vigna, were investigated by random amplified polymorphic DNA (RAPD) analysis. A total of 404 fragments amplified with 24 primers were scored and analyzed by cluster analysis. The accessions used were separated into two main groups with an average of 70% differences. Within the main groups, five subgroups were recognized, which are in complete agreement with taxonomic species. Wild forms were always grouped with their most closely related cultivated forms and they showed variation in each species. The largest intraspecific variation was found in V. radiata (mungbean), in which wild forms (V. radiata var. sublobata) were highly different from each other and from cultivated forms. V. angularis (adzuki bean) showed the least variation and thus, was probably differentiated in relatively recent times.     

Genetic diversity revealed in the apomictic fruit species Garcinia mangostana L. (mangosteen)

The novel molecular marker technique Randomly Amplified DNA Fingerprinting (RAF)was used to survey genetic relationships between 37 accessions of the tropical fruit G. mangostana (mangosteen) and among 11 accessions from eight other Garcinia species. Although mangosteen is believed to reproduce exclusively through apomixis, our results show that considerable genetic diversity exists within G. mangostana and between other Garcinia species. Among the 37G. mangostana accessions examined, nine different genotypes were identified which clustered into three distinct groups based on correspondence analysis(reciprocal averaging). For 26 (70%) of the accessions no marker variation was detected over 530 loci screened. A further eight (22%) accessions exhibited very low levels of variation (0.2–1%) suggesting at least one well conserved mangosteen genotype. The remaining three accessions (8%) showed extensive variation (22–31%)compared with the majority of accessions. The three mangosteen groups were 63–70% dissimilar to the other Garciniaspecies investigated. The genetic diversity identified in this research will assist in the conservation of Garciniagermplasm and provides a valuable framework for the genetic improvement of mangosteen. This revised version was published online in August 2006 with corrections to the Cover Date.     

Characterization of CMS and maintainer lines in indica rice (Oryza sativa L.) based on RAPD marker analysis

Total DNA from WA type CMS lines: Zhenshan 97 A, Longtepu A and theirmaintainers Zhenshan 97 B, Longtepu B wasextracted by CTAB method. One hundredprimers were used for screening RAPDmarkers to distinguish CMS line (A) andmaintainer (B) plants at seedling stage.Results showed that under the conditions of37 ^°C annealing temperature and1.5 mM MgCl₂ concentration, in Zhenshan97 A, Longtepu A there was a 1600 bp DNAfragment in product amplified by primerOPA12, while in Zhenshan 97 B, and LongtepuB no 1600 bp fragment was found. The 1600 bpfragment was also found in DA type CMS lineXieqingzao A, but was absent in XieqingzaoB. Also in the restorer line, Minghui 63the 1600 bp fragment was absent. In F₁and F₂ generation of Zhenshan97A/Minghui 63, all plants investigated hadthe 1600 bp fragment. When mitochondrial DNA(mtDNA) was isolated from the three CMS (A)and their B lines and amplified by OPA12,results showed that the 1600 fragment wasfound in all the three A lines and wasabsent in the three B lines. In DA typeXieqingzao A, two other fragments (700 bp,1000 bp) were also found except the 1600 bp.These results indicate that the 1600 bpfragment derived from CMS mitochondrial DNAcan be used as a RAPD marker to distinguishA and B plants at seedling stage, and thefragments (700 bp, 1000 bp) can be used todistinguish WA and DA cytoplasms.     

Application of DNA fingerprints for identification and genetic analysis of Carica papaya and other Carica species

Summary Various DNA fingerprint probes were applied to Carica papaya and other Carica species for both identification and genetic analysis.Each of the Carica papaya cultivars is characterized by a specific DNA fingerprints pattern. Various Carica species also have specific patterns which distinguish them from one another. Band sharing levels were used to estimate the relatedness between the various Carica species.Genetic analysis of 11 progeny from a cross between the Carica papaya cultivars 17/82 and 112 suggests that application of DNA fingerprinting to Carica papaya breeding, could make the process more efficient. Genetic analysis of the DNA fingerprint bands revealed no linkage or allelic relationship among the bands analyzed, indicating that these loci are not clustered in the Carica genome.     

Fingerprinting of alfalfa meiotic mutants using RAPD markers

Summary A calendar of female sporogenesis and gametogenesis was made for both apomictic tetraploid (2n=4x=36) Brachiaria brizantha and Brachiaria decumbens and their apomictic F₁ hybrids with sexual tetraploid (2n=4x=36) Brachiaria ruziziensis. Microgametogenesis was used as a reference. Apospory was facultative in both species and hybrids. Environmental conditions had variable effects on the level of apomixis according to each genotype. Ratios of segregation into sexuals and apomicts in the interspecific hybrids suggest an oligogenic determinism with dominant apomixis in the genus Brachiaria. Highly apomictic and partially male fertile hybrids were identified and will be used in an improvement program to transfer genes for apomixis into the sexual species B. ruziziensis.     

Genetic diversity and relationships among Lablab purpureus genotypes evaluated using RAPD as markers

Summary Genetic variation in 40 accessions of Lablab purpureus was evaluated using random amplified polymorphic DNA as markers. A high level of genetic variation in this species was detected but this was mainly restricted to the difference between cultivated and wild forms. Of the cultivated genotypes, genetic variation among Asian collections was significantly higher than that among African collections. The three most divergent cultivated genotypes were all from Asia. Four of the five wild accessions, two from Zimbabwe and the other two from Zambia, were closely related. The other one, CPI 31113 collected from Uganda, was highly divergent. The two commercial forage varieties used in Australia, Rongai from Kenya and Highworth from India, were not very different.     

Genetic relationships among cold, salt and drought tolerance during seed germination in an interspecific cross of tomato

Seed of BC₁ progeny of an interspecific cross between a slow germinating Lycopersicon esculentum breeding line(NC84173; maternal and recurrent parent) and a fast germinating L.pimpinellifolium accession (LA722) were evaluated for germination under cold stress, salt stress and drought stress, and in each treatment the most rapidly germinating seeds (first 2%) were selected. Selected individuals were grown to maturity and self-pollinated to produce BC₁S₁ progeny families. The selected BC₁S₁ progeny from each experiment were evaluated for germination rate in each of a non stress (control),cold-, salt- and drought-stress treatment, and their performances were compared with those of a non selected BC₁S₁population in the same treatments. Results indicated that selection for rapid seed germination in each of the three stress treatments was effective and significantly improved progeny germination rate under all three stress conditions. The results support the suggestion that same genes might control the rate of seed germination under cold, salt and drought stress. Furthermore, selection in each of the three stress treatments resulted in improved progeny seed germination rate under nonstress conditions, suggesting that genetic mechanisms that facilitate rapid seed germination under stress conditions might also contribute to rapid germination under nonstress conditions. In practice, therefore, selection for rapid seed germination under a single stress environment may result in progeny with improved seed germination under a wide range of environmental conditions. Furthermore, to improve germination rate under nonstress conditions, it might be more efficient to make selections under stress conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Chloroplast DNA analysis of eggplant (Solanum melongena) and related species for their taxonomic affinity

Summary Total chloroplast DNA (cpDNA) from Solanum incanum, a wild relative of eggplant, was used to probe total DNA of Solanum melongena (eggplant). The DNA fragments detected were the same as observed using purified chloroplast DNA. Chloroplast DNAs were also analysed for nine species of Solanum that are cross-compatible with eggplant: S. aethiopicum, S. anguivi, S. gilo, S. incanum, S. indicum, S. integrifolium, S. macrocarpon, S. olivare and S. panduriforme.Restriction fragments generated by eight enzymes were recorded as present or absent, and a matrix for all fragment positions, species and enzymes was used for cluster analysis. In the resulting dendrogram, the species tested formed three distinct groups: (1) S. aethiopicum, S. anguivi, S. gilo, S. indicum, S. integrifolium and S. olivare, (2) S. incanum, S. melongena and S. panduriforme, (3) S. macrocarpon. Six species of the first group belonging to section Oliganthes appears more closely related to the second group members belonging to section Melongena than does S. macrocarpon, which also belongs to section Melongena. Within the second group, S. panduriforme is slightly more like eggplant than is S. incanum.     

The genetic diversities of 4 species of subg. Lithocerasus (Prunus, Rosaceae) revealed by RAPD analysis

RAPD analysis was applied to reveal the genetic diversities of 4 speciesof subg. Lithocerasus within the genus Prunus using 40accessions representing the subgenera Prunophora, Amygdalus,Lithocerasus and Cerasus. The accession of subg. Lithocerasus are phenotypically similar to members of subgenus Cerasus but different with them in interspecific crossing tests and isozymeprofiles. Two major clusters, `Prunophora and Amygdalus group' and `Cerasus group' were constructed in the phenogram. We revealthat the examined 4 species of subg. Lithocerasus species; P.tomentosa Thunb., P. japonica Thunb., P. glandulosa Thunb.and P. besseyi Bailey, were genetically closer to the members ofsubgenera Prunophora and Amygdalus than to subg. Cerasus.     

RAPD genetic diversity of Albanian olive germplasm and its relationships with other Mediterranean countries

RAPD markers were used for the study of 19Albanian olive cultivars and two wild olives (oleasters). A total of 76polymorphic bands (4.8 polymorphic markers per primer) out of 107 reproducible were obtained using 16 primers. The number of bands per primer ranged from 4 to 10,whereas the number of polymorphic bands ranged from 1 to 9, corresponding to 71%of the total amplification products. All the accessions could be identified by the combination of four primers: OPA-19;OPA-02; OPK-16 and OPP-19. The dendrogram,based on Jaccard's index, included three major groups according to their origin: 1)most of the cultivars from the area of Berat (South of Albania) 2) cultivars from the Centre and Centre-North of Albania and3) cultivars from the Centre and North-West of Albania along with the oleaster from Elbasan. In order to evaluate the origin of Albanian cultivars they were compared to those diffused in other countries like Greece, Italy and Turkey, due to geographical and historical affinity among these countries, by using a one way AMOVA. Although most of the genetic diversity was attributable to differences among cultivars within each country (91.47%) significantφ-values among countries(φ_st = 0.085; p < 0.001)suggested the existence of RAPD phenotypic differentiation. Significant φ-values in all pairs formed by Albania with the other countries were observed. These results are consistent with the autochthonous origin of Albanian cultivars. This revised version was published online in August 2006 with corrections to the Cover Date.