Genetic fingerprinting for determining the mode of reproduction in Paspalum notatum, a subtropical apomictic forage grass

 Paspalum is an important genus of the family Gramineae that includes several valuable forage grasses. Many of the species are polyploid and either obligate or facultative apomicts. Cyto-embryological observations of several tetraploid genotypes of P. notatum were performed to determine their mode of reproduction. Afterwards, selfed progenies of the genotypes F131, Q3664 and Q4117 were analysed using RFLP and RAPD genetic fingerprints to identify maternal and non-maternal (aberrant) plants, and to establish the degree of apomictic reproduction. Five maize clones and six primers were used for detecting genetic deviations from the maternal profile. Maize clones umc379, umc384 and umc318 and primers OPG10 and OPI4 were the most informative for discriminating between maternal and aberrant individuals within the progenies of F131 and Q3664. The combined results of three RFLP clones or 4–6 RAPD primers were necessary to ascertain the mode of reproduction in plants F131 and Q3664. The results obtained with the RFLP and RAPD markers were in agreement with the cyto-embryological studies in ascertaining the mode and degree of apomictic reproduction. Plant F131 showed a completely sexual reproductive behaviour, Q3664 an elevated expression of sexuality, while Q4117 was highly apomictic. A fingerprint analysis of an outcrossing population, aimed at the identification of hybrid plants, was also performed. Maize clones um318 and umc379 and primers OPC2 and OPC9 were used. The presence of specific bands belonging to the male parent permitted a rapid and easy detection of hybrids. The methodology described here can be applied both for the characterisation of P. notatum populations and to identify hybrid progenies in Paspalum breeding programs. Received: 5 March 1997 / Accepted: 13 May 1997     

Comparative mapping reveals partial conservation of synteny at the apomixis locus in<Emphasis Type="Italic"> Paspalum</Emphasis> spp<Emphasis Type="Italic">.</Emphasis>

In plants, gametophytic apomixis is a form of asexual reproduction that leads to the formation of seed-derived offspring that are genetically identical to the mother plant. A common set of RFLP markers, including five rice anchor markers previously shown to be linked to apomixis in Paspalum simplex, were used to detect linkage with apomixis in P. notatum and P. malacophyllum. A comparative map of the region around the apomixis locus was constructed for the three Paspalum species, and compared to the rice map. The locus that controls apomixis in P. simplex was almost completely conserved in the closely related species P. malacophyllum, whereas it was only partially represented in the distantly related species P. notatum. Although strong synteny of markers was noted between this locus and a portion of rice chromosome 12 in both P. simplex and P. malacophyllum, the same locus in P. notatum was localized to a hybrid chromosome which carries markers that map to rice chromosomes 2 and 12. All three Paspalum species showed recombination suppression at the apomixis locus; in the case of P. notatum, this might be due to a heterozygosity for a translocation that most probably negatively interferes with chromosomal pairing near the locus. A common set of markers that show linkage with apomixis in all three Paspalum species define a portion of the apomixis-controlling locus that is likely to contain genes critical for apomictic reproduction.Communicated by R. Hagemann     

Genetic characterization of apospory in tetraploid Paspalum notatum based on the identification of linked molecular markers

Tetraploid Paspalum notatum (bahiagrass) is a valuable forage grass with aposporous apomictic reproduction. In a previous study, we showed that apospory in bahiagrass is under the control of a single dominant gene with a distorted segregation ratio. The objective of this work was to identify molecular markers linked to apospory in tetraploid P. notatum and establish a preliminary syntenic relationship with the genomic region associated with apospory in P. simplex. A F₁ population of 290 individuals, segregating for apospory, was generated after crossing a completely sexual plant (Q4188) with a natural aposporous apomictic plant (Q4117). The whole progeny was classified as sexual or aposporous by embryo sacs analysis. A bulked segregant analysis was carried out to identify molecular markers co-segregating with apospory. Four hundred RAPD primers, 30 AFLP primers combinations and 85 RFLP clones were screened using DNA from both parental genotypes and aposporous and sexual bulks. Linkage analysis was performed with cytological and genetic information from the complete progeny. Cytoembryological analysis showed 219 sexual and 71 aposporous F₁ individuals. Seven different molecular markers (2 RAPD, 4 AFLP and 1 RFLP) were found to be completely linked to apospory. The RFLP probe C1069, mapping to the telomeric region of the long arm of rice chromosome 12, was one of the molecular markers completely linked to apospory in P. notatum. This marker had been previously associated with apospory in P. simplex. A preliminary map of the chromosome region carrying the apospory locus was constructed.     

Patterns of plastid and nuclear variation among apomictic polyploids of Hieracium: evolutionary processes and taxonomic implications

Background and Aims

Apomictic species (with asexual seed production) make up for 20–50 % of all taxonomically recognized species in northern Europe, but the phylogenetic relationships of apomictic species and the mode of evolution and speciation remain largely unknown and their taxonomy is consequently disputed.

Methods

In the present study, plastid psbD-trnT sequences (349 accessions) and 12 nuclear microsatellite loci (478 accessions) were used to create an overview of the molecular variation in (mainly) northern European members of the most species-rich of all plant genera, Hieracium s.s. The results are discussed and interpreted in the context of morphological and cytological data on the same species.

Key Results and Conclusions

The complete psbD-trnT alignment was 1243 bp and 50 polymorphisms defined 40 haplotypes. All haplotypes found in the sections of the genus distributed in the northern European lowlands fell into one of two main groups, group H and group V, mutually separated by seven or eight polymorphisms. All accessions belonging to H. sects. Foliosa, Hieracioides (viz. H. umbellatum) and Tridentata and all but one accession of triploid species of H. sects. Oradea and Vulgata showed haplotypes of group V. Haplotypes of group H were found in all accessions of H. sects. Bifida and Hieracium and in all tetraploid representatives of H. sects. Oreadea and Vulgata. Additional haplotypes were found in accessions of the genus Pilosella and in southern European and Alpine sections of Hieracium. In contrast, the distribution of individual haplotypes in the two major groups appeared uncorrelated with morphology and current taxonomy, but polymorphisms within species were only rarely encountered. In total, 160 microsatellite alleles were identified. Levels of variation were generally high with only nine pairs of accessions being identical at all loci (in all cases representing accessions of the same species). In the neighbor-joining analysis based on the microsatellite data, accessions of the same species generally clustered together and some smaller groups of species congruent with morphology and/or current taxonomy were recovered but, except for H. sect. Oreadea, most larger groups were not correlated with morphology. Although the plastid DNA sequences show too little variation and the nuclear microsatellites are too variable to resolve relationships successfully among species or to fully understand processes of evolution, it is concluded that both species and sections as defined by morphology are largely congruent with the molecular data, that gene flow between the sections is rare or non-existent and that the tetraploid species may constitute the key to understanding evolution and speciation in this genus.     

Coordinating cell polarity and cell cycle progression: what can we learn from flies and worms?

Spatio-temporal coordination of events during cell division is crucial for animal development. In recent years, emerging data have strengthened the notion that tight coupling of cell cycle progression and cell polarity in dividing cells is crucial for asymmetric cell division and ultimately for metazoan development. Although it is acknowledged that such coupling exists, the molecular mechanisms linking the cell cycle and cell polarity machineries are still under investigation. Key cell cycle regulators control cell polarity, and thus influence cell fate determination and/or differentiation, whereas some factors involved in cell polarity regulate cell cycle timing and proliferation potential. The scope of this review is to discuss the data linking cell polarity and cell cycle progression, and the importance of such coupling for asymmetric cell division. Because studies in model organisms such as Caenorhabditis elegans and Drosophila melanogaster have started to reveal the molecular mechanisms of this coordination, we will concentrate on these two systems. We review examples of molecular mechanisms suggesting a coupling between cell polarity and cell cycle progression.     

Alien introgression in the grasses Lolium perenne (perennial ryegrass) and Festuca pratensis (meadow fescue): the development of seven monosomic substitution lines and their molecular and cytological characterization

Background and Aims

To address the issues associated with food security, environmental change and bioenergy in the context of crop plants, the production, identification and evaluation of novel plant phenotypes is fundamental. One of the major routes to this end will be wide hybridization and introgression breeding. The transfer of chromosomes and chromosome segments between related species (chromosome engineering or alien introgression) also provides an important resource for determining the genetic control of target traits. However, the realization of the full potential of chromosome engineering has previously been hampered by the inability to identify and characterize interspecific introgressions accurately.

Methods

Seven monosomic substitution lines have been generated comprising Festuca pratensis as the donor species and Lolium perenne as the recipient. Each of the seven lines has a different L. perenne chromosome replaced by the homoeologous F. pratensis chromosome (13 L. perenne + 1 F. pratensis chromosome). Molecular markers and genomic in situ hybridization (GISH) were used to assign the F. pratensis chromosomes introgressed in each of the monosomic substitutions to a specific linkage group. Cytological observations were also carried out on metaphase I of meiosis in each of the substitution lines.

Results

A significant level of synteny was found at the macro-level between L. perenne and F. pratensis. The observations at metaphase I revealed the presence of a low level of interspecific chromosomal translocations between these species.

Discussion

The isolation of the seven monosomic substitution lines provides a resource for dissecting the genetic control of important traits and for gene isolation. Parallels between the L. perenne/F. pratensis system and the Pooideae cereals such as wheat, barley, rye, oats and the model grass Brachypodium distachyon present opportunities for a comparison across the species in terms of genotype and phenotype.     

Human hair melanins: what we have learned and have not learned from mouse coat color pigmentation

Hair pigmentation is one of the most conspicuous phenotypes in humans. Melanocytes produce two distinct types of melanin pigment: brown to black, indolic eumelanin and yellow to reddish brown, sulfur‐containing pheomelanin. Biochemically, the precursor tyrosine and the key enzyme tyrosinase and the tyrosinase‐related proteins are involved in eumelanogenesis, while only the additional presence of cysteine is necessary for pheomelanogenesis. Other important proteins involved in melanogenesis include P protein, MATP protein, α‐MSH, agouti signaling protein (ASIP), MC1R (the receptor for MSH and ASIP), and SLC7A11, a cystine transporter. Many studies have examined the effects of loss‐of‐function mutations of those proteins on mouse coat color pigmentation. In contrast, much less is known regarding the effects of mutations of the corresponding proteins on human hair pigmentation except for MC1R polymorphisms that lead to pheomelanogenesis. This perspective will discuss what we have/have not learned from mouse coat color pigmentation, with special emphasis on the significant roles of pH and the level of cysteine in melanosomes in controlling melanogenesis. Based on these data, a hypothesis is proposed to explain the diversity of human hair pigmentation.     

Hydatid control in Australia: where it began, what we have achieved and where to from here

Echinococcus granulosus was imported into Australia with domestic livestock about 200 years ago. It spread rapidly through domestic animals and quickly became a public health problem in the new colony. Control was hampered by ignorance of the transmission pattern. The association between metacestodes and tapeworms was not elucidated until 63 years after the arrival of the First Fleet. Australian wildlife were highly susceptible to infection with E. granulosus and wildlife/domestic animal interaction facilated rapid infiltration of wildlife by E. granulosus. The wildlife reservoir has hampered hydatid control campaigns on mainland Australia but successful eradication has been achieved in the island state of Tasmania where there was no wildlife reservoir. The application of a new recombinant vaccine for sheep in control campaigns and the use of praziquantel baits for controlling infection in dingoes around bush campsites and picnic areas is discussed.     

Evolutionary biology of starvation resistance: what we have learned from Drosophila

Most animals face periods of food shortage and are thus expected to evolve adaptations enhancing starvation resistance (SR). Most of our knowledge of the genetic and physiological bases of those adaptations, their evolutionary correlates and trade-offs, and patterns of within- and among-population variation, comes from studies on Drosophila. In this review, we attempt to synthesize the various facets of evolutionary biology of SR emerging from those studies. Heritable variation for SR is ubiquitous in Drosophila populations, allowing for large responses to experimental selection. Individual flies can also inducibly increase their SR in response to mild nutritional stress (dietary restriction). Both the evolutionary change and the physiological plasticity involve increased accumulation of lipids, changes in carbohydrate and lipid metabolism and reduction in reproduction. They are also typically associated with greater resistance to desiccation and oxidative stress, and with prolonged development and lifespan. These responses are increasingly seen as facets of a shift of the physiology towards a 'survival mode', which helps the animal to survive hard times. The last decade has seen a great progress in revealing the molecular bases of induced responses to starvation, and the first genes contributing to genetic variation in SR have been identified. In contrast, little progress has been made in understanding the ecological significance of SR in Drosophila; in particular it remains unclear to what extent geographical variation in SR reflect differences in natural selection acting on this trait rather than correlated responses to selection on other traits. Drosophila offers a unique opportunity for an integrated study of the manifold aspects of adaptation to nutritional stress. Given that at least some major molecular mechanisms of response to nutritional stress seem common to animals, the insights from Drosophila are likely to apply more generally than just to dipterans or insects.     

Uncoupling of sexual reproduction from homologous recombination in homozygous Oenothera species

Salient features of the first meiotic division are independent segregation of chromosomes and homologous recombination (HR). In non-sexually reproducing, homozygous species studied to date HR is absent. In this study, we constructed the first linkage maps of homozygous, bivalent-forming Oenothera species and provide evidence that HR was exclusively confined to the chromosome ends of all linkage groups in our population. Co-segregation of complementary DNA-based markers with the major group of AFLP markers indicates that HR has only a minor role in generating genetic diversity of this taxon despite its efficient adaptation capability. Uneven chromosome condensation during meiosis in Oenothera may account for restriction of HR. The use of plants with ancient chromosomal arm arrangement demonstrates that limitation of HR occurred before and independent from species hybridizations and reciprocal translocations of chromosome arms-a phenomenon, which is widespread in the genus. We propose that consecutive loss of HR favored the evolution of reciprocal translocations, beneficial superlinkage groups and ultimately permanent translocation heterozygosity.     

Genetic relationship among Paspalum species of the subgenus Anachyris: Taxonomic and evolutionary implications

Paspalum is one of the most important genera of the Poaceae family due to its large number of species and diversity. The subgenus Anachyris comprises six species mainly from South America grouped together by sharing rare spikelet characteristics. A genetic analysis using ISSR markers, compared with the morphological and phenotypic variation observed in each one species, was used to establish genetic relationships among 40 accessions with several ploidy levels, belonging to 5 species of the subgenus Anachyris. Fourteen accessions of Paspalum malacophyllum (2x and 4x), 12 of P. simplex (2x, 3x, 4x and 6x), 4 of P. procurrens (2x and 4x), 4 of P. usterii (4x) and 6 of P. volcanensis (4x) were analysed. A total of 227 ISSR loci (98.7% polymorphic) were detected among all accessions, with variable loci number and percentages of polymorphism according to species delimitations. Six main groups were identified by cluster analysis based on Jaccard's genetic distance and UPGMA, four of which matched all the respective accessions of P. simplex, P. procurrens, P. usterii and P. volcanensis, while the other two were consistent with two different groups of accessions of P. malacophyllum, one involving most tetraploid accessions, and the other one grouping together a tetraploid and two diploid accessions. The distinctive morphological characteristics and the separate clustering of these tetraploid and diploid cytotypes suggest to consider a new multiploid species complex inside the subgenus Anachyris. Both cytotypes of P. procurrens, and the four co-specific cytotypes of P. simplex consistently clustered together forming two specific groups for the two multiploid taxons. This is in agreement with the existence of high phenotypic similarities between diploid and tetraploid cytotypes of P. procurrens, and among diploid, triploid, tetraploid and hexaploid cytotypes of P. simplex. Since the polyploid cytotypes of these species are reproduced by apomixis, the specific genetic clustering by ISSR markers and morphological and cytological results support the hypothesis that the two multiploid species were originated by autopolyploidy. Our results confirm previous studies suggesting a monophyletic origin for the subgenus Anachyris and are concordant with previous data regarding genomic homologies and phylogenetic analyses in the genus.     

Morphological and Ecological Characterization of Steinernema feltiae (Rhabditida: Steinernematidae) Rioja Strain Isolated from Bibio hortulanus (Diptera: Bibionidae) in Spain

A new strain of Steinernema feltiae (Rhabditida: Steinernematidae) was isolated in La Rioja (Spain) from larvae of Bibio hortulanus (Diptera: Bibionidae). A comparative morphometric analysis of this new strain and four additional S. feltiae isolates was performed. Although significant differences in morphometric measurements were observed, PCR-RFLP profiles and sequence analysis of the ITS region of rDNA confirmed the identity of the new strain as A2 RFLP type of S. feltiae. A comparative morphometric study among nematodes from three hosts, Galleria mellonella (Lepidoptera: Pyralidae), Spodoptera littoralis (Lepidoptera: Noctuidae) and B. hortulanus, was conducted. Ecological characterization of the Rioja isolate was performed in G. mellonella larvae. Larval mortality was 75.3 and 78.12% in penetration and sand column assays, respectively, and the percentage of penetrating infective juveniles was 12.0 and 2.8% in these assays. Larval mortality in the one-on-one bioassay was 4.2%, and in exposure-time bioassays, it was 50% at 11.25 hours. Relationships between morphometric characteristics and host mortality are discussed for this new strain of entomopathogenic nematode.     

Reproductive differentiation into sexual and apomictic polyploid cytotypes in Potentilla puberula (Potentilleae, Rosaceae)

Background and Aims

Intraspecific reproductive differentiation into sexual and apomictic cytotypes of differing ploidy is a common phenomenon. However, mechanisms enabling the maintenance of both reproductive modes and integrity of cytotypes in sympatry are as yet poorly understood. This study examined the association of sexual and apomictic seed formation with ploidy as well as gene flow towards sexuals within populations of purely polyploid Potentilla puberula.

Methods

The study is based on 22 populations representing various combinations of five polyploid cytotypes (tetraploid–octoploid) from East Tyrol, Austria. Embryo ploidy and the endosperm/embryo ploidy ratio obtained by a flow cytometric seed screen were used to infer reproductive modes of seed formation and to calculate the male and female genomic contributions to the embryo and endosperm. Self-incompatibility (SI) patterns were assessed and a new indirect approach was used to test for the occurrence of intercytotype matings based on the variation in the male genomic contribution to sexually derived embryos on the level of developed seed.

Key Results

Tetraploids formed seeds almost exclusively via sexual reproduction, whereas penta- to octoploids were preferentially apomictic. Non-random distribution of reproductive modes within maternal plants further revealed a tendency to separate the sexual from the apomictic mode among individuals. Self-incompatibility of sexuals indicated functionality of the gametophytic SI system despite tetraploidy of the nuclear genome. We found no indication for significant cross-fertilization of tetraploids by the high polyploids.

Conclusions

The study revealed a rare example of intraspecific differentiation into sexual and apomictic cytotypes at the polyploid level. The integrity of the sexual tetraploids was maintained due to reproductive isolation from the apomictic higher polyploids. Functionality of the gametophytic SI system suggested that the tetraploids are functional diploids.     

Tetraploidization events by chromosome doubling of nucellar cells are frequent in apomictic citrus and are dependent on genotype and environment

Background and Aims

Polyploidy is a major component of plant evolution. The citrus gene pool is essentially diploid but tetraploid plants are frequently encountered in seedlings of diploid apomictic genotypes. The main objectives of the present study were to establish the origin of these tetraploid plants and to ascertain the importance of genotypic and environmental factors on tetraploid formation.

Methods

Tetraploid seedlings from 30 diploid apomictic genotypes were selected by flow cytometry and genotyped with 24 single sequence repeat (SSR) markers to analyse their genetic origin. Embryo rescue was used to grow all embryos contained in polyembryonic seeds of ‘Tardivo di Ciaculli’ mandarin, followed by characterization of the plantlets obtained by flow cytometry and SSR markers to accurately establish the rate of tetraploidization events and their potential tissue location. Inter-annual variations in tetraploid seedling rates were analysed for seven genotypes. Variation in tetraploid plantlet rates was analysed between different seedlings of the same genotype (‘Carrizo’ citrange; Citrus sinensis × Poncirus trifoliata) from seeds collected in different tropical, subtropical and Mediterranean countries.

Key Results

Tetraploid plants were obtained for all the studied diploid genotypes, except for four mandarins. All tetraploid plants were identical to their diploid maternal line for SSR markers and were not cytochimeric. Significant genotypic and environmental effects were observed, as well as negative correlation between mean temperature during the flowering period and tetraploidy seedling rates. The higher frequencies (20 %) of tetraploids were observed for citranges cultivated in the Mediterranean area.

Conclusions

Tetraploidization by chromosome doubling of nucellar cells are frequent events in apomictic citrus, and are affected by both genotypic and environmental factors. Colder conditions in marginal climatic areas appear to favour the expression of tetraploidization. Tetraploid genotypes arising from chromosome doubling of apomictic citrus are extensively being used as parents in breeding programmes to develop seedless triploid cultivars and have potential direct use as new rootstocks.     

Genetic variability in Brazilian wheat cultivars assessed by microsatellite markers

Wheat (Triticum aestivum) is one of the most important food staples in the south of Brazil. Understanding genetic variability among the assortment of Brazilian wheat is important for breeding. The aim of this work was to molecularly characterize the thirty-six wheat cultivars recommended for various regions of Brazil, and to assess mutual genetic distances, through the use of microsatellite markers. Twenty three polymorphic microsatellite markers (PMM) delineated all 36 of the samples, revealing a total of 74 simple sequence repeat (SSR) alleles, i.e. an average of 3.2 alleles per locus. Polymorphic information content (PIC value) calculated to assess the informativeness of each marker ranged from 0.20 to 0.79, with a mean of 0.49. Genetic distances among the 36 cultivars ranged from 0.10 (between cultivars Ocepar 18 and BRS 207) to 0.88 (between cultivars CD 101 and Fudancep 46), the mean distance being 0.48. Twelve groups were obtained by using the unweighted pair-group method with arithmetic means analysis (UPGMA), and thirteen through the Tocher method. Both methods produced similar clusters, with one to thirteen cultivars per group. The results indicate that these tools may be used to protect intellectual property and for breeding and selection programs.     

Comparative Estimation of Genetic Diversity in Population Studies using Molecular Sampling and Traditional Sampling Methods

Entomopathogenic nematodes (EPN) are efficient biological pest control agents. Population genetics studies on EPN are seldom known. Therefore, it is of interest to evaluate the significance of molecular sampling method (MSM) for accuracy, time needed, and cost effectiveness over traditional sampling method (TSM). The study was conducted at the Mohican Hills golf course at the state of Ohio where the EPN H. bacteriophora has been monitored for 18 years. The nematode population occupies an area of approximately 3700 m² with density range from 0.25-2 per gram soil. Genetic diversity of EPN was studied by molecular sampling method (MSM) and traditional sampling method (TSM) using the mitochondrial gene pcox1. The MSM picked 88% in compared to TSM with only 30% of sequenced cox 1 gene. All studied genetic polymorphism measures (sequence and haplotype) showed high levels of genetic diversity of MSM over TSM. MSM minimizes the chance of mitochondrial genes amplification from non target organisms (insect or other contaminating microorganisms). Moreover, it allows the sampling of more individuals with a reliable and credible representative sample size. Thus, we show that MSM supersedes TSM in labour intensity, time consumption and requirement of no special experience and efficiency.